Combination of PKCε Activation and PTP1B Inhibition Effectively Suppresses Aβ-Induced GSK-3β Activation and Tau Phosphorylation.

PubMed

Kanno, Takeshi; Tsuchiya, Ayako; Tanaka, Akito; Nishizaki, Tomoyuki

2016-09-01

Glycogen synthase kinase-3β (GSK-3β) is a key element to phosphorylate tau and form neurofibrillary tangles (NFTs) found in tauopathies including Alzheimer's disease (AD). A current topic for AD therapy is focused upon how to prevent tau phosphorylation. In the present study, PKCε activated Akt and inactivated GSK-3β by directly interacting with each protein. Inhibition of protein tyrosine phosphatase 1B (PTP1B), alternatively, caused an enhancement in the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), allowing activation of Akt through a pathway along an IRS-1/phosphatidylinositol 3 kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt axis, to phosphorylate and inactivate GSK-3β. Combination of PKCε activation and PTP1B inhibition more sufficiently activated Akt and inactivated GSK-3β than each independent treatment, to suppress amyloid β (Aβ)-induced tau phosphorylation and ameliorate spatial learning and memory impairment in 5xFAD transgenic mice, an animal model of AD. This may represent an innovative strategy for AD therapy.

Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3'-kinase SH2 domains: a model for SH2-mediated receptor-target interactions.

PubMed Central

Reedijk, M; Liu, X; van der Geer, P; Letwin, K; Waterfield, M D; Hunter, T; Pawson, T

1992-01-01

Efficient binding of active phosphatidylinositol (PI) 3'-kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF-1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3'-kinase, the isolated CSF-1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF-1R KI bound PI 3'-kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3'-kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF-1R KI. Binding of the CSF-1R KI to PI 3'-kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF-1R with PI 3'-kinase in mammalian cells. Complex formation between the CSF-1R and PI 3'-kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha. Images PMID:1314163

Constitutively active rhodopsin mutants causing night blindness are effectively phosphorylated by GRKs but differ in arrestin-1 binding

PubMed Central

Vishnivetskiy, Sergey. A.; Ostermaier, Martin K.; Singhal, Ankita; Panneels, Valerie; Homan, Kristoff T.; Glukhova, Alisa; Sligar, Stephen G.; Tesmer, John J. G.; Schertler, Gebhard F.X.; Standfuss, Joerg; Gurevich, Vsevolod V.

2013-01-01

The effects of activating mutations associated with night blindness on the stoichiometry of rhodopsin interactions with G protein-coupled receptor kinase 1 (GRK1) and arrestin-1 have not been reported. Here we show that the monomeric form of WT rhodopsin and its constitutively active mutants M257Y, G90D, and T94I, reconstituted into HDL particles are effectively phosphorylated by GRK1, as well as two more ubiquitously expressed subtypes, GRK2 and GRK5. All versions of arrestin-1 tested (WT, pre-activated, and constitutively monomeric mutants) bind to monomeric rhodopsin and show the same selectivity for different functional forms of rhodopsin as in native disc membranes. Rhodopsin phosphorylation by GRK1 and GRK2 promotes arrestin-1 binding to a comparable extent, whereas similar phosphorylation by GRK5 is less effective, suggesting that not all phosphorylation sites on rhodopsin are equivalent in promoting arrestin-1 binding. The binding of WT arrestin-1 to phospho-opsin is comparable to the binding to its preferred target, P-Rh*, suggesting that in photoreceptors arrestin-1 only dissociates after opsin regeneration with 11-cis-retinal, which converts phospho-opsin into inactive phospho-rhodopsin that has lower affinity for arrestin-1. Reduced binding of arrestin-1 to the phospho-opsin form of G90D mutant likely contributes to night blindness caused by this mutation in humans. PMID:23872075

Constitutively active rhodopsin mutants causing night blindness are effectively phosphorylated by GRKs but differ in arrestin-1 binding.

PubMed

Vishnivetskiy, Sergey A; Ostermaier, Martin K; Singhal, Ankita; Panneels, Valerie; Homan, Kristoff T; Glukhova, Alisa; Sligar, Stephen G; Tesmer, John J G; Schertler, Gebhard F X; Standfuss, Joerg; Gurevich, Vsevolod V

2013-11-01

The effects of activating mutations associated with night blindness on the stoichiometry of rhodopsin interactions with G protein-coupled receptor kinase 1 (GRK1) and arrestin-1 have not been reported. Here we show that the monomeric form of WT rhodopsin and its constitutively active mutants M257Y, G90D, and T94I, reconstituted into HDL particles are effectively phosphorylated by GRK1, as well as two more ubiquitously expressed subtypes, GRK2 and GRK5. All versions of arrestin-1 tested (WT, pre-activated, and constitutively monomeric mutants) bind to monomeric rhodopsin and show the same selectivity for different functional forms of rhodopsin as in native disc membranes. Rhodopsin phosphorylation by GRK1 and GRK2 promotes arrestin-1 binding to a comparable extent, whereas similar phosphorylation by GRK5 is less effective, suggesting that not all phosphorylation sites on rhodopsin are equivalent in promoting arrestin-1 binding. The binding of WT arrestin-1 to phospho-opsin is comparable to the binding to its preferred target, P-Rh*, suggesting that in photoreceptors arrestin-1 only dissociates after opsin regeneration with 11-cis-retinal, which converts phospho-opsin into inactive phospho-rhodopsin that has lower affinity for arrestin-1. Reduced binding of arrestin-1 to the phospho-opsin form of G90D mutant likely contributes to night blindness caused by this mutation in humans. © 2013.

Phosphorylated Nuclear Receptor CAR Forms a Homodimer To Repress Its Constitutive Activity for Ligand Activation

PubMed Central

Shizu, Ryota; Osabe, Makoto; Perera, Lalith; Moore, Rick; Sueyoshi, Tatsuya

2017-01-01

ABSTRACT The nuclear receptor CAR (NR1I3) regulates hepatic drug and energy metabolism as well as cell fate. Its activation can be a critical factor in drug-induced toxicity and the development of diseases, including diabetes and tumors. CAR inactivates its constitutive activity by phosphorylation at threonine 38. Utilizing receptor for protein kinase 1 (RACK1) as the regulatory subunit, protein phosphatase 2A (PP2A) dephosphorylates threonine 38 to activate CAR. Here we demonstrate that CAR undergoes homodimer-monomer conversion to regulate this dephosphorylation. By coexpression of two differently tagged CAR proteins in Huh-7 cells, mouse primary hepatocytes, and mouse livers, coimmunoprecipitation and two-dimensional gel electrophoresis revealed that CAR can form a homodimer in a configuration in which the PP2A/RACK1 binding site is buried within its dimer interface. Epidermal growth factor (EGF) was found to stimulate CAR homodimerization, thus constraining CAR in its inactive form. The agonistic ligand CITCO binds directly to the CAR homodimer and dissociates phosphorylated CAR into its monomers, exposing the PP2A/RACK1 binding site for dephosphorylation. Phenobarbital, which is not a CAR ligand, binds the EGF receptor, reversing the EGF signal to monomerize CAR for its indirect activation. Thus, the homodimer-monomer conversion is the underlying molecular mechanism that regulates CAR activation, by placing phosphorylated threonine 38 as the common target for both direct and indirect activation of CAR. PMID:28265001

MOLECULAR MECHANISM OF HUMAN NRF2 ACTIVATION AND DEGRADATION: ROLE OF SEQUENTIAL PHOSPHORYLATION BY PROTEIN KINASE CK2

PubMed Central

Pi, Jingbo; Bai, Yushi; Reece, Jeffrey M.; Williams, Jason; Liu, Dianxin; Freeman, Michael L.; Fahl, William E.; Shugar, David; Liu, Jie; Qu, Wei; Collins, Sheila; Waalkes, Michael P.

2007-01-01

Nrf2 is a key transcription factor in the cellular response to oxidative stress. In this study we first identify two phosphorylated forms of endogenous human Nrf2 after chemically-induced oxidative stress and provide evidence that protein kinase CK2-mediated sequential phosphorylation plays potential role in Nrf2 activation and degradation. Human Nrf2 has a predicted molecular mass of 66 kDa. However, immunoblots showed that two bands at 98 and 118 kDa, which are identified as phosphorylated forms, are increased in response to Nrf2 inducers. In addition, human Nrf2 was found to be a substrate for CK2 which mediated two steps of phosphorylation, resulting in two forms of Nrf2 migrating with differing Mr at 98 kDa (Nrf2–98) and 118 kDa (Nrf2–118). Our results support a role in which calmodulin binding regulates CK2 activity, in that cold (25 °C) in Ca2+-free media (cold/Ca2+-free) decreased both cellular calcium levels and CK2-calmodulin binding and induced Nrf2–118 formation, the latter of which was prevented by CK2 specific inhibitors. Gel-shift assays showed that the Nrf2–118 generated under cold/Ca2+-free conditions does not bind to the antioxidant response element, indicating that Nrf2–98 has transcriptional activity. In contrast, Nrf2–118 is more susceptible to degradation. These results provide evidence for phosphorylation by CK2 as a critical controlling factor in Nrf2-mediated cellular antioxidant response. PMID:17512459

Structures of human cytosolic NADP-dependent isocitrate dehydrogenase reveal a novel self-regulatory mechanism of activity.

PubMed

Xu, Xiang; Zhao, Jingyue; Xu, Zhen; Peng, Baozhen; Huang, Qiuhua; Arnold, Eddy; Ding, Jianping

2004-08-06

Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitrate to alpha-ketoglutarate, and regulation of the enzymatic activity of IDHs is crucial for their biological functions. Bacterial IDHs are reversibly regulated by phosphorylation of a strictly conserved serine residue at the active site. Eukaryotic NADP-dependent IDHs (NADP-IDHs) have been shown to have diverse important biological functions; however, their regulatory mechanism remains unclear. Structural studies of human cytosolic NADP-IDH (HcIDH) in complex with NADP and in complex with NADP, isocitrate, and Ca2+ reveal three biologically relevant conformational states of the enzyme that differ substantially in the structure of the active site and in the overall structure. A structural segment at the active site that forms a conserved alpha-helix in all known NADP-IDH structures assumes a loop conformation in the open, inactive form of HcIDH; a partially unraveled alpha-helix in the semi-open, intermediate form; and an alpha-helix in the closed, active form. The side chain of Asp279 of this segment occupies the isocitrate-binding site and forms hydrogen bonds with Ser94 (the equivalent of the phosphorylation site in bacterial IDHs) in the inactive form and chelates the metal ion in the active form. The structural data led us to propose a novel self-regulatory mechanism for HcIDH that mimics the phosphorylation mechanism used by the bacterial homologs, consistent with biochemical and biological data. This mechanism might be applicable to other eukaryotic NADP-IDHs. The results also provide insights into the recognition and specificity of substrate and cofactor by eukaryotic NADP-IDHs.

Virus-induced apoptosis and phosphorylation form of metacaspase in the marine coccolithophorid Emiliania huxleyi.

PubMed

Liu, Jingwen; Cai, Weicong; Fang, Xian; Wang, Xueting; Li, Guiling

2018-04-01

Lytic viral infection and programmed cell death (PCD) are thought to represent two distinct death mechanisms in phytoplankton, unicellular photoautotrophs that drift with ocean currents. PCD (apoptosis) is mainly brought about by the activation of caspases, a protease family with unique substrate selectivity. Here, we demonstrated that virus infection induced apoptosis of marine coccolithophorid Emiliania huxleyi BOF92 involving activation of metacaspase. E. huxleyi cells exhibited cell death process akin to that of apoptosis when exposed to virus infection. We observed typical hallmarks of apoptosis including cell shrinkage, associated nuclear morphological changes and DNA fragmentation. Immunoblotting revealed that antibody against human active-caspase-3 shared epitopes with a protein of ≈ 23 kDa; whose pattern of expression correlated with the onset of cell death. Moreover, analysis on two-dimensional gel electrophoresis revealed that two spots of active caspase-3 co-migrated with the different isoelectric points. Phosphatase treatment of cytosolic extracts containing active caspases-3 showed a mobility shift, suggesting that phosphorylated form of this enzyme might be present in the extracts. Computational prediction of phosphorylation sites based on the amino acid sequence of E. huxleyi metacaspase showed multiple phosphorylated sites for serine, threonine and tyrosine residues. This is the first report showing that phosphorylation modification of metacaspase in E. huxleyi might be required for certain biochemical and morphological changes during virus induced apoptosis.

The Envelope Glycoprotein of Friend Spleen Focus-Forming Virus Covalently Interacts with and Constitutively Activates a Truncated Form of the Receptor Tyrosine Kinase Stk

PubMed Central

Nishigaki, Kazuo; Thompson, Delores; Hanson, Charlotte; Yugawa, Takashi; Ruscetti, Sandra

2001-01-01

The Friend spleen focus-forming virus (SFFV) encodes a unique envelope glycoprotein, gp55, which allows erythroid cells to proliferate and differentiate in the absence of erythropoietin (Epo). SFFV gp55 has been shown to interact with the Epo receptor complex, causing constitutive activation of various signal-transducing molecules. When injected into adult mice, SFFV induces a rapid erythroleukemia, with susceptibility being determined by the host gene Fv-2, which was recently shown to be identical to the gene encoding the receptor tyrosine kinase Stk/Ron. Susceptible, but not resistant, mice encode not only full-length Stk but also a truncated form of the kinase, sf-Stk, which may mediate the biological effects of SFFV infection. To determine whether expression of SFFV gp55 leads to the activation of sf-Stk, we expressed sf-Stk, with or without SFFV gp55, in hematopoietic cells expressing the Epo receptor. Our data indicate that sf-Stk interacts with SFFV gp55 as well as gp55P, the biologically active form of the viral glycoprotein, forming disulfide-linked complexes. This covalent interaction, as well as noncovalent interactions with SFFV gp55, results in constitutive tyrosine phosphorylation of sf-Stk and its association with multiple tyrosine-phosphorylated signal-transducing molecules. In contrast, neither Epo stimulation in the absence of SFFV gp55 expression nor expression of a mutant of SFFV that cannot interact with sf-Stk was able to induce tyrosine phosphorylation of sf-Stk or its association with any signal-transducing molecules. Covalent interaction of sf-Stk with SFFV gp55 and constitutive tyrosine phosphorylation of sf-Stk can also be detected in an erythroleukemia cell line derived from an SFFV-infected mouse. Our results suggest that SFFV gp55 may mediate its biological effects in vivo by interacting with and activating a truncated form of the receptor tyrosine kinase Stk. PMID:11483734

Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation*♦

PubMed Central

Yamano, Koji; Queliconi, Bruno B.; Koyano, Fumika; Saeki, Yasushi; Hirokawa, Takatsugu; Tanaka, Keiji; Matsuda, Noriyuki

2015-01-01

Damaged mitochondria are eliminated through autophagy machinery. A cytosolic E3 ubiquitin ligase Parkin, a gene product mutated in familial Parkinsonism, is essential for this pathway. Recent progress has revealed that phosphorylation of both Parkin and ubiquitin at Ser65 by PINK1 are crucial for activation and recruitment of Parkin to the damaged mitochondria. However, the mechanism by which phosphorylated ubiquitin associates with and activates phosphorylated Parkin E3 ligase activity remains largely unknown. Here, we analyze interactions between phosphorylated forms of both Parkin and ubiquitin at a spatial resolution of the amino acid residue by site-specific photo-crosslinking. We reveal that the in-between-RING (IBR) domain along with RING1 domain of Parkin preferentially binds to ubiquitin in a phosphorylation-dependent manner. Furthermore, another approach, the Fluoppi (fluorescent-based technology detecting protein-protein interaction) assay, also showed that pathogenic mutations in these domains blocked interactions with phosphomimetic ubiquitin in mammalian cells. Molecular modeling based on the site-specific photo-crosslinking interaction map combined with mass spectrometry strongly suggests that a novel binding mechanism between Parkin and ubiquitin leads to a Parkin conformational change with subsequent activation of Parkin E3 ligase activity. PMID:26260794

Tumor Necrosis Factor alpha (TNF{alpha}) regulates CD40 expression through SMAR1 phosphorylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Kamini; Sinha, Surajit; Malonia, Sunil Kumar

2010-01-08

CD40 plays an important role in mediating inflammatory response and is mainly induced by JAK/STAT phosphorylation cascade. TNF{alpha} is the key cytokine that activates CD40 during inflammation and tumorigenesis. We have earlier shown that SMAR1 can repress the transcription of Cyclin D1 promoter by forming a HDAC1 dependent repressor complex. In this study, we show that SMAR1 regulates the transcription of NF-{kappa}B target gene CD40. SMAR1 recruits HDAC1 and forms a repressor complex on CD40 promoter and keeps its basal transcription in check. Further, we show that TNF{alpha} stimulation induces SMAR1 phosphorylation at Ser-347 and promotes its cytoplasmic translocation, thusmore » releasing its negative effect. Concomitantly, TNF{alpha} induced phosphorylation of STAT1 at Tyr-701 by JAK1 facilitates its nuclear translocation and activation of CD40 through p300 recruitment and core Histone-3 acetylation. Thus, TNF{alpha} mediated regulation of CD40 expression occurs by dual phosphorylation of SMAR1 and STAT1.« less

Losartan Affects Glomerular AKT and mTOR Phosphorylation in an Experimental Model of Type 1 Diabetic Nephropathy

PubMed Central

Petrakis, Ioannis; Stylianou, Kostas; Katsarou, Theodora; Giannakakis, Konstantinos; Perakis, Kostas; Vardaki, Eleftheria; Stratigis, Spyridon; Ganotakis, Emmanuel; Papavasiliou, Stathis; Daphnis, Eugenios

2013-01-01

The AKT-mTOR pathway is activated in diabetic nephropathy. Renin-angiotensin system modulators exert beneficial effects on the diabetic kidney. We explored the action of losartan on AKT-mTOR phosphorylation in glomeruli and podocytes. Diabetes mellitus was induced to Sprague-Dawley rats by streptozotocin. Five months later, the rats were commenced on losartan and euthanized 2 months later. Kidneys were processed for immunofluorescence studies. Glomeruli were isolated for Western blot analysis. Diabetes increased activated forms of AKT and mTOR both in glomeruli and podocytes. In diabetic rats, losartan decreased phosphorylated/activated forms of AKT (Thr308) and mTOR (Ser2448) in glomeruli but decreased only activated mTOR in podocytes. However, in both glomeruli and podocytes of healthy animals, an inverse pattern was evident. In conclusion, a new body of evidence indicates the differential activation of AKT-mTOR in glomeruli and podocytes of healthy and diabetic animals in response to losartan. PMID:23456824

Yap4 PKA- and GSK3-dependent phosphorylation affects its stability but not its nuclear localization.

PubMed

Pereira, Jorge; Pimentel, Catarina; Amaral, Catarina; Menezes, Regina A; Rodrigues-Pousada, Claudina

2009-12-01

Yap4 is a nuclear-resident transcription factor induced in Saccharomyces cerevisiae when exposed to several stress conditions, which include mild hyperosmotic and oxidative stress, temperature shift or metal exposure. This protein is also phosphorylated. Here we report that this modification is driven by PKA and GSK3. In order to ascertain whether Yap4 is directly or indirectly phosphorylated by PKA, we searched for stress and PKA-related kinases that could phosphorylate Yap4. We show that phosphorylation is independent of the kinases Rim15, Yak1, Sch9, Slt2, Ste20 and Ptk2. In addition, we showed that Yap4 phosphorylation is also abrogated in the triple GSK3 mutant mck1 rim11 yol128c. Furthermore, our data reveal that Yap4 nuclear localization is independent of its phosphorylation state. This protein has several putative phosphorylation sites, but only the mutation of residues T192 and S196 impairs its phosphorylation under different stress conditions. The ability of the non-phosphorylated forms of Yap4 to partially rescue the hog1 severe sensitivity phenotype is not affected, suggesting that Yap4 activity is maintained in the absence of phosphorylation. However, this modification seems to be required for stability of the protein, as the non-phosphorylated form has a shorter half-life than the phosphorylated one.

Hydrophobic Motif Phosphorylation Coordinates Activity and Polar Localization of the Neurospora crassa Nuclear Dbf2-Related Kinase COT1

PubMed Central

Maerz, Sabine; Dettmann, Anne

2012-01-01

Nuclear Dbf2p-related (NDR) kinases and associated proteins are recognized as a conserved network that regulates eukaryotic cell polarity. NDR kinases require association with MOB adaptor proteins and phosphorylation of two conserved residues in the activation segment and hydrophobic motif for activity and function. We demonstrate that the Neurospora crassa NDR kinase COT1 forms inactive dimers via a conserved N-terminal extension, which is also required for the interaction of the kinase with MOB2 to generate heterocomplexes with basal activity. Basal kinase activity also requires autophosphorylation of the COT1-MOB2 complex in the activation segment, while hydrophobic motif phosphorylation of COT1 by the germinal center kinase POD6 fully activates COT1 through induction of a conformational change. Hydrophobic motif phosphorylation is also required for plasma membrane association of the COT1-MOB2 complex. MOB2 further restricts the membrane-associated kinase complex to the hyphal apex to promote polar cell growth. These data support an integrated mechanism of NDR kinase regulation in vivo, in which kinase activation and cellular localization of COT1 are coordinated by dual phosphorylation and interaction with MOB2. PMID:22451488

E2 superfamily of ubiquitin-conjugating enzymes: constitutively active or activated through phosphorylation in the catalytic cleft.

PubMed

Valimberti, Ilaria; Tiberti, Matteo; Lambrughi, Matteo; Sarcevic, Boris; Papaleo, Elena

2015-10-14

Protein phosphorylation is a modification that offers a dynamic and reversible mechanism to regulate the majority of cellular processes. Numerous diseases are associated with aberrant regulation of phosphorylation-induced switches. Phosphorylation is emerging as a mechanism to modulate ubiquitination by regulating key enzymes in this pathway. The molecular mechanisms underpinning how phosphorylation regulates ubiquitinating enzymes, however, are elusive. Here, we show the high conservation of a functional site in E2 ubiquitin-conjugating enzymes. In catalytically active E2s, this site contains aspartate or a phosphorylatable serine and we refer to it as the conserved E2 serine/aspartate (CES/D) site. Molecular simulations of substrate-bound and -unbound forms of wild type, mutant and phosphorylated E2s, provide atomistic insight into the role of the CES/D residue for optimal E2 activity. Both the size and charge of the side group at the site play a central role in aligning the substrate lysine toward E2 catalytic cysteine to control ubiquitination efficiency. The CES/D site contributes to the fingerprint of the E2 superfamily. We propose that E2 enzymes can be divided into constitutively active or regulated families. E2s characterized by an aspartate at the CES/D site signify constitutively active E2s, whereas those containing a serine can be regulated by phosphorylation.

Molecular dynamics simulations suggest changes in electrostatic interactions as a potential mechanism through which serine phosphorylation inhibits DNA Polymerase β's activity.

PubMed

Homouz, Dirar; Joyce-Tan, Kwee Hong; Shahir Shamsir, Mohd; Moustafa, Ibrahim M; Idriss, Haitham

2018-01-01

DNA polymerase β is a 39kDa enzyme that is a major component of Base Excision Repair in human cells. The enzyme comprises two major domains, a 31kDa domain responsible for the polymerase activity and an 8kDa domain, which bind ssDNA and has a deoxyribose phosphate (dRP) lyase activity. DNA polymerase β was shown to be phosphorylated in vitro with protein kinase C (PKC) at serines 44 and 55 (S44 and S55), resulting in loss of its polymerase enzymic activity, but not its ability to bind ssDNA. In this study, we investigate the potential phosphorylation-induced structural changes for DNA polymerase β using molecular dynamics. The simulations show drastic conformational changes of the polymerase structure as a result of S44 phosphorylation. Phosphorylation-induced conformational changes transform the closed (active) enzyme structure into an open one. Further analysis of the results points to a key hydrogen bond and newly formed salt bridges as potential drivers of these structural fluctuations. The changes observed with S44/55 and S55 phosphorylation were less dramatic than S44 and the integrity of the H-bond was not compromised. Thus the phosphorylation of S44 is likely the major contributor to structural fluctuations that lead to loss of enzymatic activity. Copyright © 2017. Published by Elsevier Inc.

Endogenous overexpression of an active phosphorylated form of DNA polymerase β under oxidative stress in Trypanosoma cruzi.

PubMed

Rojas, Diego A; Urbina, Fabiola; Moreira-Ramos, Sandra; Castillo, Christian; Kemmerling, Ulrike; Lapier, Michel; Maya, Juan Diego; Solari, Aldo; Maldonado, Edio

2018-02-01

Trypanosoma cruzi is exposed during its life to exogenous and endogenous oxidative stress, leading to damage of several macromolecules such as DNA. There are many DNA repair pathways in the nucleus and mitochondria (kinetoplast), where specific protein complexes detect and eliminate damage to DNA. One group of these proteins is the DNA polymerases. In particular, Tc DNA polymerase β participates in kinetoplast DNA replication and repair. However, the mechanisms which control its expression under oxidative stress are still unknown. Here we describe the effect of oxidative stress on the expression and function of Tc DNA polymerase β To this end parasite cells (epimastigotes and trypomastigotes) were exposed to peroxide during short periods of time. Tc DNA polymerase β which was associated physically with kinetoplast DNA, showed increased protein levels in response to peroxide damage in both parasite forms analyzed. Two forms of DNA polymerase β were identified and overexpressed after peroxide treatment. One of them was phosphorylated and active in DNA synthesis after renaturation on polyacrylamide electrophoresis gel. This phosphorylated form showed 3-4-fold increase in both parasite forms. Our findings indicate that these increments in protein levels are not under transcriptional control because the level of Tc DNA polymerase β mRNA is maintained or slightly decreased during the exposure to oxidative stress. We propose a mechanism where a DNA repair pathway activates a cascade leading to the increment of expression and phosphorylation of Tc DNA polymerase β in response to oxidative damage, which is discussed in the context of what is known in other trypanosomes which lack transcriptional control.

Phosphorylation at the Homotypic Interface Regulates Nucleoprotein Oligomerization and Assembly of the Influenza Virus Replication Machinery

PubMed Central

Mondal, Arindam; Potts, Gregory K.; Dawson, Anthony R.; Coon, Joshua J.; Mehle, Andrew

2015-01-01

Negative-sense RNA viruses assemble large ribonucleoprotein (RNP) complexes that direct replication and transcription of the viral genome. Influenza virus RNPs contain the polymerase, genomic RNA and multiple copies of nucleoprotein (NP). During RNP assembly, monomeric NP oligomerizes along the length of the genomic RNA. Regulated assembly of the RNP is essential for virus replication, but how NP is maintained as a monomer that subsequently oligomerizes to form RNPs is poorly understood. Here we elucidate a mechanism whereby NP phosphorylation regulates oligomerization. We identified new evolutionarily conserved phosphorylation sites on NP and demonstrated that phosphorylation of NP decreased formation of higher-order complexes. Two phosphorylation sites were located on opposite sides of the NP:NP interface. In both influenza A and B virus, mutating or mimicking phosphorylation at these residues blocked homotypic interactions and drove NP towards a monomeric form. Highlighting the central role of this process during infection, these mutations impaired RNP formation, polymerase activity and virus replication. Thus, dynamic phosphorylation of NP regulates RNP assembly and modulates progression through the viral life cycle. PMID:25867750

Sustained phosphorylation of mutated FGFR3 is a crucial feature of genetic dwarfism and induces apoptosis in the ATDC5 chondrogenic cell line via PLCgamma-activated STAT1.

PubMed

Harada, Daisuke; Yamanaka, Yoshitaka; Ueda, Koso; Nishimura, Riko; Morishima, Tsuneo; Seino, Yoshiki; Tanaka, Hiroyuki

2007-08-01

The most frequent type of rhizomelic dwarfism, achondroplasia (ACH), is caused by mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. Mutations in FGFR3 result in skeletal dysplasias of variable severity, including mild phenotypic effects in hypochondroplasia (HCH), severe phenotypic effects in thanatophoric dysplasia types I (TDI) and II (TDII), and severe but survivable phenotypic effects in severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN). To explore the molecular mechanisms that result in the different phenotypes, we investigated the kinetics of mutated versions of FGFR3. First, we assayed the phosphorylation states of the mutated FGFR3s and found that the level of phosphorylation in TDI-FGFR3 was lower than in ACH-FGFR3, although the other mutants were phosphorylated according to phenotypic severity. Second, we analyzed the duration of the phosphorylation. TDI-FGFR3 was not highly phosphorylated under ligand-free conditions, but the peak phosphorylation levels of TDI-FGFR3 and ACH-FGFR3 were maintained for 30 min after stimulation with FGF-1. Moreover, ligand-dependent phosphorylation of TDI-FGFR3, but not ACH-FGFR3, lasted for more than 8 h after FGF-1 administration. The other mutant proteins showed sustained phosphorylation independent of ligand presence. Third, we investigated the intracellular localization of the mutant proteins. Immunofluorescence analysis showed accumulations of TDII-FGFR3, SADDAN-FGFR3, and a portion of TDI-FGFR3 in the endoplasmic reticulum (ER). Based on these data, we concluded that sustained phosphorylation of FGFR3 causes chondrodysplasia, and the phenotypic severity depends on the proportion of ER-localized mutant FGFR3. In FGFR3 signaling, the transcription factor, signal transducer and activator of transcription 1 (STAT1) inhibit proliferation and induce apoptosis of chondrocytes. Here we reveal that phospholipase C gamma (PLCgamma) mediates FGFR3-induced STAT1 activation. Both PLCgamma and STAT1 were activated by FGFR3 signaling, but a dominant-negative form of PLCgamma (DN-PLCgamma) remarkably reduced STAT1 phosphorylation. Apoptosis assays revealed that the constitutively active forms of FGFR3 (TDII-FGFR3) and STAT1 (STAT1-C) induce apoptosis of chondrogenic ATDC5 cells via caspase activity. DN-PLCgamma reduced the apoptosis of ATDC5 cells expressing TDII-FGFR3, but over-expression of both DN-PLCgamma and STAT1-C induced apoptosis. Therefore, we conclude that a PLCgamma-STAT1 pathway mediates apoptotic signaling by FGFR3.

Transactivation activity and nucleocytoplasmic transport of β-catenin are independently regulated by its C-terminal end.

PubMed

Maturana, J L; Niechi, I; Silva, E; Huerta, H; Cataldo, R; Härtel, S; Barros, L F; Galindo, M; Tapia, J C

2015-11-15

The key protein in the canonical Wnt pathway is β-catenin, which is phosphorylated both in absence and presence of Wnt signals by different kinases. Upon activation in the cytoplasm, β-catenin can enter into the nucleus to transactivate target gene expression, many of which are cancer-related genes. The mechanism governing β-catenin's nucleocytoplasmic transport has been recently unvealed, although phosphorylation at its C-terminal end and its functional consequences are not completely understood. Serine 646 of β-catenin is a putative CK2 phosphorylation site and lies in a region which has been proposed to be important for its nucleocytoplasmic transport and transactivation activity. This residue was mutated to aspartic acid mimicking CK2-phosphorylation and its effects on β-catenin activity as well as localization were explored. β-Catenin S6464D did not show significant differences in both transcriptional activity and nuclear localization compared to the wild-type form, but displayed a characteristic granular nuclear pattern. Three-dimensional models of nuclei were constructed which showed differences in number and volume of granules, being those from β-catenin S646D more and smaller than the wild-type form. FRAP microscopy was used to compare nuclear export of both proteins which showed a slightly higher but not significant retention of β-catenin S646D. Altogether, these results show that C-terminal phosphorylation of β-catenin seems to be related with its nucleocytoplasmic transport but not transactivation activity. Copyright © 2015 Elsevier B.V. All rights reserved.

Protein phosphorylation in plant immunity: insights into the regulation of pattern recognition receptor-mediated signaling

PubMed Central

Park, Chang-Jin; Caddell, Daniel F.; Ronald, Pamela C.

2012-01-01

Plants are continuously challenged by pathogens including viruses, bacteria, and fungi. The plant immune system recognizes invading pathogens and responds by activating an immune response. These responses occur rapidly and often involve post-translational modifications (PTMs) within the proteome. Protein phosphorylation is a common and intensively studied form of these PTMs and regulates many plant processes including plant growth, development, and immunity. Most well-characterized pattern recognition receptors (PRRs), including Xanthomonas resistance 21, flagellin sensitive 2, and elongation factor-Tu receptor, possess intrinsic protein kinase activity and regulate downstream signaling through phosphorylation events. Here, we focus on the phosphorylation events of plant PRRs that play important roles in the immune response. We also discuss the role of phosphorylation in regulating mitogen-associated protein kinase cascades and transcription factors in plant immune signaling. PMID:22876255

Effects of endoplasmic reticulum stressors on maturation and signaling of hemizygous and heterozygous wild-type and mutant forms of KIT.

PubMed

Brahimi-Adouane, Sabrina; Bachet, Jean-Baptiste; Tabone-Eglinger, Séverine; Subra, Frédéric; Capron, Claude; Blay, Jean-Yves; Emile, Jean-François

2013-06-01

Gain of function mutations of KIT are frequent in some human tumors, and are sensible to tyrosine kinase inhibitors. In most tumors, oncogenic mutations are heterozygous, however most in vitro data of KIT activation have been obtained with hemizygous mutation. This study aimed to investigate the maturation and activation of wild-type (WT) and mutant (M) forms of KIT in hemizygous and heterozygous conditions. WT and two types of exon 11 deletions M forms of human KIT were expressed in NIH3T3 cell lines. Membrane expression of KIT was quantified by flow cytometry. Quantification of glycosylated forms of KIT and phosphorylated forms of AKT and ERK were performed by western blot. Simultaneous activation of WT KIT and treatment with endoplasmic reticulum (ER) inhibitors, tunicamycin or brefeldin A induced a complete inhibition of membrane expression of the 145 kDa form of KIT. By contrast activation or ER inhibitors alone, only partly inhibited this form. ER inhibitors also inhibited KIT activation-dependent phosphorylation of AKT and ERK1/2. Brefeldin A induced a complete down regulation of the 145 kDa form in hemizygous M, and induced an intra-cellular accumulation of the 125 kDa form in WT but not in hemizygous M. Heterozygous cells had glycosylation and response to ER inhibitors patterns more similar to WT than to hemizygous M. Phosphorylated AKT was reduced in hemizygous cells in comparison to WT KIT cells and heterozygous cells, and in the presence of brefeldin A in all cell lines. Effects of ER inhibitors are significantly different in hemizygous and heterozygous mutants. Differences in intra-cellular trafficking of KIT forms result in differences in downstream signaling pathways, and activation of PI3K/AKT pathway appears to be tied to the presence of the mature 145 kDa form of KIT at the membrane surface. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Activity and structure of human acetyl-CoA carboxylase targeted by a specific inhibitor.

PubMed

Jang, SoRi; Gornicki, Piotr; Marjanovic, Jasmina; Bass, Ethan; Lurcotta, Toni; Rodriguez, Pedro; Austin, Jotham; Haselkorn, Robert

2018-05-17

We have studied a series of human acetyl CoA-carboxylase (ACC) 1 and ACC2 proteins with deletions and/or Ser to Ala substitutions of the known phosphorylation sites. In vitro dephosphorylation/phosphorylation experiments reveal a substantial level of phosphorylation of human ACCs produced in insect cells. Our results are consistent with AMPK phosphorylation of Ser 29, Ser 80 , Ser 1,201 and Ser 1,216 . Phosphorylation of the N-terminal regulatory domain decreases ACC1 activity, while phosphorylation of residues in the ACC central domain has no effect. Inhibition of the activity by phosphorylation is significantly more profound at citrate concentrations below 2 mM. Furthermore, deletion of the N-terminal domain facilitates structural changes induced by citrate, including conversion of ACC dimers to linear polymers. We have also identified ACC2 amino acid mutations affecting specific inhibition of the isozyme by compound CD-017-0191. They form two clusters separated by 60-90 Å: one located in the vicinity of the BC active site and the other one in the vicinity of the ACC1 phosphorylation sites in the central domain, suggesting a contribution of the interface of two ACC dimers in the polymer to the inhibitor binding site. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Sedimentation studies reveal a direct role of phosphorylation in Smad3:Smad4 homo- and hetero-trimerization.

PubMed

Correia, J J; Chacko, B M; Lam, S S; Lin, K

2001-02-06

SMAD proteins are known to oligomerize and hetero-associate during their activation and translocation to the nucleus for transcriptional control. Analytical ultracentrifuge studies on Smad3 and Smad4 protein constructs are presented to clarify the model of homo- and hetero-oligomerization and the role of phosphorylation in the activation process. These constructs all exhibit a tendency to form disulfide cross-linked aggregates, primarily dimers, and a strong reducing agent, TCEP, was found to be required to determine the best estimates for reversible association models and equilibrium constants. A Smad4 construct, S4AF, consisting of the middle linker (L) domain and the C-terminal (C) domain, is shown to be a monomer, while a Smad3 construct, S3LC, consisting of the LC domains, is shown to form a trimer with an affinity K(3) = (1.2-3.1) x 10(9) M(-2). A Smad3 construct that mimics phosphorylation at the C-terminal target sequence, S3LC(3E), has 17--35-fold enhanced ability to form trimer over that of the wild-type construct, S3LC. S4AF associates with either S3LC or S3LC(3E) to form a hetero-trimer. In each case, the hetero-trimer is favored over the formation of the homo-trimer. Despite high sequence homology between Smad3 and Smad4, a chimeric Smad4 construct with an engineered Smad3 C-terminal pseudo-phosphorylation sequence, S4AF(3E), shows no tendency to form trimer. This suggests a Smad4-specific sequence insert inhibits homo-trimer formation, or other domains or sequences in S3LC are required in addition to the target sequence to mediate the formation of trimer. These results represent a direct molecular measure of the importance of hetero-trimerization and phosphorylation in the TGF-beta-activated Smad protein signal transduction process.

Heat-shock-specific phosphorylation and transcriptional activity of RNA polymerase II.

PubMed

Egyházi, E; Ossoinak, A; Lee, J M; Greenleaf, A L; Mäkelä, T P; Pigon, A

1998-07-10

The carboxyl-terminal domain (CTD) of the largest RNA polymerase II (pol II) subunit is a target for extensive phosphorylation in vivo. Using in vitro kinase assays it was found that several different protein kinases can phosphorylate the CTD including the transcription factor IIH-associated CDK-activating CDK7 kinase (R. Roy, J. P. Adamczewski, T. Seroz, W. Vermeulen, J. P. Tassan, L. Schaeffer, E. A. Nigg, J. H. Hoeijmakers, and J. M. Egly, 1994, Cell 79, 1093-1101). Here we report the colocalization of CDK7 and the phosphorylated form of CTD (phosphoCTD) to actively transcribing genes in intact salivary gland cells of Chironomus tentans. Following a heat-shock treatment, both CDK7 and pol II staining disappear from non-heat-shock genes concomitantly with the abolishment of transcriptional activity of these genes. In contrast, the actively transcribing heat-shock genes, manifested as chromosomal puff 5C on chromosome IV (IV-5C), stain intensely for phosphoCTD, but are devoid of CDK7. Furthermore, the staining of puff IV-5C with anti-PCTD antibodies was not detectably influenced by the TFIIH kinase and transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). Following heat-shock treatment, the transcription of non-heat-shock genes was completely eliminated, while newly formed heat-shock gene transcripts emerged in a DRB-resistant manner. Thus, heat shock in these cells induces a rapid clearance of CDK7 from the non-heat-shock genes, indicating a lack of involvement of CDK7 in the induction and function of the heat-induced genes. The results taken together suggest the existence of heat-shock-specific CTD phosphorylation in living cells. This phosphorylation is resistant to DRB treatment, suggesting that not only phosphorylation but also transcription of heat-shock genes is DRB resistant and that CDK7 in heat shock cells is not associated with TFIIH.

Erythroblast Transformation by the Friend Spleen Focus-Forming Virus Is Associated with a Block in Erythropoietin-Induced STAT1 Phosphorylation and DNA Binding and Correlates with High Expression of the Hematopoietic Phosphatase SHP-1

PubMed Central

Nishigaki, Kazuo; Hanson, Charlotte; Ohashi, Takashi; Spadaccini, Angelo; Ruscetti, Sandra

2006-01-01

Infection of mice with Friend spleen focus-forming virus (SFFV) results in a multistage erythroleukemia. In the first stage, the SFFV envelope glycoprotein interacts with the erythropoietin receptor and a short form of the receptor tyrosine kinase sf-Stk, resulting in constitutive activation of signal transducing molecules and the development of erythropoietin (Epo)-independent erythroid hyperplasia and polycythemia. The second stage results from the outgrowth of a rare virus-infected erythroid cell that expresses nonphysiological levels of the myeloid transcription factor PU.1. These cells exhibit a differentiation block and can be grown as murine erythroleukemia (MEL) cell lines. In this study, we examined SFFV MEL cells to determine whether their transformed phenotype was associated with a block in the activation of any Epo signal-transducing molecules. Our studies indicate that Epo- or SFFV-induced activation of STAT1/3 DNA binding activity is blocked in SFFV MEL cells. The block is at the level of tyrosine phosphorylation of STAT1, although Jak2 phosphorylation is not blocked in these cells. In contrast to Epo, alpha interferon can induce STAT1 phosphorylation and DNA binding in SFFV MEL cells. The SFFV-transformed cells were shown to express elevated levels of the hematopoietic phosphatase SHP-1, and treatment of the cells with a phosphatase inhibitor restored STAT1 tyrosine phosphorylation. MEL cells derived from Friend murine leukemia virus (MuLV) or ME26 MuLV-infected mice, which do not express PU.1, express lower levels of SHP-1 and are not blocked in STAT1/3 DNA-binding activity. Our studies suggest that SFFV-infected erythroid cells become transformed when differentiation signals activated by STAT1/3 are blocked due to high SHP-1 levels induced by inappropriate expression of the PU.1 protein. PMID:16731906

G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs).

PubMed

Li, Lingyong; Homan, Kristoff T; Vishnivetskiy, Sergey A; Manglik, Aashish; Tesmer, John J G; Gurevich, Vsevolod V; Gurevich, Eugenia V

2015-04-24

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in homologous desensitization of GPCRs. It is widely assumed that most GRKs selectively phosphorylate only active GPCRs. Here, we show that although this seems to be the case for the GRK2/3 subfamily, GRK5/6 effectively phosphorylate inactive forms of several GPCRs, including β2-adrenergic and M2 muscarinic receptors, which are commonly used as representative models for GPCRs. Agonist-independent GPCR phosphorylation cannot be explained by constitutive activity of the receptor or membrane association of the GRK, suggesting that it is an inherent ability of GRK5/6. Importantly, phosphorylation of the inactive β2-adrenergic receptor enhanced its interactions with arrestins. Arrestin-3 was able to discriminate between phosphorylation of the same receptor by GRK2 and GRK5, demonstrating preference for the latter. Arrestin recruitment to inactive phosphorylated GPCRs suggests that not only agonist activation but also the complement of GRKs in the cell regulate formation of the arrestin-receptor complex and thereby G protein-independent signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The human gastrin precursor. Characterization of phosphorylated forms and fragments.

PubMed Central

Varro, A; Desmond, H; Pauwels, S; Gregory, H; Young, J; Dockray, G J

1988-01-01

There is a potential phosphorylation site in the C-terminal region of the precursor for the acid-stimulating hormone gastrin, which is immediately adjacent to an important cleavage point. In the present study we have sought to identify, separate, quantify and characterize phosphorylated and unphosphorylated forms of human progastrin and its fragments. Identification was made by two radioimmunoassays: (a) a novel assay employing an antibody raised to intact human progastrin; and (b) an assay using antibody reacting with the C-terminal tryptic fragment of human progastrin, as well as progastrin itself. Two forms of human progastrin isolated from a gastrinoma were separated by ion-exchange h.p.l.c., and had similar elution positions on reverse-phase h.p.l.c. and on gel filtration. The more acidic peptide contained close to equimolar amounts of phosphate. On trypsinization, peptides were released that co-eluted on ion-exchange h.p.l.c. with, and had the immunochemical properties of, naturally occurring C-terminal fragments of progastrin. One of the latter was isolated and shown by Edman degradation after derivatization with ethanethiol to have the sequence Ser (P)-Ala-Glu-Asp-Glu-Asn. Similar peptides occur in antral mucosa resected from ulcer patients. The unphosphorylated forms of progastrin predominated, whereas the phosphorylated forms of the C-terminal fragments were predominant. This distribution could be explained by preferential cleavage of phosphorylated progastrin. We conclude that in human progastrin, Ser-96 can occur in the phosphorylated form; this residue immediately follows a pair of basic residues (Arg-Arg) that are cleaved during synthesis of the biologically active product. PMID:3223964

Phosphorylation regulates the water channel activity of the seed-specific aquaporin alpha-TIP.

PubMed

Maurel, C; Kado, R T; Guern, J; Chrispeels, M J

1995-07-03

The vacuolar membrane protein alpha-TIP is a seed-specific protein of the Major Intrinsic Protein family. Expression of alpha-TIP in Xenopus oocytes conferred a 4- to 8-fold increase in the osmotic water permeability (Pf) of the oocyte plasma membrane, showing that alpha-TIP forms water channels and is thus a new aquaporin. alpha-TIP has three putative phosphorylation sites on the cytoplasmic side of the membrane (Ser7, Ser23 and Ser99), one of which (Ser7) has been shown to be phosphorylated. We present several lines of evidence that the activity of this aquaporin is regulated by phosphorylation. First, mutation of the putative phosphorylation sites in alpha-TIP (Ser7Ala, Ser23Ala and Ser99Ala) reduced the apparent water transport activity of alpha-TIP in oocytes, suggesting that phosphorylation of alpha-TIP occurs in the oocytes and participates in the control of water channel activity. Second, exposure of oocytes to the cAMP agonists 8-bromoadenosine 3',5'-cyclic monophosphate, forskolin and 3-isobutyl-1-methylxanthine, which stimulate endogenous protein kinase A (PKA), increased the water transport activity of alpha-TIP by 80-100% after 60 min. That the protein can be phosphorylated by PKA was demonstrated by phosphorylating alpha-TIP in isolated oocyte membranes with the bovine PKA catalytic subunit. Third, the integrity of the three sites at positions 7, 23 and 99 was necessary for the cAMP-dependent increase in the Pf of oocytes expressing alpha-TIP, as well as for in vitro phosphorylation of alpha-TIP. These findings demonstrate that the alpha-TIP water channel can be modulated via phosphorylation of Ser7, Ser23 and Ser99.(ABSTRACT TRUNCATED AT 250 WORDS)

Myosin Light Chain Kinase and the Role of Myosin Light Chain Phosphorylation in Skeletal Muscle

PubMed Central

Stull, James T.; Kamm, Kristine E.; Vandenboom, Rene

2011-01-01

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca2+/calmodulin-dependent serine-threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca2+ binding to calmodulin forming a (Ca2+)4•calmodulin complex sufficient for activation with a diffusion limited, stoichiometic binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca2+ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca2+/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca2+-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue. PMID:21284933

c-Abl interacts with the WAVE2 signaling complex to induce membrane ruffling and cell spreading.

PubMed

Stuart, Jeremy R; Gonzalez, Francis H; Kawai, Hidehiko; Yuan, Zhi-Min

2006-10-20

The Wiskott-Aldrich syndrome-related protein WAVE2 promotes Arp2/3-dependent actin polymerization downstream of Rho-GTPase activation. The Abelson-interacting protein-1 (Abi-1) forms the core of the WAVE2 complex and is necessary for proper stimulation of WAVE2 activity. Here we have shown that the Abl-tyrosine kinase interacts with the WAVE2 complex and that Abl kinase activity facilitates interaction between Abl and WAVE2 complex members. We have characterized various interactions between Abl and members of the WAVE2 complex and revealed that Abi-1 promotes interaction between Abl and WAVE2 members. We have demonstrated that Abl-dependent phosphorylation of WAVE2 is necessary for its activation in vivo, which is highlighted by the findings that RNA interference of WAVE2 expression in Abl/Arg-/- cells has no additive effect on the amount of membrane ruffling. Furthermore, Abl phosphorylates WAVE2 on tyrosine 150, and WAVE2-deficient cells rescued with a Y150F mutant fail to regain their ability to ruffle and form microspikes, unlike cells rescued with wild-type WAVE2. Together, these data show that c-Abl activates WAVE2 via tyrosine phosphorylation to promote actin remodeling in vivo and that Abi-1 forms the crucial link between these two factors.

Structure of phosphorylated UBL domain and insights into PINK1-orchestrated parkin activation.

PubMed

Aguirre, Jacob D; Dunkerley, Karen M; Mercier, Pascal; Shaw, Gary S

2017-01-10

Mutations in PARK2 and PARK6 genes are responsible for the majority of hereditary Parkinson's disease cases. These genes encode the E3 ubiquitin ligase parkin and the protein kinase PTEN-induced kinase 1 (PINK1), respectively. Together, parkin and PINK1 regulate the mitophagy pathway, which recycles damaged mitochondria following oxidative stress. Native parkin is inactive and exists in an autoinhibited state mediated by its ubiquitin-like (UBL) domain. PINK1 phosphorylation of serine 65 in parkin's UBL and serine 65 of ubiquitin fully activate ubiquitin ligase activity; however, a structural rationale for these observations is not clear. Here, we report the structure of the phosphorylated UBL domain from parkin. We find that destabilization of the UBL results from rearrangements to hydrophobic core packing that modify its structure. Altered surface electrostatics from the phosphoserine group disrupt its intramolecular association, resulting in poorer autoinhibition in phosphorylated parkin. Further, we show that phosphorylation of both the UBL domain and ubiquitin are required to activate parkin by releasing the UBL domain, forming an extended structure needed to facilitate E2-ubiquitin binding. Together, the results underscore the importance of parkin activation by the PINK1 phosphorylation signal and provide a structural picture of the unraveling of parkin's ubiquitin ligase potential.

Smad3 allostery links TGF-β receptor kinase activation to transcriptional control

PubMed Central

Qin, Bin Y.; Lam, Suvana S.; Correia, John J.; Lin, Kai

2002-01-01

Smad3 transduces the signals of TGF-βs, coupling transmembrane receptor kinase activation to transcriptional control. The membrane-associated molecule SARA (Smad Anchor for Receptor Activation) recruits Smad3 for phosphorylation by the receptor kinase. Upon phosphorylation, Smad3 dissociates from SARA and enters the nucleus, in which its transcriptional activity can be repressed by Ski. Here, we show that SARA and Ski recognize specifically the monomeric and trimeric forms of Smad3, respectively. Thus, trimerization of Smad3, induced by phosphorylation, simultaneously activates the TGF-β signal by driving Smad3 dissociation from SARA and sets up the negative feedback mechanism by Ski. Structural models of the Smad3/SARA/receptor kinase complex and Smad3/Ski complex provide insights into the molecular basis of regulation. PMID:12154125

The Atypical Response Regulator Protein ChxR Has Structural Characteristics and Dimer Interface Interactions That Are Unique within the OmpR/PhoB Subfamily

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hickey, John M.; Lovell, Scott; Battaile, Kevin P.

2013-05-29

Typically as a result of phosphorylation, OmpR/PhoB response regulators form homodimers through a receiver domain as an integral step in transcriptional activation. Phosphorylation stabilizes the ionic and hydrophobic interactions between monomers. Recent studies have shown that some response regulators retain functional activity in the absence of phosphorylation and are termed atypical response regulators. The two currently available receiver domain structures of atypical response regulators are very similar to their phospho-accepting homologs, and their propensity to form homodimers is generally retained. An atypical response regulator, ChxR, from Chlamydia trachomatis, was previously reported to form homodimers; however, the residues critical to thismore » interaction have not been elucidated. We hypothesize that the intra- and intermolecular interactions involved in forming a transcriptionally competent ChxR are distinct from the canonical phosphorylation (activation) paradigm in the OmpR/PhoB response regulator subfamily. To test this hypothesis, structural and functional studies were performed on the receiver domain of ChxR. Two crystal structures of the receiver domain were solved with the recently developed method using triiodo compound I3C. These structures revealed many characteristics unique to OmpR/PhoB subfamily members: typical or atypical. Included was the absence of two {alpha}-helices present in all other OmpR/PhoB response regulators. Functional studies on various dimer interface residues demonstrated that ChxR forms relatively stable homodimers through hydrophobic interactions, and disruption of these can be accomplished with the introduction of a charged residue within the dimer interface. A gel shift study with monomeric ChxR supports that dimerization through the receiver domain is critical for interaction with DNA.« less

Endogenous overexpression of an active phosphorylated form of DNA polymerase β under oxidative stress in Trypanosoma cruzi

PubMed Central

Moreira-Ramos, Sandra; Castillo, Christian; Kemmerling, Ulrike; Lapier, Michel; Maya, Juan Diego; Solari, Aldo

2018-01-01

Trypanosoma cruzi is exposed during its life to exogenous and endogenous oxidative stress, leading to damage of several macromolecules such as DNA. There are many DNA repair pathways in the nucleus and mitochondria (kinetoplast), where specific protein complexes detect and eliminate damage to DNA. One group of these proteins is the DNA polymerases. In particular, Tc DNA polymerase β participates in kinetoplast DNA replication and repair. However, the mechanisms which control its expression under oxidative stress are still unknown. Here we describe the effect of oxidative stress on the expression and function of Tc DNA polymerase β To this end parasite cells (epimastigotes and trypomastigotes) were exposed to peroxide during short periods of time. Tc DNA polymerase β which was associated physically with kinetoplast DNA, showed increased protein levels in response to peroxide damage in both parasite forms analyzed. Two forms of DNA polymerase β were identified and overexpressed after peroxide treatment. One of them was phosphorylated and active in DNA synthesis after renaturation on polyacrylamide electrophoresis gel. This phosphorylated form showed 3-4-fold increase in both parasite forms. Our findings indicate that these increments in protein levels are not under transcriptional control because the level of Tc DNA polymerase β mRNA is maintained or slightly decreased during the exposure to oxidative stress. We propose a mechanism where a DNA repair pathway activates a cascade leading to the increment of expression and phosphorylation of Tc DNA polymerase β in response to oxidative damage, which is discussed in the context of what is known in other trypanosomes which lack transcriptional control. PMID:29432450

Phosphorylation of the IDP KID Modulates Affinity for KIX by Increasing the Lifetime of the Complex.

PubMed

Dahal, Liza; Shammas, Sarah L; Clarke, Jane

2017-12-19

Intrinsically disordered proteins (IDPs) are known to undergo a range of posttranslational modifications, but by what mechanism do such modifications affect the binding of an IDP to its partner protein? We investigate this question using one such IDP, the kinase inducible domain (KID) of the transcription factor CREB, which interacts with the KIX domain of CREB-binding protein upon phosphorylation. As with many other IDPs, KID undergoes coupled folding and binding to form α-helical structure upon interacting with KIX. This single site phosphorylation plays an important role in the control of transcriptional activation in vivo. Here we show that, contrary to expectation, phosphorylation has no effect on association rates-unphosphorylated KID binds just as rapidly as pKID, the phosphorylated form-but rather, acts by increasing the lifetime of the complex. We propose that by controlling the lifetime of the bound complex of pKID:KIX via altering the dissociation rate, phosphorylation can facilitate effective control of transcription regulation. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

NIK and Cot cooperate to trigger NF-kappaB p65 phosphorylation.

PubMed

Wittwer, Tobias; Schmitz, M Lienhard

2008-06-27

The serine/threonine kinase Cot triggers NF-kappaB-dependent transactivation and activation of various MAPKinases. Here we identify Cot as a novel p65 interacting protein kinase. Cot expression induces p65 phosphorylation at serines 536 and 468 in dependence from its kinase function. Accordingly, shRNA-mediated knockdown of Cot expression interferes with TNF-induced NF-kappaB-dependent gene expression. Also the C-terminally truncated, oncogenic form of Cot is able to trigger p65 phosphorylation. In vitro kinase assays and dominant negative mutants revealed that NIK functions downstream of Cot to mediate p65 phosphorylation.

One-step purification of a functional, constitutively activated form of visual arrestin.

PubMed

Huang, Li; Mao, Xiang; Abdulaev, Najmoutin G; Ngo, Tony; Liu, Wei; Ridge, Kevin D

2012-03-01

Desensitization of agonist-activated G protein-coupled receptors (GPCRs) requires phosphorylation followed by the binding of arrestin, a ~48 kDa soluble protein. While crystal structures for the inactive, 'basal' state of various arrestins are available, the conformation of 'activated' arrestin adopted upon interaction with activated GPCRs remains unknown. As a first step towards applying high-resolution structural methods to study arrestin conformation and dynamics, we have utilized the subtilisin prodomain/Profinity eXact™ fusion-tag system for the high-level bacterial expression and one-step purification of wild-type visual arrestin (arrestin 1) as well as a mutant form (R175E) of the protein that binds to non-phosphorylated, light-activated rhodopsin (Rho∗). The results show that both prodomain/Profinity eXact™ fusion-tagged wild-type and R175E arrestins can be expressed to levels approaching 2-3 mg/l in Luria-Bertani media, and that the processed, tag-free mature forms can be purified to near homogeneity using a Bio-Scale™ Mini Profinity eXact™ cartridge on the Profinia™ purification system. Functional analysis of R175E arrestin generated using this approach shows that it binds to non-phosphorylated rhodopsin in a light-dependent manner. These findings should facilitate the structure determination of this 'constitutively activated' state of arrestin 1 as well as the monitoring of conformational changes upon interaction with Rho∗. Copyright © 2011 Elsevier Inc. All rights reserved.

A new TAO kinase inhibitor reduces tau phosphorylation at sites associated with neurodegeneration in human tauopathies.

PubMed

Giacomini, Caterina; Koo, Chuay-Yeng; Yankova, Natalia; Tavares, Ignatius A; Wray, Selina; Noble, Wendy; Hanger, Diane P; Morris, Jonathan D H

2018-05-07

In Alzheimer's disease (AD) and related tauopathies, the microtubule-associated protein tau is highly phosphorylated and aggregates to form neurofibrillary tangles that are characteristic of these neurodegenerative diseases. Our previous work has demonstrated that the thousand-and-one amino acid kinases (TAOKs) 1 and 2 phosphorylate tau on more than 40 residues in vitro. Here we show that TAOKs are phosphorylated and active in AD brain sections displaying mild (Braak stage II), intermediate (Braak stage IV) and advanced (Braak stage VI) tau pathology and that active TAOKs co-localise with both pre-tangle and tangle structures. TAOK activity is also enriched in pathological tau containing sarkosyl-insoluble extracts prepared from AD brain. Two new phosphorylated tau residues (T123 and T427) were identified in AD brain, which appear to be targeted specifically by TAOKs. A new small molecule TAOK inhibitor (Compound 43) reduced tau phosphorylation on T123 and T427 and also on additional pathological sites (S262/S356 and S202/T205/S208) in vitro and in cell models. The TAOK inhibitor also decreased tau phosphorylation in differentiated primary cortical neurons without affecting markers of synapse and neuron health. Notably, TAOK activity also co-localised with tangles in post-mortem frontotemporal lobar degeneration (FTLD) brain tissue. Furthermore, the TAOK inhibitor decreased tau phosphorylation in induced pluripotent stem cell derived neurons from FTLD patients, as well as cortical neurons from a transgenic mouse model of tauopathy (Tau35 mice). Our results demonstrate that abnormal TAOK activity is present at pre-tangles and tangles in tauopathies and that TAOK inhibition effectively decreases tau phosphorylation on pathological sites. Thus, TAOKs may represent a novel target to reduce or prevent tau-associated neurodegeneration in tauopathies.

Increased expression of phosphorylated forms of RNA-dependent protein kinase and eukaryotic initiation factor 2α may signal skeletal muscle atrophy in weight-losing cancer patients

PubMed Central

Eley, H L; Skipworth, R J E; Deans, D A C; Fearon, K C H; Tisdale, M J

2007-01-01

Previous studies suggest that the activation (autophosphorylation) of dsRNA-dependent protein kinase (PKR) can stimulate protein degradation, and depress protein synthesis in skeletal muscle through phosphorylation of the translation initiation factor 2 (eIF2) on the α-subunit. To understand whether these mediators are important in muscle wasting in cancer patients, levels of the phospho forms of PKR and eIF2α have been determined in rectus abdominus muscle of weight losing patients with oesophago-gastric cancer, in comparison with healthy controls. Levels of both phospho PKR and phospho eIF2α were significantly enhanced in muscle of cancer patients with weight loss irrespective of the amount and there was a linear relationship between phosphorylation of PKR and phosphorylation of eIF2α (correlation coefficient 0.76, P=0.005). This suggests that phosphorylation of PKR led to phosphorylation of eIF2α. Myosin levels decreased as the weight loss increased, and there was a linear relationship between myosin expression and the extent of phosphorylation of eIF2α (correlation coefficient 0.77, P=0.004). These results suggest that phosphorylation of PKR may be an important initiator of muscle wasting in cancer patients. PMID:18087277

The Parkinson Disease-linked LRRK2 Protein Mutation I2020T Stabilizes an Active State Conformation Leading to Increased Kinase Activity*

PubMed Central

Ray, Soumya; Bender, Samantha; Kang, Stephanie; Lin, Regina; Glicksman, Marcie A.; Liu, Min

2014-01-01

The effect of leucine-rich repeat kinase 2 (LRRK2) mutation I2020T on its kinase activity has been controversial, with both increased and decreased effects being reported. We conducted steady-state and pre-steady-state kinetic studies on LRRKtide and its analog LRRKtideS. Their phosphorylation differs by the rate-limiting steps: product release is rate-limiting for LRRKtide and phosphoryl transfer is rate-limiting for LRRKtideS. As a result, we observed that the I2020T mutant is more active than wild type (WT) LRRK2 for LRRKtideS phosphorylation, whereas it is less active than WT for LRRKtide phosphorylation. Our pre-steady-state kinetic data suggest that (i) the I2020T mutant accelerates the rates of phosphoryl transfer of both reactions by 3–7-fold; (ii) this increase is masked by a rate-limiting product release step for LRRKtide phosphorylation; and (iii) the observed lower activity of the mutant for LRRKtide phosphorylation is a consequence of its instability: the concentration of the active form of the mutant is 3-fold lower than WT. The I2020T mutant has a dramatically low KATP and therefore leads to resistance to ATP competitive inhibitors. Two well known DFG-out or type II inhibitors are also weaker toward the mutant because they inhibit the mutant in an unexpected ATP competitive mechanism. The I2020 residue lies next to the DYG motif of the activation loop of the LRRK2 kinase domain. Our modeling and metadynamic simulations suggest that the I2020T mutant stabilizes the DYG-in active conformation and creates an unusual allosteric pocket that can bind type II inhibitors but in an ATP competitive fashion. PMID:24695735

The kinase activity of fibroblast growth factor receptor 3 with activation loop mutations affects receptor trafficking and signaling.

PubMed

Lievens, Patricia M-J; Mutinelli, Chiara; Baynes, Darcie; Liboi, Elio

2004-10-08

Amino acid substitutions at the Lys-650 codon within the activation loop kinase domain of fibroblast growth factor receptor 3 (FGFR3) result in graded constitutive phosphorylation of the receptor. Accordingly, the Lys-650 mutants are associated with dwarfisms with graded clinical severity. To assess the importance of the phosphorylation level on FGFR3 maturation along the secretory pathway, hemagglutinin A-tagged derivatives were studied. The highly activated SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans) mutant accumulates in its immature and phosphorylated form in the endoplasmic reticulum (ER), which fails to be degraded. Furthermore, the Janus kinase (Jak)/STAT pathway is activated from the ER by direct recruitment of Jak1. Abolishing the autocatalytic property of the mutated FGFR3 by replacing the critical Tyr-718 reestablishes the receptor full maturation and inhibits signaling. Differently, the low activated hypochondroplasia mutant is present as a mature phosphorylated form on the plasma membrane, although with a delayed transition in the ER, and is completely processed. Signaling does not occur in the presence of brefeldin A; instead, STAT1 is activated when protein secretion is blocked with monensin, suggesting that the hypochondroplasia receptor signals at the exit from the ER. Our results suggest that kinase activity affects FGFR3 trafficking and determines the spatial segregation of signaling pathways. Consequently, the defect in down-regulation of the highly activated receptors results in the increased signaling capacity from the intracellular compartments, and this may determine the severity of the diseases.

Secretion of the Phosphorylated Form of S100A9 from Neutrophils Is Essential for the Proinflammatory Functions of Extracellular S100A8/A9.

PubMed

Schenten, Véronique; Plançon, Sébastien; Jung, Nicolas; Hann, Justine; Bueb, Jean-Luc; Bréchard, Sabrina; Tschirhart, Eric J; Tolle, Fabrice

2018-01-01

S100A8 and S100A9 are members of the S100 family of cytoplasmic EF-hand Ca 2+ -binding proteins and are abundantly expressed in the cytosol of neutrophils. In addition to their intracellular roles, S100A8/A9 can be secreted in the extracellular environment and are considered as alarmins able to amplify the inflammatory response. The intracellular activity of S100A8/A9 was shown to be regulated by S100A9 phosphorylation, but the importance of this phosphorylation on the extracellular activity of S100A8/A9 has not yet been extensively studied. Our work focuses on the impact of the phosphorylation state of secreted S100A9 on the proinflammatory function of neutrophils. In a first step, we characterized the secretion of S100A8/A9 in different stimulatory conditions and investigated the phosphorylation state of secreted S100A9. Our results on neutrophil-like differentiated HL-60 (dHL-60) cells and purified human neutrophils showed a time-dependent secretion of S100A8/A9 when induced by phorbol 12-myristoyl 13-acetate and this secreted S100A9 was found in a phosphorylated form. Second, we evaluated the impact of this phosphorylation on proinflammatory cytokine expression and secretion in dHL-60 cells. Time course experiments with purified unphosphorylated or phosphorylated S100A8/A9 were performed and the expression and secretion levels of interleukin (IL)-1α, IL-1β, IL-6, tumor necrosis factor alpha, CCL2, CCL3, CCL4, and CXCL8 were measured by real-time PCR and cytometry bead array, respectively. Our results demonstrate that only the phosphorylated form of the complex induces proinflammatory cytokine expression and secretion. For the first time, we provide evidence that S100A8/PhosphoS100A9 is inducing cytokine secretion through toll-like receptor 4 signaling.

Secretion of the Phosphorylated Form of S100A9 from Neutrophils Is Essential for the Proinflammatory Functions of Extracellular S100A8/A9

PubMed Central

Schenten, Véronique; Plançon, Sébastien; Jung, Nicolas; Hann, Justine; Bueb, Jean-Luc; Bréchard, Sabrina; Tschirhart, Eric J.; Tolle, Fabrice

2018-01-01

S100A8 and S100A9 are members of the S100 family of cytoplasmic EF-hand Ca2+-binding proteins and are abundantly expressed in the cytosol of neutrophils. In addition to their intracellular roles, S100A8/A9 can be secreted in the extracellular environment and are considered as alarmins able to amplify the inflammatory response. The intracellular activity of S100A8/A9 was shown to be regulated by S100A9 phosphorylation, but the importance of this phosphorylation on the extracellular activity of S100A8/A9 has not yet been extensively studied. Our work focuses on the impact of the phosphorylation state of secreted S100A9 on the proinflammatory function of neutrophils. In a first step, we characterized the secretion of S100A8/A9 in different stimulatory conditions and investigated the phosphorylation state of secreted S100A9. Our results on neutrophil-like differentiated HL-60 (dHL-60) cells and purified human neutrophils showed a time-dependent secretion of S100A8/A9 when induced by phorbol 12-myristoyl 13-acetate and this secreted S100A9 was found in a phosphorylated form. Second, we evaluated the impact of this phosphorylation on proinflammatory cytokine expression and secretion in dHL-60 cells. Time course experiments with purified unphosphorylated or phosphorylated S100A8/A9 were performed and the expression and secretion levels of interleukin (IL)-1α, IL-1β, IL-6, tumor necrosis factor alpha, CCL2, CCL3, CCL4, and CXCL8 were measured by real-time PCR and cytometry bead array, respectively. Our results demonstrate that only the phosphorylated form of the complex induces proinflammatory cytokine expression and secretion. For the first time, we provide evidence that S100A8/PhosphoS100A9 is inducing cytokine secretion through toll-like receptor 4 signaling. PMID:29593718

Complementary phosphorylation sites in the adaptor protein SLP-76 promote synergistic activation of natural killer cells.

PubMed

Kim, Hun Sik; Long, Eric O

2012-07-10

The cytotoxic effects of natural killer (NK) cells and their ability to secrete cytokines require synergistic signals from specific pairs of co-activation receptors, such as CD314 (also known as NKG2D) and CD244 (2B4), which bind to distinct ligands present on target cells. These signals are required to overcome inhibition mediated by the E3 ubiquitin ligase c-Cbl of the guanine nucleotide exchange factor Vav1, which promotes activation of NK cells. Here, we showed that the adaptor protein SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kilodaltons) was required for this synergy and that distinct tyrosine residues in SLP-76 were phosphorylated by each member of a pair of synergistic receptors. Selective phosphorylation of tyrosine 113 or tyrosine 128 in SLP-76 enabled binding of SLP-76 to Vav1. Selective phosphorylation of SLP-76 at these residues was restricted to receptors that stimulated ligand-dependent target cell killing; antibody-dependent stimulation of the Fc receptor CD16 promoted phosphorylation at both sites. Knockdown and reconstitution experiments with SLP-76 mutant proteins showed the distinct role of each tyrosine in the synergistic mobilization of Ca2+, revealing an unexpected degree of selectivity in the phosphorylation of SLP-76 by NK cell co-activation receptors. Together, these data suggest that combined phosphorylation of separate tyrosine residues in SLP-76 forms the basis of synergistic NK cell activation.

Crystal structure of human IRAK1.

PubMed

Wang, Li; Qiao, Qi; Ferrao, Ryan; Shen, Chen; Hatcher, John M; Buhrlage, Sara J; Gray, Nathanael S; Wu, Hao

2017-12-19

Interleukin 1 (IL-1) receptor-associated kinases (IRAKs) are serine/threonine kinases that play critical roles in initiating innate immune responses against foreign pathogens and other types of dangers through their role in Toll-like receptor (TLR) and interleukin 1 receptor (IL-1R) mediated signaling pathways. Upon ligand binding, TLRs and IL-1Rs recruit adaptor proteins, such as myeloid differentiation primary response gene 88 (MyD88), to the membrane, which in turn recruit IRAKs via the death domains in these proteins to form the Myddosome complex, leading to IRAK kinase activation. Despite their biological and clinical significance, only the IRAK4 kinase domain structure has been determined among the four IRAK family members. Here, we report the crystal structure of the human IRAK1 kinase domain in complex with a small molecule inhibitor. The structure reveals both similarities and differences between IRAK1 and IRAK4 and is suggestive of approaches to develop IRAK1- or IRAK4-specific inhibitors for potential therapeutic applications. While the IRAK4 kinase domain is capable of homodimerization in the unphosphorylated state, we found that the IRAK1 kinase domain is constitutively monomeric regardless of its phosphorylation state. Additionally, the IRAK1 kinase domain forms heterodimers with the phosphorylated, but not unphosphorylated, IRAK4 kinase domain. Collectively, these data indicate a two-step kinase activation process in which the IRAK4 kinase domain first homodimerizes in the Myddosome, leading to its trans -autophosphorylation and activation. The phosphorylated IRAK4 kinase domain then forms heterodimers with the IRAK1 kinase domain within the Myddosome, leading to its subsequent phosphorylation and activation.

Functional map of arrestin binding to phosphorylated opsin, with and without agonist.

PubMed

Peterhans, Christian; Lally, Ciara C M; Ostermaier, Martin K; Sommer, Martha E; Standfuss, Jörg

2016-06-28

Arrestins desensitize G protein-coupled receptors (GPCRs) and act as mediators of signalling. Here we investigated the interactions of arrestin-1 with two functionally distinct forms of the dim-light photoreceptor rhodopsin. Using unbiased scanning mutagenesis we probed the individual contribution of each arrestin residue to the interaction with the phosphorylated apo-receptor (Ops-P) and the agonist-bound form (Meta II-P). Disruption of the polar core or displacement of the C-tail strengthened binding to both receptor forms. In contrast, mutations of phosphate-binding residues (phosphosensors) suggest the phosphorylated receptor C-terminus binds arrestin differently for Meta II-P and Ops-P. Likewise, mutations within the inter-domain interface, variations in the receptor-binding loops and the C-edge of arrestin reveal different binding modes. In summary, our results indicate that arrestin-1 binding to Meta II-P and Ops-P is similarly dependent on arrestin activation, although the complexes formed with these two receptor forms are structurally distinct.

JAK1 kinase forms complexes with interleukin-4 receptor and 4PS/insulin receptor substrate-1-like protein and is activated by interleukin-4 and interleukin-9 in T lymphocytes.

PubMed

Yin, T; Tsang, M L; Yang, Y C

1994-10-28

Interleukin (IL)-4 and IL-9 regulate the proliferation of T lymphocytes through interactions with their receptors. Previous studies have shown that unknown tyrosine kinases are involved in the proliferative signaling triggered by IL-4 and IL-9. Here we show that IL-4 and IL-9 induce overlapping (170, 130, and 125 kilodalton (kDa)) and distinct (45 and 88/90 kDa, respectively) protein tyrosine phosphorylation in T lymphocytes. We further identify the 170-kDa tyrosine-phosphorylated protein as 4PS/insulin receptor substrate-1-like (IRS-1L) protein and 130-kDa protein as JAK1 kinase. Furthermore, we demonstrate for the first time that JAK1 forms complexes with the IL-4 receptor and 4PS/IRS-1L protein following ligand-receptor interaction. In addition, we demonstrate that IL-9, but not IL-4, induced tyrosine phosphorylation of Stat 91 transcriptional factor. The overlapping and distinct protein tyrosine phosphorylation and activation of the same JAK1 kinase in T lymphocytes strongly suggests that IL-4 and IL-9 share the common signal transduction pathways and that the specificity for each cytokine could be achieved through the unique tyrosine-phosphorylated proteins triggered by individual cytokines.

The phosphorylated C-terminus of cAR1 plays a role in cell-type-specific gene expression and STATa tyrosine phosphorylation.

PubMed

Briscoe, C; Moniakis, J; Kim, J Y; Brown, J M; Hereld, D; Devreotes, P N; Firtel, R A

2001-05-01

cAMP receptors mediate some signaling pathways via coupled heterotrimeric G proteins, while others are G-protein-independent. This latter class includes the activation of the transcription factors GBF and STATa. Within the cellular mounds formed by aggregation of Dictyostelium, micromolar levels of cAMP activate GBF function, thereby inducing the transcription of postaggregative genes and initiating multicellular differentiation. Activation of STATa, a regulator of culmination and ecmB expression, results from cAMP receptor-dependent tyrosine phosphorylation and nuclear localization, also in mound-stage cells. During mound development, the cAMP receptor cAR1 is in a low-affinity state and is phosphorylated on multiple serine residues in its C-terminus. This paper addresses possible roles of cAMP receptor phosphorylation in the cAMP-mediated stimulation of GBF activity, STATa tyrosine phosphorylation, and cell-type-specific gene expression. To accomplish this, we have expressed cAR1 mutants in a strain in which the endogenous cAMP receptors that mediate postaggregative gene expression in vivo are deleted. We then examined the ability of these cells to undergo morphogenesis and induce postaggregative and cell-type-specific gene expression and STATa tyrosine phosphorylation. Analysis of cAR1 mutants in which the C-terminal tail is deleted or the ligand-mediated phosphorylation sites are mutated suggests that the cAR1 C-terminus is not essential for GBF-mediated postaggregative gene expression or STATa tyrosine phosphorylation, but may play a role in regulating cell-type-specific gene expression and morphogenesis. A mutant receptor, in which the C-terminal tail is constitutively phosphorylated, exhibits constitutive activation of STATa tyrosine phosphorylation in pulsed cells in suspension and a significantly impaired ability to induce cell-type-specific gene expression. The constitutively phosphorylated receptor also exerts a partial dominant negative effect on multicellular development when expressed in wild-type cells. These findings suggest that the phosphorylated C-terminus of cAR1 may be involved in regulating aspects of receptor-mediated processes, is not essential for GBF function, and may play a role in mediating subsequent development. Copyright 2001 Academic Press.

Arabidopsis Receptor of Activated C Kinase1 Phosphorylation by WITH NO LYSINE8 KINASE

DOE PAGES

Urano, Daisuke; Czarnecki, Olaf; Wang, Xiaoping; ...

2014-12-08

Receptor of activated C kinase1 (RACK1) is a versatile scaffold protein that binds to numerous proteins to regulate diverse cellular pathways in mammals. In Arabidopsis (Arabidopsis thaliana), RACK1 has been shown to regulate plant hormone signaling, stress responses, and multiple processes of growth and development. However, little is known about the molecular mechanism underlying these regulations. In this paper, we show that an atypical serine (Ser)/threonine (Thr) protein kinase, WITH NO LYSINE8 (WNK8), phosphorylates RACK1. WNK8 physically interacted with and phosphorylated RACK1 proteins at two residues: Ser-122 and Thr-162. Genetic epistasis analysis of rack1 wnk8 double mutants indicated that RACK1more » acts downstream of WNK8 in the glucose responsiveness and flowering pathways. The phosphorylation-dead form, RACK1AS122A/T162A, but not the phosphomimetic form, RACK1AS122D/T162E, rescued the rack1a null mutant, implying that phosphorylation at Ser-122 and Thr-162 negatively regulates RACK1A function. The transcript of RACK1AS122D/T162E accumulated at similar levels as those of RACK1S122A/T162A. However, although the steady-state level of the RACK1AS122A/T162A protein was similar to wild-type RACK1A protein, the RACK1AS122D/T162E protein was nearly undetectable, suggesting that phosphorylation affects the stability of RACK1A proteins. In conclusion, these results suggest that RACK1 is phosphorylated by WNK8 and that phosphorylation negatively regulates RACK1 function by influencing its protein stability.« less

Phosphorylation of deoxycytidine kinase on Ser-74: impact on kinetic properties and nucleoside analog activation in cancer cells.

PubMed

Amsailale, Rachid; Van Den Neste, Eric; Arts, Angélique; Starczewska, Eliza; Bontemps, Françoise; Smal, Caroline

2012-07-01

Deoxycytidine kinase (dCK) (EC 2.7.1.74) is a key enzyme in the activation of several therapeutic nucleoside analogs (NA). Its activity can be increased in vivo by Ser-74 phosphorylation, a property that could be used for enhancing NA activation and clinical efficacy. In line with this, studies with recombinant dCK showed that mimicking Ser-74 phosphorylation by a S74E mutation increases its activity toward pyrimidine analogs. However, purine analogs had not been investigated. Here, we show that the S74E mutation increased the k(cat) for cladribine (CdA) by 8- or 3-fold, depending on whether the phosphoryl donor was ATP or UTP, for clofarabine (CAFdA) by about 2-fold with both ATP and UTP, and for fludarabine (F-Ara-A) by 2-fold, but only with UTP. However, the catalytic efficiencies (k(cat)/Km) were not, or slightly, increased. The S74E mutation also sensitized dCK to feed-back inhibition by dCTP, regardless of the phosphoryl donor. Importantly, we did not observe an increase of endogenous dCK activity toward purine analogs after in vivo-induced increase of Ser-74 phosphorylation. Accordingly, treatment of CLL cells with aphidicolin, which enhances dCK activity through Ser-74 phosphorylation, did not modify the conversion of CdA or F-Ara-A into their active triphosphate form. Nevertheless, the same treatment enhanced activation of gemcitabine (dFdC) into dFdCTP in CLL as well as in HCT-116 cells and produced synergistic cytotoxicity. We conclude that increasing phosphorylation of dCK on Ser-74 might constitute a valuable strategy to enhance the clinical efficacy of some NA, like dFdC, but not of CdA or F-Ara-A. Copyright © 2012 Elsevier Inc. All rights reserved.

The utility of vitamin K3 (menadione) against pancreatic cancer.

PubMed

Osada, Shinji; Tomita, Hiroyuki; Tanaka, Yoshihiro; Tokuyama, Yasuharu; Tanaka, Hidenori; Sakashita, Fumio; Takahashi, Takao

2008-01-01

To evaluate the efficacy of vitamin K3 (VK3) against pancreatic cancer, the molecular mechanism of VK3 or gemcitabine (GEM)-induced inhibition of proliferation was characterized. The cell viability was determined using the 3-[4,5-dimethylthiazol]-2,5-diphenyl tetrazolium bromide (MTT) test method. The expressions of cellular proteins were evaluated by Western blot analysis. For morphological studies of the in vivo transplanted cancer cells, the tissues were stained with hematoxylin and eosin. The IC50 of VK3 for pancreatic cancer cells was calculated for 42.1 +/- 3.5 microM. Western blot analysis showed that VK3 induced rapid phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) 30 minutes after application. ERK but not JNK phosphorylation was maintained for at least 12 hours. Activation of apoptosis by VK3, as shown by molecular weight shifts of the pro-activated 32-kDa form of caspase-3 and poly(ADP-ribose)polymerase (PARP) cleavage of the 112-kDa form, was found. Treatment with the thiol antioxidant, L-cysteine (>0.2 mM), completely abrogated the VK3-induced phosphorylation of ERK, but not the JNK, and inhibition of proliferation. A caspase-3 inhibitor antagonized caspase-3 activation, but had no inhibitory effect on the proliferative activity of VK3. GEM at concentrations >0.1 microg/ml was found to inhibit cell proliferation after 24 hours. GEM also induced phosphorylation of JNK, activation of caspase-3 and accumulation of cyclin B1. Local application of VK3 was found to induce extensive tumor tissue necrosis, but slight hematemesis without necrosis was observed 48 hours after GEM injection. In Western blot, ERK but not JNK phosphorylation, was clearly detected in response to VK3 injection into the tumor tissue. The action of VK3 may lead to a favorable outcome against pancreatic cancer, and the detection of ERK phosphorylation in the tissue is important for predicting this effect.

Activation of phosphatidylinositol-3 kinase by nerve growth factor involves indirect coupling of the trk proto-oncogene with src homology 2 domains.

PubMed

Ohmichi, M; Decker, S J; Saltiel, A R

1992-10-01

Growth factor receptor tyrosine kinases can form stable associations with intracellular proteins that contain src homology (SH) 2 domains, including the p85 regulatory subunit of phosphatidylinositol (PI)-3 kinase. The activation of this enzyme by growth factors is evaluated in PC12 pheochromocytoma cells and NIH 3T3 fibroblasts expressing the pp140c-trk nerve growth factor (NGF) receptor (3T3-c-trk). NGF causes the rapid stimulation of PI-3 kinase activity detected in anti-phosphotyrosine, but not in anti-trk, immunoprecipitates. This effect coincides with the tyrosine phosphorylation of two proteins, with molecular masses of of 100 kd and 110 kd, that coimmunoprecipitate with p85. Similar phosphorylation patterns are induced when an immobilized fusion protein containing the amino-terminal SH2 domain of p85 is used to precipitate tyrosine-phosphorylated proteins. Thus, although NGF produces the rapid activation of PI-3 kinase through a mechanism that involves tyrosine phosphorylation, there is no evidence for tyrosine phosphorylation of p85, or for its ligand-dependent association with the NGF receptor. Perhaps another phosphoprotein may link the NGF receptor to this enzyme.

Regulatory domain phosphorylation to distinguish the mechanistic basis underlying acute CFTR modulators

PubMed Central

Pyle, Louise C.; Ehrhardt, Annette; Mitchell, Lisa High; Fan, LiJuan; Ren, Aixia; Naren, Anjaparavanda P.; Li, Yao; Clancy, J. P.; Bolger, Graeme B.; Sorscher, Eric J.

2011-01-01

Modulator compounds intended to overcome disease-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) show significant promise in clinical testing for cystic fibrosis. However, the mechanism(s) of action underlying these compounds are not fully understood. Activation of CFTR ion transport requires PKA-regulated phosphorylation of the regulatory domain (R-D) and dimerization of the nucleotide binding domains. Using a newly developed assay, we evaluated nine compounds including both CFTR potentatiators and activators discovered via various high-throughput screening strategies to acutely augment CFTR activity. We found considerable differences in the effects on R-D phosphorylation. Some (including UCCF-152) stimulated robust phosphorylation, and others had little effect (e.g., VRT-532 and VX-770). We then compared CFTR activation by UCCF-152 and VRT-532 in Ussing chamber studies using two epithelial models, CFBE41o− and Fischer rat thyroid cells, expressing various CFTR forms. UCCF-152 activated wild-type-, G551D-, and rescued F508del-CFTR currents but did not potentiate cAMP-mediated CFTR activation. In contrast, VRT-532 moderately activated CFTR short-circuit current and strongly potentiated forskolin-mediated current. Combined with the result that UCCF-152, but not VRT-532 or VX-770, acts by increasing CFTR R-D phosphorylation, these findings indicate that potentiation of endogenous cAMP-mediated activation of mutant CFTR is not due to a pathway involving augmented R-D phosphorylation. This study presents an assay useful to distinguish preclinical compounds by a crucial mechanism underlying CFTR activation, delineates two types of compound able to acutely augment CFTR activity (e.g., activators and potentiators), and demonstrates that a number of different mechanisms can be successfully employed to activate mutant CFTR. PMID:21724857

Reactive oxygen species contribute to arsenic-induced EZH2 phosphorylation in human bronchial epithelial cells and lung cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Lingzhi; Qiu, Ping; Chen, Bailing

Our previous studies suggested that arsenic is able to induce serine 21 phosphorylation of the EZH2 protein through activation of JNK, STAT3, and Akt signaling pathways in the bronchial epithelial cell line, BEAS-2B. In the present report, we further demonstrated that reactive oxygen species (ROS) were involved in the arsenic-induced protein kinase activation that leads to EZH2 phosphorylation. Several lines of evidence supported this notion. First, the pretreatment of the cells with N-acetyl-L-cysteine (NAC), a potent antioxidant, abolishes arsenic-induced EZH2 phosphorylation along with the inhibition of JNK, STAT3, and Akt. Second, H{sub 2}O{sub 2}, the most important form of ROSmore » in the cells in response to extracellular stress signals, can induce phosphorylation of the EZH2 protein and the activation of JNK, STAT3, and Akt. By ectopic expression of the myc-tagged EZH2, we additionally identified direct interaction and phosphorylation of the EZH2 protein by Akt in response to arsenic and H{sub 2}O{sub 2}. Furthermore, both arsenic and H{sub 2}O{sub 2} were able to induce the translocation of ectopically expressed or endogenous EZH2 from nucleus to cytoplasm. In summary, the data presented in this report indicate that oxidative stress due to ROS generation plays an important role in the arsenic-induced EZH2 phosphorylation. - Highlights:: • Arsenic (As{sup 3+}) induces EZH phosphorylation. • JNK, STAT3, and Akt contribute to EZH2 phosphorylation. • Oxidative stress is involved in As{sup 3+}-induced EZH2 phosphorylation. • As{sup 3+} induces direct interaction of Akt and EZH2. • Phosphorylated EZH2 localized in cytoplasm.« less

Cell biology of Smad2/3 linker region phosphorylation in vascular smooth muscle.

PubMed

Rezaei, Hossein B; Kamato, Danielle; Ansari, Ghazaleh; Osman, Narin; Little, Peter J

2012-08-01

The transforming growth factor (TGF)-β superfamily of ligands regulates a diverse set of cellular functions. Transforming growth factor-β induces its biological effects through Type I and Type II transmembrane receptors that have serine/threonine kinase activities and weak tyrosine kinase activity. In vascular smooth muscle, TGF-β binds to the TGF-β Type II receptor (TβRII) at the cell surface, recruiting the Type I receptor (TβRI) to form a heterocomplex. Consequently, after phosphorylation and activation of TβRI, the transcription factors receptor activated (R-) Smad2 and Smad3 are recruited and activated through phosphorylation of C terminal residues. Overall, Smad2/3 and co-Smad4 have similar structures consisting of three regions an N-terminal MH1 domain, a C-terminal MH2 domain and a central linker region. Phosphorylation of the Smad linker region appears to have an important role in the regulation of Smad activity and function. The mitogen-activated protein kinase (MAPK) family, CDK2, CDK4 and calcium-calmodulin dependent kinase are the main kinases that phosphorylate sites in the linker region. The role of the linker region includes enabling the formation of Smad homo-oligomers and provision of phosphorylation sites for MAPK and other kinases. In some instances, linker region phosphorylation regulates the inhibition of the nuclear translocation of Smads. In the present review, we describe TGF-β signalling through Smad2/3 and the importance of the linker region in the regulation and expression of genes induced by TGF-β superfamily ligands in the context of vascular smooth muscle. © 2011 The Authors. Clinical and Experimental Pharmacology and Physiology © 2011 Blackwell Publishing Asia Pty Ltd.

p21 Activated Kinase 5 Activates Raf-1 and Targets it to Mitochondria

PubMed Central

Wu, Xiaochong; Carr, Heather S.; Dan, Ippeita; Ruvolo, Peter P.; Frost, Jeffrey A.

2008-01-01

Raf-1 is an important effector of Ras mediated signaling and is a critical regulator of the ERK/MAPK pathway. Raf-1 activation is controlled in part by phosphorylation on multiple residues, including an obligate phosphorylation site at serine 338. Previously PAK1 and casein kinase II have been implicated as serine 338 kinases. To identify novel kinases that phosphorylate this site, we tested the ability of group II PAKs (PAKs 4-6) to control serine 338 phosphorylation. We observed that all group II PAKs were efficient serine 338 kinases, although only PAK1 and PAK5 significantly stimulated Raf-1 kinase activity. We also showed that PAK5 forms a tight complex with Raf-1 in the cell, but not A-Raf or B-Raf. Importantly, we also demonstrated that the association of Raf-1 with PAK5 targets a subpopulation of Raf-1 to mitochondria. These data indicate that PAK5 is a potent regulator of Raf-1 activity and may control Raf-1 dependent signaling at the mitochondria. PMID:18465753

Regulatory Phosphorylation of Ikaros by Bruton's Tyrosine Kinase

PubMed Central

Zhang, Jian; Ishkhanian, Rita; Uckun, Fatih M.

2013-01-01

Diminished Ikaros function has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL), the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros is of paramount importance for normal lymphocyte ontogeny. Here we provide genetic and biochemical evidence for a previously unknown function of Bruton's tyrosine kinase (BTK) as a partner and posttranslational regulator of Ikaros, a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis. We demonstrate that BTK phosphorylates Ikaros at unique phosphorylation sites S214 and S215 in the close vicinity of its zinc finger 4 (ZF4) within the DNA binding domain, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Our results further demonstrate that BTK-induced activating phosphorylation is critical for the optimal transcription factor function of Ikaros. PMID:23977012

Effect of binding in cyclic phosphorylation-dephosphorylation process and in energy transformation.

PubMed

Sarkar, A; Beard, D A; Franza, B R

2006-07-01

The effects of binding on the phosphorylation-dephosphorylation cycle (PDPC) - one of the key components of the signal transduction processes - is analyzed based on a mathematical model. The model shows that binding of proteins, forming a complex, diminishes the ultrasensitivity of the PDPC to the differences in activity between kinase and phosphatase in the cycle. It is also found that signal amplification depends upon the strength of the binding affinity of the protein (phosphorylated or dephosphorylated) to other proteins . It is also observed that the amplification of signal is not only dependent on phosphorylation potential but also on binding properties and resulting adjustments in binding energies.

Regulation of the Incorporation of Tissue Factor into Microparticles by Serine Phosphorylation of the Cytoplasmic Domain of Tissue Factor*

PubMed Central

Collier, Mary E. W.; Ettelaie, Camille

2011-01-01

The mechanisms that regulate the incorporation and release of tissue factors (TFs) into cell-derived microparticles are as yet unidentified. In this study, we have explored the regulation of TF release into microparticles by the phosphorylation of serine residues within the cytoplasmic domain of TF. Wild-type and mutant forms of TF, containing alanine and aspartate substitutions at Ser253 and Ser258, were overexpressed in coronary artery and dermal microvascular endothelial cells and microparticle release stimulated with PAR2 agonist peptide (PAR2-AP). The release of TF antigen and activity was then monitored. In addition, the phosphorylation state of the two serine residues within the released microparticles and the cells was monitored for 150 min. The release of wild-type TF as procoagulant microparticles peaked at 90 min and declined thereafter in both cell types. The TF within these microparticles was phosphorylated at Ser253 but not at Ser258. Aspartate substitution of Ser253 resulted in rapid release of TF antigen but not activity, whereas TF release was reduced and delayed by alanine substitution of Ser253 or aspartate substitution of Ser258. Alanine substitution of Ser258 prolonged the release of TF following PAR2-AP activation. The release of TF was concurrent with phosphorylation of Ser253 and was followed by dephosphorylation at 120 min and phosphorylation of Ser258. We propose a sequential mechanism in which the phosphorylation of Ser253 through PAR2 activation results in the incorporation of TF into microparticles, simultaneously inducing Ser258 phosphorylation. Phosphorylation of Ser258 in turn promotes the dephosphorylation of Ser253 and suppresses the release of TF. PMID:21310953

Fasting and Systemic Insulin Signaling Regulate Phosphorylation of Brain Proteins That Modulate Cell Morphology and Link to Neurological Disorders*

PubMed Central

Li, Min; Quan, Chao; Toth, Rachel; Campbell, David G.; MacKintosh, Carol; Wang, Hong Yu; Chen, Shuai

2015-01-01

Diabetes is strongly associated with cognitive decline, but the molecular reasons are unknown. We found that fasting and peripheral insulin promote phosphorylation and dephosphorylation, respectively, of specific residues on brain proteins including cytoskeletal regulators such as slit-robo GTPase-activating protein 3 (srGAP3) and microtubule affinity-regulating protein kinases (MARKs), in which deficiency or dysregulation is linked to neurological disorders. Fasting activates protein kinase A (PKA) but not PKB/Akt signaling in the brain, and PKA can phosphorylate the purified srGAP3. The phosphorylation of srGAP3 and MARKs were increased when PKA signaling was activated in primary neurons. Knockdown of PKA decreased the phosphorylation of srGAP3. Furthermore, WAVE1, a protein kinase A-anchoring protein, formed a complex with srGAP3 and PKA in the brain of fasted mice to facilitate the phosphorylation of srGAP3 by PKA. Although brain cells have insulin receptors, our findings are inconsistent with the down-regulation of phosphorylation of target proteins being mediated by insulin signaling within the brain. Rather, our findings infer that systemic insulin, through a yet unknown mechanism, inhibits PKA or protein kinase(s) with similar specificity and/or activates an unknown phosphatase in the brain. Ser858 of srGAP3 was identified as a key regulatory residue in which phosphorylation by PKA enhanced the GAP activity of srGAP3 toward its substrate, Rac1, in cells, thereby inhibiting the action of this GTPase in cytoskeletal regulation. Our findings reveal novel mechanisms linking peripheral insulin sensitivity with cytoskeletal remodeling in neurons, which may help to explain the association of diabetes with neurological disorders such as Alzheimer disease. PMID:26499801

Tyrosine phosphorylation of 3BP2 is indispensable for the interaction with VAV3 in chicken DT40 cells.

PubMed

Chihara, Kazuyasu; Kimura, Yukihiro; Honjoh, Chisato; Yamauchi, Shota; Takeuchi, Kenji; Sada, Kiyonao

2014-03-10

Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2) is known to play regulatory roles in immunoreceptor-mediated signal transduction. We have previously demonstrated that Tyr(174), Tyr(183) and Tyr(446) in mouse 3BP2 are predominantly phosphorylated by Syk, and the phosphorylation of Tyr(183) and the Src homology 2 (SH2) domain of mouse 3BP2 are critical for B cell receptor (BCR)-induced activation of nuclear factor of activated T cells (NFAT) in human B cells. In this report, we have shown that Syk, but not Abl family protein-tyrosine kinases, is critical for BCR-mediated tyrosine phosphorylation of 3BP2 in chicken DT40 cells. Mutational analysis showed that Tyr(174), Tyr(183) and Tyr(426) of chicken 3BP2 are the major phosphorylation sites by Syk and the SH2 domain of 3BP2 is critical for tyrosine phosphorylation. In addition, phosphorylation of Tyr(426) is required for the inducible interaction with the SH2 domain of Vav3. Moreover, the expression of the mutant form of 3BP2 in which Tyr(426) was substituted to Phe resulted in the reduction in BCR-mediated Rac1 activation, when compared with the case of wild-type. Altogether, these data suggest that 3BP2 is involved in the activation of Rac1 through the regulation of Vav3 by Syk-dependent phosphorylation of Tyr(426) following BCR stimulation. Copyright © 2014 Elsevier Inc. All rights reserved.

Tyrosine phosphorylation of 3BP2 is indispensable for the interaction with VAV3 in chicken DT40 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chihara, Kazuyasu; Organization for Life Science Advancement Programs, University of Fukui, Fukui 910-1193; Kimura, Yukihiro

Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2) is known to play regulatory roles in immunoreceptor-mediated signal transduction. We have previously demonstrated that Tyr{sup 174}, Tyr{sup 183} and Tyr{sup 446} in mouse 3BP2 are predominantly phosphorylated by Syk, and the phosphorylation of Tyr{sup 183} and the Src homology 2 (SH2) domain of mouse 3BP2 are critical for B cell receptor (BCR)-induced activation of nuclear factor of activated T cells (NFAT) in human B cells. In this report, we have shown that Syk, but not Abl family protein-tyrosine kinases, is critical for BCR-mediated tyrosine phosphorylation of 3BP2 in chicken DT40 cells. Mutationalmore » analysis showed that Tyr{sup 174}, Tyr{sup 183} and Tyr{sup 426} of chicken 3BP2 are the major phosphorylation sites by Syk and the SH2 domain of 3BP2 is critical for tyrosine phosphorylation. In addition, phosphorylation of Tyr{sup 426} is required for the inducible interaction with the SH2 domain of Vav3. Moreover, the expression of the mutant form of 3BP2 in which Tyr{sup 426} was substituted to Phe resulted in the reduction in BCR-mediated Rac1 activation, when compared with the case of wild-type. Altogether, these data suggest that 3BP2 is involved in the activation of Rac1 through the regulation of Vav3 by Syk-dependent phosphorylation of Tyr{sup 426} following BCR stimulation. - Highlights: • 3BP2 is phosphorylated by Syk, but not Abl family kinases in BCR signaling. • Tyr183 and Tyr426 in chicken 3BP2 are the major phosphorylation sites by Syk. • The SH2 domain of 3BP2 is critical for tyrosine phosphorylation of 3BP2. • Phosphorylation of Tyr426 in 3BP2 is required for the inducible binding with Vav3. • 3BP2 is involved in the regulation of BCR-mediated Rac1 activation.« less

Phosphorylation of Stats at Ser727 in renal proximal tubular epithelial cells exposed to cadmium.

PubMed

Nakagawa, Junko; Nishitai, Gen; Inageda, Kiyoshi; Matsuoka, Masato

2007-11-01

The effects of cadmium exposure on serine phosphorylation of signal transducers and activators of transcription (Stats) and an upstream kinase were examined in renal proximal tubular cells. In porcine LLC-PK1 cells treated with cadmium, Stat1 and Stat3 proteins were phosphorylated at Ser727 without changing total Stat protein levels. While phosphorylated forms of the members of mitogen-activated protein kinases (MAPKs) increased in response to cadmium exposure, treatment with a p38 inhibitor, SB203580 reduced Ser727 phosphorylation of Stat1 and Stat3 markedly in LLC-PK1 cells. The expression of human matrix metalloproteinase-3 (MMP-3), a Stats-inducible gene, was found to be up-regulated in human HK-2 cells exposed to cadmium, and suppressed by preincubation with SB203580. These results suggest that cadmium might induce the phosphorylation of Stat1 and Stat3 at Ser727 via the p38 pathway at least in part, and modulate gene expression in these proximal tubular cells. Copyright © 2007 Elsevier B.V. All rights reserved.

Aluminum interaction with human brain tau protein phosphorylation by various kinases

DOE Office of Scientific and Technical Information (OSTI.GOV)

El-Sebae; Abou Zeid, M.M.; Saleh, M.A.

1993-01-01

Phosphorylation is an indispensable process for energy and signal transduction in biological systems. AlCl[sub 3] at 10 nM to 10 [mu]M range activated in-vitro [[gamma][sup [minus]32]P]ATP phosphorylation of the brain ([tau]) [Gamma] protein in both normal human or E.coli expressed [Gamma] forms; in the presence of the kinases P34,PKP, and PKC. However, higher concentrations of AlCl[sub 3] inhibited the [Gamma] phosphorylation with P34, PKP, and PKC to a maximum at 1 mM level. AlCl[sub 3] at 100 [mu]M to 500 [mu]M range induced non-enzymatic phosphorylation of [Gamma] with [gamma]-ATP, [gamma]-GTP, and [alpha]-GRP. AlCl[sub 3] activated histone phosphorylation by P34 inmore » a similar pattern. The hyperphosphorylation of [Gamma] by Al[sup 3+] was accompanied in molecular shift and mobility retardation in SDS-PAGE. This may demonstrate the mechanism of the long term neurological effect of Al[sub 3+] in human brain leading to the formation of the neutrofibrillary tangles related to Alzeheimer's disease.« less

Stimulation of hepatocytic AMP-activated protein kinase by okadaic acid and other autophagy-suppressive toxins

PubMed Central

2004-01-01

Autophagic activity in isolated rat hepatocytes is strongly suppressed by OA (okadaic acid) and other PP (protein phosphatase)-inhibitory toxins as well as by AICAR (5-aminoimidazole-4-carboxamide riboside), a direct activator of AMPK (AMP-activated protein kinase). To investigate whether AMPK is a mediator of the effects of the toxin, a phosphospecific antibody directed against the activation of phosphorylation of the AMPK α (catalytic)-subunit at Thr172 was used to assess the activation status of this enzyme. AICAR as well as all the toxins tested (OA, microcystin-LR, calyculin A, cantharidin and tautomycin) induced strong, dose-dependent AMPKα phosphorylation, correlating with AMPK activity in situ (in intact hepatocytes) as measured by the AMPK-dependent phosphorylation of acetyl-CoA carboxylase at Ser79. All treatments induced the appearance of multiple, phosphatase-sensitive, low-mobility forms of the AMPK α-subunit, consistent with phosphorylation at several sites other than Thr172. The flavonoid naringin, an effective antagonist of OA-induced autophagy suppression, inhibited the AMPK phosphorylation and mobility shifting induced by AICAR, OA or microcystin, but not the changes induced by calyculin A or cantharidin. AMPK may thus be activated both by a naringin-sensitive and a naringin-resistant mechanism, probably involving the PPs PP2A and PP1 respectively. Neither the Thr172-phosphorylating protein kinase LKB1 nor the Thr172-dephosphorylating PP, PP2C, were mobility-shifted after treatment with toxins or AICAR, whereas a slight mobility shifting of the regulatory AMPK β-subunit was indicated. Immunoblotting with a phosphospecific antibody against pSer108 at the β-subunit revealed a naringin-sensitive phosphorylation induced by OA, microcystin and AICAR and a naringin-resistant phosphorylation induced by calyculin A and cantharidin, suggesting that β-subunit phosphorylation could play a role in AMPK activation. Naringin antagonized the autophagy-suppressive effects of AICAR and OA, but not the autophagy suppression caused by cantharidin, consistent with AMPK-mediated inhibition of autophagy by toxins as well as by AICAR. PMID:15461583

Putting one step before the other: distinct activation pathways for Cdk1 and Cdk2 bring order to the mammalian cell cycle

PubMed Central

Merrick, Karl A.; Fisher, Robert P.

2010-01-01

Eukaryotic cell division is controlled by the activity of cyclin-dependent kinases (CDKs). Cdk1 and Cdk2, which function at different stages of the mammalian cell cycle, both require cyclin-binding and phosphorylation of the activation (T-) loop for full activity, but differ with respect to the order in which the two steps occur in vivo. To form stable complexes with either of its partners—cyclins A and B—Cdk1 must be phosphorylated on its T-loop, but that phosphorylation in turn depends on the presence of cyclin. Cdk2 can follow a kinetically distinct path to activation in which T-loop phosphorylation precedes cyclin-binding, and thereby out-compete the more abundant Cdk1 for limiting amounts of cyclin A. Mathematical modeling suggests this could be a principal basis for the temporal ordering of CDK activation during S phase, which may dictate the sequence in which replication origins fire. Still to be determined are how: 1) the activation machinery discriminates between closely related CDKs, and 2) coordination of the cell cycle is affected when this mechanism of pathway insulation breaks down. PMID:20139727

Age- and brain region-dependent α-synuclein oligomerization is attributed to alterations in intrinsic enzymes regulating α-synuclein phosphorylation in aging monkey brains.

PubMed

Chen, Min; Yang, Weiwei; Li, Xin; Li, Xuran; Wang, Peng; Yue, Feng; Yang, Hui; Chan, Piu; Yu, Shun

2016-02-23

We previously reported that the levels of α-syn oligomers, which play pivotal pathogenic roles in age-related Parkinson's disease (PD) and dementia with Lewy bodies, increase heterogeneously in the aging brain. Here, we show that exogenous α-syn incubated with brain extracts from older cynomolgus monkeys and in Lewy body pathology (LBP)-susceptible brain regions (striatum and hippocampus) forms higher amounts of phosphorylated and oligomeric α-syn than that in extracts from younger monkeys and LBP-insusceptible brain regions (cerebellum and occipital cortex). The increased α-syn phosphorylation and oligomerization in the brain extracts from older monkeys and in LBP-susceptible brain regions were associated with higher levels of polo-like kinase 2 (PLK2), an enzyme promoting α-syn phosphorylation, and lower activity of protein phosphatase 2A (PP2A), an enzyme inhibiting α-syn phosphorylation, in these brain extracts. Further, the extent of the age- and brain-dependent increase in α-syn phosphorylation and oligomerization was reduced by inhibition of PLK2 and activation of PP2A. Inversely, phosphorylated α-syn oligomers reduced the activity of PP2A and showed potent cytotoxicity. In addition, the activity of GCase and the levels of ceramide, a product of GCase shown to activate PP2A, were lower in brain extracts from older monkeys and in LBP-susceptible brain regions. Our results suggest a role for altered intrinsic metabolic enzymes in age- and brain region-dependent α-syn oligomerization in aging brains.

Amyloid-β oligomer Aβ*56 induces specific alterations of tau phosphorylation and neuronal signaling

PubMed Central

Amar, Fatou; Sherman, Mathew A.; Rush, Travis; Larson, Megan; Boyle, Gabriel; Chang, Liu; Götz, Jürgen; Buisson, Alain; Lesné, Sylvain E.

2018-01-01

Oligomeric forms of amyloid-forming proteins are believed to be the principal initiating bioactive species in many neurodegenerative disorders, including Alzheimer’s disease (AD). Amyloid-β (Aβ) oligomers are implicated in pathological modification and aggregation of the microtubule-associated protein tau. To investigate the specific molecular pathways activated by different assemblies, we isolated various forms of Aβ from Tg2576 mice. We found that the Aβ*56, which is linked with preclinical AD, interacted with NMDA receptors (NMDARs) in primary cortical neurons, increased NMDAR-dependent Ca2+ influx and, consequently, increased intracellular calcium concentrations and the activation of Ca2+-dependent calmodulin kinase IIα (CaMKIIα). In neurons in mice and in culture, activated CaMKIIα induced increased phosphorylation and missorting of tau, which is associated with AD pathology. In contrast, exposure of cultured primary cortical neurons to other oligomeric Aβ forms (dimers and trimers) did not trigger these effects. Our results indicate that distinct Aβ assemblies activate neuronal signaling pathways in a selective manner, and that dissecting the molecular events caused by each may inform more effective therapeutic strategies. PMID:28487416

Inflammation kinase PKR phosphorylates α-synuclein and causes α-synuclein-dependent cell death.

PubMed

Reimer, Lasse; Vesterager, Louise Buur; Betzer, Cristine; Zheng, Jin; Nielsen, Lærke Dalsgaard; Kofoed, Rikke Hahn; Lassen, Louise Berkhoudt; Bølcho, Ulrik; Paludan, Søren Riis; Fog, Karina; Jensen, Poul Henning

2018-07-01

Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy comprise a group of neurodegenerative diseases termed synucleinopathies. Synucleinopathie are, characterized by presence of inclusion bodies in degenerating brain cells which contain aggregated α-synuclein phosphorylated on Ser129. Although the inflammation-associated serine-threonine kinase, PKR (EIF2AK2), promotes cellular protection against infection, we demonstrate a pro-degenerative role of activated PKR in an α-synuclein-dependent cell model of multiple system atrophy, where inhibition and silencing of PKR decrease cellular degeneration. In vitro phosphorylation demonstrates that PKR can directly bind and phosphorylate monomeric and filamenteous α-synuclein on Ser129. Inhibition and knockdown of PKR reduce Ser129 phosphorylation in different models (SH-SY5Y ASYN cells, OLN-AS7 cells, primary mouse hippocampal neurons, and acute brain slices), while overexpression of constitutively active PKR increases Ser129 α-syn phosphorylation. Treatment with pre-formed α-synuclein fibrils, proteostatic stress-promoting MG-132 and known PKR activators, herpes simplex virus-1-∆ICP34.5 and LPS, as well as PKR inducer, IFN-β-1b, lead to increased levels of phosphorylated Ser129 α-synuclein that is completely blocked by simultaneous PKR inhibition. These results reveal a direct link between PKR and the phosphorylation and toxicity of α-synuclein, and they support that neuroinflammatory processes play a role in modulating the pathogenicity of α-synuclein. Copyright © 2018 Elsevier Inc. All rights reserved.

The Golgi localization of phosphatidylinositol transfer protein beta requires the protein kinase C-dependent phosphorylation of serine 262 and is essential for maintaining plasma membrane sphingomyelin levels.

PubMed

van Tiel, Claudia M; Westerman, Jan; Paasman, Marten A; Hoebens, Martha M; Wirtz, Karel W A; Snoek, Gerry T

2002-06-21

Recombinant mouse phosphatidylinositol transfer protein (PI-TP)beta is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser(262) was identified as the major site of phosphorylation and Ser(165) as a minor phosphorylation site. The phospholipid transfer activities of wild-type PI-TP beta and PI-TP beta(S262A) were identical, whereas PI-TP beta(S165A) was completely inactive. PKC-dependent phosphorylation of Ser(262) also had no effect on the transfer activity of PI-TP beta. To investigate the role of Ser(262) in the functioning of PI-TP beta, wtPI-TP beta and PI-TP beta(S262A) were overexpressed in NIH3T3 fibroblast cells. Two-dimensional PAGE analysis of cell lysates was used to separate PI-TP beta from its phosphorylated form. After Western blotting, wtPI-TP beta was found to be 85% phosphorylated, whereas PI-TP beta(S262A) was not phosphorylated. In the presence of the PKC inhibitor GF 109203X, the phosphorylated form of wtPI-TP beta was strongly reduced. Immunolocalization showed that wtPI-TP beta was predominantly associated with the Golgi membranes. In the presence of the PKC inhibitor, wtPI-TP beta was distributed throughout the cell similar to what was observed for PI-TP beta(S262A). In contrast to wtPI-TP beta overexpressors, cells overexpressing PI-TP beta(S262A) were unable to rapidly replenish sphingomyelin in the plasma membrane upon degradation by sphingomyelinase. This implies that PKC-dependent association with the Golgi complex is a prerequisite for PI-TP beta to express its effect on sphingomyelin metabolism.

The phosphoproteome of bloodstream form Trypanosoma brucei, causative agent of African sleeping sickness.

PubMed

Nett, Isabelle R E; Martin, David M A; Miranda-Saavedra, Diego; Lamont, Douglas; Barber, Jonathan D; Mehlert, Angela; Ferguson, Michael A J

2009-07-01

The protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and related animal diseases, and it has over 170 predicted protein kinases. Protein phosphorylation is a key regulatory mechanism for cellular function that, thus far, has been studied in T.brucei principally through putative kinase mRNA knockdown and observation of the resulting phenotype. However, despite the relatively large kinome of this organism and the demonstrated essentiality of several T. brucei kinases, very few specific phosphorylation sites have been determined in this organism. Using a gel-free, phosphopeptide enrichment-based proteomics approach we performed the first large scale phosphorylation site analyses for T.brucei. Serine, threonine, and tyrosine phosphorylation sites were determined for a cytosolic protein fraction of the bloodstream form of the parasite, resulting in the identification of 491 phosphoproteins based on the identification of 852 unique phosphopeptides and 1204 phosphorylation sites. The phosphoproteins detected in this study are predicted from their genome annotations to participate in a wide variety of biological processes, including signal transduction, processing of DNA and RNA, protein synthesis, and degradation and to a minor extent in metabolic pathways. The analysis of phosphopeptides and phosphorylation sites was facilitated by in-house developed software, and this automated approach was validated by manual annotation of spectra of the kinase subset of proteins. Analysis of the cytosolic bloodstream form T. brucei kinome revealed the presence of 44 phosphorylated protein kinases in our data set that could be classified into the major eukaryotic protein kinase groups by applying a multilevel hidden Markov model library of the kinase catalytic domain. Identification of the kinase phosphorylation sites showed conserved phosphorylation sequence motifs in several kinase activation segments, supporting the view that phosphorylation-based signaling is a general and fundamental regulatory process that extends to this highly divergent lower eukaryote.

Gating connexin 43 channels reconstituted in lipid vesicles by mitogen-activated protein kinase phosphorylation.

PubMed

Kim, D Y; Kam, Y; Koo, S K; Joe, C O

1999-02-26

The regulation of gap junctional permeability by phosphorylation was examined in a model system in which connexin 43 (Cx43) gap junction hemichannels were reconstituted in lipid vesicles. Cx43 was immunoaffinity-purified from rat brain, and Cx43 channels were reconstituted into unilamellar phospholipid liposomes. The activities of the reconstituted channels were measured by monitoring liposome permeability. Liposomes containing the Cx43 protein were fractionated on the basis of permeability to sucrose using sedimentation in an iso-osmolar density gradient. The gradient allowed separation of the sucrose-permeable and -impermeable liposomes. Liposomes that were permeable to sucrose were also permeable to the communicating dye molecule lucifer yellow. Permeability, and therefore activity of the reconstituted Cx43 channels, were directly dependent on the state of Cx43 phosphorylation. The permeability of liposomes containing Cx43 channels was increased by treatment of liposomes with calf intestinal phosphatase. Moreover, liposomes formed with Cx43 that had been dephosphorylated by calf intestinal phosphatase treatment showed increased permeability to sucrose. The role of phosphorylation in the gating mechanism of Cx43 channels was supported further by the observation that phosphorylation of Cx43 by mitogen-activated protein kinase reversibly reduced the permeability of liposomes containing dephosphorylated Cx43. Our results show a direct correlation between gap junctional permeability and the phosphorylation state of Cx43.

Parkin-catalyzed Ubiquitin-Ester Transfer Is Triggered by PINK1-dependent Phosphorylation*

PubMed Central

Iguchi, Masahiro; Kujuro, Yuki; Okatsu, Kei; Koyano, Fumika; Kosako, Hidetaka; Kimura, Mayumi; Suzuki, Norihiro; Uchiyama, Shinichiro; Tanaka, Keiji; Matsuda, Noriyuki

2013-01-01

PINK1 and PARKIN are causal genes for autosomal recessive familial Parkinsonism. PINK1 is a mitochondrial Ser/Thr kinase, whereas Parkin functions as an E3 ubiquitin ligase. Under steady-state conditions, Parkin localizes to the cytoplasm where its E3 activity is repressed. A decrease in mitochondrial membrane potential triggers Parkin E3 activity and recruits it to depolarized mitochondria for ubiquitylation of mitochondrial substrates. The molecular basis for how the E3 activity of Parkin is re-established by mitochondrial damage has yet to be determined. Here we provide in vitro biochemical evidence for ubiquitin-thioester formation on Cys-431 of recombinant Parkin. We also report that Parkin forms a ubiquitin-ester following a decrease in mitochondrial membrane potential in cells, and that this event is essential for substrate ubiquitylation. Importantly, the Parkin RING2 domain acts as a transthiolation or acyl-transferring domain rather than an E2-recruiting domain. Furthermore, formation of the ubiquitin-ester depends on PINK1 phosphorylation of Parkin Ser-65. A phosphorylation-deficient mutation completely inhibited formation of the Parkin ubiquitin-ester intermediate, whereas phosphorylation mimics, such as Ser to Glu substitution, enabled partial formation of the intermediate irrespective of Ser-65 phosphorylation. We propose that PINK1-dependent phosphorylation of Parkin leads to the ubiquitin-ester transfer reaction of the RING2 domain, and that this is an essential step in Parkin activation. PMID:23754282

The insulin and IGF1 receptor kinase domains are functional dimers in the activated state

NASA Astrophysics Data System (ADS)

Cabail, M. Zulema; Li, Shiqing; Lemmon, Eric; Bowen, Mark E.; Hubbard, Stevan R.; Miller, W. Todd

2015-03-01

The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are highly related receptor tyrosine kinases with a disulfide-linked homodimeric architecture. Ligand binding to the receptor ectodomain triggers tyrosine autophosphorylation of the cytoplasmic domains, which stimulates catalytic activity and creates recruitment sites for downstream signalling proteins. Whether the two phosphorylated tyrosine kinase domains within the receptor dimer function independently or cooperatively to phosphorylate protein substrates is not known. Here we provide crystallographic, biophysical and biochemical evidence demonstrating that the phosphorylated kinase domains of IR and IGF1R form a specific dimeric arrangement involving an exchange of the juxtamembrane region proximal to the kinase domain. In this dimer, the active position of α-helix C in the kinase N lobe is stabilized, which promotes downstream substrate phosphorylation. These studies afford a novel strategy for the design of small-molecule IR agonists as potential therapeutic agents for type 2 diabetes.

Evidence for a role of platelet endothelial cell adhesion molecule-1 in endothelial cell mechanosignal transduction: is it a mechanoresponsive molecule?

PubMed

Osawa, Masaki; Masuda, Michitaka; Kusano, Ken-ichi; Fujiwara, Keigi

2002-08-19

Fluid shear stress (FSS) induces many forms of responses, including phosphorylation of extracellular signal-regulated kinase (ERK) in endothelial cells (ECs). We have earlier reported rapid tyrosine phosphorylation of platelet endothelial cell adhesion molecule-1 (PECAM-1) in ECs exposed to FSS. Osmotic changes also induced similar PECAM-1 and ERK phosphorylation with nearly identical kinetics. Because both FSS and osmotic changes should mechanically perturb the cell membrane, they might activate the same mechanosignaling cascade. When PECAM-1 is tyrosine phosphorylated by FSS or osmotic changes, SHP-2 binds to it. Here we show that ERK phosphorylation by FSS or osmotic changes depends on PECAM-1 tyrosine phosphorylation, SHP-2 binding to phospho-PECAM-1, and SHP-2 phosphatase activity. In ECs under flow, detectable amounts of SHP-2 and Gab1 translocated from the cytoplasm to the EC junction. When magnetic beads coated with antibodies against the extracellular domain of PECAM-1 were attached to ECs and tugged by magnetic force for 10 min, PECAM-1 associated with the beads was tyrosine phosphorylated. ERK was also phosphorylated in these cells. Binding of the beads by itself or pulling on the cell surface using poly-l-coated beads did not induce phosphorylation of PECAM-1 and ERK. These results suggest that PECAM-1 is a mechanotransduction molecule.

A phosphatase threshold sets the level of Cdk1 activity in early mitosis in budding yeast

PubMed Central

Harvey, Stacy L.; Enciso, Germán; Dephoure, Noah; Gygi, Steven P.; Gunawardena, Jeremy; Kellogg, Douglas R.

2011-01-01

Entry into mitosis is initiated by synthesis of cyclins, which bind and activate cyclin-dependent kinase 1 (Cdk1). Cyclin synthesis is gradual, yet activation of Cdk1 occurs in a stepwise manner: a low level of Cdk1 activity is initially generated that triggers early mitotic events, which is followed by full activation of Cdk1. Little is known about how stepwise activation of Cdk1 is achieved. A key regulator of Cdk1 is the Wee1 kinase, which phosphorylates and inhibits Cdk1. Wee1 and Cdk1 show mutual regulation: Cdk1 phosphorylates Wee1, which activates Wee1 to inhibit Cdk1. Further phosphorylation events inactivate Wee1. We discovered that a specific form of protein phosphatase 2A (PP2ACdc55) opposes the initial phosphorylation of Wee1 by Cdk1. In vivo analysis, in vitro reconstitution, and mathematical modeling suggest that PP2ACdc55 sets a threshold that limits activation of Wee1, thereby allowing a low constant level of Cdk1 activity to escape Wee1 inhibition in early mitosis. These results define a new role for PP2ACdc55 and reveal a systems-level mechanism by which dynamically opposed kinase and phosphatase activities can modulate signal strength. PMID:21849476

Multiple Protein Kinases Determine the Phosphorylated State of the Small Heat Shock Protein, HSP27, in SH-SY5Y Neuroblastoma Cells

PubMed Central

Dokas, Linda A.; Malone, Amy M.; Williams, Frederick E.; Nauli, Surya M.; Messer, William S.

2011-01-01

In SH-SY5Y human neuroblastoma cells, the cholinergic agonist, carbachol, stimulates phosphorylation of the small heat shock protein 27 (HSP27). Carbachol increases phosphorylation of both Ser-82 and Ser-78 while the phorbol ester, phorbol-12, 13-dibutyrate (PDB) affects only Ser-82. Muscarinic receptor activation by carbachol was confirmed by sensitivity of Ser-82 phosphorylation to hyoscyamine with no effect of nicotine or bradykinin. This response to carbachol is partially reduced by inhibition of protein kinase C (PKC) with GF 109203X and p38 mitogen-activated protein kinase (MAPK) with SB 203580. In contrast, phosphorylation produced by PDB is completely reversed by GF 109203X or CID 755673, an inhibitor of PKD. Inhibition of phosphatidylinositol 3-kinase or Akt with LY 294002 or Akti-1/2 stimulates HSP27 phosphorylation while rapamycin, which inhibits mTORC1, does not. The stimulatory effect of Akti-1/2 is reversed by SB 203580 and correlates with increased p38 MAPK phosphorylation. SH-SY5Y cells differentiated with a low concentration of PDB and basic fibroblast growth factor to a more neuronal phenotype retain carbachol-, PDB- and Akti-1/2-responsive HSP27 phosphorylation. Immunofluorescence microscopy confirms increased HSP27 phosphorylation in response to carbachol or PDB. At cell margins, PDB causes f-actin to reorganize forming lamellipodial structures from which phospho-HSP27 is segregated. The resultant phenotypic change in cell morphology is dependent upon PKC, but not PKD, activity. The major conclusion from this study is that the phosphorylated state of HSP27 in SH-SY5Y cells results from integrated signaling involving PKC, p38 MAPK and Akt. PMID:21338617

Applying the Brakes to Multi-Site SR Protein Phosphorylation: Substrate-Induced Effects on the Splicing Kinase SRPK1†

PubMed Central

Aubol, Brandon E.; Adams, Joseph A.

2011-01-01

To investigate how a protein kinase interacts with its protein substrate during extended, multi-site phosphorylation, the kinetic mechanism of a protein kinase involved in mRNA splicing control was investigated using rapid quench flow techniques. The protein kinase SRPK1 phosphorylates approximately 10 serines in the arginine-serine-rich domain (RS domain) of the SR protein SRSF1 in a C-to-N-terminal direction, a modification that directs this essential splicing factor from the cytoplasm to the nucleus. Transient-state kinetic experiments illustrate that the first phosphate is added rapidly onto the RS domain of SRSF1 (t1/2 = 0.1 sec) followed by slower, multi-site phosphorylation at the remaining serines (t1/2 = 15 sec). Mutagenesis experiments suggest that efficient phosphorylation rates are maintained by an extensive hydrogen bonding and electrostatic network between the RS domain of the SR protein and the active site and docking groove of the kinase. Catalytic trapping and viscosometric experiments demonstrate that while the phosphoryl transfer step is fast, ADP release limits multi-site phosphorylation. By studying phosphate incorporation into selectively pre-phosphorylated forms of the enzyme-substrate complex, the kinetic mechanism for site-specific phosphorylation along the reaction coordinate was assessed. The binding affinity of the SR protein, the phosphoryl transfer rate and ADP exchange rate were found to decline significantly as a function of progressive phosphorylation in the RS domain. These findings indicate that the protein substrate actively modulates initiation, extension and termination events associated with prolonged, multi-site phosphorylation. PMID:21728354

Ralstonia eutropha's Poly(3-hydroxybutyrate)(PHB) polymerase PhaC1 and PHB depolymerase PhaZa1 are phosphorylated in vivo.

PubMed

Jüngert, Janina R; Patterson, Cameron; Jendrossek, Dieter

2018-04-20

In this study, we screened PHB synthase PhaC1 and PHB depolymerase PhaZa1 of Ralstonia eutropha for the presence of phosphorylated residues during the PHB accumulation and PHB degradation phase. Thr373 of PHB synthase PhaC1 was phosphorylated in the stationary growth phase but was not modified in the exponential and PHB accumulation phases. Ser35 of PHB depolymerase PhaZa1 was identified in phosphorylated form both in the exponential and in the stationary growth phase. Additional phosphosites were identified for both proteins in sample-dependent forms. Site-directed mutagenesis of the codon for Thr373 and other phosphosites of PhaC1 revealed a strong negative impact on PHB synthase activity. Modification of Thr26 and Ser35 of PhaZa1 reduced the ability of R. eutropha to mobilize PHB in the stationary growth phase. Our results show that phosphorylation of PhaC1 and PhaZa1 can be important for modulation of the activities of PHB synthase and PHB depolymerase. Importance Polyhydroxybutyrate (PHB) and related polyhydroxyalkanoates (PHAs) are important intracellular carbon and energy storage compounds in many prokaryotes. The accumulation of PHB or PHAs increases the fitness of cells during periods of starvation and other stress conditions. The simultaneous presence of poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) and PHB depolymerase (PhaZa1) on synthesized PHB granules in Ralstonia eutropha (alternative designation Cupriavidus necator ) has been previously shown in several laboratories. These findings imply that the activities of PHB synthase and PHB depolymerase should be regulated to avoid a futile cycle of simultaneous synthesis and degradation of PHB. Here, we addressed this question by identifying phosphorylation sites on PhaC1 and PhaZa1 and by site-directed mutagenesis of identified residues. Furthermore, we conducted in vitro and in vivo analysis of PHB synthase activity and PHB contents. Copyright © 2018 American Society for Microbiology.

Inhibition of AMPK catabolic action by GSK3

PubMed Central

Suzuki, Tsukasa; Bridges, Dave; Nakada, Daisuke; Skiniotis, Georgios; Morrison, Sean J.; Lin, Jiandie; Saltiel, Alan R.; Inoki, Ken

2013-01-01

SUMMARY AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis by inhibiting anabolic and activating catabolic processes. While AMPK activation has been extensively studied, mechanisms that inhibit AMPK remain elusive. Here we report that glycogen synthase kinase 3 (GSK3) inhibits AMPK function. GSK3 forms a stable complex with AMPK through interactions with the AMPK β regulatory subunit and phosphorylates the AMPK α catalytic subunit. This phosphorylation enhances the accessibility of the activation loop of the α subunit to phosphatases, thereby inhibiting AMPK kinase activity. Surprisingly, PI3K-Akt signaling, which is a major anabolic signaling and normally inhibits GSK3 activity, promotes GSK3 phosphorylation and inhibition of AMPK, thus revealing how AMPK senses anabolic environments in addition to cellular energy levels. Consistently, disrupting GSK3 function within the AMPK complex sustains higher AMPK activity and cellular catabolic processes even under anabolic conditions, indicating that GSK3 acts as a critical sensor for anabolic signaling to regulate AMPK. PMID:23623684

SKI promotes Smad3 linker phosphorylations associated with the tumor-promoting trait of TGFbeta.

PubMed

Lin, Qiushi; Chen, Dahu; Timchenko, Nikolai A; Medrano, Estela E

2010-05-01

The transcriptional co-regulator SKI is a potent inhibitor of TGFbeta-growth inhibitory signals. SKI binds to receptor-activated Smads in the nucleus, forming repressor complexes containing HDACs, mSin3, NCoR, and other protein partners. Alternatively, SKI binds to activated Smads in the cytoplasm, preventing their nuclear translocation. SKI is necessary for anchorage-independent growth of melanoma cells in vitro, and most important, for human melanoma xenograft growth in vivo. We recently identified a novel role of SKI in TGFbeta signaling. SKI promotes the switch of Smad3 from repressor of proliferation to activator of oncogenesis by facilitating phosphorylations in the linker domain. High levels of endogenous SKI are required by the tumor promoting trait of TGFbeta to induce expression of the plasminogen-activator inhibitor-1 (PAI-1), sustained expression of C-Myc and for aborting upregulation of p21(Waf-1). Here we discuss how SKI diversifies and amplifies its functions by associating with multiple protein partners and by promoting Smad3 linker phosphorylation(s) in response to TGFbeta signaling in melanoma cells.

Phosphorylated mesoporous carbon as effective catalyst for the selective fructose dehydration to HMF

DOE Office of Scientific and Technical Information (OSTI.GOV)

Villa, Alberto; Schiavoni, Marco; Fulvio, Pasquale F

Phosphorylated mesoporous carbons (PMCs) have been synthesized using an already reported one pot methodology. These materials have been applied as acidic catalysts in the dehydration of fructose to hydroxymethylfurfural (HMF). PMCs showed better selectivity to HMF compared to sulfonated carbon catalyst (SC) despite lower activity. The concentration of P-O groups correlates to the activity/selectivity of the catalysts; the higher the P-O concentration the higher the activity. However, the higher the P-O content the lower the selectivity to HMF. Indeed a lower concentration of the P-O groups (and even the acidic groups) minimized the degradation of HMF to levulinic acid andmore » the formation of by-products, such as humines. Stability tests showed that these systems deactivate due to the formation of humines, water insoluble by-products derived from the dehydration of fructose, blocking the active site of the catalyst. Increasing the amount of P-O groups, higher amount of humines are formed; therefore carbons containing lower amount of phosphorylated groups, such as P/N-0.25, are less prone to deactivation. Keywords: Phosphorylated mesoporous carbons; fructose dehydration; HMF« less

The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2.

PubMed Central

Poon, R Y; Yamashita, K; Adamczewski, J P; Hunt, T; Shuttleworth, J

1993-01-01

Activation of the cyclin-dependent protein kinases p34cdc2 and p33cdk2 requires binding with a cyclin partner and phosphorylation on the first threonine residue in the sequence THEVVTLWYRAPE. We present evidence that this threonine residue, number 160 in p33cdk2, can be specifically phosphorylated by a cdc2-related protein kinase from Xenopus oocytes called p40MO15. Binding to cyclin A and phosphorylation of this threonine are both required to activate fully the histone H1 kinase activity of p33cdk2. In cell extracts, a portion of p40MO15 is found in a high molecular weight complex that is considerably more active than a lower molecular weight form. Wild-type MO15 protein expressed in bacteria does not possess kinase activity, but acquires p33cdk2-T160 kinase activity after incubation with cell extract and ATP. We conclude that p40MO15 corresponds to CAK (cdc2/cdk2 activating kinase) and speculate that, like p33cdk2 and p34cdc2, p40MO15 requires activation by phosphorylation and association with a companion subunit. Images PMID:8393783

The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2.

PubMed

Poon, R Y; Yamashita, K; Adamczewski, J P; Hunt, T; Shuttleworth, J

1993-08-01

Activation of the cyclin-dependent protein kinases p34cdc2 and p33cdk2 requires binding with a cyclin partner and phosphorylation on the first threonine residue in the sequence THEVVTLWYRAPE. We present evidence that this threonine residue, number 160 in p33cdk2, can be specifically phosphorylated by a cdc2-related protein kinase from Xenopus oocytes called p40MO15. Binding to cyclin A and phosphorylation of this threonine are both required to activate fully the histone H1 kinase activity of p33cdk2. In cell extracts, a portion of p40MO15 is found in a high molecular weight complex that is considerably more active than a lower molecular weight form. Wild-type MO15 protein expressed in bacteria does not possess kinase activity, but acquires p33cdk2-T160 kinase activity after incubation with cell extract and ATP. We conclude that p40MO15 corresponds to CAK (cdc2/cdk2 activating kinase) and speculate that, like p33cdk2 and p34cdc2, p40MO15 requires activation by phosphorylation and association with a companion subunit.

Crystal Structures of the Receiver Domain of the Response Regulator PhoP from Escherichia coli in the Absence and Presence of the Phosphoryl Analog Beryllofluoride▿

PubMed Central

Bachhawat, Priti; Stock, Ann M.

2007-01-01

The response regulator PhoP is part of the PhoQ/PhoP two-component system involved in responses to depletion of extracellular Mg2+. Here, we report the crystal structures of the receiver domain of Escherichia coli PhoP determined in the absence and presence of the phosphoryl analog beryllofluoride. In the presence of beryllofluoride, the active receiver domain forms a twofold symmetric dimer similar to that seen in structures of other regulatory domains from the OmpR/PhoB family, providing further evidence that members of this family utilize a common mode of dimerization in the active state. In the absence of activating agents, the PhoP receiver domain crystallizes with a similar structure, consistent with the previous observation that high concentrations can promote an active state of PhoP independent of phosphorylation. PMID:17545283

Uncoupling of acetylation from phosphorylation regulates FoxO1 function independent of its subcellular localization.

PubMed

Qiang, Li; Banks, Alexander S; Accili, Domenico

2010-08-27

The activity of transcription factor FoxO1 is regulated by phosphorylation-dependent nuclear exclusion and deacetylation-dependent nuclear retention. It is unclear whether and how these two post-translational modifications affect each other. To answer this question, we expressed FoxO1 cDNAs with combined mutations of phosphorylation and acetylation sites in HEK-293 cells and analyzed their subcellular localization patterns. We show that mutations mimicking the acetylated state (KQ series) render FoxO1 more sensitive to Akt-mediated phosphorylation and nuclear exclusion and can reverse the constitutively nuclear localization of phosphorylation-defective FoxO1. Conversely, mutations mimicking the deacetylated state (KR series) promote FoxO1 nuclear retention. Oxidative stress and the Sirt1 activator resveratrol are thought to promote FoxO1 deacetylation and nuclear retention, thus increasing its activity. Accordingly, FoxO1 deacetylation was required for the effect of oxidative stress (induced by H(2)O(2)) to retain FoxO1 in the nucleus. H(2)O(2) also inhibited FoxO1 phosphorylation on Ser-253 and Thr-24, the key insulin-regulated sites, irrespective of its acetylation. In contrast, the effect of resveratrol was independent of FoxO1 acetylation and its phosphorylation on Ser-253 and Thr-24, suggesting that resveratrol acts on FoxO1 in a Sirt1- and Akt-independent manner. The dissociation of deacetylation from dephosphorylation in H(2)O(2)-treated cells indicates that the two modifications can occur independently of each other. It can be envisaged that FoxO1 exists in multiple nuclear forms with distinct activities depending on the balance of acetylation and phosphorylation.

Two widely expressed plasma membrane H(+)-ATPase isoforms of Nicotiana tabacum are differentially regulated by phosphorylation of their penultimate threonine.

PubMed

Bobik, Krzysztof; Duby, Geoffrey; Nizet, Yannick; Vandermeeren, Caroline; Stiernet, Patrick; Kanczewska, Justyna; Boutry, Marc

2010-04-01

The plasma membrane H(+)-ATPases PMA2 and PMA4 are the most widely expressed in Nicotiana plumbaginifolia, and belong to two different subfamilies. Both are activated by phosphorylation of a Thr at the penultimate position and the subsequent binding of 14-3-3 proteins. Their expression in Saccharomyces cerevisiae revealed functional and regulatory differences. To determine whether different regulatory properties between PMA2 and PMA4 exist in plants, we generated two monoclonal antibodies able to detect phosphorylation of the penultimate Thr of either PMA2 or PMA4 in a total protein extract. We also raised Nicotiana tabacum transgenic plants expressing 6-His-tagged PMA2 or PMA4, enabling their individual purification. Using these tools we showed that phosphorylation of the penultimate Thr of both PMAs was high during the early exponential growth phase of an N. tabacum cell culture, and then progressively declined. This decline correlated with decreased 14-3-3 binding and decreased plasma membrane ATPase activity. However, the rate and extent of the decrease differed between the two isoforms. Cold stress of culture cells or leaf tissues reduced the Thr phosphorylation of PMA2, whereas no significant changes in Thr phosphorylation of PMA4 were seen. These results strongly suggest that PMA2 and PMA4 are differentially regulated by phosphorylation. Analysis of the H(+)-ATPase phosphorylation status in leaf tissues indicated that no more than 44% (PMA2) or 32% (PMA4) was in the activated state under normal growth conditions. Purification of either isoform showed that, when activated, the two isoforms did not form hetero-oligomers, which is further support for these two H(+)-ATPase subfamilies having different properties.

Global regulation of post-translational modifications on core histones.

PubMed

Galasinski, Scott C; Louie, Donna F; Gloor, Kristen K; Resing, Katheryn A; Ahn, Natalie G

2002-01-25

Full-length masses of histones were analyzed by mass spectrometry to characterize post-translational modifications of bulk histones and their changes induced by cell stimulation. By matching masses of unique peptides with full-length masses, H4 and the variants H2A.1, H2B.1, and H3.1 were identified as the main histone forms in K562 cells. Mass changes caused by covalent modifications were measured in a dose- and time-dependent manner following inhibition of phosphatases by okadaic acid. Histones H2A, H3, and H4 underwent changes in mass consistent with altered acetylation and phosphorylation, whereas H2B mass was largely unchanged. Unexpectedly, histone H4 became almost completely deacetylated in a dose-dependent manner that occurred independently of phosphorylation. Okadaic acid also partially blocked H4 hyperacetylation induced by trichostatin-A, suggesting that the mechanism of deacetylation involves inhibition of H4 acetyltransferase activity, following perturbation of cellular phosphatases. In addition, mass changes in H3 in response to okadaic acid were consistent with phosphorylation of methylated, acetylated, and phosphorylated forms. Finally, kinetic differences were observed with respect to the rate of phosphorylation of H2A versus H4, suggesting differential regulation of phosphorylation at sites on these proteins, which are highly related by sequence. These results provide novel evidence that global covalent modifications of chromatin-bound histones are regulated through phosphorylation-dependent mechanisms.

β-Arrestins Negatively Regulate the Toll Pathway in Shrimp by Preventing Dorsal Translocation and Inhibiting Dorsal Transcriptional Activity*

PubMed Central

Sun, Jie-Jie; Lan, Jiang-Feng; Shi, Xiu-Zhen; Yang, Ming-Chong; Niu, Guo-Juan; Ding, Ding; Zhao, Xiao-Fan; Yu, Xiao-Qiang; Wang, Jin-Xing

2016-01-01

The Toll signaling pathway plays an important role in the innate immunity of Drosophila melanogaster and mammals. The activation and termination of Toll signaling are finely regulated in these animals. Although the primary components of the Toll pathway were identified in shrimp, the functions and regulation of the pathway are seldom studied. We first demonstrated that the Toll signaling pathway plays a central role in host defense against Staphylococcus aureus by regulating expression of antimicrobial peptides in shrimp. We then found that β-arrestins negatively regulate Toll signaling in two different ways. β-Arrestins interact with the C-terminal PEST domain of Cactus through the arrestin-N domain, and Cactus interacts with the RHD domain of Dorsal via the ankyrin repeats domain, forming a heterotrimeric complex of β-arrestin·Cactus·Dorsal, with Cactus as the bridge. This complex prevents Cactus phosphorylation and degradation, as well as Dorsal translocation into the nucleus, thus inhibiting activation of the Toll signaling pathway. β-Arrestins also interact with non-phosphorylated ERK (extracellular signal-regulated protein kinase) through the arrestin-C domain to inhibit ERK phosphorylation, which affects Dorsal translocation into the nucleus and phosphorylation of Dorsal at Ser276 that impairs Dorsal transcriptional activity. Our study suggests that β-arrestins negatively regulate the Toll signaling pathway by preventing Dorsal translocation and inhibiting Dorsal phosphorylation and transcriptional activity. PMID:26846853

Maintained activity of glycogen synthase kinase-3{beta} despite of its phosphorylation at serine-9 in okadaic acid-induced neurodegenerative model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lim, Yong-Whan; Yoon, Seung-Yong, E-mail: ysy@amc.seoul.kr; Institute for Biomacromolecules, University of Ulsan College of Medicine, Seoul

2010-04-30

Glycogen synthase kinase-3{beta} (GSK3{beta}) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3{beta}. However, the inactive form of GSK3{beta} which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3{beta} substrates, such as {beta}-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly,more » OA also induces phosphorylation of GSK3{beta} at serine-9 and other substrates including tau, {beta}-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3{beta} inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3{beta} may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3{beta} inhibitors could be a valuable drug candidate in AD.« less

Phosphorylation of Mitochondrial Polyubiquitin by PINK1 Promotes Parkin Mitochondrial Tethering

PubMed Central

Shiba-Fukushima, Kahori; Arano, Taku; Matsumoto, Gen; Inoshita, Tsuyoshi; Yoshida, Shigeharu; Ishihama, Yasushi; Ryu, Kwon-Yul; Nukina, Nobuyuki; Hattori, Nobutaka; Imai, Yuzuru

2014-01-01

The kinase PINK1 and the E3 ubiquitin (Ub) ligase Parkin participate in mitochondrial quality control. The phosphorylation of Ser65 in Parkin's ubiquitin-like (UBl) domain by PINK1 stimulates Parkin activation and translocation to damaged mitochondria, which induces mitophagy generating polyUb chain. However, Parkin Ser65 phosphorylation is insufficient for Parkin mitochondrial translocation. Here we report that Ser65 in polyUb chain is also phosphorylated by PINK1, and that phosphorylated polyUb chain on mitochondria tethers Parkin at mitochondria. The expression of Tom70MTS-4xUb SE, which mimics phospho-Ser65 polyUb chains on the mitochondria, activated Parkin E3 activity and its mitochondrial translocation. An E3-dead form of Parkin translocated to mitochondria with reduced membrane potential in the presence of Tom70MTS-4xUb SE, whereas non-phospho-polyUb mutant Tom70MTS-4xUb SA abrogated Parkin translocation. Parkin binds to the phospho-polyUb chain through its RING1-In-Between-RING (IBR) domains, but its RING0-linker is also required for mitochondrial translocation. Moreover, the expression of Tom70MTS-4xUb SE improved mitochondrial degeneration in PINK1-deficient, but not Parkin-deficient, Drosophila. Our study suggests that the phosphorylation of mitochondrial polyUb by PINK1 is implicated in both Parkin activation and mitochondrial translocation, predicting a chain reaction mechanism of mitochondrial phospho-polyUb production by which rapid translocation of Parkin is achieved. PMID:25474007

The CK2 Kinase Stabilizes CLOCK and Represses Its Activity in the Drosophila Circadian Oscillator

PubMed Central

Szabó, Áron; Papin, Christian; Zorn, Daniela; Ponien, Prishila; Weber, Frank; Raabe, Thomas; Rouyer, François

2013-01-01

Phosphorylation is a pivotal regulatory mechanism for protein stability and activity in circadian clocks regardless of their evolutionary origin. It determines the speed and strength of molecular oscillations by acting on transcriptional activators and their repressors, which form negative feedback loops. In Drosophila, the CK2 kinase phosphorylates and destabilizes the PERIOD (PER) and TIMELESS (TIM) proteins, which inhibit CLOCK (CLK) transcriptional activity. Here we show that CK2 also targets the CLK activator directly. Downregulating the activity of the catalytic α subunit of CK2 induces CLK degradation, even in the absence of PER and TIM. Unexpectedly, the regulatory β subunit of the CK2 holoenzyme is not required for the regulation of CLK stability. In addition, downregulation of CK2α activity decreases CLK phosphorylation and increases per and tim transcription. These results indicate that CK2 inhibits CLK degradation while reducing its activity. Since the CK1 kinase promotes CLK degradation, we suggest that CLK stability and transcriptional activity result from counteracting effects of CK1 and CK2. PMID:24013921

Phosphorylation of mononucleotides and formation of cytidine 5'-diphosphate-choline and sugar nucleotides by respiration-deficient mutants of yeasts.

PubMed Central

Kimura, A; Hirose, K; Kariya, Y; Nagai, S

1976-01-01

Respiration-deficient mutants (Rho-, petite) of Saccharomyces carlsbergensis were obtained by treatment with trypaflavin (euflavine). Dried cells of these mutants phosphorylated mononucleotides to their triphosphates and further formed not only cytidine 5'-diphosphate-choline, but also sugar nucleotides, such as uridine 5'-diphosphate-glucose, guanosine 5'-diphosphate-mannose, etc. The activities were the same or slightly greater than those of the wild strain. These results showed that energy (adenosine 5'-triphosphate) necessary for phosphorylation of mononucleotides was sufficiently supplied by the glycolysis system. PMID:1245470

Protein Kinase C Controls Binding of Igo/ENSA Proteins to Protein Phosphatase 2A in Budding Yeast.

PubMed

Thai, Vu; Dephoure, Noah; Weiss, Amit; Ferguson, Jacqueline; Leitao, Ricardo; Gygi, Steven P; Kellogg, Douglas R

2017-03-24

Protein phosphatase 2A (PP2A) plays important roles in controlling mitosis in all eukaryotic cells. The form of PP2A that controls mitosis is associated with a conserved regulatory subunit that is called B55 in vertebrates and Cdc55 in budding yeast. The activity of this form of PP2A can be inhibited by binding of conserved Igo/ENSA proteins. Although the mechanisms that activate Igo/ENSA to bind and inhibit PP2A are well understood, little is known about how Igo/Ensa are inactivated. Here, we have analyzed regulation of Igo/ENSA in the context of a checkpoint pathway that links mitotic entry to membrane growth in budding yeast. Protein kinase C (Pkc1) relays signals in the pathway by activating PP2A Cdc55 We discovered that constitutively active Pkc1 can drive cells through a mitotic checkpoint arrest, which suggests that Pkc1-dependent activation of PP2A Cdc55 plays a critical role in checkpoint signaling. We therefore used mass spectrometry to determine how Pkc1 modifies the PP2A Cdc55 complex. This revealed that Pkc1 induces changes in the phosphorylation of multiple subunits of the complex, as well as dissociation of Igo/ENSA. Pkc1 directly phosphorylates Cdc55 and Igo/ENSA, and phosphorylation site mapping and mutagenesis indicate that phosphorylation of Cdc55 contributes to Igo/ENSA dissociation. Association of Igo2 with PP2A Cdc55 is regulated during the cell cycle, yet mutation of Pkc1-dependent phosphorylation sites on Cdc55 and Igo2 did not cause defects in mitotic progression. Together, the data suggest that Pkc1 controls PP2A Cdc55 by multiple overlapping mechanisms. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Role of CBS and Bateman Domains in Phosphorylation-Dependent Regulation of a CLC Anion Channel.

PubMed

Yamada, Toshiki; Krzeminski, Mickael; Bozoky, Zoltan; Forman-Kay, Julie D; Strange, Kevin

2016-11-01

Eukaryotic CLC anion channels and transporters are homodimeric proteins composed of multiple α-helical membrane domains and large cytoplasmic C-termini containing two cystathionine-β-synthase domains (CBS1 and CBS2) that dimerize to form a Bateman domain. The Bateman domains of adjacent CLC subunits interact to form a Bateman domain dimer. The functions of CLC CBS and Bateman domains are poorly understood. We utilized the Caenorhabditis elegans CLC-1/2/Ka/Kb anion channel homolog CLH-3b to characterize the regulatory roles of CLC cytoplasmic domains. CLH-3b activity is reduced by phosphorylation or deletion of a 14-amino-acid activation domain (AD) located on the linker connecting CBS1 and CBS2. We demonstrate here that phosphorylation-dependent reductions in channel activity require an intact Bateman domain dimer and concomitant phosphorylation or deletion of both ADs. Regulation of a CLH-3b AD deletion mutant is reconstituted by intracellular perfusion with recombinant 14-amino-acid AD peptides. The sulfhydryl reactive reagent 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET) alters in a phosphorylation-dependent manner the activity of channels containing single cysteine residues that are engineered into the short intracellular loop connecting membrane α-helices H and I (H-I loop), the AD, CBS1, and CBS2. In contrast, MTSET has no effect on channels in which cysteine residues are engineered into intracellular regions that are dispensable for regulation. These studies together with our previous work suggest that binding and unbinding of the AD to the Bateman domain dimer induces conformational changes that are transduced to channel membrane domains via the H-I loop. Our findings provide new, to our knowledge, insights into the roles of CLC Bateman domains and the structure-function relationships that govern the regulation of CLC protein activity by diverse ligands and signaling pathways. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Phosphorylation of Dgk1 Diacylglycerol Kinase by Casein Kinase II Regulates Phosphatidic Acid Production in Saccharomyces cerevisiae.

PubMed

Qiu, Yixuan; Hassaninasab, Azam; Han, Gil-Soo; Carman, George M

2016-12-16

In the yeast Saccharomyces cerevisiae, Dgk1 diacylglycerol (DAG) kinase catalyzes the CTP-dependent phosphorylation of DAG to form phosphatidic acid (PA). The enzyme in conjunction with Pah1 PA phosphatase controls the levels of PA and DAG for the synthesis of triacylglycerol and membrane phospholipids, the growth of the nuclear/endoplasmic reticulum membrane, and the formation of lipid droplets. Little is known about how DAG kinase activity is regulated by posttranslational modification. In this work, we examined the phosphorylation of Dgk1 DAG kinase by casein kinase II (CKII). When phosphate groups were globally reduced using nonspecific alkaline phosphatase, Triton X-100-solubilized membranes from DGK1-overexpressing cells showed a 7.7-fold reduction in DAG kinase activity; the reduced enzyme activity could be increased 5.5-fold by treatment with CKII. Dgk1(1-77) expressed heterologously in Escherichia coli was phosphorylated by CKII on a serine residue, and its phosphorylation was dependent on time as well as on the concentrations of CKII, ATP, and Dgk1(1-77). We used site-specific mutagenesis, coupled with phosphorylation analysis and phosphopeptide mapping, to identify Ser-45 and Ser-46 of Dgk1 as the CKII target sites, with Ser-46 being the major phosphorylation site. In vivo, the S46A and S45A/S46A mutations of Dgk1 abolished the stationary phase-dependent stimulation of DAG kinase activity. In addition, the phosphorylation-deficient mutations decreased Dgk1 function in PA production and in eliciting pah1Δ phenotypes, such as the expansion of the nuclear/endoplasmic reticulum membrane, reduced lipid droplet formation, and temperature sensitivity. This work demonstrates that the CKII-mediated phosphorylation of Dgk1 regulates its function in the production of PA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural Investigation of a Phosphorylation-Catalyzed, Isoaspartate-Free, Protein Succinimide: Crystallographic Structure of Post-Succinimide His15Asp Histidine-Containing Protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Napper, Scott; Prasad, Lata; Delbaere, Louis T.J.

2008-09-08

Aspartates and asparagines can spontaneously cyclize with neighboring main-chain amides to form succinimides. These succinimides hydrolyze to a mixture of isoaspartate and aspartate products. Phosphorylation of aspartates is a common mechanism of protein regulation and increases the propensity for succinimide formation. Although typically regarded as a form of protein damage, we hypothesize succinimides could represent an effective mechanism of phosphoaspartate autophosphatase activity, provided hydrolysis is limited to aspartate products. We previously reported the serendipitous creation of a protein, His15Asp histidine-containing protein (HPr), which undergoes phosphorylation-catalyzed formation of a succinimide whose hydrolysis is seemingly exclusive for aspartate formation. Here, through themore » high-resolution structure of postsuccinimide His15Asp HPr, we confirm the absence of isoaspartate residues and propose mechanisms for phosphorylation-catalyzed succinimide formation and its directed hydrolysis to aspartate. His15Asp HPr represents the first characterized protein example of an isoaspartate-free succinimide and lends credence to the hypothesis that intramolecular cyclization could represent a physiological mechanism of autophosphatase activity. Furthermore, this indicates that current strategies for succinimide evaluation, based on isoaspartate detection, underestimate the frequencies of these reactions. This is considerably significant for evaluation of protein stability and integrity.« less

Identification of phosphorylated butyrylcholinesterase in human plasma using immunoaffinity purification and mass spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aryal, Uma K.; Lin, Chiann Tso; Kim, Jong Seo

2012-04-20

Paraoxon (diethyl 4-nitrophenyl phosphate) is an active metabolite of the common insecticide parathion and is acutely toxic due to the inhibition of cholinesterase (ChE) activity in the nervous systems. The Inhibition of butyrylcholinesterase (BChE) activity by paraoxon is due to the formation of phosphorylated BChE adduct, and the detection of the phosphorylated BChE adduct in human plasma can serve as an exposure biomarker of organophosphate pesticides and nerve agents. In this study, we performed immunoaffinity purification and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis for identifying phosphorylated BChE in human plasma treated by paraoxon. BChE was captured by biotinylated anti-BChEmore » polyclonal antibodies conjugated to streptavidin magnetic beads. Western blot analysis showed that the antibody was effective to recognize both native and modified BChE with high specificity. The exact phosphorylation site of BChE was confirmed on Serine 198 by MS/MS with a 108 Da modification mass and accurately measured parent ion masses. The phosphorylated BChE peptide was also successfully detected in the immunoaffinity purified sample from paraoxon treated human plasma. Thus, immunoaffinity purification combined with mass spectrometry represents a viable approach for the detection of paraoxon-modified BChE and other forms of modified BChE as exposure biomarkers of organophosphates and nerve agents.« less

Coordinate regulation of the mother centriole component nlp by nek2 and plk1 protein kinases.

PubMed

Rapley, Joseph; Baxter, Joanne E; Blot, Joelle; Wattam, Samantha L; Casenghi, Martina; Meraldi, Patrick; Nigg, Erich A; Fry, Andrew M

2005-02-01

Mitotic entry requires a major reorganization of the microtubule cytoskeleton. Nlp, a centrosomal protein that binds gamma-tubulin, is a G(2)/M target of the Plk1 protein kinase. Here, we show that human Nlp and its Xenopus homologue, X-Nlp, are also phosphorylated by the cell cycle-regulated Nek2 kinase. X-Nlp is a 213-kDa mother centriole-specific protein, implicating it in microtubule anchoring. Although constant in abundance throughout the cell cycle, it is displaced from centrosomes upon mitotic entry. Overexpression of active Nek2 or Plk1 causes premature displacement of Nlp from interphase centrosomes. Active Nek2 is also capable of phosphorylating and displacing a mutant form of Nlp that lacks Plk1 phosphorylation sites. Importantly, kinase-inactive Nek2 interferes with Plk1-induced displacement of Nlp from interphase centrosomes and displacement of endogenous Nlp from mitotic spindle poles, while active Nek2 stimulates Plk1 phosphorylation of Nlp in vitro. Unlike Plk1, Nek2 does not prevent association of Nlp with gamma-tubulin. Together, these results provide the first example of a protein involved in microtubule organization that is coordinately regulated at the G(2)/M transition by two centrosomal kinases. We also propose that phosphorylation by Nek2 may prime Nlp for phosphorylation by Plk1.

Crystal structure of the inactive state of the receiver domain of Spo0A from Paenisporosarcina sp. TG-14, a psychrophilic bacterium isolated from an Antarctic glacier.

PubMed

Lee, Chang Woo; Park, Sun-Ha; Lee, Sung Gu; Shin, Seung Chul; Han, Se Jong; Kim, Han-Woo; Park, Hyun Ho; Kim, Sunghwan; Kim, Hak Jun; Park, Hyun; Park, HaJeung; Lee, Jun Hyuck

2017-06-01

The two-component phosphorelay system is the most prevalent mechanism for sensing and transducing environmental signals in bacteria. Spore formation, which relies on the two-component phosphorelay system, enables the long-term survival of the glacial bacterium Paenisporosarcina sp. TG-14 in the extreme cold environment. Spo0A is a key response regulator of the phosphorelay system in the early stage of spore formation. The protein is composed of a regulatory N-terminal phospho-receiver domain and a DNA-binding C-terminal activator domain. We solved the three-dimensional structure of the unphosphorylated (inactive) form of the receiver domain of Spo0A (PaSpo0A-R) from Paenisporosarcina sp. TG-14. A structural comparison with phosphorylated (active form) Spo0A from Bacillus stearothermophilus (BsSpo0A) showed minor notable differences. A molecular dynamics study of a model of the active form and the crystal structures revealed significant differences in the α4 helix and the preceding loop region where phosphorylation occurs. Although an oligomerization study of PaSpo0A-R by analytical ultracentrifugation (AUC) has shown that the protein is in a monomeric state in solution, both crosslinking and crystal-packing analyses indicate the possibility of weak dimer formation by a previously undocumented mechanism. Collectively, these observations provide insight into the mechanism of phosphorylation-dependent activation unique to Spo0A.

Subcellular localization and logistics of integral membrane protein biogenesis in Escherichia coli.

PubMed

Bogdanov, Mikhail; Aboulwafa, Mohammad; Saier, Milton H

2013-01-01

Transporters catalyze entry and exit of molecules into and out of cells and organelles, and protein-lipid interactions influence their activities. The bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS) catalyzes transport-coupled sugar phosphorylation as well as nonvectorial sugar phosphorylation in the cytoplasm. The vectorial process is much more sensitive to the lipid environment than the nonvectorial process. Moreover, cytoplasmic micellar forms of these enzyme-porters have been identified, and non-PTS permeases have similarly been shown to exist in 'soluble' forms. The latter porters exhibit lipid-dependent activities and can adopt altered topologies by simply changing the lipid composition. Finally, intracellular membranes and vesicles exist in Escherichia coli leading to the following unanswered questions: (1) what determines whether a PTS permease catalyzes vectorial or nonvectorial sugar phosphorylation? (2) How do phospholipids influence relative amounts of the plasma membrane, intracellular membrane, inner membrane-derived vesicles and cytoplasmic micelles? (3) What regulates the route(s) of permease insertion and transfer into and between the different subcellular sites? (4) Do these various membranous forms have distinct physiological functions? (5) What methods should be utilized to study the biogenesis and interconversion of these membranous structures? While research concerning these questions is still in its infancy, answers will greatly enhance our understanding of protein-lipid interactions and how they control the activities, conformations, cellular locations and biogenesis of integral membrane proteins. Copyright © 2013 S. Karger AG, Basel.

Two Naturally Occurring Terpenes, Dehydrocostuslactone and Costunolide, Decrease Intracellular GSH Content and Inhibit STAT3 Activation

PubMed Central

Butturini, Elena; Cavalieri, Elisabetta; Carcereri de Prati, Alessandra; Darra, Elena; Rigo, Antonella; Shoji, Kazuo; Murayama, Norie; Yamazaki, Hiroshi; Watanabe, Yasuo; Suzuki, Hisanori; Mariotto, Sofia

2011-01-01

The main purpose of the present study is to envisage the molecular mechanism of inhibitory action ofdehydrocostuslactone (DCE) andcostunolide (CS), two naturally occurring sesquiterpene lactones, towards the activation of signal transducer and activator of transcription 3 (STAT3). We report that, in human THP-1 cell line, they inhibit IL-6-elicited tyrosine phosphorylation of STAT3 and its DNA binding activity with EC50 of 10 µM with concomitantdown-regulation ofthe phosphorylation of the tyrosine Janus kinases JAK1, JAK2 and Tyk2. Furthermore, these compounds that contain an α-β-unsatured carbonyl moiety and function as potent Michael reaction acceptor, induce a rapid drop in intracellular glutathione (GSH) concentration by direct interaction with it, thereby triggering S-glutathionylation of STAT3. Dehydrocostunolide (HCS), the reduced form of CS lacking only the α-β-unsaturated carbonyl group, fails to exert any inhibitory action. Finally, the glutathione ethylene ester (GEE), the cell permeable GSH form, reverts the inhibitory action of DCE and CS on STAT3 tyrosine phosphorylation. We conclude that these two sesquiterpene lactones are able to induce redox-dependent post-translational modification of cysteine residues of STAT3 protein in order to regulate its function. PMID:21625597

Regulation of spinogenesis in mature Purkinje cells via mGluR/PKC-mediated phosphorylation of CaMKIIβ

PubMed Central

Sugawara, Takeyuki; Hisatsune, Chihiro; Miyamoto, Hiroyuki; Ogawa, Naoko; Mikoshiba, Katsuhiko

2017-01-01

Dendritic spines of Purkinje cells form excitatory synapses with parallel fiber terminals, which are the primary sites for cerebellar synaptic plasticity. Nevertheless, how density and morphology of these spines are properly maintained in mature Purkinje cells is not well understood. Here we show an activity-dependent mechanism that represses excessive spine development in mature Purkinje cells. We found that CaMKIIβ promotes spine formation and elongation in Purkinje cells through its F-actin bundling activity. Importantly, activation of group I mGluR, but not AMPAR, triggers PKC-mediated phosphorylation of CaMKIIβ, which results in dissociation of the CaMKIIβ/F-actin complex. Defective function of the PKC-mediated CaMKIIβ phosphorylation promotes excess F-actin bundling and leads to abnormally numerous and elongated spines in mature IP3R1-deficient Purkinje cells. Thus, our data suggest that phosphorylation of CaMKIIβ through the mGluR/IP3R1/PKC signaling pathway represses excessive spine formation and elongation in mature Purkinje cells. PMID:28607044

Phosphorylation-regulated Binding of RNA Polymerase II to Fibrous Polymers of Low Complexity Domains

PubMed Central

Xiang, Siheng; Wu, Leeju; Theodoropoulos, Pano; Mirzaei, Hamid; Han, Tina; Xie, Shanhai; Corden, Jeffry L.; McKnight, Steven L.

2014-01-01

SUMMARY The low complexity (LC) domains of the products of the fused in sarcoma (FUS), Ewings sarcoma (EWS) and TAF15 genes are translocated onto a variety of different DNA-binding domains and thereby assist in driving the formation of cancerous cells. In the context of the translocated fusion proteins, these LC sequences function as transcriptional activation domains. Here we show that polymeric fibers formed from these LC domains directly bind the C-terminal domain (CTD) of RNA polymerase II in a manner reversible by phosphorylation of the iterated, heptad repeats of the CTD. Mutational analysis indicates that the degree of binding between the CTD and the LC domain polymers correlates with the strength of transcriptional activation. These studies offer a simple means of conceptualizing how RNA polymerase II is recruited to active genes in its unphosphorylated state, and released for elongation following phosphorylation of the CTD. PMID:24267890

Skeletal muscle Ca(2+)-independent kinase activity increases during either hypertrophy or running

NASA Technical Reports Server (NTRS)

Fluck, M.; Waxham, M. N.; Hamilton, M. T.; Booth, F. W.

2000-01-01

Spikes in free Ca(2+) initiate contractions in skeletal muscle cells, but whether and how they might signal to transcription factors in skeletal muscles of living animals is unknown. Since previous studies in non-muscle cells have shown that serum response factor (SRF) protein, a transcription factor, is phosphorylated rapidly by Ca(2+)/calmodulin (CaM)-dependent protein kinase after rises in intracellular Ca(2+), we measured enzymatic activity that phosphorylates SRF (designated SRF kinase activity). Homogenates from 7-day-hypertrophied anterior latissimus dorsi muscles of roosters had more Ca(2+)-independent SRF kinase activity than their respective control muscles. However, no differences were noted in Ca(2+)/CaM-dependent SRF kinase activity between control and trained muscles. To determine whether the Ca(2+)-independent and Ca(2+)/CaM-dependent forms of Ca(2+)/CaM-dependent protein kinase II (CaMKII) might contribute to some of the SRF kinase activity, autocamtide-3, a synthetic substrate that is specific for CaMKII, was employed. While the Ca(2+)-independent form of CaMKII was increased, like the Ca(2+)-independent form of SRF kinase, no alteration in CaMKII occurred at 7 days of stretch overload. These observations suggest that some of SRF phosphorylation by skeletal muscle extracts could be due to CaMKII. To determine whether this adaptation was specific to the exercise type (i.e., hypertrophy), similar measurements were made in the white vastus lateralis muscle of rats that had completed 2 wk of voluntary running. Although Ca(2+)-independent SRF kinase was increased, no alteration occurred in Ca(2+)/CaM-dependent SRF kinase activity. Thus any role of Ca(2+)-independent SRF kinase signaling has downstream modulators specific to the exercise phenotype.

Helix formation in arrestin accompanies recognition of photoactivated rhodopsin.

PubMed

Feuerstein, Sophie E; Pulvermüller, Alexander; Hartmann, Rudolf; Granzin, Joachim; Stoldt, Matthias; Henklein, Peter; Ernst, Oliver P; Heck, Martin; Willbold, Dieter; Koenig, Bernd W

2009-11-17

Binding of arrestin to photoactivated phosphorylated rhodopsin terminates the amplification of visual signals in photoreceptor cells. Currently, there is no crystal structure of a rhodopsin-arrestin complex available, although structures of unbound rhodopsin and arrestin have been determined. High-affinity receptor binding is dependent on distinct arrestin sites responsible for recognition of rhodopsin activation and phosphorylation. The loop connecting beta-strands V and VI in rod arrestin has been implicated in the recognition of active rhodopsin. We report the structure of receptor-bound arrestin peptide Arr(67-77) mimicking this loop based on solution NMR data. The peptide binds photoactivated rhodopsin in the unphosphorylated and phosphorylated form with similar affinities and stabilizes the metarhodopsin II photointermediate. A largely alpha-helical conformation of the receptor-bound peptide is observed.

Hormonal regulation of circulating insulin-like growth factor-binding protein-1 phosphorylation status.

PubMed

Westwood, M; Gibson, J M; Williams, A C; Clayton, P E; Hamberg, O; Flyvbjerg, A; White, A

1995-12-01

Insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) normally circulates as a single, highly phosphorylated species. However, IGFBP-1 phosphorylation status can be altered, such as in pregnancy where non- and lesser phosphorylated isoforms are also present. We have examined how hormonal regulators of circulating IGFBP-1 influence its phosphorylation status and, hence, its ability to modulate IGF activity. In response to insulin-induced hypoglycemia (0.2 U/kg, iv), an increase in the highly phosphorylated isoform was observed after 5 h [16 (range, 11.5-35.5) to 77 (range, 63-250) microgram/L; 4.8-fold increase; P = 0.009], but no non- or lesser phosphorylated variants could be detected. Glucagon (1 mg, sc), increased IGFBP-1 from 27 (range, 13-36.5) to 112 (range, 100.5-129) micrograms/L (4.1-fold increase; P = 0.009) after 90 min despite preceding insulin concentrations of more than 500 pmol/L, but again the IGFBP-1 remained in the highly phosphorylated form. Regulation of IGFBP-1 phosphorylation by sex steroids was studied by comparing women receiving a combined oral contraceptive with women on no medication. Although plasma IGFBP-1 levels were significantly elevated in the treatment group [120 (range, 97.5-237.5) vs. 52 (range, 38-70) micrograms/L; P < 0.004], there was no difference in the form of IGFBP-1 present. The acute effect of somatostatin (500 micrograms/h) on IGFBP-1 phosphorylation status was also studied. Somatostatin only increased the phosphoform characteristic of normal subjects; the appearance of non- or lesser phosphorylated variants was not induced. The effect of rhIGF-I (80 or 120 micrograms, sc) on plasma IGFBP-1 was studied in three subjects with Laron's syndrome. A transient increase in the highly phosphorylated isoform of IGFBP-1 was noted; there was no rise in the non- and lesser phosphorylated isoforms also found in the plasma of Laron's syndrome subjects. These data suggest that only the highly phosphorylated species of IGFBP-1 is under hormonal control; regulation of the non- and lesser phosphorylated variants remains to be determined.

Eukaryotic polypeptide elongation system and its sensitivity to the inhibitory substances of plant origin.

PubMed

Gałasiński, W

1996-05-01

The structural and functional characteristics of the elongation system (ribosomes and elongation factors) are presented. The immunochemical and diagnostic meaning of the ribosome investigations is considered. Evidence of the participation of ribosomes in the first step of protein glycosylation is presented. The heterogeneous elongation factor eEF-1, isolated from Guerin epithelioma, can be separated into three fractions: one of them functionally corresponds to EF-1 alpha, the second on to EF-1 beta gamma, and the third is an unidentified, active aggregate named EF-1B, which contains the subunit forms EF-1 alpha and EF-1 beta gamma, and other polypeptides showing protein kinase activity. The aggregate EF-1B can be autophosphorylated, while the subunit forms EF-1 alpha and EF-1 beta gamma can neither become autophosphorylated nor phosphorylate other polypeptides. The subunit form EF-beta gamma consists from two polypeptides of 32 and 51 kDa, corresponding to other eukaryotic beta and gamma polypeptides, respectively. EF-1 beta gamma is thermostable and protects against thermal inactivation of EF-1 alpha in the EF-1 alpha-EF-1 beta gamma complex. Pure eEF-2 preparations isolated from normal and neoplastic tissues show different structural features. The existence of eEF-2 in multiple forms, differing in molecular mass, have been found. The eEF-2 with molecular weight of about 100 kDa can be phosphorylated, while eEF-2 of about 65 kDa was not phosphorylated by protein kinase eEF-2. The phosphorylated eEF-2 lost its activity, and this effect was reversed by dephosphorylation. The eEF-2 (65 kDa) was isolated from the active polyribosomes, and it may directly participate in the translocation step of the peptide elongation. It was noted that the components of elongation system can be inhibited, in separate steps, by the substances isolated from various sources of plant origin. Alkaloids emetine and cepheline, cardiac remedy digoxin, saponin glycoside, and its aglycon directly inactivated ribosomes. Quercetin inhibited eEF-1 activity by directly influencing its subunit form EF-1 alpha. eEF-2 was shown to be a target site of the inhibitory action of the glycoside isolated from Melissa officinalis leaves.

Endothelial nitric-oxide synthase (eNOS) is activated through G-protein-coupled receptor kinase-interacting protein 1 (GIT1) tyrosine phosphorylation and Src protein.

PubMed

Liu, Songling; Premont, Richard T; Rockey, Don C

2014-06-27

Nitric oxide (NO) is a critical regulator of vascular tone and plays an especially prominent role in liver by controlling portal blood flow and pressure within liver sinusoids. Synthesis of NO in sinusoidal endothelial cells by endothelial nitric-oxide synthase (eNOS) is regulated in response to activation of endothelial cells by vasoactive signals such as endothelins. The endothelin B (ETB) receptor is a G-protein-coupled receptor, but the mechanisms by which it regulates eNOS activity in sinusoidal endothelial cells are not well understood. In this study, we built on two previous strands of work, the first showing that G-protein βγ subunits mediated activation of phosphatidylinositol 3-kinase and Akt to regulate eNOS and the second showing that eNOS directly bound to the G-protein-coupled receptor kinase-interacting protein 1 (GIT1) scaffold protein, and this association stimulated NO production. Here we investigated the mechanisms by which the GIT1-eNOS complex is formed and regulated. GIT1 was phosphorylated on tyrosine by Src, and Y293F and Y554F mutations reduced GIT1 phosphorylation as well as the ability of GIT1 to bind to and activate eNOS. Akt phosphorylation activated eNOS (at Ser(1177)), and Akt also regulated the ability of Src to phosphorylate GIT1 as well as GIT1-eNOS association. These pathways were activated by endothelin-1 through the ETB receptor; inhibiting receptor-activated G-protein βγ subunits blocked activation of Akt, GIT1 tyrosine phosphorylation, and ET-1-stimulated GIT1-eNOS association but did not affect Src activation. These data suggest a model in which Src and Akt cooperate to regulate association of eNOS with the GIT1 scaffold to facilitate NO production. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Phosphorylated and Nonphosphorylated PfMAP2 Are Localized in the Nucleus, Dependent on the Stage of Plasmodium falciparum Asexual Maturation

PubMed Central

Dahalan, Farah Aida; Sidek, Hasidah Mohd; Murtey, Mogana Das; Embi, Mohammed Noor; Ibrahim, Jamaiah; Fei Tieng, Lim; Zakaria, Nurul Aiezzah

2016-01-01

Plasmodium falciparum mitogen-activated protein (MAP) kinases, a family of enzymes central to signal transduction processes including inflammatory responses, are a promising target for antimalarial drug development. Our study shows for the first time that the P. falciparum specific MAP kinase 2 (PfMAP2) is colocalized in the nucleus of all of the asexual erythrocytic stages of P. falciparum and is particularly elevated in its phosphorylated form. It was also discovered that PfMAP2 is expressed in its highest quantity during the early trophozoite (ring form) stage and significantly reduced in the mature trophozoite and schizont stages. Although the phosphorylated form of the kinase is always more prevalent, its ratio relative to the nonphosphorylated form remained constant irrespective of the parasites' developmental stage. We have also shown that the TSH motif specifically renders PfMAP2 genetically divergent from the other plasmodial MAP kinase activation sites using Neighbour Joining analysis. Furthermore, TSH motif-specific designed antibody is crucial in determining the location of the expression of the PfMAP2 protein. However, by using immunoelectron microscopy, PPfMAP2 were detected ubiquitously in the parasitized erythrocytes. In summary, PfMAP2 may play a far more important role than previously thought and is a worthy candidate for research as an antimalarial. PMID:27525262

Co-clustering of Fcgamma and B cell receptors induces dephosphorylation of the Grb2-associated binder 1 docking protein.

PubMed

Koncz, G; Tóth, G K; Bökönyi, G; Kéri, G; Pecht, I; Medgyesi, D; Gergely, J; Sármay, G

2001-07-01

The immunoreceptor tyrosine-based inhibitory motif (ITIM) of human type IIb Fcgamma receptor (FcgammaRIIb) is phosphorylated on its tyrosine upon co-clustering with the B cell receptor (BCR). The phosphorylated ITIM (p-ITIM) binds to the SH2 domains of polyphosphoinositol 5-phosphatase (SHIP) and the tyrosine phosphatase, SHP-2. We investigated the involvement of the molecular complex composed of the phosphorylated SHIP and FcgammaRIIb in the activation of SHP-2. As a model compound, we synthesized a bisphosphopeptide, combining the sequences of p-ITIM and the N-terminal tyrosine phosphorylated motif of SHIP with a flexible spacer. This compound bound to the recombinant SH2 domains of SHP-2 with high affinity and activated the phosphatase in an in vitro assay. These data suggest that the phosphorylated FcgammaRII-SHIP complexes formed in the intact cells may also activate SHP-2. Grb2-associated binder 1 (Gab1) is a multisite docking protein, which becomes tyrosine-phosphorylated in response to various types of signaling, including BCR. In turn it binds to the SH2 domains of SHP-2, SHIP and the p85 subunit of phosphatidyl inositol 3-kinase (PtdIns3-K) and may regulate their activity. Gab1 is a potential substrate of SHP-2, thus its binding to FcgammaRIIb may modify the Gab1-bound signaling complex. We show here that Gab1 is part of the multiprotein complex assembled by FcgammaRIIb upon its co-clustering with BCR. Gab1 may recruit SH2 domain-containing molecules to the phosphorylated FcgammaRIIb. SHP-2, activated upon the binding to FcgammaRIIb-SHIP complex, partially dephosphorylates Gab1, resulting in the release of PtdIns3-K and ultimately in the inhibition of downstream activation pathways in BCR/FcgammaRIIb co-aggregated cells.

Phosphorylation-mediated negative regulation of RIG-I antiviral activity.

PubMed

Gack, Michaela U; Nistal-Villán, Estanislao; Inn, Kyung-Soo; García-Sastre, Adolfo; Jung, Jae U

2010-04-01

Recognition of invading viruses by the host is elicited by cellular sensors which trigger signaling cascades that lead to type I interferon (IFN) gene expression. Retinoic acid-inducible gene I (RIG-I) has emerged as a key receptor for the detection of viral RNA in the cytosol, inducing IFN-mediated innate immune responses to limit viral replication through its interaction with MAVS (also called IPS-1, CARDIF, or VISA). Upon the recognition of viral RNA, the Lys-172 residue of RIG-I undergoes ubiquitination induced by tripartite motif protein 25 (TRIM25), an essential protein for antiviral signal transduction. Here we demonstrate that phosphorylation represents another regulatory mechanism for RIG-I-mediated antiviral activity. Using protein purification and mass spectrometry analysis, we identified three phosphorylation sites in the amino-terminal caspase recruitment domains (CARDs) of RIG-I. One of these residues, Thr-170, is located in close proximity to Lys-172, and we speculated that its phosphorylation may affect Lys-172 ubiquitination and functional activation of RIG-I. Indeed, a RIG-I mutant carrying a phosphomimetic Glu residue in place of Thr-170 loses TRIM25 binding, Lys-172 ubiquitination, MAVS binding, and downstream signaling ability. This suggests that phosphorylation of RIG-I at Thr-170 inhibits RIG-I-mediated antiviral signal transduction. Immunoblot analysis with a phospho-specific antibody showed that the phosphorylation of the RIG-I Thr-170 residue is present under normal conditions but rapidly declines upon viral infection. Our results indicate that Thr-170 phosphorylation and TRIM25-mediated Lys-172 ubiquitination of RIG-I functionally antagonize each other. While Thr-170 phosphorylation keeps RIG-I latent, Lys-172 ubiquitination enables RIG-I to form a stable complex with MAVS, thereby inducing IFN signal transduction.

Methionine sulfoximine-treatment and carbon starvation elicit Snf1-independent phosphorylation of the transcription activator Gln3 in Saccharomyces cerevisiae

PubMed Central

Tate, Jennifer J.; Rai, Rajendra; Cooper, Terrance G.

2008-01-01

SUMMARY Tor proteins are global regulators situated at the top of a signal transduction pathway conserved from yeast to humans. Specific inhibition of the two S. cerevisiae Tor proteins by rapamycin alters many cellular processes and the expression of hundreds of genes. Among the regulated genes are those whose expression is activated by the GATA-family transcription activator, Gln3. The extent of Gln3 phosphorylation has been thought to determine its intracellular localization, with phosphorylated and dephosphorylated forms accumulating in the cytoplasm and nucleus, respectively. Data presented here demonstrate that rapamycin and the glutamine synthetase inhibitor, methionine sulfoximine (MSX), although eliciting the same outcomes with respect to Gln3-Myc13 nuclear accumulation and NCR-sensitive transcription, generate diametrically opposite effects on Gln3-Myc13 phosphorylation. MSX increases Gln3-Myc13 phosphorylation while rapamycin decreases it. Gln3-Myc13 phosphorylation levels are regulated by at least three mechanisms: (i) one, observed during carbon starvation, depends on Snf1 kinase, (ii) another, observed during both carbon-starvation and MSX-treatment, is Snf1-independent, and (iii) the last is rapamycin-induced dephosphorylation. MSX and rapamycin act additively on Gln3-Myc13 phosphorylation, but MSX clearly predominates. These results suggest that MSX- and rapamycin-inhibited proteins are more likely to function in separate regulatory pathways than they are to function tandemly in a single pathway as previously thought. Further, Gln3 phosphorylation/dephosphorylation, that we and others have detected thus far, is not a demonstrably required step in achieving Gln3 nuclear localization and NCR-sensitive transcription in response to MSX- or rapamycin-treatment. PMID:15911613

EphA2 Receptor Unliganded Dimers Suppress EphA2 Pro-tumorigenic Signaling*

PubMed Central

Singh, Deo R.; Ahmed, Fozia; King, Christopher; Gupta, Nisha; Salotto, Matt; Pasquale, Elena B.; Hristova, Kalina

2015-01-01

The EphA2 receptor tyrosine kinase promotes cell migration and cancer malignancy through a ligand- and kinase-independent distinctive mechanism that has been linked to high Ser-897 phosphorylation and low tyrosine phosphorylation. Here, we demonstrate that EphA2 forms dimers in the plasma membrane of HEK293T cells in the absence of ephrin ligand binding, suggesting that the current seeding mechanism model of EphA2 activation is incomplete. We also characterize a dimerization-deficient EphA2 mutant that shows enhanced ability to promote cell migration, concomitant with increased Ser-897 phosphorylation and decreased tyrosine phosphorylation compared with EphA2 wild type. Our data reveal a correlation between unliganded dimerization and tumorigenic signaling and suggest that EphA2 pro-tumorigenic activity is mediated by the EphA2 monomer. Thus, a therapeutic strategy that aims at the stabilization of EphA2 dimers may be beneficial for the treatment of cancers linked to EphA2 overexpression. PMID:26363067

IGF1 regulates PKM2 function through Akt phosphorylation

PubMed Central

Salani, Barbara; Ravera, Silvia; Amaro, Adriana; Salis, Annalisa; Passalacqua, Mario; Millo, Enrico; Damonte, Gianluca; Marini, Cecilia; Pfeffer, Ulrich; Sambuceti, Gianmario; Cordera, Renzo; Maggi, Davide

2015-01-01

Pyruvate kinase M2 (PKM2) acts at the crossroad of growth and metabolism pathways in cells. PKM2 regulation by growth factors can redirect glycolytic intermediates into key biosynthetic pathway. Here we show that IGF1 can regulate glycolysis rate, stimulate PKM2 Ser/Thr phosphorylation and decrease cellular pyruvate kinase activity. Upon IGF1 treatment we found an increase of the dimeric form of PKM2 and the enrichment of PKM2 in the nucleus. This effect was associated to a reduction of pyruvate kinase enzymatic activity and was reversed using metformin, which decreases Akt phosphorylation. IGF1 induced an increased nuclear localization of PKM2 and STAT3, which correlated with an increased HIF1α, HK2, and GLUT1 expression and glucose entrapment. Metformin inhibited HK2, GLUT1, HIF-1α expression and glucose consumption. These findings suggest a role of IGFIR/Akt axis in regulating glycolysis by Ser/Thr PKM2 phosphorylation in cancer cells. PMID:25790097

Enhanced Phosphorylation-Independent Arrestins and Gene Therapy

PubMed Central

Gurevich, Vsevolod V.; Song, Xiufeng; Vishnivetskiy, Sergey A.; Gurevich, Eugenia V.

2015-01-01

A variety of heritable and acquired disorders is associated with excessive signaling by mutant or overstimulated GPCRs. Since any conceivable treatment of diseases caused by gain-of-function mutations requires gene transfer, one possible approach is functional compensation. Several structurally distinct forms of enhanced arrestins that bind phosphorylated and even non-phosphorylated active GPCRs with much higher affinity than parental wild-type proteins have the ability to dampen the signaling by hyperactive GPCR, pushing the balance closer to normal. In vivo this approach was so far tested only in rod photoreceptors deficient in rhodopsin phosphorylation, where enhanced arrestin improved the morphology and light sensitivity of rods, prolonged their survival, and accelerated photoresponse recovery. Considering that rods harbor the fastest, as well as the most demanding and sensitive GPCR-driven signaling cascade, even partial success of functional compensation of defect in rhodopsin phosphorylation by enhanced arrestin demonstrates the feasibility of this strategy and its therapeutic potential. PMID:24292828

Identification of Multiple Phosphorylation Sites on Maize Endosperm Starch Branching Enzyme IIb, a Key Enzyme in Amylopectin Biosynthesis

PubMed Central

Makhmoudova, Amina; Williams, Declan; Brewer, Dyanne; Massey, Sarah; Patterson, Jenelle; Silva, Anjali; Vassall, Kenrick A.; Liu, Fushan; Subedi, Sanjeena; Harauz, George; Siu, K. W. Michael; Tetlow, Ian J.; Emes, Michael J.

2014-01-01

Starch branching enzyme IIb (SBEIIb) plays a crucial role in amylopectin biosynthesis in maize endosperm by defining the structural and functional properties of storage starch and is regulated by protein phosphorylation. Native and recombinant maize SBEIIb were used as substrates for amyloplast protein kinases to identify phosphorylation sites on the protein. A multidisciplinary approach involving bioinformatics, site-directed mutagenesis, and mass spectrometry identified three phosphorylation sites at Ser residues: Ser649, Ser286, and Ser297. Two Ca2+-dependent protein kinase activities were partially purified from amyloplasts, termed K1, responsible for Ser649 and Ser286 phosphorylation, and K2, responsible for Ser649 and Ser297 phosphorylation. The Ser286 and Ser297 phosphorylation sites are conserved in all plant branching enzymes and are located at opposite openings of the 8-stranded parallel β-barrel of the active site, which is involved with substrate binding and catalysis. Molecular dynamics simulation analysis indicates that phospho-Ser297 forms a stable salt bridge with Arg665, part of a conserved Cys-containing domain in plant branching enzymes. Ser649 conservation appears confined to the enzyme in cereals and is not universal, and is presumably associated with functions specific to seed storage. The implications of SBEIIb phosphorylation are considered in terms of the role of the enzyme and the importance of starch biosynthesis for yield and biotechnological application. PMID:24550386

Requirement for tyrosine phosphatase during serotonergic neuromodulation by protein kinase C.

PubMed

Catarsi, S; Drapeau, P

1997-08-01

Tyrosine kinases and phosphatases are abundant in the nervous system, where they signal cellular differentiation, mediate the responses to growth factors, and direct neurite outgrowth during development. Tyrosine phosphorylation can also alter ion channel activity, but its physiological significance remains unclear. In an identified leech mechanosensory neuron, the ubiquitous neuromodulator serotonin increases the activity of a cation channel by activating protein kinase C (PKC), resulting in membrane depolarization and modulation of the receptive field properties. We observed that the effects on isolated neurons and channels were blocked by inhibiting tyrosine phosphatases. Serotonergic stimulation of PKC thus activates a tyrosine phosphatase activity associated with the channels, which reverses their constitutive inhibition by tyrosine phosphorylation, representing a novel form of neuromodulation.

Complementary Phosphorylation Sites in the Adaptor Protein SLP-76 Promote Synergistic Activation of Natural Killer Cells

PubMed Central

Kim, Hun Sik; Long, Eric O.

2013-01-01

The cytotoxic effects of natural killer (NK) cells and their ability to secrete cytokines require the induction of synergistic signals from co-activation receptors, such as CD314 (NKG2D) and CD244 (2B4), which bind to ligands expressed on target cells. Synergy is required to overcome inhibition of the guanine nucleotide exchange factor (GEF) Vav1, a central regulator of NK cell activation, by the E3 ubiquitin ligase c-Cbl. However, the molecular basis for this synergy is unknown. Here, we showed that the adaptor protein Src homology 2 (SH2) domain–containing leukocyte phosphoprotein of 76 kD (SLP-76) was required for this synergy, and that distinct tyrosine residues in SLP-76 were phosphorylated by each receptor of a synergistic pair. Selective phosphorylation of tyrosine 113 or tyrosine 128 in SLP-76, each of which enables binding of SLP-76 to Vav1, was unique to receptors that stimulate ligand-dependent target cell killing, because antibody-dependent stimulation by Fc receptor CD16 promoted phosphorylation at both sites. Knockdown and reconstitution experiments with SLP-76 showed the distinct role of each tyrosine in the synergistic mobilization of Ca2+, revealing an unexpected degree of selectivity in the phosphorylation of SLP-76 by NK cell co-activation receptors. Together, these data suggest that complementation of separate phospho-tyrosine targets in SLP-76 forms the basis of synergistic NK cell activation. PMID:22786724

Expression of Human CTP Synthetase in Saccharomyces cerevisiae Reveals Phosphorylation by Protein Kinase A*

PubMed Central

Han, Gil-Soo; Sreenivas, Avula; Choi, Mal-Gi; Chang, Yu-Fang; Martin, Shelley S.; Baldwin, Enoch P.; Carman, George M.

2005-01-01

CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7Δ ura8Δ mutant lacking CTP synthetase activity. The expression of the CTPS1-and CTPS2-encoded human CTP synthetase enzymes in the ura7Δ ura8Δ mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from 32Pi-labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose- and time-dependent, and dependent on the concentrations of CTP synthetase1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation. PMID:16179339

Nitrate and ammonium lead to distinct global dynamic phosphorylation patterns when resupplied to nitrogen-starved Arabidopsis seedlings.

PubMed

Engelsberger, Wolfgang R; Schulze, Waltraud X

2012-03-01

Nitrogen is an essential macronutrient for plant growth and development. Inorganic nitrogen and its assimilation products control various metabolic, physiological and developmental processes. Although the transcriptional responses induced by nitrogen have been extensively studied in the past, our work here focused on the discovery of candidate proteins for regulatory events that are complementary to transcriptional changes. Most signaling pathways involve modulation of protein abundance and/or activity by protein phosphorylation. Therefore, we analyzed the dynamic changes in protein phosphorylation in membrane and soluble proteins from plants exposed to rapid changes in nutrient availability over a time course of 30 min. Plants were starved of nitrogen and subsequently resupplied with nitrogen in the form of nitrate or ammonium. Proteins with maximum change in their phosphorylation level at up to 5 min after nitrogen resupply (fast responses) included GPI-anchored proteins, receptor kinases and transcription factors, while proteins with maximum change in their phosphorylation level after 10 min of nitrogen resupply (late responses) included proteins involved in protein synthesis and degradation, as well as proteins with functions in central metabolism and hormone metabolism. Resupply of nitrogen in the form of nitrate or ammonium resulted in distinct phosphorylation patterns, mainly of proteins with signaling functions, transcription factors and transporters. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lorenzato, Annalisa; Biolatti, Marta; Institute for Cancer Research at Candiolo, Candiolo, Torino

The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancermore » cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. - highlights: • CSE1L is a key player in nucleocytoplasmic traffic by forming complex with Ran. • AKT phosphorylates RanBP3 that regulates the nucleocytoplasmic gradient of Ran. • The activated oncogenic AKT drives the nuclear accumulation of CSE1L. • CSE1L in the nucleus up-regulates genes conveying pro-oncogenic signals. • CSE1L might contribute to tumor progression driven by the activated oncogenic AKT.« less

AMP-activated protein kinase is involved in the activation of the Fanconi anemia/BRCA pathway in response to DNA interstrand crosslinks.

PubMed

Chun, Min Jeong; Kim, Sunshin; Hwang, Soo Kyung; Kim, Bong Sub; Kim, Hyoun Geun; Choi, Hae In; Kim, Jong Heon; Goh, Sung Ho; Lee, Chang-Hun

2016-08-16

Fanconi anemia complementation group (FANC) proteins constitute the Fanconi Anemia (FA)/BRCA pathway that is activated in response to DNA interstrand crosslinks (ICLs). We previously performed yeast two-hybrid screening to identify novel FANC-interacting proteins and discovered that the alpha subunit of AMP-activated protein kinase (AMPKα1) was a candidate binding partner of the FANCG protein, which is a component of the FA nuclear core complex. We confirmed the interaction between AMPKα and both FANCG using co-immunoprecipitation experiments. Additionally, we showed that AMPKα interacted with FANCA, another component of the FA nuclear core complex. AMPKα knockdown in U2OS cells decreased FANCD2 monoubiquitination and nuclear foci formation upon mitomycin C-induced ICLs. Furthermore, AMPKα knockdown enhanced cellular sensitivity to MMC. MMC treatment resulted in an increase in AMPKα phosphorylation/activation, indicating AMPK is involved in the cellular response to ICLs. FANCA was phosphorylated by AMPK at S347 and phosphorylation increased with MMC treatment. MMC-induced FANCD2 monoubiquitination and nuclear foci formation were compromised in a U2OS cell line that stably overexpressed the S347A mutant form of FANCA compared to wild-type FANCA-overexpressing cells, indicating a requirement for FANCA phosphorylation at S347 for proper activation of the FA/BRCA pathway. Our data suggest AMPK is involved in the activation of the FA/BRCA pathway.

mTOR Complex 2 mediates Akt Phosphorylation that Requires PKCε in Adult Cardiac Muscle Cells

PubMed Central

Moschella, Phillip C.; McKillop, John; Pleasant, Dorea L.; Harston, Rebecca K.; Balasubramanian, Sundaravadivel; Kuppuswamy, Dhandapani

2013-01-01

Our earlier work showed that mammalian target of rapamycin (mTOR) is essential to the development of various hypertrophic responses, including cardiomyocyte survival. mTOR forms two independent complexes, mTORC1 and mTORC2, by associating with common and distinct cellular proteins. Both complexes are sensitive to a pharmacological inhibitor, torin1, although only mTORC1 is inhibited by rapamycin. Since mTORC2 is known to mediate the activation of a prosurvival kinase, Akt, we analyzed whether mTORC2 directly mediates Akt activation or whether it requires the participation of another prosurvival kinase, PKC ε (epsilon isoform of protein kinase-C). Our studies reveal that treatment of adult feline cardiomyocytes in vitro with insulin results in Akt phosphorylation at S473 for its activation which could be augmented with rapamycin but blocked by torin1. Silencing the expression of Rictor (rapamycin-insensitive companion of mTOR), an mTORC2 component, with a sh-RNA in cardiomyocytes lowers both insulin-stimulated Akt and PKC ε phosphorylation. Furthermore, phosphorylation of PKC ε and Akt at the critical S729 and S473 sites respectively was blocked by torin1 or Rictor knockdown but not by rapamycin, indicating that the phosphorylation at these specific sites occurs downstream of mTORC2. Additionally, expression of DN-PKC ε significantly lowered the insulin-stimulated Akt S473 phosphorylation, indicating an upstream role for PKC ε in the Akt activation. Biochemical analyses also revealed that PKC ε was part of Rictor but not Raptor (a binding partner and component of mTORC1). Together, these studies demonstrate that mTORC2 mediates prosurvival signaling in adult cardiomyocytes where PKC ε functions downstream of mTORC2 leading to Akt activation. PMID:23673367

mTOR complex 2 mediates Akt phosphorylation that requires PKCε in adult cardiac muscle cells.

PubMed

Moschella, Phillip C; McKillop, John; Pleasant, Dorea L; Harston, Rebecca K; Balasubramanian, Sundaravadivel; Kuppuswamy, Dhandapani

2013-09-01

Our earlier work showed that mammalian target of rapamycin (mTOR) is essential to the development of various hypertrophic responses, including cardiomyocyte survival. mTOR forms two independent complexes, mTORC1 and mTORC2, by associating with common and distinct cellular proteins. Both complexes are sensitive to a pharmacological inhibitor, torin1, although only mTORC1 is inhibited by rapamycin. Since mTORC2 is known to mediate the activation of a prosurvival kinase, Akt, we analyzed whether mTORC2 directly mediates Akt activation or whether it requires the participation of another prosurvival kinase, PKCε (epsilon isoform of protein kinase-C). Our studies reveal that treatment of adult feline cardiomyocytes in vitro with insulin results in Akt phosphorylation at S473 for its activation which could be augmented with rapamycin but blocked by torin1. Silencing the expression of Rictor (rapamycin-insensitive companion of mTOR), an mTORC2 component, with a sh-RNA in cardiomyocytes lowers both insulin-stimulated Akt and PKCε phosphorylation. Furthermore, phosphorylation of PKCε and Akt at the critical S729 and S473 sites respectively was blocked by torin1 or Rictor knockdown but not by rapamycin, indicating that the phosphorylation at these specific sites occurs downstream of mTORC2. Additionally, expression of DN-PKCε significantly lowered the insulin-stimulated Akt S473 phosphorylation, indicating an upstream role for PKCε in the Akt activation. Biochemical analyses also revealed that PKCε was part of Rictor but not Raptor (a binding partner and component of mTORC1). Together, these studies demonstrate that mTORC2 mediates prosurvival signaling in adult cardiomyocytes where PKCε functions downstream of mTORC2 leading to Akt activation. Copyright © 2013 Elsevier Inc. All rights reserved.

A Radical Pathway for Organic Phosphorylation during Schreibersite Corrosion with Implications for the Origin of Life

NASA Technical Reports Server (NTRS)

Pasek, Matthew A.; Dworkin, Jason P.; Lauretta, Dante S.

2007-01-01

Phosphorylated compounds (e.g. DNA, RNA, phospholipids, and many coenzymes) are critical to biochemistry. Thus, their origin is of prime interest to origin of life studies. The corrosion of the meteoritic mineral schreibersite ((Fe,Ni)3P) may have significantly contributed to the origin of phosphorylated biomolecules. Corrosion of synthetic schreibersite in a variety of solutions was analyzed by nuclear magnetic resonance spectroscopy, mass spectrometry, and electron paramagnetic resonance spectroscopy. These methods suggest a radical reaction pathway for the corrosion of schreibersite to form phosphite radicals (raised dot PO3 sup 2-)) aqueous solution. These radicals can form activated polyphosphates and can phosphorylate organic compounds such as acetate (3% yield). Phosphonates (O3P-C) are found in the organic P inventory of the carbonaceous meteorite Murchison. While phosphonates are rare in biochemistry, the ubiquity of corroding iron meteorites on the early Earth could have provided an accessible source of organophosphorous for the origin of life allowing the invention of the organophosphates in modern biology as a product of early evolution.

Protein kinase D1 phosphorylates HDAC7 and induces its nuclear export after T-cell receptor activation.

PubMed

Parra, Maribel; Kasler, Herbert; McKinsey, Timothy A; Olson, Eric N; Verdin, Eric

2005-04-08

HDAC7, a class II histone deacetylase that is highly expressed in thymocytes, inhibits both transcription of the orphan steroid nuclear receptor Nur77 and induction of apoptosis in response to activation of the T-cell receptor (TCR). Here, we report that HDAC7 is exported to the cytoplasm by a calcium-independent signaling pathway after TCR activation. Protein kinase D1 (PKD1) was activated after TCR engagement, interacted with HDAC7, and phosphorylated three serines (Ser155, Ser318, and Ser448) at its N terminus, leading to its export from the nucleus. Mutation of Ser155, Ser318, and Ser448 blocked the nucleocytoplasmic shuttling of HDAC7 in response to TCR activation, as did overexpression of a kinase-inactive form of PKD1. Consistent with the regulatory role of HDAC7 in Nur77 expression, PKD1 activation led to the transcriptional activation of Nur77 via myocyte enhancer factor 2-binding sites in its promoter. In a mouse model of negative selection, PKD1 was activated during thymocyte activation. These observations indicate that PKD1 regulates the expression of Nur77 during thymocyte activation at least in part by phosphorylating HDAC7.

Localization of phosphorylated forms of Bcl-2 in mitosis: co-localization with Ki-67 and nucleolin in nuclear structures and on mitotic chromosomes.

PubMed

Barboule, Nadia; Truchet, Isabelle; Valette, Annie

2005-04-01

Bcl-2 phosphorylation is a normal physiological process occurring at mitosis or during mitotic arrest induced by microtubule damaging agents. The consequences of Bcl-2 phosphorylation on its function are still controversial. To better understand the role of Bcl-2 phosphorylation in mitosis, we studied the subcellular localization of phosphorylated forms of Bcl-2. Immunofluorescence experiments performed in synchronized HeLa cells indicate for the first time that mitotic phosphorylated forms of Bcl-2 can be detected in nuclear structures in prophase cells together with nucleolin and Ki-67. In later mitotic stages, as previously described, phosphorylated forms of Bcl-2 are localized on mitotic chromosomes. In addition, we demonstrate that Bcl-2 in these structures is at least in part phosphorylated on the T56 residue. Then, coimmunoprecipitation experiments reveal that, in cells synchronized at the onset of mitosis, Bcl-2 is present in a complex with nucleolin, cdc2 kinase and PP1 phosphatase. Taken together, these data further support the idea that Bcl-2 could have a new function at mitosis.

Signaling pathways of interleukin-1 actions in the brain: anatomical distribution of phospho-ERK1/2 in the brain of rat treated systemically with interleukin-1beta.

PubMed

Nadjar, A; Combe, C; Busquet, P; Dantzer, R; Parnet, P

2005-01-01

Interleukin-1beta is released at the periphery during infection and acts on the nervous system to induce fever, neuroendocrine activation, and behavioral changes. These effects are mediated by brain type I IL-1 receptors. In vitro studies have shown the ability of interleukin-1beta to activate mitogen-activated protein kinase signaling pathways including p38, c-Jun N-terminal kinase and extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). In contrast to other mitogen-activated protein kinases, little is known about ERK1/2 activation in the rat brain in response to interleukin-1beta. The aim of the present study was therefore to investigate spatial and temporal activation of ERK1/2 in the rat brain after peripheral administration of interleukin-1beta using immunohistochemistry to detect the phosphorylated form of the kinase. In non-stimulated conditions, phosphorylated ERK1/2 immunoreactivity was observed in neurons throughout the brain. Administration of interleukin-1beta (60 microg/kg, i.p.) induced the phosphorylation of ERK1/2 in areas at the interface between brain and blood or cerebrospinal fluid: meninges, circumventricular organs, endothelial like cells of the blood vessels, and in brain nuclei involved in behavioral depression, fever and neuroendocrine activation: paraventricular nucleus of the hypothalamus, supraoptic nucleus, central amygdala and arcuate nucleus. Double labeling of phosphorylated ERK1/2 and cell markers revealed the expression of phosphorylated ERK1/2 in neurons, astrocytes and microglia. Since phosphorylated ERK1/2 was found in structures in which type I IL-1 receptor has already been identified as well as in structures lacking this receptor, activation of ERK1/2 is likely to occur in response to both direct and indirect action of interleukin-1beta on its target cells.

Role of Rac1 in Escherichia coli K1 invasion of human brain microvascular endothelial cells.

PubMed

Rudrabhatla, Rajyalakshmi S; Selvaraj, Suresh K; Prasadarao, Nemani V

2006-02-01

Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC) requires the reorganization of host cytoskeleton at the sites of bacterial entry. Both actin and myosin constitute the cytoskeletal architecture. We have previously shown that myosin light chain (MLC) phosphorylation by MLC kinase is regulated during E. coli invasion by an upstream kinase, p21-activated kinase 1 (PAK1), which is an effector protein of Rac and Cdc42 GTPases, but not of RhoA. Here, we report that the binding of only Rac1 to PAK1 decreases in HBMEC upon infection with E. coli K1, which resulted in increased phosphorylation of MLC. Overexpression of a constitutively active (cAc) form of Rac1 in HBMEC blocked the E. coli invasion significantly, whereas overexpression of a dominant negative form had no effect. Increased PAK1 phosphorylation was observed in HBMEC expressing cAc-Rac1 with a concomitant reduction in the phosphorylation of MLC. Immunocytochemistry studies demonstrated that the inhibition of E. coli invasion into cAc-Rac1/HBMEC is due to lack of phospho-MLC recruitment to the sites of E. coli entry. Taken together the data suggest that E. coli modulates the binding of Rac1, but not Cdc42, to PAK1 during the invasion of HBMEC.

Effects of active and inactive phospholipase D2 on signal transduction, adhesion, migration, invasion, and metastasis in EL4 lymphoma cells.

PubMed

Knoepp, Stewart M; Chahal, Manpreet S; Xie, Yuhuan; Zhang, Zhihong; Brauner, Daniel J; Hallman, Mark A; Robinson, Stephanie A; Han, Shujie; Imai, Masaki; Tomlinson, Stephen; Meier, Kathryn E

2008-09-01

The phosphatidylcholine-using phospholipase D (PLD) isoform PLD2 is widely expressed in mammalian cells and is activated in response to a variety of promitogenic agonists. In this study, active and inactive hemagglutinin-tagged human PLD2 (HA-PLD2) constructs were stably expressed in an EL4 cell line lacking detectable endogenous PLD1 or PLD2. The overall goal of the study was to examine the roles of PLD2 in cellular signal transduction and cell phenotype. HA-PLD2 confers PLD activity that is activated by phorbol ester, ionomycin, and okadaic acid. Proliferation and Erk activation are unchanged in cells transfected with active PLD2; proliferation rate is decreased in cells expressing inactive PLD2. Basal tyrosine phosphorylation of focal adhesion kinase (FAK) is increased in cells expressing active PLD2, as is phosphorylation of Akt; inactive PLD2 has no effect. Expression of active PLD2 is associated with increased spreading and elongation of cells on tissue culture plastic, whereas inactive PLD2 inhibits cell spreading. Inactive PLD2 also inhibits cell adhesion, migration, and serum-induced invasion. Cells expressing active PLD2 form metastases in syngeneic mice, as do the parental cells; cells expressing inactive PLD2 form fewer metastases than parental cells. In summary, active PLD2 enhances FAK phosphorylation, Akt activation, and cell invasion in EL4 lymphoma cells, whereas inactive PLD2 exerts inhibitory effects on adhesion, migration, invasion, and tumor formation. Overall, expression of active PLD2 enhances processes favorable to lymphoma cell metastasis, whereas expression of inactive PLD2 inhibits metastasis.

MEK-1 Activates C-Raf Through a Ras-Independent Mechanism

PubMed Central

Leicht, Deborah T.; Balan, Vitaly; Zhu, Jun; Kaplun, Alexander; Bronisz, Agnieszka; Rana, Ajay; Tzivion, Guri

2013-01-01

C-Raf is a member of the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) signaling pathway that plays key roles in diverse physiological processes and is upregulated in many human cancers. C-Raf activation involves binding to Ras, increased phosphorylation and interactions with co-factors. Here, we describe a Ras-independent in vivo pathway for C-Raf activation by its downstream target MEK. Using 32P-metabolic labeling and 2D-phosphopeptide mapping experiments, we show that MEK increases C-Raf phosphorylation by up-to 10-fold. This increase was associated with C-Raf kinase activation, matching the activity seen with growth factor stimulation. Consequently, coexpression of wildtype C-Raf and MEK was sufficient for full and constitutive activation of ERK. Notably, the ability of MEK to activate C-Raf was completely Ras independent, since mutants impaired in Ras binding that are irresponsive to growth factors or Ras were fully activated by MEK. The ability of MEK to activate C-Raf was only partially dependent on MEK kinase activity but required MEK binding to C-Raf, suggesting that the binding results in a conformational change that increases C-Raf susceptibility to phosphorylation and activation or in the stabilization of the phosphorylated-active form. These findings propose a novel Ras-independent mechanism for activating C-Raf and the MAPK pathway without the need for mutations in the pathway. This mechanism could be of significance in pathological conditions or cancers overexpressing C-Raf and MEK or in conditions where C-Raf-MEK interaction is enhanced due to the downregulation of RKIP and MST2. PMID:23360980

Differential phosphorylation of azidothymidine, dideoxycytidine, and dideoxyinosine in resting and activated peripheral blood mononuclear cells.

PubMed Central

Gao, W Y; Shirasaka, T; Johns, D G; Broder, S; Mitsuya, H

1993-01-01

The antiviral activity of azidothymidine (AZT), dideoxycytidine (ddC), and dideoxyinosine (ddI) against HIV-1 was comparatively evaluated in PHA-stimulated PBM. The mean drug concentration which yielded 50% p24 Gag negative cultures were substantially different: 0.06, 0.2, and 6 microM for AZT, ddC, and ddI, respectively. We found that AZT was preferentially phosphorylated to its triphosphate (TP) form in PHA-PBM rather than unstimulated, resting PBM (R-PBM), producing 10- to 17-fold higher ratios of AZTTP/dTTP in PHA-PBM than in R-PBM. The phosphorylation of ddC and ddI to their TP forms was, however, much less efficient in PHA-PBM, resulting in approximately 5-fold and approximately 15-fold lower ratios of ddCTP/dCTP and ddATP/dATP, respectively, in PHA-PBM than in R-PBM. The comparative order of PHA-induced increase in cellular enzyme activities examined was: thymidine kinase > uridine kinase > deoxycytidine kinase > adenosine kinase > 5'-nucleotidase. We conclude that AZT, ddC, and ddI exert disproportionate antiviral effects depending on the activation state of the target cells, i.e., ddI and ddC exert antiviral activity more favorably in resting cells than in activated cells, while AZT preferentially protects activated cells against HIV infection. Considering that HIV-1 proviral DNA synthesis in resting lymphocytes is reportedly initiated at levels comparable with those of activated lymphocytes, the current data should have practical relevance in the design of anti-HIV chemotherapy, particularly combination chemotherapy. PMID:8387546

The LDL Receptor-Related Protein 1 (LRP1) Regulates the PDGF Signaling Pathway by Binding the Protein Phosphatase SHP-2 and Modulating SHP-2- Mediated PDGF Signaling Events

PubMed Central

Craig, Julie; Mikhailenko, Irina; Noyes, Nathaniel; Migliorini, Mary; Strickland, Dudley K.

2013-01-01

Background The PDGF signaling pathway plays a major role in several biological systems, including vascular remodeling that occurs following percutaneous transluminal coronary angioplasty. Recent studies have shown that the LDL receptor-related protein 1 (LRP1) is a physiological regulator of the PDGF signaling pathway. The underlying mechanistic details of how this regulation occurs have yet to be resolved. Activation of the PDGF receptor β (PDGFRβ) leads to tyrosine phosphorylation of the LRP1 cytoplasmic domain within endosomes and generates an LRP1 molecule with increased affinity for adaptor proteins such as SHP-2 that are involved in signaling pathways. SHP-2 is a protein tyrosine phosphatase that positively regulates the PDGFRβ pathway, and is required for PDGF-mediated chemotaxis. We investigated the possibility that LRP1 may regulate the PDGFRβ signaling pathway by binding SHP-2 and competing with the PDGFRβ for this molecule. Methodology/Principal Findings To quantify the interaction between SHP-2 and phosphorylated forms of the LRP1 intracellular domain, we utilized an ELISA with purified recombinant proteins. These studies revealed high affinity binding of SHP-2 to phosphorylated forms of both LRP1 intracellular domain and the PDGFRβ kinase domain. By employing the well characterized dynamin inhibitor, dynasore, we established that PDGF-induced SHP-2 phosphorylation primarily occurs within endosomal compartments, the same compartments in which LRP1 is tyrosine phosphorylated by activated PDGFRβ. Immunofluorescence studies revealed colocalization of LRP1 and phospho-SHP-2 following PDGF stimulation of fibroblasts. To define the contribution of LRP1 to SHP-2-mediated PDGF chemotaxis, we employed fibroblasts expressing LRP1 and deficient in LRP1 and a specific SHP-2 inhibitor, NSC-87877. Our results reveal that LRP1 modulates SHP-2-mediated PDGF-mediated chemotaxis. Conclusions/Significance Our data demonstrate that phosphorylated forms of LRP1 and PDGFRβ compete for SHP-2 binding, and that expression of LRP1 attenuates SHP-2-mediated PDGF signaling events. PMID:23922991

Expression, purification and characterization of recombinant mitogen-activated protein kinase kinases.

PubMed

Dent, P; Chow, Y H; Wu, J; Morrison, D K; Jove, R; Sturgill, T W

1994-10-01

Mitogen-activated protein (MAP) kinase kinases (MKKs) are dual-specificity protein kinases which activate p42mapk and p44mapk by phosphorylation of regulatory tyrosine and threonine residues. cDNAs for two isotypes of MKK, MKK1 and MKK2, have been isolated from several species. Here we describe construction of recombinant baculoviruses for high-level expression of histidine-tagged rat MKK1 and MKK2, and procedures for production of nearly homogeneous MKK1 and MKK2 fusion proteins, in both inactive and active forms. Co-infection of Sf9 cells with either MKK1 or MKK2 virus together with recombinant viruses for Raf-1, pp60src (Y527F) and c-Ha-Ras resulted in activations of 250-fold and 150-fold for MKK1 and MKK2 respectively. Specific activities towards kinase-defective p42mapk were of the order of several hundred nanomoles of phosphate transferred/min per mg of MKK protein. The Michaelis constants for both enzymes were approx. 1 microM. Preparations of activated MKK were apparently free of Raf-1 as assessed by Western blotting. Raf-1 phosphorylated MKK1 on one major tryptic phosphopeptide, the phosphorylation of which increased with time. This phosphopeptide contained only phosphoserine and possessed neutral overall charge at pH 1.9 on two-dimensional peptide mapping. Phosphorylation of MKK1 by Raf-1 correlated with activation and reached a plateau of approximately 2 mol/mol.

Ser123 Is Essential for the Water Channel Activity of McPIP2;1 from Mesembryanthemum crystallinum*

PubMed Central

Amezcua-Romero, Julio C.; Pantoja, Omar; Vera-Estrella, Rosario

2010-01-01

The increased expression of McPIP2;1 (MipC), a root-specific aquaporin (AQP) from Mesembryanthemum crystallinum, under salt stress has suggested a role for this AQP in the salt tolerance of the plant. However, whether McPIP2;1 transports water or another solute and how its activity is regulated are so far unknown. Therefore, wild type (wt) or mutated McPIP2;1 protein was expressed in Xenopus laevis oocytes. Then, the osmotic water permeability (Pf) of the oocytes membrane was assessed by hypotonic challenges. Selectivity of McPIP2;1 to water was determined by radiolabeled glycerol or urea uptake assays. Moreover, swelling and in vitro phosphorylation assays revealed that both water permeation and phosphorylation status of McPIP2;1 were significantly increased by the phosphorylation agonists okadaic acid (OA), phorbol myristate acetate (PMA), and 8-Br-cAMP, and markedly decreased by the inhibitory peptides PKI 14-22 and PKC 20-28, inhibitors of protein kinases A (PKA) and C (PKC), respectively. Substitution of Ser123 or both, Ser123 and Ser282, abolished the water channel activity of McPIP2;1 while substitution of Ser282 only partially inhibited it (51.9% inhibition). Despite lacking Ser123 and/or Ser282, the McPIP2;1 mutant forms were still phosphorylated in vitro, which suggests that phosphorylation may have a dual role on this AQP. Our results indicate that McPIP2;1 water permeability depends completely on Ser123 and is positively regulated by PKA- and PKC-mediated phosphorylation. Regulation of the phosphorylation status of McPIP2;1 may contribute to control water transport through root cells when the plant is subjected to high salinity conditions. PMID:20332086

Tyrosine phosphorylation of P-selectin in intact platelets and in a disulphide-linked complex with immunoprecipitated pp60c-src.

PubMed Central

Modderman, P W; von dem Borne, A E; Sonnenberg, A

1994-01-01

P-selectin is a 140 kDa membrane glycoprotein found in secretory granules of platelets and endothelial cells where it is rapidly translocated to the plasma membrane upon cell activation. It then functions as a receptor for various types of leucocytes. Metabolic labelling of resting platelets with 32Pi showed that P-selectin is primarily phosphorylated on serine residues, although some tyrosine phosphorylation was observed as well. However, tyrosine phosphorylation of P-selectin was greatly stimulated by treatment with the permeating phosphatase inhibitor, pervanadate. When P-selectin immunoprecipitates were incubated with [gamma-32P]ATP (in vitro kinase assay), a fraction of P-selectin was phosphorylated on its tyrosine residues by a co-precipitated kinase. P-selectin phosphorylated in vitro co-migrated with 140 kDa surface-labelled 125I-P-selectin during SDS/PAGE under reducing conditions. Under non-reducing conditions, however, phosphorylated P-selectin was disulphide-linked to unknown protein(s) in a 205 kDa complex. In vitro kinase assays of the most abundant platelet tyrosine kinase, pp60c-src, demonstrated the presence of similar 140 and 205 kDa phosphorylated proteins in SDS/PAGE under reducing and non-reducing conditions respectively. Extraction and reprecipitation studies with proteins phosphorylated in vitro indicated that P-selectin and pp60c-src form a 205 kDa 1:1 disulphide-linked complex. In the complex, pp60c-src autophosphorylation is inhibited and P-selectin is phosphorylated on tyrosine residues. As protein disulphides in the cytoplasm of intact cells are extremely rare, our results suggest that P-selectin and pp60c-src, which co-localize in platelet dense granules, may be non-covalently associated and spontaneously form disulphide bridges during lysis. In addition, the observed tyrosine phosphorylation of P-selectin in intact platelets suggests that its function might be regulated by phosphorylation by pp60c-src. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7514867

The mitosis-regulating and protein-protein interaction activities of astrin are controlled by aurora-A-induced phosphorylation.

PubMed

Chiu, Shao-Chih; Chen, Jo-Mei Maureen; Wei, Tong-You Wade; Cheng, Tai-Shan; Wang, Ya-Hui Candice; Ku, Chia-Feng; Lian, Chiao-Hsuan; Liu, Chun-Chih Jared; Kuo, Yi-Chun; Yu, Chang-Tze Ricky

2014-09-01

Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser(115). The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis. Copyright © 2014 the American Physiological Society.

Crystal structure of a complex between the phosphorelay protein YPD1 and the response regulator domain of SLN1 bound to a phosphoryl analog

PubMed Central

Zhao, Xiaodong; Copeland, Daniel M.; Soares, Alexei S.; West, Ann H.

2008-01-01

Summary The crystal structure of the yeast SLN1 response regulator domain bound to both a phosphoryl analog (BeF3−) and Mg2+ ion in complex with its downstream phosphorelay signaling partner YPD1 has been determined at a resolution of 1.70 Å. Comparisons between the beryllium fluoride-activated complex and the unliganded (or apo) complex determined previously reveal modest but important differences. The SLN1-R1•Mg2+•BeF3− structure from the complex provides evidence for the first time that the mechanism of phosphorylation-induced activation is highly conserved between bacterial response regulator domains and this example from a eukaryotic organism. Residues in and around the active site undergo slight rearrangements in order to form bonds to the essential divalent cation and fluorine atoms of BeF3−. Two conserved switch-like residues (Thr 1173 and Phe 1192) occupy distinctly different positions in the apo- versus BeF3−-bound structures consistent with the “Y-T” coupling mechanism proposed for activation of CheY and other bacterial response regulators. Several loop regions and the α4-β5-α5 surface of the SLN1-R1 domain undergo subtle conformational changes (∼1-3 Å displacements relative to the apo-structure) that lead to significant changes in terms of contacts that are formed with YPD1. Detailed structural comparisons of protein-protein interactions in the apo- and BeF3−-bound complexes suggest at least a two-state equilibrium model for formation of a transient encounter complex, in which phosphorylation of the response regulator promotes the formation of a phosphotransfer-competent complex. In the BeF3−-activated complex, the position of His 64 from YPD1 is within ideal distance and near linear geometry with Asp 1144 from the SLN1-R1 domain for phosphotransfer to occur. The ground state structure presented here suggests that phosphoryl transfer will likely proceed through an associative mechanism involving formation of a pentacoordinate phosphorus intermediate. PMID:18076904

Cot kinase promotes Ca2+ oscillation/calcineurin-independent osteoclastogenesis by stabilizing NFATc1 protein.

PubMed

Kuroda, Yukiko; Hisatsune, Chihiro; Mizutani, Akihiro; Ogawa, Naoko; Matsuo, Koichi; Mikoshiba, Katsuhiko

2012-07-01

Osteoclasts are multinuclear bone-resorbing cells formed by the fusion of monocyte/macrophage-lineage precursor cells. Activation of the transcription factor NFATc1 (nuclear factor of activated T cells c1) by the receptor activator of NF-κB ligand (RANKL) is critical for osteoclast differentiation. In our previous report (Y. Kuroda, C. Hisatsune, T. Nakamura, K. Matsuo, and K. Mikoshiba. Proc. Natl. Acad. Sci. U. S. A. 105:8643, 2008), we demonstrated that osteoblasts induce osteoclast differentiation via Ca(2+) oscillation/calcineurin-dependent and -independent NFATc1 activation pathways; however, the mechanism underlying the latter remained unclear. Here we show that Cot, a serine/threonine kinase also known as tumor progression locus 2 (Tpl-2), directly phosphorylates all Ca(2+)/calcineurin-regulated NFAT family members (NFATc1 through NFATc4) and increases their protein levels. Moreover, Cot activity in osteoclasts was enhanced via cell-cell interaction with osteoblasts, and Cot promoted Ca(2+) oscillation/calcineurin-independent osteoclastogenesis by increasing NFATc1 stability through phosphorylation. We propose that NFAT activation in vivo occurs via phosphorylation-induced protein stabilization, even in the absence of Ca(2+) oscillation and calcineurin activity.

Cot Kinase Promotes Ca2+ Oscillation/Calcineurin-Independent Osteoclastogenesis by Stabilizing NFATc1 Protein

PubMed Central

Kuroda, Yukiko; Hisatsune, Chihiro; Mizutani, Akihiro; Ogawa, Naoko

2012-01-01

Osteoclasts are multinuclear bone-resorbing cells formed by the fusion of monocyte/macrophage-lineage precursor cells. Activation of the transcription factor NFATc1 (nuclear factor of activated T cells c1) by the receptor activator of NF-κB ligand (RANKL) is critical for osteoclast differentiation. In our previous report (Y. Kuroda, C. Hisatsune, T. Nakamura, K. Matsuo, and K. Mikoshiba. Proc. Natl. Acad. Sci. U. S. A. 105:8643, 2008), we demonstrated that osteoblasts induce osteoclast differentiation via Ca2+ oscillation/calcineurin-dependent and -independent NFATc1 activation pathways; however, the mechanism underlying the latter remained unclear. Here we show that Cot, a serine/threonine kinase also known as tumor progression locus 2 (Tpl-2), directly phosphorylates all Ca2+/calcineurin-regulated NFAT family members (NFATc1 through NFATc4) and increases their protein levels. Moreover, Cot activity in osteoclasts was enhanced via cell-cell interaction with osteoblasts, and Cot promoted Ca2+ oscillation/calcineurin-independent osteoclastogenesis by increasing NFATc1 stability through phosphorylation. We propose that NFAT activation in vivo occurs via phosphorylation-induced protein stabilization, even in the absence of Ca2+ oscillation and calcineurin activity. PMID:22615493

Prolonged seizure activity leads to increased Protein Kinase A activation in the rat pilocarpine model of status epilepticus.

PubMed

Bracey, James M; Kurz, Jonathan E; Low, Brian; Churn, Severn B

2009-08-04

Status epilepticus is a life-threatening form of seizure activity that represents a major medical emergency associated with significant morbidity and mortality. Protein Kinase A is an important regulator of synaptic strength that may play an important role in the development of status epilepticus-induced neuronal pathology. This study demonstrated an increase in PKA activity against exogenous and endogenous substrates during later stages of SE. As SE progressed, a significant increase in PKA-mediated phosphorylation of an exogenous peptide substrate was demonstrated in cortical structures. The increased activity was not due to altered expression of either regulatory or catalytic subunits of the enzyme. Through the use of phospho-specific antibodies, this study also investigated the effects of SE on the phosphorylation of the GluR1 subunit of the AMPA subtype of glutamate receptor. After the onset of continuous seizure activity, an increase in phosphorylation of the PKA site on the GluR1 subunit of the AMPA receptor was observed. These data suggest a potential mechanism by which SE may increase neuronal excitability in the cortex, potentially leading to maintenance of seizure activity or long-term neuronal pathology.

Cercospora beticola Toxins (X. Inhibition of Plasma Membrane H+-ATPase by Beticolin-1).

PubMed Central

Simon-Plas, F.; Gomes, E.; Milat, M. L.; Pugin, A.; Blein, J. P.

1996-01-01

Beticolin-1 is a toxin produced by the fungus Cercospora beticola. The chemical structure of this toxin was previously elucidated. The effects of beticolin-1 on purified corn root plasma membrane H+-ATPase were studied in a solubilized form or were reconstituted into liposome membranes. The ATP hydrolysis activity of the purified solubilized enzyme was inhibited by micromolar concentrations of beticolin-1, and this inhibition was noncompetitive with respect to ATP. When this purified enzyme was inserted into liposome membranes, a competitive inhibition of the H+-ATPase hydrolysis activity by beticolin-1 was observed. The effect of beticolin-1 on the formation of H+-ATPase-phosphorylated intermediate was also studied. With the purified enzyme in its solubilized form, the level of phosphorylated intermediate was not affected by the presence of beticolin-1, whereas micromolar concentrations of the toxin led to a marked inhibition of its formation when the enzyme was reconstituted into liposomes. These data suggest that (a) the plasma membrane H+-ATPase is a direct target for beticolin-1, and (b) the kinetics of inhibition and the effect on the phosphorylated intermediate are linked and both depend on the lipid environment of the enzyme. PMID:12226329

AMP-activated protein kinase is involved in the activation of the Fanconi anemia/BRCA pathway in response to DNA interstrand crosslinks

PubMed Central

Hwang, Soo Kyung; Kim, Bong Sub; Kim, Hyoun Geun; Choi, Hae In; Kim, Jong Heon; Goh, Sung Ho; Lee, Chang-Hun

2016-01-01

Fanconi anemia complementation group (FANC) proteins constitute the Fanconi Anemia (FA)/BRCA pathway that is activated in response to DNA interstrand crosslinks (ICLs). We previously performed yeast two-hybrid screening to identify novel FANC-interacting proteins and discovered that the alpha subunit of AMP-activated protein kinase (AMPKα1) was a candidate binding partner of the FANCG protein, which is a component of the FA nuclear core complex. We confirmed the interaction between AMPKα and both FANCG using co-immunoprecipitation experiments. Additionally, we showed that AMPKα interacted with FANCA, another component of the FA nuclear core complex. AMPKα knockdown in U2OS cells decreased FANCD2 monoubiquitination and nuclear foci formation upon mitomycin C-induced ICLs. Furthermore, AMPKα knockdown enhanced cellular sensitivity to MMC. MMC treatment resulted in an increase in AMPKα phosphorylation/activation, indicating AMPK is involved in the cellular response to ICLs. FANCA was phosphorylated by AMPK at S347 and phosphorylation increased with MMC treatment. MMC-induced FANCD2 monoubiquitination and nuclear foci formation were compromised in a U2OS cell line that stably overexpressed the S347A mutant form of FANCA compared to wild-type FANCA-overexpressing cells, indicating a requirement for FANCA phosphorylation at S347 for proper activation of the FA/BRCA pathway. Our data suggest AMPK is involved in the activation of the FA/BRCA pathway. PMID:27449087

Various stressors rapidly activate the p38-MAPK signaling pathway in Mytilus galloprovincialis (Lam.).

PubMed

Gaitanaki, Catherine; Kefaloyianni, Erene; Marmari, Athina; Beis, Isidoros

2004-05-01

The stimulation of p38-MAPK signal transduction pathway by various stressful stimuli was investigated in the marine bivalve M. galloprovincialis. Oxidative stress (5 microM H2O2) induced a biphasic pattern of p38-MAPK phosphorylation with maximal values attained at 15 min (8.1-fold) and 1 h (8.0-fold) of treatment respectively. Furthermore, 1 microM SB203580 abolished the p38-MAPK phosphorylation induced by oxidative stress. Aerial exposure also induced a biphasic pattern of p38-MAPK phosphorylation, with maximal values attained at 1 h (6.8-fold) and 8 h (4.9-fold) respectively. Re-oxygenation following a 15 min of aerial exposure resulted in the progressive dephosphorylation of the kinase. Treatment with 0.5 M sorbitol (in normal seawater) induced the rapid kinase phosphorylation (9.2-fold) and this effect was reversible. Seawater salinities varying between 100-60% had no effect, whereas a salinity of 50% induced a significant p38-MAPK phosphorylation. Furthermore, hypertonicity (120% seawater) resulted in a moderate kinase phosphorylation. All the above results demonstrate for the first time in a marine invertebrate imposed to environmental and other forms of stress as an intact, living organism, that the p38-MAPK pathway is specifically activated by various stressful stimuli which this animal can often face and sustain in vivo.

Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

PubMed Central

Zhou, Yue; Yamada, Naoki; Tanaka, Tomohiro; Hori, Takashi; Yokoyama, Satoru; Hayakawa, Yoshihiro; Yano, Seiji; Fukuoka, Junya; Koizumi, Keiichi; Saiki, Ikuo; Sakurai, Hiroaki

2015-01-01

Crosstalk between inflammatory signalling pathways and receptor tyrosine kinases has been revealed as an indicator of cancer malignant progression. In the present study, we focus on EphA2 receptor tyrosine kinase, which is overexpressed in many human cancers. It has been reported that ligand-independent phosphorylation of EphA2 at Ser-897 is induced by Akt. We show that inflammatory cytokines promote RSK-, not Akt-, dependent phosphorylation of EphA2 at Ser-897. In addition, the RSK–EphA2 signalling pathway controls cell migration and invasion of metastatic breast cancer cells. Moreover, Ser-897-phosphorylated EphA2 co-localizes with phosphorylated active form of RSK in various human tumour specimens, and this double positivity is related to poor survival in lung cancer patients, especially those with a smoking history. Taken together, these results indicate that the phosphorylation of EphA2 at Ser-897 is controlled by RSK and the RSK–EphA2 axis might contribute to cell motility and promote tumour malignant progression. PMID:26158630

Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2.

PubMed

Zhou, Yue; Yamada, Naoki; Tanaka, Tomohiro; Hori, Takashi; Yokoyama, Satoru; Hayakawa, Yoshihiro; Yano, Seiji; Fukuoka, Junya; Koizumi, Keiichi; Saiki, Ikuo; Sakurai, Hiroaki

2015-07-09

Crosstalk between inflammatory signalling pathways and receptor tyrosine kinases has been revealed as an indicator of cancer malignant progression. In the present study, we focus on EphA2 receptor tyrosine kinase, which is overexpressed in many human cancers. It has been reported that ligand-independent phosphorylation of EphA2 at Ser-897 is induced by Akt. We show that inflammatory cytokines promote RSK-, not Akt-, dependent phosphorylation of EphA2 at Ser-897. In addition, the RSK-EphA2 signalling pathway controls cell migration and invasion of metastatic breast cancer cells. Moreover, Ser-897-phosphorylated EphA2 co-localizes with phosphorylated active form of RSK in various human tumour specimens, and this double positivity is related to poor survival in lung cancer patients, especially those with a smoking history. Taken together, these results indicate that the phosphorylation of EphA2 at Ser-897 is controlled by RSK and the RSK-EphA2 axis might contribute to cell motility and promote tumour malignant progression.

The RISC component VIG is a target for dsRNA-independent protein kinase activity in Drosophila S2 cells.

PubMed

Ivanov, Konstantin I; Tselykh, Timofey V; Heino, Tapio I; Mäkinen, Kristiina

2005-07-27

RNA interference (RNAi) is mediated by a multicomponent RNA-induced silencing complex (RISC). Here we examine the phosphorylation state of three Drosophila RISC-associated proteins, VIG, R2D2 and a truncated form of Argonaute2 devoid of the nonconserved N-terminal glutamine-rich domain. We show that of the three studied proteins, only VIG is phosphorylated in cultured Drosophila cells. We also demonstrate that the phosphorylation state of VIG remains unchanged after cell transfection with exogenous dsRNA. A sequence similarity search revealed that VIG shares significant similarity with the human phosphoprotein Ki-1/57, a known in vivo substrate for protein kinase C (PKC). In vitro kinase assays followed by tryptic phosphopeptide mapping showed that PKC could efficiently phosphorylate VIG on multiple sites, suggesting PKC as a candidate kinase for VIG phosphorylation in vivo. Taken together, our results identify the RISC component VIG as a novel kinase substrate in cultured Drosophila cells and suggest a possible involvement of PKC in its phosphorylation.

Ser1928 phosphorylation by PKA stimulates the L-type Ca2+ channel CaV1.2 and vasoconstriction during acute hyperglycemia and diabetes.

PubMed

Nystoriak, Matthew A; Nieves-Cintrón, Madeline; Patriarchi, Tommaso; Buonarati, Olivia R; Prada, Maria Paz; Morotti, Stefano; Grandi, Eleonora; Fernandes, Julia Dos Santos; Forbush, Katherine; Hofmann, Franz; Sasse, Kent C; Scott, John D; Ward, Sean M; Hell, Johannes W; Navedo, Manuel F

2017-01-24

Hypercontractility of arterial myocytes and enhanced vascular tone during diabetes are, in part, attributed to the effects of increased glucose (hyperglycemia) on L-type Ca V 1.2 channels. In murine arterial myocytes, kinase-dependent mechanisms mediate the increase in Ca V 1.2 activity in response to increased extracellular glucose. We identified a subpopulation of the Ca V 1.2 channel pore-forming subunit (α1 C ) within nanometer proximity of protein kinase A (PKA) at the sarcolemma of murine and human arterial myocytes. This arrangement depended upon scaffolding of PKA by an A-kinase anchoring protein 150 (AKAP150) in mice. Glucose-mediated increases in Ca V 1.2 channel activity were associated with PKA activity, leading to α1 C phosphorylation at Ser 1928 Compared to arteries from low-fat diet (LFD)-fed mice and nondiabetic patients, arteries from high-fat diet (HFD)-fed mice and from diabetic patients had increased Ser 1928 phosphorylation and Ca V 1.2 activity. Arterial myocytes and arteries from mice lacking AKAP150 or expressing mutant AKAP150 unable to bind PKA did not exhibit increased Ser 1928 phosphorylation and Ca V 1.2 current density in response to increased glucose or to HFD. Consistent with a functional role for Ser 1928 phosphorylation, arterial myocytes and arteries from knockin mice expressing a Ca V 1.2 with Ser 1928 mutated to alanine (S1928A) lacked glucose-mediated increases in Ca V 1.2 activity and vasoconstriction. Furthermore, the HFD-induced increases in Ca V 1.2 current density and myogenic tone were prevented in S1928A knockin mice. These findings reveal an essential role for α1 C phosphorylation at Ser 1928 in stimulating Ca V 1.2 channel activity and vasoconstriction by AKAP-targeted PKA upon exposure to increased glucose and in diabetes. Copyright © 2017, American Association for the Advancement of Science.

Ser1928 phosphorylation by PKA stimulates the L-type Ca2+ channel CaV1.2 and vasoconstriction during acute hyperglycemia and diabetes

PubMed Central

Nystoriak, Matthew A.; Nieves-Cintrón, Madeline; Patriarchi, Tommaso; Buonarati, Olivia R.; Prada, Maria Paz; Morotti, Stefano; Grandi, Eleonora; Fernandes, Julia Dos Santos; Forbush, Katherine; Hofmann, Franz; Sasse, Kent C.; Scott, John D.; Ward, Sean M.; Hell, Johannes W.; Navedo, Manuel F.

2017-01-01

Hypercontractility of arterial myocytes and enhanced vascular tone during diabetes are, in part, attributed to the effects of increased glucose (hyperglycemia) on L-type CaV1.2 channels. In murine arterial myocytes, kinase-dependent mechanisms mediate the increase in CaV1.2 activity in response to increased extracellular glucose. We identified a subpopulation of the CaV1.2 channel pore-forming subunit (α1C) within nanometer proximity of protein kinase A (PKA) at the sarcolemma of murine and human arterial myocytes. This arrangement depended upon scaffolding of PKA by an A-kinase anchoring protein 150 (AKAP150) in mice. Glucose-mediated increases in CaV1.2 channel activity were associated with PKA activity, leading to α1C phosphorylation at Ser1928. Compared to arteries from low-fat diet (LFD)–fed mice and nondiabetic patients, arteries from high-fat diet (HFD)–fed mice and from diabetic patients had increased Ser1928 phosphorylation and CaV1.2 activity. Arterial myocytes and arteries from mice lacking AKAP150 or expressing mutant AKAP150 unable to bind PKA did not exhibit increased Ser1928 phosphorylation and CaV1.2 current density in response to increased glucose or to HFD. Consistent with a functional role for Ser1928 phosphorylation, arterial myocytes and arteries from knockin mice expressing a CaV1.2 with Ser1928 mutated to alanine (S1928A) lacked glucose-mediated increases in CaV1.2 activity and vasoconstriction. Furthermore, the HFD-induced increases in CaV1.2 current density and myogenic tone were prevented in S1928A knockin mice. These findings reveal an essential role for α1C phosphorylation at Ser1928 in stimulating CaV1.2 channel activity and vasoconstriction by AKAP-targeted PKA upon exposure to increased glucose and in diabetes. PMID:28119464

The Role of Dimerization in Raf Signaling | Center for Cancer Research

Cancer.gov

One frequently mutated pathway in a variety of cancers and developmental disorders is the Ras-Raf-MEK-ERK cascade. Normally, binding of a growth factor to its receptor switches on Ras, which, in turn, activates one or more of the Raf kinase family members, A-Raf, B-Raf, and C-Raf. Rafs perpetuate the signal by phosphorylating and activating MEK, another kinase that phosphorylates a third kinase, ERK. ERK then phosphorylates a number of key growth-, survival-, or differentiation-promoting targets. Of the proteins in the cascade, Rafs have the most complex regulatory mechanisms, including the ability to form dimers. Because the role that dimerization plays in Raf function has been unclear, researchers working with Deborah Morrison, Ph.D., Chief of CCR’s Laboratory of Cell and Developmental Signaling, decided to investigate its significance in normal and disease-associated Raf signaling.

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria†

PubMed Central

Deutscher, Josef; Francke, Christof; Postma, Pieter W.

2006-01-01

The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens. PMID:17158705

EphA2 Receptor Unliganded Dimers Suppress EphA2 Pro-tumorigenic Signaling.

PubMed

Singh, Deo R; Ahmed, Fozia; King, Christopher; Gupta, Nisha; Salotto, Matt; Pasquale, Elena B; Hristova, Kalina

2015-11-06

The EphA2 receptor tyrosine kinase promotes cell migration and cancer malignancy through a ligand- and kinase-independent distinctive mechanism that has been linked to high Ser-897 phosphorylation and low tyrosine phosphorylation. Here, we demonstrate that EphA2 forms dimers in the plasma membrane of HEK293T cells in the absence of ephrin ligand binding, suggesting that the current seeding mechanism model of EphA2 activation is incomplete. We also characterize a dimerization-deficient EphA2 mutant that shows enhanced ability to promote cell migration, concomitant with increased Ser-897 phosphorylation and decreased tyrosine phosphorylation compared with EphA2 wild type. Our data reveal a correlation between unliganded dimerization and tumorigenic signaling and suggest that EphA2 pro-tumorigenic activity is mediated by the EphA2 monomer. Thus, a therapeutic strategy that aims at the stabilization of EphA2 dimers may be beneficial for the treatment of cancers linked to EphA2 overexpression. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution.

PubMed Central

Colwill, K; Pawson, T; Andrews, B; Prasad, J; Manley, J L; Bell, J C; Duncan, P I

1996-01-01

Mammalian Clk/Sty is the prototype for a family of dual specificity kinases (termed LAMMER kinases) that have been conserved in evolution, but whose physiological substrates are unknown. In a yeast two-hybrid screen, the Clk/Sty kinase specifically interacted with RNA binding proteins, particularly members of the serine/arginine-rich (SR) family of splicing factors. Clk/Sty itself has an serine/arginine-rich non-catalytic N-terminal region which is important for its association with SR splicing factors. In vitro, Clk/Sty efficiently phosphorylated the SR family member ASF/SF2 on serine residues located within its serine/arginine-rich region (the RS domain). Tryptic phosphopeptide mapping demonstrated that the sites on ASF/SF2 phosphorylated in vitro overlap with those phosphorylated in vivo. Immunofluorescence studies showed that a catalytically inactive form of Clk/Sty co-localized with SR proteins in nuclear speckles. Overexpression of the active Clk/Sty kinase caused a redistribution of SR proteins within the nucleus. These results suggest that Clk/Sty kinase directly regulates the activity and compartmentalization of SR splicing factors. Images PMID:8617202

Discovery of genetic variants of the kinases that activate tenofovir among individuals in the United States, Thailand, and South Africa: HPTN067.

PubMed

Figueroa, Dominique B; Tillotson, Joseph; Li, Maoji; Piwowar-Manning, Estelle; Hendrix, Craig W; Holtz, Timothy H; Bokoch, Kevin; Bekker, Linda-Gail; van Griensven, Frits; Mannheimer, Sharon; Hughes, James P; Grant, Robert M; Bumpus, Namandjé N

2018-01-01

Tenofovir (TFV), a nucleotide reverse transcriptase inhibitor, requires two phosphorylation steps to form a competitive inhibitor of HIV reverse transcriptase. Adenylate kinase 2 (AK2) has been previously demonstrated to phosphorylate tenofovir to tenofovir-monophosphate, while creatine kinase, muscle (CKM), pyruvate kinase, muscle (PKM) and pyruvate kinase, liver and red blood cell (PKLR) each have been found to phosphorylate tenofovir-monophosphate to the pharmacologically active tenofovir-diphosphate. In the present study, genomic DNA isolated from dried blood spots collected from 505 participants from Bangkok, Thailand; Cape Town, South Africa; and New York City, USA were examined for variants in AK2, CKM, PKM, and PKLR using next-generation sequencing. The bioinformatics tools SIFT and PolyPhen predicted that 19 of the 505 individuals (3.7% frequency) carried variants in at least one kinase that would result in a decrease or loss of enzymatic activity. To functionally test these predictions, AK2 and AK2 variants were expressed in and purified from E. coli, followed by investigation of their activities towards tenofovir. Interestingly, we found that purified AK2 had the ability to phosphorylate tenofovir-monophosphate to tenofovir-diphosphate in addition to phosphorylating tenofovir to tenofovir-monophosphate. Further, four of the six AK2 variants predicted to result in a loss or decrease of enzyme function exhibited a ≥30% decrease in activity towards tenofovir in our in vitro assays. Of note, an AK2 K28R variant resulted in a 72% and 81% decrease in the formation of tenofovir-monophosphate and tenofovir-diphosphate, respectively. These data suggest that there are naturally occurring genetic variants that could potentially impact TFV activation.

Site-specific Phosphorylation Protects Glycogen Synthase Kinase-3β from Calpain-mediated Truncation of Its N and C Termini*

PubMed Central

Ma, Shanshan; Liu, Shaojun; Huang, Qiaoying; Xie, Bo; Lai, Bingquan; Wang, Chong; Song, Bin; Li, Mingtao

2012-01-01

Glycogen synthase kinase-3β (GSK-3β), a key regulator of neuronal apoptosis, is inhibited by the phosphorylation of Ser-9/Ser-389 and was recently shown to be cleaved by calpain at the N terminus, leading to its subsequent activation. In this study calpain was found to cleave GSK-3β not only at the N terminus but also at the C terminus, and cleavage sites were identified at residues Thr-38–Thr-39 and Ile-384–Gln-385. Furthermore, the cleavage of GSK-3β occurred in tandem with Ser-9 dephosphorylation during cerebellar granule neuron apoptosis. Increasing Ser-9 phosphorylation of GSK-3β by inhibiting phosphatase 1/2A or pretreating with purified active Akt inhibited calpain-mediated cleavage of GSK-3β at both N and C termini, whereas non-phosphorylatable mutant GSK-3β S9A facilitated its cleavage. In contrast, Ser-389 phosphorylation selectively inhibited the cleavage of GSK-3β at the C terminus but not the N terminus. Calpain-mediated cleavage resulted in three truncated products, all of which contained an intact kinase domain: ΔN-GSK-3β (amino acids 39–420), ΔC-GSK-3β (amino acids 1–384), and ΔN/ΔC-GSK-3β (amino acids 39–384). All three truncated products showed increased kinase and pro-apoptotic activity, with ΔN/ΔC-GSK-3β being the most active form. This observation suggests that the GSK-3β C terminus acts as an autoinhibitory domain similar to the N terminus. Taken together, these findings demonstrate that calpain-mediated cleavage activates GSK-3β by removing its N- and C-terminal autoinhibitory domains and that Ser-9 phosphorylation inhibits the cleavage of GSK-3β at both termini. In contrast, Ser-389 phosphorylation inhibits only C-terminal cleavage but not N-terminal cleavage. These findings also identify a mechanism by which site-specific phosphorylation and calpain-mediated cleavage operate in concert to regulate GSK-3β activity. PMID:22496446

Site-specific phosphorylation protects glycogen synthase kinase-3β from calpain-mediated truncation of its N and C termini.

PubMed

Ma, Shanshan; Liu, Shaojun; Huang, Qiaoying; Xie, Bo; Lai, Bingquan; Wang, Chong; Song, Bin; Li, Mingtao

2012-06-29

Glycogen synthase kinase-3β (GSK-3β), a key regulator of neuronal apoptosis, is inhibited by the phosphorylation of Ser-9/Ser-389 and was recently shown to be cleaved by calpain at the N terminus, leading to its subsequent activation. In this study calpain was found to cleave GSK-3β not only at the N terminus but also at the C terminus, and cleavage sites were identified at residues Thr-38-Thr-39 and Ile-384-Gln-385. Furthermore, the cleavage of GSK-3β occurred in tandem with Ser-9 dephosphorylation during cerebellar granule neuron apoptosis. Increasing Ser-9 phosphorylation of GSK-3β by inhibiting phosphatase 1/2A or pretreating with purified active Akt inhibited calpain-mediated cleavage of GSK-3β at both N and C termini, whereas non-phosphorylatable mutant GSK-3β S9A facilitated its cleavage. In contrast, Ser-389 phosphorylation selectively inhibited the cleavage of GSK-3β at the C terminus but not the N terminus. Calpain-mediated cleavage resulted in three truncated products, all of which contained an intact kinase domain: ΔN-GSK-3β (amino acids 39-420), ΔC-GSK-3β (amino acids 1-384), and ΔN/ΔC-GSK-3β (amino acids 39-384). All three truncated products showed increased kinase and pro-apoptotic activity, with ΔN/ΔC-GSK-3β being the most active form. This observation suggests that the GSK-3β C terminus acts as an autoinhibitory domain similar to the N terminus. Taken together, these findings demonstrate that calpain-mediated cleavage activates GSK-3β by removing its N- and C-terminal autoinhibitory domains and that Ser-9 phosphorylation inhibits the cleavage of GSK-3β at both termini. In contrast, Ser-389 phosphorylation inhibits only C-terminal cleavage but not N-terminal cleavage. These findings also identify a mechanism by which site-specific phosphorylation and calpain-mediated cleavage operate in concert to regulate GSK-3β activity.

Analysis of simian virus 40 small t antigen-induced progression of rat F111 cells minimally transformed by large T antigen.

PubMed Central

Zerrahn, J; Deppert, W

1993-01-01

Minimal transformants of rat F111 fibroblasts were established after infection with the large T antigen (large T)-encoding retroviral expression vector pZIPTEX (M. Brown, M. McCormack, K. Zinn, M. Farrell, I. Bikel, and D. Livingston, J. Virol. 60:290-293, 1986). Coexpression of small t antigen (small t) in these cells efficiently led to their progression toward a significantly enhanced transformed phenotype. Small t forms a complex with phosphatase 2A and thereby might influence cellular phosphorylation processes, including the phosphorylation of large T. Since phosphorylation can modulate the transforming activity of large T, we asked whether the phosphorylation status of large T in minimally transformed cells might differ from that of large T in maximally transformed FR(wt648) cells and whether it might be altered by coexpression of small t. We found the phosphate turnover on large T in minimally transformed cells significantly different from that in fully transformed cells. This resulted in underphosphorylation of large T in minimally transformed cells at phosphorylation sites previously shown to be involved in the regulation of the transforming activity of large T. However, coexpression of small t in the minimally transformed cells did not alter the phosphate turnover on large T during progression; i.e., it did not induce a change in the steady-state phosphorylation of large T. This suggests that the helper function of small t during the progression of these cells was not mediated by modulating phosphatase 2A activity toward large T. Images PMID:8382310

Calcium-calmodulin-dependent kinase II modulates Kv4.2 channel expression and upregulates neuronal A-type potassium currents.

PubMed

Varga, Andrew W; Yuan, Li-Lian; Anderson, Anne E; Schrader, Laura A; Wu, Gang-Yi; Gatchel, Jennifer R; Johnston, Daniel; Sweatt, J David

2004-04-07

Calcium-calmodulin-dependent kinase II (CaMKII) has a long history of involvement in synaptic plasticity, yet little focus has been given to potassium channels as CaMKII targets despite their importance in repolarizing EPSPs and action potentials and regulating neuronal membrane excitability. We now show that Kv4.2 acts as a substrate for CaMKII in vitro and have identified CaMKII phosphorylation sites as Ser438 and Ser459. To test whether CaMKII phosphorylation of Kv4.2 affects channel biophysics, we expressed wild-type or mutant Kv4.2 and the K(+) channel interacting protein, KChIP3, with or without a constitutively active form of CaMKII in Xenopus oocytes and measured the voltage dependence of activation and inactivation in each of these conditions. CaMKII phosphorylation had no effect on channel biophysical properties. However, we found that levels of Kv4.2 protein are increased with CaMKII phosphorylation in transfected COS cells, an effect attributable to direct channel phosphorylation based on site-directed mutagenesis studies. We also obtained corroborating physiological data showing increased surface A-type channel expression as revealed by increases in peak K(+) current amplitudes with CaMKII phosphorylation. Furthermore, endogenous A-currents in hippocampal pyramidal neurons were increased in amplitude after introduction of constitutively active CaMKII, which results in a decrease in neuronal excitability in response to current injections. Thus CaMKII can directly modulate neuronal excitability by increasing cell-surface expression of A-type K(+) channels.

Inositol pyrophosphates mediate the DNA-PK/ATM-p53 cell death pathway by regulating CK2 phosphorylation of Tti1/Tel2

PubMed Central

Rao, Feng; Cha, Jiyoung; Xu, Jing; Xu, Risheng; Vandiver, M. Scott; Tyagi, Richa; Tokhunts, Robert; Koldobskiy, Michael A.; Fu, Chenglai; Barrow, Roxanne; Wu, Mingxuan; Fiedler, Dorothea; Barrow, James C.; Snyder, Solomon H.

2014-01-01

The apoptotic actions of p53 require its phosphorylation by a family of phosphoinositide-3-kinase-related-kinases (PIKKs), which include DNA-PKcs and ATM. These kinases are stabilized by the TTT (Tel2, Tti1, Tti2) co-chaperone family, whose actions are mediated by CK2 phosphorylation. The inositol pyrophosphates, such as 5-diphosphoinositol pentakisphosphate (IP7), are generated by a family of inositol hexakisphosphate kinases (IP6Ks) of which IP6K2 has been implicated in p53-associated cell death. In the present study we report a novel apoptotic signaling cascade linking CK2, TTT, the PIKKs, and p53. We demonstrate that IP7, formed by IP6K2, binds CK2 to enhance its phosphorylation of the TTT complex thereby stabilizing DNA-PKcs and ATM. This process stimulates p53 phosphorylation at serine-15 to activate the cell death program in human cancer cells and in murine B cells. PMID:24657168

NECAPs are negative regulators of the AP2 clathrin adaptor complex.

PubMed

Beacham, Gwendolyn M; Partlow, Edward A; Lange, Jeffrey J; Hollopeter, Gunther

2018-01-18

Eukaryotic cells internalize transmembrane receptors via clathrin-mediated endocytosis, but it remains unclear how the machinery underpinning this process is regulated. We recently discovered that membrane-associated muniscin proteins such as FCHo and SGIP initiate endocytosis by converting the AP2 clathrin adaptor complex to an open, active conformation that is then phosphorylated (Hollopeter et al., 2014). Here we report that loss of ncap-1 , the sole C. elegans gene encoding an adaptiN Ear-binding Coat-Associated Protein (NECAP), bypasses the requirement for FCHO-1. Biochemical analyses reveal AP2 accumulates in an open, phosphorylated state in ncap-1 mutant worms, suggesting NECAPs promote the closed, inactive conformation of AP2. Consistent with this model, NECAPs preferentially bind open and phosphorylated forms of AP2 in vitro and localize with constitutively open AP2 mutants in vivo. NECAPs do not associate with phosphorylation-defective AP2 mutants, implying that phosphorylation precedes NECAP recruitment. We propose NECAPs function late in endocytosis to inactivate AP2. © 2018, Beacham et al.

NECAPs are negative regulators of the AP2 clathrin adaptor complex

PubMed Central

Beacham, Gwendolyn M; Partlow, Edward A; Lange, Jeffrey J

2018-01-01

Eukaryotic cells internalize transmembrane receptors via clathrin-mediated endocytosis, but it remains unclear how the machinery underpinning this process is regulated. We recently discovered that membrane-associated muniscin proteins such as FCHo and SGIP initiate endocytosis by converting the AP2 clathrin adaptor complex to an open, active conformation that is then phosphorylated (Hollopeter et al., 2014). Here we report that loss of ncap-1, the sole C. elegans gene encoding an adaptiN Ear-binding Coat-Associated Protein (NECAP), bypasses the requirement for FCHO-1. Biochemical analyses reveal AP2 accumulates in an open, phosphorylated state in ncap-1 mutant worms, suggesting NECAPs promote the closed, inactive conformation of AP2. Consistent with this model, NECAPs preferentially bind open and phosphorylated forms of AP2 in vitro and localize with constitutively open AP2 mutants in vivo. NECAPs do not associate with phosphorylation-defective AP2 mutants, implying that phosphorylation precedes NECAP recruitment. We propose NECAPs function late in endocytosis to inactivate AP2. PMID:29345618

Physical basis behind achondroplasia, the most common form of human dwarfism.

PubMed

He, Lijuan; Horton, William; Hristova, Kalina

2010-09-24

Fibroblast growth factor receptor 3 (FGFR3) is a receptor tyrosine kinase that plays an important role in long bone development. The G380R mutation in FGFR3 transmembrane domain is known as the genetic cause for achondroplasia, the most common form of human dwarfism. Despite many studies, there is no consensus about the exact mechanism underlying the pathology. To gain further understanding into the physical basis behind the disorder, here we measure the activation of wild-type and mutant FGFR3 in mammalian cells using Western blots, and we analyze the activation within the frame of a physical-chemical model describing dimerization, ligand binding, and phosphorylation probabilities within the dimers. The data analysis presented here suggests that the mutation does not increase FGFR3 dimerization, as proposed previously. Instead, FGFR3 activity in achondroplasia is increased due to increased probability for phosphorylation of the unliganded mutant dimers. This finding has implications for the design of targeted molecular treatments for achondroplasia.

Physical Basis behind Achondroplasia, the Most Common Form of Human Dwarfism*

PubMed Central

He, Lijuan; Horton, William; Hristova, Kalina

2010-01-01

Fibroblast growth factor receptor 3 (FGFR3) is a receptor tyrosine kinase that plays an important role in long bone development. The G380R mutation in FGFR3 transmembrane domain is known as the genetic cause for achondroplasia, the most common form of human dwarfism. Despite many studies, there is no consensus about the exact mechanism underlying the pathology. To gain further understanding into the physical basis behind the disorder, here we measure the activation of wild-type and mutant FGFR3 in mammalian cells using Western blots, and we analyze the activation within the frame of a physical-chemical model describing dimerization, ligand binding, and phosphorylation probabilities within the dimers. The data analysis presented here suggests that the mutation does not increase FGFR3 dimerization, as proposed previously. Instead, FGFR3 activity in achondroplasia is increased due to increased probability for phosphorylation of the unliganded mutant dimers. This finding has implications for the design of targeted molecular treatments for achondroplasia. PMID:20624921

Expression pattern and phosphorylation status of Smad2/3 in different subtypes of human first trimester trophoblast.

PubMed

Haider, S; Kunihs, V; Fiala, C; Pollheimer, J; Knöfler, M

2017-09-01

TGF-β superfamily members are thought to play a pivotal role in placental development and differentiation. However, their downstream effectors, the Smad transcription factors, have been poorly investigated in human trophoblasts. Expression and localisation of the canonical TGF-β targets Smad2/3 and their regulators (Smad4 and Smad7) were investigated in first trimester placenta and purified cytotrophoblast (CTB) subtypes using immunofluorescence, western blotting and qPCR. Canonical and non-canonical activation was analysed in nuclear/cytoplasmic extracts of trophoblast subtypes as well as in tissue sections using antibodies against Smad2/3, phosphorylated either at the C-terminus (pSmad2C/3C) or in their linker regions (pSmad2L/3L). Smad phosphorylation was also examined in differentiating extravillous trophoblasts (EVTs) in the absence or presence of decidual stromal cell (DSC)-conditioned medium. Smad2, Smad4 and Smad7 protein were uniformly expressed between 6th and 12th week placentae and the different isolated CTB subtypes. Activated pSmad2L was mainly detected in nuclei and cytoplasm of villous CTBs, whereas pSmad2C was absent from these cells. In contrast, pSmad2C could be detected in the cytoplasm of cell column trophoblasts and in the cytoplasm/nuclei of EVTs. Smad3 and its phosphorylated forms pSmad3C and pSmad3L specifically localised to EVT nuclei. During EVT differentiation autocrine activation of pSmad2C/3C and pSmad3L was observed. DSC-conditioned medium further increased Smad2/3 phosphorylation in EVTs. The lack of pSmad2C in villous CTBs suggests that other mitogens than TGF-β could promote Smad2 linker phosphorylation under homeostatic conditions. Whereas autocrine signalling activates Smad2/3 in differentiating EVTs, paracrine factors contribute to Smad phosphorylation in these cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

The oncoprotein Ski acts as an antagonist of transforming growth factor-beta signaling by suppressing Smad2 phosphorylation.

PubMed

Prunier, Celine; Pessah, Marcia; Ferrand, Nathalie; Seo, Su Ryeon; Howe, Philip; Atfi, Azeddine

2003-07-11

The phosphorylation of Smad2 and Smad3 by the transforming growth factor (TGF)-beta-activated receptor kinases and their subsequent heterodimerization with Smad4 and translocation to the nucleus form the basis for a model how Smad proteins work to transmit TGF-beta signals. The transcriptional activity of Smad2-Smad4 or Smad3-Smad4 complexes can be limited by the corepressor Ski, which is believed to interact with Smad complexes on TGF-beta-responsive promoters and represses their ability to activate TGF-beta target genes by assembling on DNA a repressor complex containing histone deacetylase. Here we show that Ski can block TGF-beta signaling by interfering with the phosphorylation of Smad2 and Smad3 by the activated TGF-beta type I receptor. Furthermore, we demonstrate that overexpression of Ski induces the assembly of Smad2-Smad4 and Smad3-Smad4 complexes independent of TGF-beta signaling. The ability of Ski to engage Smad proteins in nonproductive complexes provides new insights into the molecular mechanism used by Ski for disabling TGF-beta signaling.

Dynamic control of Hsf1 during heat shock by a chaperone switch and phosphorylation

PubMed Central

Zheng, Xu; Krakowiak, Joanna; Patel, Nikit; Beyzavi, Ali; Ezike, Jideofor; Khalil, Ahmad S; Pincus, David

2016-01-01

Heat shock factor (Hsf1) regulates the expression of molecular chaperones to maintain protein homeostasis. Despite its central role in stress resistance, disease and aging, the mechanisms that control Hsf1 activity remain unresolved. Here we show that in budding yeast, Hsf1 basally associates with the chaperone Hsp70 and this association is transiently disrupted by heat shock, providing the first evidence that a chaperone repressor directly regulates Hsf1 activity. We develop and experimentally validate a mathematical model of Hsf1 activation by heat shock in which unfolded proteins compete with Hsf1 for binding to Hsp70. Surprisingly, we find that Hsf1 phosphorylation, previously thought to be required for activation, in fact only positively tunes Hsf1 and does so without affecting Hsp70 binding. Our work reveals two uncoupled forms of regulation - an ON/OFF chaperone switch and a tunable phosphorylation gain - that allow Hsf1 to flexibly integrate signals from the proteostasis network and cell signaling pathways. DOI: http://dx.doi.org/10.7554/eLife.18638.001 PMID:27831465

Effect of phosphorylation on hydrogen-bonding interactions of the active site histidine of the phosphocarrier protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system determined by sup 15 N NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

van Dijk, A.A.; de Lange, L.C.M.; Robillard, G.T.

1990-09-04

The phosphocarrier protein HPr of the phosphoenolpyruvate-dependent sugar transport system of Escherichia coli can exist in a phosphorylated and a nonphosphorylated form. During phosphorylation, the phosphoryl group is carried on a histidine residue, His15. The hydrogen-bonding state of this histidine was examined with {sup 15}N NMR. For this purpose we selectively enriched the histidine imidazole nitrogens with {sup 15}N by supplying an E. coli histidine auxotroph with the amino acid labeled either at the N{delta}1 and N{epsilon}2 positions or at only the N{delta}1 position. {sup 15}N NMR spectra of two synthesized model compound, phosphoimidazole and phosphomethylimidazole, were also recorded. Themore » authors show that, prior to phosphorylation, the protonated His15 N{epsilon}2 is strongly hydrogen bonded, most probably to a carboxylate moiety. The H-bond should strengthen the nucleophilic character of the deprotonated N{delta}1, resulting in a good acceptor for the phosphoryl group. The hydrogen bond to the His15 N{delta}1 breaks upon phosphorylation of the residue. Implications of the H-bond structure for the mechanism of phosphorylation of HPr are discussed.« less

Phosphorylation of the rat hepatic polymeric IgA receptor.

PubMed Central

Larkin, J M; Sztul, E S; Palade, G E

1986-01-01

In vivo labeling with [35S]cysteine has identified three transmembrane forms of the rat hepatic polymeric IgA receptor: (i) a 105-kDa core glycosylated precursor; (ii) a terminally glycosylated 116-kDa intermediate; and (iii) a mature 120-kDa form. In the current study we show that the 120-kDa form is phosphorylated. After in vivo labeling with [32P]orthophosphate, all receptor forms were immunoprecipitated from hepatic total microsomes (TM) (with an antireceptor antiserum), separated by NaDodSO4/PAGE, and detected by autoradiography. The 120-kDa form was selectively phosphorylated, whereas the 116- and 105-kDa forms incorporated no detectable 32P. To determine the topology of the phosphorylation sites, hepatic TM isolated from rats labeled in vivo with either [35S]cysteine or [32P]orthophosphate were treated with trypsin. TM were solubilized and receptors were immunoprecipitated from lysates. With increasing trypsin concentrations, the [35S]cysteine-labeled receptor triplet was degraded to a trypsin-resistant doublet of approximately 95 and 85 kDa, indicating that approximately 20 kDa was removed from the receptor endodomain by trypsin. The same treatment removed all detectable 32P from labeled receptors. Furthermore, no 32P was detected in the 80-kDa biliary form of the receptor. Serine was identified as the only phosphorylated residue in acid hydrolysates of 32P-labeled immunoprecipitated receptor. These findings indicate that (i) the 120-kDa form is the only phosphorylated species of the receptor; and (ii) the phosphorylated residues are serine(s) located in the endodomain of the protein. Images PMID:3460069

Protein kinase C (PKC) phosphorylates human platelet inositol trisphosphate 5/sup +/-/-phosphomonoesterase (IP/sub 3/ 5'-p'tase) increasing phosphatase activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Connolly, T.M.; Majerus, P.W.

1986-05-01

Phosphoinositide breakdown in response to thrombin stimulation of human platelets generates messenger molecules that activate PKC (diglyceride) and mobilize Ca/sup + +/ (inositol tris-phosphates). The water soluble products of phospholipase C-mediated metabolism of phosphatidylinositol 4,5-diphosphate are inositol 1,4,5 P/sub 3/ (IP/sub 3/) and inositol 1:2-cyclic 4,5 P/sub 3/ (cIP/sub 3/). A specific phosphatase, IP/sub 3/ 5'-p'tase, cleaves the 5 phosphate from IP/sub 3/ or cIP/sub 3/ to form IP/sub 2/ or cIP/sub 2/ and P/sub i/, none of which mobilizes Ca/sup + +/. Thus, the IP/sub 3/ 5'-p'tase may regulate cellular responses to IP/sub 3/ or cIP/sub 3/. The authorsmore » find that IP/sub 3/ 5'-p'tase isolated from human platelets is phosphorylated by rat brain PKC, resulting in a 4-fold increase in IP/sub 3/ 5'-p'tase activity. The authors phosphorylated IP/sub 3/ 5'-p'tase using ..gamma.. /sup 32/P-ATP and found that the labeled enzyme comigrated on SDS-PAGE with the previously described 40K protein phosphorylated in response to thrombin stimulation of platelets. The similarity of the PKC-phosphorylated IP/sub 3/ 5'-p'tase observed in vitro and the thrombin-stimulated phosphorylated 40K protein known to be phosphorylated by PKC in vivo, suggests that these proteins may be the same. These results suggest that platelet Ca/sup + +/ mobilization maybe regulated by PKC phosphorylation of the IP/sub 3/ 5'-p'tase and can explain the observation that phorbol ester treatment of intact human platelets results in decreased production of IP/sub 3/ and decreased Ca/sup + +/ mobilization upon subsequent thrombin addition.« less

Structural Characterizations of Glycerol Kinase: Unraveling Phosphorylation-Induced Long-Range Activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yeh, Joanne I.; Kettering, Regina; Saxl, Ruth

2009-09-11

Glycerol metabolism provides a central link between sugar and fatty acid catabolism. In most bacteria, glycerol kinase plays a crucial role in regulating channel/facilitator-dependent uptake of glycerol into the cell. In the firmicute Enterococcus casseliflavus, this enzyme's activity is enhanced by phosphorylation of the histidine residue (His232) located in its activation loop, approximately 25 A from its catalytic cleft. We reported earlier that some mutations of His232 altered enzyme activities; we present here the crystal structures of these mutant GlpK enzymes. The structure of a mutant enzyme with enhanced enzymatic activity, His232Arg, reveals that residues at the catalytic cleft aremore » more optimally aligned to bind ATP and mediate phosphoryl transfer. Specifically, the position of Arg18 in His232Arg shifts by approximately 1 A when compared to its position in wild-type (WT), His232Ala, and His232Glu enzymes. This new conformation of Arg18 is more optimally positioned at the presumed gamma-phosphate location of ATP, close to the glycerol substrate. In addition to structural changes exhibited at the active site, the conformational stability of the activation loop is decreased, as reflected by an approximately 35% increase in B factors ('thermal factors') in a mutant enzyme displaying diminished activity, His232Glu. Correlating conformational changes to alteration of enzymatic activities in the mutant enzymes identifies distinct localized regions that can have profound effects on intramolecular signal transduction. Alterations in pairwise interactions across the dimer interface can communicate phosphorylation states over 25 A from the activation loop to the catalytic cleft, positioning Arg18 to form favorable interactions at the beta,gamma-bridging position with ATP. This would offset loss of the hydrogen bonds at the gamma-phosphate of ATP during phosphoryl transfer to glycerol, suggesting that appropriate alignment of the second substrate of glycerol kinase, the ATP molecule, may largely determine the rate of glycerol 3-phosphate production.« less

Phosphorylation of Pex11p does not regulate peroxisomal fission in the yeast Hansenula polymorpha

PubMed Central

Thomas, Ann S.; Krikken, Arjen M.; van der Klei, Ida J.; Williams, Chris P.

2015-01-01

Pex11p plays a crucial role in peroxisomal fission. Studies in Saccharomyces cerevisiae and Pichia pastoris indicated that Pex11p is activated by phosphorylation, which results in enhanced peroxisome proliferation. In S. cerevisiae but not in P. pastoris, Pex11p phosphorylation was shown to regulate the protein’s trafficking to peroxisomes. However, phosphorylation of PpPex11p was proposed to influence its interaction with Fis1p, another component of the organellar fission machinery. Here, we have examined the role of Pex11p phosphorylation in the yeast Hansenula polymorpha. Employing mass spectrometry, we demonstrate that HpPex11p is also phosphorylated on a Serine residue present at a similar position to that of ScPex11p and PpPex11p. Furthermore, through the use of mutants designed to mimic both phosphorylated and unphosphorylated forms of HpPex11p, we have investigated the role of this post-translational modification. Our data demonstrate that mutations to the phosphorylation site do not disturb the function of Pex11p in peroxisomal fission, nor do they alter the localization of Pex11p. Also, no effect on peroxisome inheritance was observed. Taken together, these data lead us to conclude that peroxisomal fission in H. polymorpha is not modulated by phosphorylation of Pex11p. PMID:26099236

Escitalopram Ameliorates Tau Hyperphosphorylation and Spatial Memory Deficits Induced by Protein Kinase A Activation in Sprague Dawley Rats.

PubMed

Ren, Qing-Guo; Wang, Yan-Juan; Gong, Wei-Gang; Xu, Lin; Zhang, Zhi-Jun

2015-01-01

Here, we investigated the effect of escitalopram pretreatment on protein kinase A (PKA)-induced tau hyperphosphorylation and spatial memory deficits in rats using western blot and behavioral tests, respectively. We demonstrated that escitalopram effectively ameliorated tau hyperphosphorylation and the spatial memory deficits induced by PKA activation. We measured the total and activity-dependent Ser9-phosphorylated levels of glycogen synthase kinase (GSK)-3β in hippocampal extracts. No significant change in the total level of GSK-3β was observed between the different groups. However, compared with forskolin injection alone, pretreatment with escitalopram increased the level of Ser9-phosphorylated GSK-3β. We also demonstrated that escitalopram increased Akt phosphorylation at Ser473 (the active form of Akt). Furthermore, we identified other important kinases and phosphatases, such as protein phosphatase 2A, extracellular signal-regulated kinases 1 and 2, and MAP kinase kinase-1/2, that have previously been reported to play a crucial role in tau phosphorylation; however, we did not detect any significant change in the activation of these kinases or phosphatases in our study. We unexpectedly demonstrated that forskolin caused anxiety-like behavior in rats, and pretreatment with escitalopram did not significantly ameliorate the anxiety-like behavior induced by forskolin. These data provide the first evidence that escitalopram ameliorates forskolin-induced tau hyperphosphorylation and spatial memory impairment in rats; these effects do not occur via the anti-anxiety activity of escitalopram but may involve the Akt/GSK-3β signaling pathway.

UBE4B targets phosphorylated p53 at serines 15 and 392 for degradation

PubMed Central

Du, Cheng; Wu, Hong; Leng, Roger P.

2016-01-01

Phosphorylation of p53 is a key mechanism responsible for the activation of its tumor suppressor functions in response to various stresses. In unstressed cells, p53 is rapidly turned over and is maintained at a low basal level. After DNA damage or other forms of cellular stress, the p53 level increases, and the protein becomes metabolically stable. However, the mechanism of phosphorylated p53 regulation is unclear. In this study, we studied the kinetics of UBE4B, Hdm2, Pirh2, Cop1 and CHIP induction in response to p53 activation. We show that UBE4B coimmunoprecipitates with phosphorylated p53 at serines 15 and 392. Notably, the affinity between UBE4B and Hdm2 is greatly decreased after DNA damage. Furthermore, we observe that UBE4B promotes endogenous phospho-p53(S15) and phospho-p53(S392) degradation in response to IR. We demonstrate that UBE4B and Hdm2 repress p53S15A, p53S392A, and p53-2A(S15A, S392A) functions, including p53-dependent transactivation and growth inhibition. Overall, our results reveal that UBE4B plays an important role in regulating phosphorylated p53 following DNA damage. PMID:26673821

A Link between Dimerization and Autophosphorylation of the Response Regulator PhoB*

PubMed Central

Creager-Allen, Rachel L.; Silversmith, Ruth E.; Bourret, Robert B.

2013-01-01

Response regulator proteins within two-component signal transduction systems are activated by phosphorylation and can catalyze their own covalent phosphorylation using small molecule phosphodonors. To date, comprehensive kinetic characterization of response regulator autophosphorylation is limited to CheY, which follows a simple model of phosphodonor binding followed by phosphorylation. We characterized autophosphorylation of the response regulator PhoB, known to dimerize upon phosphorylation. In contrast to CheY, PhoB time traces exhibited an initial lag phase and gave apparent pseudo-first order rate constants that increased with protein concentration. Furthermore, plots of the apparent autophosphorylation rate constant versus phosphodonor concentration were sigmoidal, as were PhoB binding isotherms for the phosphoryl group analog BeF3−. Successful mathematical modeling of the kinetic data necessitated inclusion of the formation of a PhoB heterodimer (one phosphorylated and one unphosphorylated monomer) with an enhanced rate of phosphorylation. Specifically, dimerization constants for the PhoB heterodimer and homodimer (two phosphorylated monomers) were similar, but the rate constant for heterodimer phosphorylation was ∼10-fold higher than for the monomer. In a test of the model, disruption of the known PhoBN dimerization interface by mutation led to markedly slower and noncooperative autophosphorylation kinetics. Furthermore, phosphotransfer from the sensor kinase PhoR was enhanced by dimer formation. Phosphorylation-mediated dimerization allows many response regulators to bind to tandem DNA-binding sites and regulate transcription. Our data challenge the notion that response regulator dimers primarily form between two phosphorylated monomers and raise the possibility that response regulator heterodimers containing one phosphoryl group may participate in gene regulation. PMID:23760278

Hepatic p38α regulates gluconeogenesis by suppressing AMPK.

PubMed

Jing, Yanyan; Liu, Wei; Cao, Hongchao; Zhang, Duo; Yao, Xuan; Zhang, Shengjie; Xia, Hongfeng; Li, Dan; Wang, Yu-cheng; Yan, Jun; Hui, Lijian; Ying, Hao

2015-06-01

It is proposed that p38 is involved in gluconeogenesis, however, the genetic evidence is lacking and precise mechanisms remain poorly understood. We sought to delineate the role of hepatic p38α in gluconeogenesis during fasting by applying a loss-of-function genetic approach. We examined fasting glucose levels, performed pyruvate tolerance test, imaged G6Pase promoter activity, as well as determined the expression of gluconeogenic genes in mice with a targeted deletion of p38α in liver. Results were confirmed both in vivo and in vitro by using an adenoviral dominant-negative form of p38α (p38α-AF) and the constitutively active mitogen-activated protein kinase 6, respectively. Adenoviral dominant-negative form of AMP-activated protein kinase α (DN-AMPKα) was employed to test our proposed model. Mice lacking hepatic p38α exhibited reduced fasting glucose level and impaired gluconeogenesis. Interestingly, hepatic deficiency of p38α did not result in an alteration in CREB phosphorylation, but led to an increase in AMPKα phosphorylation. Adenoviral DN-AMPKα could abolish the effect of p38α-AF on gluconeogenesis. Knockdown of up-steam transforming growth factor β-activated kinase 1 decreased the AMPKα phosphorylation induced by p38α-AF, suggesting a negative feedback loop. Consistently, inverse correlations between p38 and AMPKα phosphorylation were observed during fasting and in diabetic mouse models. Importantly, adenoviral p38α-AF treatment ameliorated hyperglycemia in diabetic mice. Our study provides evidence that hepatic p38α functions as a negative regulator of AMPK signaling in maintaining gluconeogenesis, dysregulation of this regulatory network contributes to unrestrained gluconeogenesis in diabetes, and hepatic p38α could be a drug target for hyperglycemia. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Cadmium induces phosphorylation and stabilization of c-Fos in HK-2 renal proximal tubular cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iwatsuki, Mamiko; Inageda, Kiyoshi; Matsuoka, Masato, E-mail: matsuoka@research.twmu.ac.jp

2011-03-15

We examined the effects of cadmium chloride (CdCl{sub 2}) exposure on the expression and phosphorylation status of members of the Fos family, components of the activator protein-1 transcription factor, in HK-2 human renal proximal tubular cells. Following the exposure to CdCl{sub 2}, the expression of c-fos, fosB, fra-1, and fra-2 increased markedly, with different magnitudes and time courses. The levels of Fos family proteins (c-Fos, FosB, Fra-1, and Fra-2) also increased in response to CdCl{sub 2} exposure. Although the elevation of c-fos transcripts was transient, c-Fos protein levels increased progressively with lower electrophoretic mobility, suggesting stabilization of c-Fos through post-translationalmore » modifications. Consistently, we observed phosphorylation of c-Fos at Ser362 and Ser374 in HK-2 cells treated with CdCl{sub 2}. Phosphorylated forms of mitogen-activated protein kinases (MAPKs)-including extracellular signal-regulated protein kinase (ERK), c-Jun NH{sub 2}-terminal kinase, and p38-increased after CdCl{sub 2} exposure, whereas treatment with the MAPK/ERK kinase inhibitor U0126 and the p38 inhibitor SB203580 suppressed the accumulation and phosphorylation of c-Fos. We mutated Ser362 to alanine (S362A), Ser374 to alanine (S374A), and both residues to alanines (S362A/S374A) to inhibit potential phosphorylation of c-Fos at these sites. S374A or double S362A/S374A mutations reduced c-Fos level markedly, but S362A mutation did not. On the other hand, S362A/S374A mutations induced a more pronounced reduction in c-Fos DNA-binding activity than S374A mutation. These results suggest that while Ser374 phosphorylation seems to play a role in c-Fos stabilization, phosphorylation at two C-terminal serine residues is required for the transcriptional activation of c-Fos in HK-2 cells treated with CdCl{sub 2}.« less

Ligand-independent Dimer Formation of Epidermal Growth Factor Receptor (EGFR) Is a Step Separable from Ligand-induced EGFR Signaling

PubMed Central

Yu, Xiaochun; Sharma, Kailash D.; Takahashi, Tsuyoshi; Iwamoto, Ryo; Mekada, Eisuke

2002-01-01

Dimerization and phosphorylation of the epidermal growth factor (EGF) receptor (EGFR) are the initial and essential events of EGF-induced signal transduction. However, the mechanism by which EGFR ligands induce dimerization and phosphorylation is not fully understood. Here, we demonstrate that EGFRs can form dimers on the cell surface independent of ligand binding. However, a chimeric receptor, comprising the extracellular and transmembrane domains of EGFR and the cytoplasmic domain of the erythropoietin receptor (EpoR), did not form a dimer in the absence of ligands, suggesting that the cytoplasmic domain of EGFR is important for predimer formation. Analysis of deletion mutants of EGFR showed that the region between 835Ala and 918Asp of the EGFR cytoplasmic domain is required for EGFR predimer formation. In contrast to wild-type EGFR ligands, a mutant form of heparin-binding EGF-like growth factor (HB2) did not induce dimerization of the EGFR-EpoR chimeric receptor and therefore failed to activate the chimeric receptor. However, when the dimerization was induced by a monoclonal antibody to EGFR, HB2 could activate the chimeric receptor. These results indicate that EGFR can form a ligand-independent inactive dimer and that receptor dimerization and activation are mechanistically distinct and separable events. PMID:12134089

Interaction between ROCK II and nucleophosmin/B23 in the regulation of centrosome duplication.

PubMed

Ma, Zhiyong; Kanai, Masayuki; Kawamura, Kenji; Kaibuchi, Kozo; Ye, Keqiang; Fukasawa, Kenji

2006-12-01

Nucleophosmin (NPM)/B23 has been implicated in the regulation of centrosome duplication. NPM/B23 localizes between two centrioles in the unduplicated centrosome. Upon phosphorylation on Thr(199) by cyclin-dependent kinase 2 (CDK2)/cyclin E, the majority of centrosomal NPM/B23 dissociates from centrosomes, but some NPM/B23 phosphorylated on Thr(199) remains at centrosomes. It has been shown that Thr(199) phosphorylation of NPM/B23 is critical for the physical separation of the paired centrioles, an initial event of the centrosome duplication process. Here, we identified ROCK II kinase, an effector of Rho small GTPase, as a protein that localizes to centrosomes and physically interacts with NPM/B23. Expression of the constitutively active form of ROCK II promotes centrosome duplication, while down-regulation of ROCK II expression results in the suppression of centrosome duplication, especially delaying the initiation of centrosome duplication during the cell cycle. Moreover, ROCK II regulates centrosome duplication in its kinase and centrosome localization activity-dependent manner. We further found that ROCK II kinase activity is significantly enhanced by binding to NPM/B23 and that NPM/B23 acquires a higher binding affinity to ROCK II upon phosphorylation on Thr(199). Moreover, physical interaction between ROCK II and NPM/B23 in vivo occurs in association with CDK2/cyclin E activation and the emergence of Thr(199)-phosphorylated NPM/B23. All these findings point to ROCK II as the effector of the CDK2/cyclin E-NPM/B23 pathway in the regulation of centrosome duplication.

In Vitro Stretch Injury Induces Time- and Severity-Dependent Alterations of STEP Phosphorylation and Proteolysis in Neurons

PubMed Central

Mesfin, Mahlet N.; von Reyn, Catherine R.; Mott, Rosalind E.; Putt, Mary E.

2012-01-01

Abstract Striatal-enriched tyrosine phosphatase (STEP) has been identified as a component of physiological and pathophysiological signaling pathways mediated by N-methyl-d-aspartate (NMDA) receptor/calcineurin/calpain activation. Activation of these pathways produces a subsequent change in STEP isoform expression or activation via dephosphorylation. In this study, we evaluated changes in STEP phosphorylation and proteolysis in dissociated cortical neurons after sublethal and lethal mechanical injury using an in vitro stretch injury device. Sublethal stretch injury produces minimal changes in STEP phosphorylation at early time points, and increased STEP phosphorylation at 24 h that is blocked by the NMDA-receptor antagonist APV, the calcineurin-inhibitor FK506, and the sodium channel blocker tetrodotoxin. Lethal stretch injury produces rapid STEP dephosphorylation via NR2B-containing NMDA receptors, but not calcineurin, and a subsequent biphasic phosphorylation pattern. STEP61 expression progressively increases after sublethal stretch with no change in calpain-mediated STEP33 formation, while lethal stretch injury results in STEP33 formation via a NR2B-containing NMDA receptor pathway within 1 h of injury. Blocking calpain activation in the initial 30 min after stretch injury increases the ratio of active STEP in cells and blocks STEP33 formation, suggesting that STEP is an early substrate of calpain after mechanical injury. There is a strong correlation between the amount of STEP33 formed and the degree of cell death observed after lethal stretch injury. In summary, these data demonstrate that previously characterized pathways of STEP regulation via the NMDA receptor are generally conserved in mechanical injury, and suggest that calpain-mediated cleavage of STEP33 should be further examined as an early marker of neuronal fate after stretch injury. PMID:22435660

Amplification and oscillations in the FAK/Src kinase system during integrin signaling.

PubMed

Caron-Lormier, G; Berry, H

2005-01-21

Integrin signaling is a major pathway of cell adhesion to extracellular matrices that regulates many physiological cell behaviors such as cell proliferation, migration or differentiation and is implied in pathologies such as tumor invasion. In this paper, we focused on the molecular system formed by the two kinases FAK (focal adhesion kinase) and Src, which undergo auto- and co-activation during early steps of integrin signaling. The system is modelled using classical kinetic equations and yields a set of three nonlinear ordinary differential equations describing the dynamics of the different phosphorylation forms of FAK. Analytical and numerical analysis of these equations show that this system may in certain cases amplify incoming signals from the integrins. A quantitative condition is obtained, which indicates that the total FAK charge in the system acts as a critical mass that must be exceeded for amplification to be effective. Furthermore, we show that when FAK activity is lower than Src activity, spontaneous oscillations of FAK phosphorylation forms may appear. The oscillatory behavior is studied using bifurcation and stability diagrams. We finally discuss the significance of this behavior with respect to recent experimental results evidencing FAK dynamics.

An Unbiased Approach to Identifying Tau Kinases That Phosphorylate Tau at Sites Associated with Alzheimer Disease

PubMed Central

Cavallini, Annalisa; Brewerton, Suzanne; Bell, Amanda; Sargent, Samantha; Glover, Sarah; Hardy, Clare; Moore, Roger; Calley, John; Ramachandran, Devaki; Poidinger, Michael; Karran, Eric; Davies, Peter; Hutton, Michael; Szekeres, Philip; Bose, Suchira

2013-01-01

Neurofibrillary tangles, one of the hallmarks of Alzheimer disease (AD), are composed of paired helical filaments of abnormally hyperphosphorylated tau. The accumulation of these proteinaceous aggregates in AD correlates with synaptic loss and severity of dementia. Identifying the kinases involved in the pathological phosphorylation of tau may identify novel targets for AD. We used an unbiased approach to study the effect of 352 human kinases on their ability to phosphorylate tau at epitopes associated with AD. The kinases were overexpressed together with the longest form of human tau in human neuroblastoma cells. Levels of total and phosphorylated tau (epitopes Ser(P)-202, Thr(P)-231, Ser(P)-235, and Ser(P)-396/404) were measured in cell lysates using AlphaScreen assays. GSK3α, GSK3β, and MAPK13 were found to be the most active tau kinases, phosphorylating tau at all four epitopes. We further dissected the effects of GSK3α and GSK3β using pharmacological and genetic tools in hTau primary cortical neurons. Pathway analysis of the kinases identified in the screen suggested mechanisms for regulation of total tau levels and tau phosphorylation; for example, kinases that affect total tau levels do so by inhibition or activation of translation. A network fishing approach with the kinase hits identified other key molecules putatively involved in tau phosphorylation pathways, including the G-protein signaling through the Ras family of GTPases (MAPK family) pathway. The findings identify novel tau kinases and novel pathways that may be relevant for AD and other tauopathies. PMID:23798682

Updates on the sporulation process in Clostridium species.

PubMed

Talukdar, Prabhat K; Olguín-Araneda, Valeria; Alnoman, Maryam; Paredes-Sabja, Daniel; Sarker, Mahfuzur R

2015-05-01

Sporulation is an important strategy for certain bacterial species within the phylum Firmicutes to survive longer periods of time in adverse conditions. All spore-forming bacteria have two phases in their life; the vegetative form, where they can maintain all metabolic activities and replicate to increase numbers, and the spore form, where no metabolic activities exist. Although many essential components of sporulation are conserved among the spore-forming bacteria, there are differences in the regulation and the pathways among different genera, even at the species level. While we have gained much information from the most studied spore-forming bacterial genus, Bacillus, we still lack an in-depth understanding of spore formation in the genus Clostridium. Clostridium and Bacillus share the master regulator of sporulation, Spo0A, and its downstream pathways, but there are differences in the activation of the Spo0A pathway. While Bacillus species use a multi-component phosphorylation pathway for phosphorylation of Spo0A, termed phosphorelay, such a phosphorelay system is absent in Clostridium. On the other hand, a number of genes regulated by the different sporulation-specific transcription factors are conserved between different Clostridium and Bacillus species. In this review, we discuss the recent findings on Clostridium sporulation and compare the sporulation mechanism in Clostridium and Bacillus. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Phosphorylation of STAT-1 Serine 727 Is Prolonged in HLA-B27-Expressing Human Monocytic Cells

PubMed Central

Ruuska, Marja; Sahlberg, Anna S.; Granfors, Kaisa; Penttinen, Markus A.

2013-01-01

A tissue antigen, HLA-B27, is strongly associated with a group of rheumatic diseases called spondyloarthritides. Despite the intensive research, the exact role of HLA-B27 in the pathogenesis of these diseases is still unclear. Here we studied whether HLA-B27 modulates the phosphorylation of signal transducer and activator of transcription 1 (STAT-1) serine 727 residue and the localization of STAT-1 in Salmonella-infected human monocytic cells. In addition, we studied the role of signaling molecule double-stranded RNA activated protein kinase (PKR) in these modulatory effects. U937 human monocytic cell transfectants stably expressing wild type HLA-B27 or mutated HLA-B27 heavy chains with amino acid substitutions in the B pocket were prepared. The PMA-differentiated cells were infected with S. enteritidis. Western blotting was used to detect the phosphorylation of STAT-1, and to visualize the localization of STAT-1 in the cells confocal microscopy was used. Specific inhibitors were employed to study the role of PKR in STAT-1 phosphorylation. We discovered that the phosphorylation of STAT-1 serine 727 is prolonged in cells expressing misfolding forms of HLA-B27 after S. enteritidis infection, whereas in mock cells and in cells expressing mutated, non-misfolding HLA-B27 the phosphorylation of serine 727 is transient. Interestingly, STAT-1 serine 727 phosphorylation is partly dependent on PKR. In addition, more STAT-1 is localized in the nucleus of HLA-B27-expressing cells, even before an external trigger, when compared to mock cells. In conclusion, our results show that the phosphorylation of STAT-1 serine 727 residue is prolonged in HLA-B27-expressing monocyte-macrophage U937 cells after bacterial infection. This is of interest since the phosphorylation of serine 727 on STAT-1 is suggested to contribute to macrophage activation and promote inflammatory responses. Therefore, our results provide a mechanism which explains how the expression of an HLA-B27 molecule can impact the course of Salmonella infection and reactive arthritis. PMID:23349666

AKT-dependent phosphorylation of the SAM domain induces oligomerization and activation of the scaffold protein CNK1.

PubMed

Fischer, Adrian; Weber, Wilfried; Warscheid, Bettina; Radziwill, Gerald

2017-01-01

Scaffold proteins are hubs for the coordination of intracellular signaling networks. The scaffold protein CNK1 promotes several signal transduction pathway. Here we demonstrate that sterile motif alpha (SAM) domain-dependent oligomerization of CNK1 stimulates CNK1-mediated signaling in growth factor-stimulated cells. We identified Ser22 located within the SAM domain as AKT-dependent phosphorylation site triggering CNK1 oligomerization. Oligomeric CNK1 increased the affinity for active AKT indicating a positive AKT feedback mechanism. A CNK1 mutant lacking the SAM domain and the phosphorylation-defective mutant CNK1 S22A antagonizes oligomerization and prevents CNK1-driven cell proliferation and matrix metalloproteinase 14 promoter activation. The phosphomimetic mutant CNK1 S22D constitutively oligomerizes and stimulates CNK1 downstream signaling. Searching the COSMIC database revealed Ser22 as putative target for oncogenic activation of CNK1. Like the phosphomimetic mutant CNK1 S22D , the oncogenic mutant CNK1 S22F forms clusters in serum-starved cells comparable to clusters of CNK1 in growth factor-stimulated cells. CNK1 clusters induced by activating Ser22 mutants correlate with enhanced cell invasion and binding to and activation of ADP ribosylation factor 1 associated with tumor formation. Mutational analysis indicate that EGF-triggered phosphorylation of Thr8 within the SAM domain prevents AKT binding and antagonizes CNK1-mediated AKT signaling. Our findings reveal SAM domain-dependent oligomerization by AKT as switch for CNK1 activation. Copyright © 2016 Elsevier B.V. All rights reserved.

Interactions and phosphorylation of postsynaptic density 93 (PSD-93) by extracellular signal-regulated kinase (ERK).

PubMed

Guo, Ming-Lei; Xue, Bing; Jin, Dao-Zhong; Mao, Li-Min; Wang, John Q

2012-07-17

Postsynaptic density 93 (PSD-93) is a protein enriched at postsynaptic sites. As a key scaffolding protein, PSD-93 forms complexes with the clustering of various synaptic proteins to construct postsynaptic signaling networks and control synaptic transmission. Extracellular signal-regulated kinase (ERK) is a prototypic member of a serine/threonine protein kinase family known as mitogen-activated protein kinase (MAPK). This kinase, especially ERK2 isoform, noticeably resides in peripheral structures of neurons, such as dendritic spines and postsynaptic density areas, in addition to its distribution in the cytoplasm and nucleus, although little is known about specific substrates of ERK at synaptic sites. In this study, we found that synaptic PSD-93 is a direct target of ERK. This was demonstrated by direct protein-protein interactions between purified ERK2 and PSD-93 in vitro. The accurate ERK2-binding region seems to locate at an N-terminal region of PSD-93. In adult rat striatal neurons in vivo, native ERK from synaptosomal fractions also associated with PSD-93. In phosphorylation assays, active ERK2 phosphorylated PSD-93. An accurate phosphorylation site was identified at a serine site (S323). In striatal neurons, immunoprecipitated PSD-93 showed basal phosphorylation at an ERK-sensitive site. Our data provide evidence supporting PSD-93 as a new substrate of the synaptic species of ERK. ERK2 possesses the ability to interact with PSD-93 and phosphorylate PSD-93 at a specific site. Published by Elsevier B.V.

Phosphorylation of GSK-3β and reduction of apoptosis as targets of troxerutin effect on reperfusion injury of diabetic myocardium.

PubMed

Mokhtari, Behnaz; Badalzadeh, Reza; Alihemmati, Alireza; Mohammadi, Mustafa

2015-10-15

Diabetes has various interactions with ischemic heart diseases. Troxerutin, a flavonoid, owns outstanding pharmacological potentials in cardiovascular medicine. The purpose of this study was to investigate the effects of troxerutin on phosphorylation of GSK-3β protein and apoptosis induced by myocardial ischemia-reperfusion injury in healthy and diabetic hearts. Male Wistar rats (n=36, 250-300 g) were randomly divided into four groups: healthy, diabetic, healthy-troxerutin and diabetic-troxerutin. Diabetes was induced by a single injection of streptozotocin (50 mg/kg; ip) and the diabetic period was lasted for ten weeks. Six weeks after induction of diabetes, troxerutin-treated groups received 150 mg/kg/day troxerutin by oral gavage for 4 weeks. The rats' hearts were transferred to the Langendorff apparatus and then subjected to 30 min regional ischemia followed by 45 min reperfusion. Supernatants of the left ventricle were used to measure the levels of cardiac troponin-I (cTnI) by ELISA, total and phosphorylated form of GSK-3β by western blotting and tissue apoptosis by TUNEL assay. Troxerutin administration significantly decreased the cTnI levels in healthy and diabetic groups, as compared to the corresponding controls (P<0.05). In addition, troxerutin significantly increased the level of phosphorylated form and the ratio of phosphorylated to total form of GSK-3β in diabetic and control groups (P<0.05). Tissue apoptosis level and apoptotic index also showed a significant decrease after administration of troxerutin in control and diabetic groups (P<0.05). The findings indicated that the attenuation of GSK-3β activity and subsequent reduction of apoptosis by troxerutin play significant roles in its cardioprotection on reperfusion injuries. Copyright © 2015. Published by Elsevier B.V.

Detection of phosphorylated forms of moloney murine leukemia virus major capsid protein p30 by immunoprecipitation and two-dimensional gel electrophoresis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ikuta, K.; Luftig, R.B.

1988-01-01

The authors detected phosphorylation of the major Moloney murine leukemia virus (M-MuLV) capsid polypeptide, p30, by using /sup 32/P/sub i/-labeled virions. This was observed both on two-dimensional polyacrylamide gels directly or on one-dimensional gels of viral lysates that had been immunoprecipitated with monospecific goat anti-p30 serum. The phosphorylation event had been difficult to detect because pp12 the major virion phosphoprotein incorporates almost all of the /sup 32/P label added to infected cells. When immunoprecipitates from M-MuLV lysates labeled with /sup 32/P/sub i/ were compared with those labeled with (/sup 35/S)methionine, it was calculated that the degree of phosphorylation at themore » p30 domain of Pr65/sup gag/ was only 0.22 to 0.54% relative to phosphorylation at the p12 domain. Two-dimensional gel electrophoresis of the /sup 32/P-labeled p30 immunoprecipitates showed that there were three phosphorylated p30 forms with isoelectric points (pIs) of 5.7, 5.8, and 6.0. These forms were generally more acidic than the (/sup 35/S) methionine-labeled p30 forms, which had pIs of 6.0, 6.1, 6.3 (the major constituent with > 80% of the label), and 6.6. The predominant phosphoamino acid of the major phosphorylated p30 form (pI 5.8) was phosphoserine. Further, tryptic peptide analysis of this p30 form showed that only one peptide was predominantly phosphorylated. Based on a comparison of specific labeling of p30 tryptic peptides with (/sup 14/C)sesrine, (/sup 35/S)methionine, and /sup 32/P/sub i/, we tentatively assigned the phosphorylation site to a 2.4-kilodalton NH/sub 2/-terminal peptide containing triple tandem serines spanning the region from amino acids 4 to 24.« less

The Fanconi Anemia C Protein Binds to and Regulates Stathmin-1 Phosphorylation

PubMed Central

Magron, Audrey; Elowe, Sabine; Carreau, Madeleine

2015-01-01

The Fanconi anemia (FA) proteins are involved in a signaling network that assures the safeguard of chromosomes. To understand the function of FA proteins in cellular division events, we investigated the interaction between Stathmin-1 (STMN1) and the FA group C (FANCC) protein. STMN1 is a ubiquitous cytosolic protein that regulates microtubule dynamics. STMN1 activities are regulated through phosphorylation-dephosphorylation mechanisms that control assembly of the mitotic spindle, and dysregulation of STMN1 phosphorylation is associated with mitotic aberrancies leading to chromosome instability and cancer progression. Using different biochemical approaches, we showed that FANCC interacts and co-localizes with STMN1 at centrosomes during mitosis. We also showed that FANCC is required for STMN1 phosphorylation, as mutations in FANCC reduced serine 16- and 38-phosphorylated forms of STMN1. Phosphorylation of STMN1 at serine 16 is likely an event dependent on a functional FA pathway, as it is reduced in FANCA- and FANCD2-mutant cells. Furthermore, FA-mutant cells exhibited mitotic spindle anomalies such as supernumerary centrosomes and shorter mitotic spindles. These results suggest that FA proteins participate in the regulation of cellular division via the microtubule-associated protein STMN1. PMID:26466335

Escitalopram Ameliorates Forskolin-Induced Tau Hyperphosphorylation in HEK239/tau441 Cells.

PubMed

Ren, Qing-Guo; Wang, Yan-Juan; Gong, Wei-Gang; Zhou, Qi-Da; Xu, Lin; Zhang, Zhi-Jun

2015-06-01

To investigate the effect of escitalopram (a widely used and highly efficacious antidepressant from the SSRI class) on tau hyperphosphorylation, HEK293/tau441 cells were pretreated with 4 μM of forskolin for 2 h. Then we treated the cells with different doses of escitalopram (0, 5, 10, 20, 40, 80 μM) for 22 h. We measured the phosphorylation level of tau by Western blotting. It was shown that escitalopram could protect tau from hyperphosphorylation induced by pharmacological activation of protein kinase A (PKA) at a dose of 20, 40, and 80 μM in vitro. Interestingly, the same dose of escitalopram could also increase the level of serine-9-phosphorylated GSK-3β (inactive form) and the phosphorylation level of Akt at Ser473 (active form) with no significant change in the level of total GSK-3β and Akt. Unexpectedly, 5-hydroxytryptamine 1A receptor (5-HT1A) agonist 8-OH-DPAT did not decrease forskolin-induced tau hyperphosphorylation. Our results suggest that escitalopram can ameliorate forskolin-induced tau hyperphosphorylation, which is not through the typical 5-HT1A pathway, and Akt/GSK-3β signaling pathway is involved. These findings may support an effective role of antidepressants in the prevention of dementia associated with depression in patients.

Identification of EloR (Spr1851) as a regulator of cell elongation in Streptococcus pneumoniae.

PubMed

Stamsås, Gro Anita; Straume, Daniel; Ruud Winther, Anja; Kjos, Morten; Frantzen, Cyril Alexander; Håvarstein, Leiv Sigve

2017-09-01

In a screen for mutations suppressing the lethal loss of PBP2b in Streptococcus pneumoniae we identified Spr1851 (named EloR), a cytoplasmic protein of unknown function whose inactivation removed the requirement for PBP2b as well as RodA. It follows from this that EloR and the two elongasome proteins must be part of the same functional network. This network also includes StkP, as this serine/threonine kinase phosphorylates EloR on threonine 89 (T89). We found that ΔeloR cells, and cells expressing the phosphoablative form of EloR (EloR T89A ), are significantly shorter than wild-type cells. Furthermore, the phosphomimetic form of EloR (EloR T89E ) is not tolerated unless the cell in addition acquires a truncated MreC or non-functional RodZ protein. By itself, truncation of MreC as well as inactivation of RodZ gives rise to less elongated cells, demonstrating that the stress exerted by the phosphomimetic form of EloR is relieved by suppressor mutations that reduce or abolish the activity of the elongasome. Of note, it was also found that loss of elongasome activity caused by truncation of MreC elicits increased StkP-mediated phosphorylation of EloR. Together, the results support a model in which phosphorylation of EloR stimulates cell elongation, while dephosphorylation has an inhibitory effect. © 2017 John Wiley & Sons Ltd.

Dysregulation of Ezrin phosphorylation prevents metastasis and alters cellular metabolism in osteosarcoma

PubMed Central

Ren, Ling; Hong, Sung-Hyeok; Chen, Qing-Rong; Briggs, Joseph; Cassavaugh, Jessica; Srinivasan, Satish; Lizardo, Michael M.; Mendoza, Arnulfo; Xia, Ashley Y.; Avadhani, Narayan; Khan, Javed; Khanna, Chand

2013-01-01

Ezrin links the plasma membrane to the actin cytoskeleton where it plays a pivotal role in the metastatic progression of several human cancers (1, 2), however, the precise mechanistic basis for its role remains unknown. Here we define transitions between active (phosphorylated open) and inactive (dephosphorylated closed) forms of Ezrin that occur during metastatic progression in osteosarcoma. In our evaluation of these conformations we expressed C-terminal mutant forms of Ezrin that are open (phosphomimetic T567D) or closed (phosphodeficient T567A) and compared their biological characteristics to full length wild-type Ezrin in osteosarcoma cells. Unexpectedly, cells expressing open, active Ezrin could form neither primary orthotopic tumors nor lung metastases. In contrast, cells expressing closed, inactive Ezrin were also deficient in metastasis but were unaffected in their capacity for primary tumor growth. By imaging single metastatic cells in the lung, we found that cells expressing either open or closed Ezrin displayed increased levels of apoptosis early after their arrival in the lung. Gene expression analysis suggested dysregulation of genes that are functionally linked to carbohydrate and amino acid metabolism. In particular, cells expressing closed, inactive Ezrin exhibited reduced lactate production and basal or ATP-dependent oxygen consumption. Collectively, our results suggest that dynamic regulation of Ezrin phosphorylation at amino acid T567 that controls structural transitions of this protein plays a pivotal role in tumor progression and metastasis, possibly in part by altering cellular metabolism. PMID:22147261

The plastid casein kinase 2 phosphorylates Rubisco activase at the Thr-78 site but is not essential for regulation of Rubisco activation state

USDA-ARS?s Scientific Manuscript database

Rubisco activase (RCA) is essential for the activation of Rubisco, the carboxylating enzyme of photosynthesis. In Arabidopsis, RCA is encoded by a single gene (At2g39730) that is alternatively spliced to form a large alpha-RCA and small beta-RCA isoform. The activity of Rubisco is controlled in res...

Mechanism of phosphoryl transfer and protein-protein interaction in the PTS system-an NMR study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rajagopal, P.; Klevit, R.E.

1994-12-01

HPr and Enzyme IIA{sup Glc} are two of the components of the bacterial PTS (phosphoenolpyruvate: sugar phosphotranferase system) and are involved in the phosphorylation and concomitant translocation of sugars across the membrane. These PTS protein complexes also regulate sugar transport. HPr, phosphorylated at a histidine N1 site by Enzyme I and phosphoenol pyruvate, transfers the phosphoryl group to a histidine N3 position in Enzyme IIA{sup Glc}. HPrs from Gram-positive bacteria undergo regulatory phosphorylation at Ser{sup 46}, whereby phosphorylation of the histidine residue is inhibited. Conversely, histidine phosphorylation inhibits phosphorylation at Ser{sup 46}. HPrs from Gram-negative bacteria possess a serine residuemore » at position 46, but do not undergo regulatory phosphorylation. HPr forms an open-faced sandwich structure with a four-strand S-sheet and 2 to 3 helices lying on top of the sheet. The active-site histidine and Ser{sup 46} occur in conformationally flexible regions. P-His-HPr from the Gram-positive bacterium Bacillus subtilus has been investigated by both homonuclear and heteronuclear two-dimensional and three-dimensional NMR experiments using an in-situ enzymatic regeneration system to maintain a constant level of P-His-HPr. The results show that localized conformational changes occur in the vicinity of the active-site histidine and also near Ser{sup 46}. HPr-Enzyme IIA{sup Glc} complexes from both Bacillus subtilis and Gram-negative Escherichia coli were also studied by a variety of {sup 15}N-edited two-dimensional NMR experiments, which were performed on uniformly {sup 15}N-labeled HPr complexed to unlabeled Enzyme IIA{sup Glc}. The complex is in fast exchange with a molecular weight of about 27 kDa. The focus of our work is to assess the changes undergone by HPr (the smaller of the two components), and so all the experiments were performed with excess Enzyme IIA present in the system.« less

Polo-like kinase 1-mediated phosphorylation stabilizes Pin1 by inhibiting its ubiquitination in human cells.

PubMed

Eckerdt, Frank; Yuan, Juping; Saxena, Krishna; Martin, Bernd; Kappel, Sven; Lindenau, Christine; Kramer, Andrea; Naumann, Steffen; Daum, Sebastian; Fischer, Gunter; Dikic, Ivan; Kaufmann, Manfred; Strebhardt, Klaus

2005-11-04

The Polo-like kinase 1 (Plk1) is a key regulator of mitosis. It is reported that the human peptidyl-prolyl cis/trans-isomerase Pin1 binds to Plk1 from mitotic cell extracts in vitro. Here we demonstrate that Ser-65 in Pin1 is the major site for Plk1-specific phosphorylation, and the polo-box domain of Plk1 is required for this phosphorylation. Interestingly, the phosphorylation of Pin1 by Plk1 does not affect its isomerase activity but rather is linked to its protein stability. Pin1 is ubiquitinated in HeLa S3 cells, and substitution of Glu for Ser-65 reduces the ubiquitination of Pin1. Furthermore, inhibition of Plk1 activity by expression of a dominant negative form of Plk1 or by transfection of small interfering RNA targeted to Plk1 enhances the ubiquitination of Pin1 and subsequently reduces the amount of Pin1 in human cancer cells. Since previous reports suggested that Plk1 is a substrate of Pin1, our work adds a new dimension to this interaction of two important mitotic regulators.

ERK3 signals through SRC-3 coactivator to promote human lung cancer cell invasion

PubMed Central

Long, Weiwen; Foulds, Charles E.; Qin, Jun; Liu, Jian; Ding, Chen; Lonard, David M.; Solis, Luisa M.; Wistuba, Ignacio I.; Qin, Jun; Tsai, Sophia Y.; Tsai, Ming-Jer; O’Malley, Bert W.

2012-01-01

In contrast to the well-studied classic MAPKs, such as ERK1/2, little is known concerning the regulation and substrates of the atypical MAPK ERK3 signaling cascade and its function in cancer progression. Here, we report that ERK3 interacted with and phosphorylated steroid receptor coactivator 3 (SRC-3), an oncogenic protein overexpressed in multiple human cancers at serine 857 (S857). This ERK3-mediated phosphorylation at S857 was essential for interaction of SRC-3 with the ETS transcription factor PEA3, which promotes upregulation of MMP gene expression and proinvasive activity in lung cancer cells. Importantly, knockdown of ERK3 or SRC-3 inhibited the ability of lung cancer cells to invade and form tumors in the lung in a xenograft mouse model. In addition, ERK3 was found to be highly upregulated in human lung carcinomas. Our study identifies a previously unknown role for ERK3 in promoting lung cancer cell invasiveness by phosphorylating SRC-3 and regulating SRC-3 proinvasive activity by site-specific phosphorylation. As such, ERK3 protein kinase may be an attractive target for therapeutic treatment of invasive lung cancer. PMID:22505454

Differential phosphorylation of Smad1 integrates BMP and neurotrophin pathways through Erk/Dusp in axon development.

PubMed

Finelli, Mattéa J; Murphy, Kevin J; Chen, Lei; Zou, Hongyan

2013-05-30

Sensory axon development requires concerted actions of growth factors for the precise control of axonal outgrowth and target innervation. How developing sensory neurons integrate different cues is poorly understood. We demonstrate here that Smad1 activation is required for neurotrophin-mediated sensory axon growth in vitro and in vivo. Through differential phosphorylation, Smad1 exerts transcriptional selectivity to regulate the expression and activity of Erk1 and Erk2-two key neurotrophin effectors. Specifically, bone morphogenetic proteins (BMPs) signal through carboxy-terminal phosphorylation of Smad1 (pSmad1C) to induce Erk1/2 transcription for enhanced neurotrophin responsiveness. Meanwhile, neurotrophin signaling results in linker phosphorylation of Smad1 (pSmad1L), which in turn upregulates an Erk-specific dual-specificity phosphatase, Dusp6, leading to reduced pErk1/2 and constituting a negative-feedback loop for the prevention of axon overgrowth. Together, the BMP and neurotrophin pathways form a tightly regulated signaling network with a balanced ratio of Erk1/2 and pErk1/2 to direct the precise connections between sensory neurons and peripheral targets. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Dimer monomer transition and dimer re-formation play important role for ATM cellular function during DNA repair

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Fengxia; Zhang, Minjie; University of Chinese Academy of Sciences, Beijing 100049

2014-10-03

Highlights: • ATM phosphorylates the opposite strand of the dimer in response to DNA damage. • The PETPVFRLT box of ATM plays a key role in its dimer dissociation in DNA repair. • The dephosphorylation of ATM is critical for dimer re-formation after DNA repair. - Abstract: The ATM protein kinase, is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks, mediates responses to ionizing radiation in mammalian cells. Here we show that ATM is held inactive in unirradiated cells as a dimer and phosphorylates the opposite strand of the dimer in response to DNA damage.more » Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. ATM cannot phosphorylate the substrates when it could not undergo dimer monomer transition. After DNA repair, the active monomer will undergo dephosphorylation to form dimer again and dephosphorylation is critical for dimer re-formation. Our work reveals novel function of ATM dimer monomer transition and explains why ATM dimer monomer transition plays such important role for ATM cellular activity during DNA repair.« less

Coincident regulation of PKCδ in human platelets by phosphorylation of Tyr311 and Tyr565 and phospholipase C signalling

PubMed Central

Hall, Kellie J.; Jones, Matthew L.; Poole, Alastair W.

2007-01-01

PKC (protein kinase C)δ plays a complex role in platelets, having effects on both positive and negative signalling functions. It is phosphorylated on tyrosine residues in response to thrombin and collagen, and it has recently been shown that Tyr311 is phosphorylated in response to PAR (protease-activated receptor) 1 and PAR4 receptor activation. In the present study, we show that Tyr311 and Tyr565 are phosphorylated in response to thrombin, and have examined the interplay between phosphorylation and the classical lipid-mediated activation of PKCδ. Phosphorylation of both Tyr311 and Tyr565 is dependent on Src kinase and PLC (phospholipase C) activity in response to thrombin. Importantly, direct allosteric activation of PKCδ with PMA also induced phosphorylation of Tyr311 and Tyr565, and this was dependent on the activity of Src kinases, but not PLC. Membrane recruitment of PKCδ is essential for phosphorylation of this tyrosine residue, but tyrosine phosphorylation is not required for membrane recruitment of PKCδ. Both thrombin and PMA induce recruitment of PKCδ to the membrane, and for thrombin, this recruitment is a PLC-dependent process. In order to address the functional role of tyrosine residue phosphorylation of PKCδ, we demonstrate that phosphorylation can potentiate the activity of the kinase, although phosphorylation does not play a role in membrane recruitment of the kinase. PKCδ is therefore regulated in a coincident fashion, PLC-dependent signals recruiting it to the plasma membrane and by phosphorylation on tyrosine residues, potentiating its activity. PMID:17570831

Role of calcium activated kinases and phosphatases in heat shock factor-1 activation.

PubMed

Soncin, F; Asea, A; Zhang, X; Stevenson, M A; Calderwood, S K

2000-12-01

HSF-1 is regulated at multiple molecular levels through intra- and intermolecular protein-protein interactions as well as by post-translational modification through phosphorylation. We have found that elevating intracellular calcium ion levels by exposure to the ionophore A23187 or thapsigargin inhibits the conversion of HSF-1 from a latent cytoplasmic form to its nuclear/DNA binding form. To examine a role for calcium/calmodulin regulated enzymes in this process, we examined the ability of specific inhibitors to abrogate the effects of calcium elevation. While the inhibitor of calmodulin dependent kinase II, KCN62 enhanced activation of HSF-1 during heat shock, it failed to block the inhibitory effects of calcium increase. By contrast, the immunosuppresant drugs cyclosporin A and FK506 abolished the effects of calcium elevation on HSF-1 activation. As the biological effects of the drugs are effected through inhibition of the calcium/calmodulin regulated phosphatase calcineurin, this suggests a role for calcineurin in antagonizing HSF-1 activity. The experiments suggest the existence of phosphorylated residue(s) in HSF-1 important in one or more of the processes that lead to activation (trimerization, nuclear localization, DNA binding) and which becomes dephosphorylated due to the activation of a calcium/calmodulin/calcineurin complex.

Interdependency and phosphorylation of KIF4 and condensin I are essential for organization of chromosome scaffold.

PubMed

Poonperm, Rawin; Takata, Hideaki; Uchiyama, Susumu; Fukui, Kiichi

2017-01-01

Kinesin family member 4 (KIF4) and condensins I and II are essential chromosomal proteins for chromosome organization by locating primarily to the chromosome scaffold. However, the mechanism of how KIF4 and condensins localize to the chromosome scaffold is poorly understood. Here, we demonstrate a close relationship between the chromosome localization of KIF4 and condensin I, but not condensin II, and show that KIF4 and condensin I assist each other for stable scaffold formation by forming a stable complex. Moreover, phosphorylation of KIF4 and condensin I by Aurora B and polo-like kinase 1 (Plk1) is important for KIF4 and condensin I localization to the chromosome. Aurora B activity facilitates the targeting of KIF4 and condensin I to the chromosome, whereas Plk1 activity promotes the dissociation of these proteins from the chromosome. Thus, the interdependency between KIF4 and condensin I, and their phosphorylation states play important roles in chromosome scaffold organization during mitosis.

Interdependency and phosphorylation of KIF4 and condensin I are essential for organization of chromosome scaffold

PubMed Central

Uchiyama, Susumu; Fukui, Kiichi

2017-01-01

Kinesin family member 4 (KIF4) and condensins I and II are essential chromosomal proteins for chromosome organization by locating primarily to the chromosome scaffold. However, the mechanism of how KIF4 and condensins localize to the chromosome scaffold is poorly understood. Here, we demonstrate a close relationship between the chromosome localization of KIF4 and condensin I, but not condensin II, and show that KIF4 and condensin I assist each other for stable scaffold formation by forming a stable complex. Moreover, phosphorylation of KIF4 and condensin I by Aurora B and polo-like kinase 1 (Plk1) is important for KIF4 and condensin I localization to the chromosome. Aurora B activity facilitates the targeting of KIF4 and condensin I to the chromosome, whereas Plk1 activity promotes the dissociation of these proteins from the chromosome. Thus, the interdependency between KIF4 and condensin I, and their phosphorylation states play important roles in chromosome scaffold organization during mitosis. PMID:28817632

Halofuginone inhibits Smad3 phosphorylation via the PI3K/Akt and MAPK/ERK pathways in muscle cells: Effect on myotube fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roffe, Suzy; Hagai, Yosey; Institute of Animal Sciences, Volcani Center, Bet Dagan 50250

2010-04-01

Halofuginone, a novel inhibitor of Smad3 phosphorylation, has been shown to inhibit muscle fibrosis and to improve cardiac and skeletal muscle functions in the mdx mouse model of Duchenne muscular dystrophy. Here, we demonstrate that halofuginone promotes the phosphorylation of Akt and mitogen-activated protein kinase (MAPK) family members in a C2 muscle cell line and in primary myoblasts derived from wild-type and mdx mice diaphragms. Halofuginone enhanced the association of phosphorylated Akt and MAPK/extracellular signal-regulated protein kinase (ERK) with the non-phosphorylated form of Smad3, accompanied by a reduction in Smad3 phosphorylation levels. This reduction was reversed by inhibitors of themore » phosphoinositide 3'-kinase/Akt (PI3K/Akt) and MAPK/ERK pathways, suggesting their specific role in mediating halofuginone's inhibitory effect on Smad3 phosphorylation. Halofuginone enhanced Akt, MAPK/ERK and p38 MAPK phosphorylation and inhibited Smad3 phosphorylation in myotubes, all of which are crucial for myotube fusion. In addition, halofuginone increased the association Akt and MAPK/ERK with Smad3. As a consequence, halofuginone promoted myotube fusion, as reflected by an increased percentage of C2 and mdx myotubes containing high numbers of nuclei, and this was reversed by specific inhibitors of the PI3K and MAPK/ERK pathways. Together, the data suggest a role, either direct or via inhibition of Smad3 phosphorylation, for Akt or MAPK/ERK in halofuginone-enhanced myotube fusion, a feature which is crucial to improving muscle function in muscular dystrophies.« less

Oncogenic Receptor Tyrosine Kinases Directly Phosphorylate Focal Adhesion Kinase (FAK) as a Resistance Mechanism to FAK-kinase Inhibitors

PubMed Central

Marlowe, Timothy A.; Lenzo, Felicia L.; Figel, Sheila A.; Grapes, Abigail T.; Cance, William G.

2016-01-01

Focal adhesion kinase (FAK) is a major drug target in cancer and current inhibitors targeted to the ATP-binding pocket of the kinase domain have entered clinical trials. However, preliminary results have shown limited single-agent efficacy in patients. Despite these unfavorable data, the molecular mechanisms which drive intrinsic and acquired resistance to FAK-kinase inhibitors are largely unknown. We have demonstrated that receptor tyrosine kinases (RTKs) can directly bypass FAK-kinase inhibition in cancer cells through phosphorylation of FAK’s critical tyrosine 397 (Y397). We also showed that HER2 forms a direct protein-protein interaction with the FAK-FERM-F1 lobe, promoting direct phosphorylation of Y397. Additionally, FAK-kinase inhibition induced two forms of compensatory RTK reprogramming: 1) the rapid phosphorylation and activation of RTK signaling pathways in RTKHigh cells and 2) the long-term acquisition of RTKs novel to the parental cell line in RTKLow cells. Finally, HER2+ cancer cells displayed resistance to FAK-kinase inhibition in 3D–growth assays using a HER2 isogenic system and HER2+ cancer cell lines. Our data indicate a novel drug resistance mechanism to FAK-kinase inhibitors whereby HER2 and other RTKs can rescue and maintain FAK activation (pY397) even in the presence of FAK-kinase inhibition. These data may have important ramifications for existing clinical trials of FAK inhibitors and suggest that individual tumor stratification by RTK expression would be important to predict patient response to FAK-kinase inhibitors. PMID:27638858

FGFR3 Heterodimerization in Achondroplasia, the Most Common Form of Human Dwarfism*

PubMed Central

He, Lijuan; Shobnam, Nadia; Wimley, William C.; Hristova, Kalina

2011-01-01

The G380R mutation in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) causes achondroplasia, the most common form of human dwarfism. Achondroplasia is a heterozygous disorder, and thus the affected individuals express both wild-type and mutant FGFR3. Yet heterodimerization in achondroplasia has not been characterized thus far. To investigate the formation of FGFR3 heterodimers in cellular membranes, we designed an FGFR3 construct that lacks the kinase domain, and we monitored the formation of inactive heterodimers between this construct and wild-type and mutant FGFR3. The formation of the inactive heterodimers depleted the pool of full-length receptors capable of forming active homodimers and ultimately reduced their phosphorylation. By analyzing the effect of the truncated FGFR3 on full-length receptor phosphorylation, we demonstrated that FGFR3 WT/G380R heterodimers form with lower probability than wild-type FGFR3 homodimers at low ligand concentration. These results further our knowledge of FGFR3-associated bone disorders. PMID:21324899

Folic acid supplementation during high-fat diet feeding restores AMPK activation via an AMP-LKB1-dependent mechanism

PubMed Central

Sid, Victoria; Wu, Nan; Sarna, Lindsei K.; Siow, Yaw L.; House, James D.

2015-01-01

AMPK is an endogenous energy sensor that regulates lipid and carbohydrate metabolism. Nonalcoholic fatty liver disease (NAFLD) is regarded as a hepatic manifestation of metabolic syndrome with impaired lipid and glucose metabolism and increased oxidative stress. Our recent study showed that folic acid supplementation attenuated hepatic oxidative stress and lipid accumulation in high-fat diet-fed mice. The aim of the present study was to investigate the effect of folic acid on hepatic AMPK during high-fat diet feeding and the mechanisms involved. Male C57BL/6J mice were fed a control diet (10% kcal fat), a high-fat diet (60% kcal fat), or a high-fat diet supplemented with folic acid (26 mg/kg diet) for 5 wk. Mice fed a high-fat diet exhibited hyperglycemia, hepatic cholesterol accumulation, and reduced hepatic AMPK phosphorylation. Folic acid supplementation restored AMPK phosphorylation (activation) and reduced blood glucose and hepatic cholesterol levels. Activation of AMPK by folic acid was mediated through an elevation of its allosteric activator AMP and activation of its upstream kinase, namely, liver kinase B1 (LKB1) in the liver. Consistent with in vivo findings, 5-methyltetrahydrofolate (bioactive form of folate) restored phosphorylation (activation) of both AMPK and LKB1 in palmitic acid-treated HepG2 cells. Activation of AMPK by folic acid might be responsible for AMPK-dependent phosphorylation of HMG-CoA reductase, leading to reduced hepatic cholesterol synthesis during high-fat diet feeding. These results suggest that folic acid supplementation may improve cholesterol and glucose metabolism by restoration of AMPK activation in the liver. PMID:26400185

Propofol restores TRPV1 sensitivity via a TRPA1-, nitric oxide synthase-dependent activation of PKCε

PubMed Central

Sinharoy, Pritam; Zhang, Hongyu; Sinha, Sayantani; Prudner, Bethany C; Bratz, Ian N; Damron, Derek S

2015-01-01

We previously demonstrated that the intravenous anesthetic, propofol, restores the sensitivity of transient receptor potential vanilloid channel subtype-1 (TRPV1) receptors via a protein kinase C epsilon (PKCε)-dependent and transient receptor potential ankyrin channel subtype-1 (TRPA1)-dependent pathway in sensory neurons. The extent to which the two pathways are directly linked or operating in parallel has not been determined. Using a molecular approach, our objectives of the current study were to confirm that TRPA1 activation directly results in PKCε activation and to elucidate the cellular mechanism by which this occurs. F-11 cells were transfected with complimentary DNA (cDNA) for TRPV1 only or both TRPV1 and TRPA1. Intracellular Ca2+ concentration was measured in individual cells via fluorescence microscopy. An immunoblot analysis of the total and phosphorylated forms of PKCε, nitric oxide synthase (nNOS), and TRPV1 was also performed. In F-11 cells containing both channels, PKCε inhibition prevented the propofol- and allyl isothiocyanate (AITC)-induced restoration of TRPV1 sensitivity to agonist stimulation as well as increased phosphorylation of PKCε and TRPV1. In cells containing TRPV1 only, neither agonist induced PKCε or TRPV1 phosphorylation. Moreover, NOS inhibition blocked propofol-and AITC-induced restoration of TRPV1 sensitivity and PKCε phosphorylation, and PKCε inhibition prevented the nitric oxide donor, SNAP, from restoring TRPV1 sensitivity. Also, propofol-and AITC-induced phosphorylation of nNOS and nitric oxide (NO) production were blocked with the TRPA1-antagonist, HC-030031. These data indicate that the AITC- and propofol-induced restoration of TRPV1 sensitivity is mediated by a TRPA1-dependent, nitric oxide synthase-dependent activation of PKCε. PMID:26171233

Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

PubMed Central

Grabinski, Tessa; Kanaan, Nicholas M.

2016-01-01

Glycogen synthase kinase 3 (GSK3) β and α are serine/threonine kinases involved in many biological processes. A primary mechanism of GSK3 activity regulation is phosphorylation of N-terminal serine (S) residues (S9 in GSK3β, S21 in GSK3α). Phosphorylation is inhibitory to GSK3 kinase activity because the phosphorylated N-terminus acts as a competitive inhibitor for primed substrates. Despite widespread interest in GSK3 across most fields of biology, the research community does not have reagents that specifically react with nonphosphoS9/21 GSK3β/α (the so-called “active” form). Here, we describe two novel monoclonal antibodies that specifically react with nonphosphoS9/21 GSK3β/α in multiple species (human, mouse, and rat). One of the antibodies is specific for nonphospho-S9 GSK3β (clone 12B2) and one for nonphospho-S9/21 GSK3β/α (clone 15C2). These reagents were validated for specificity and reactivity in several biochemical and immunochemical assays, and they show linear detection of nonphosphoS GSK3. Finally, these reagents provide significant advantages in studying GSK3β regulation. We used both antibodies to study the regulation of S9 phosphorylation by Akt and protein phosphatases. We used 12B2 (due to its specificity for GSK3β) and to demonstrate that protein phosphatase inhibition reduces nonphospho-S9 GSK3β levels and lowers kinase activity within cells. The ability to use the same reagent across biochemical, immunohistological and kinase activity assays provides a powerful approach for studying serine-dependent regulation of GSK3β/α. PMID:27909397

Phosphorylation by CK2 regulates MUS81/EME1 in mitosis and after replication stress.

PubMed

Palma, Anita; Pugliese, Giusj Monia; Murfuni, Ivana; Marabitti, Veronica; Malacaria, Eva; Rinalducci, Sara; Minoprio, Anna; Sanchez, Massimo; Mazzei, Filomena; Zolla, Lello; Franchitto, Annapaola; Pichierri, Pietro

2018-06-01

The MUS81 complex is crucial for preserving genome stability through the resolution of branched DNA intermediates in mitosis. However, untimely activation of the MUS81 complex in S-phase is dangerous. Little is known about the regulation of the human MUS81 complex and how deregulated activation affects chromosome integrity. Here, we show that the CK2 kinase phosphorylates MUS81 at Serine 87 in late-G2/mitosis, and upon mild replication stress. Phosphorylated MUS81 interacts with SLX4, and this association promotes the function of the MUS81 complex. In line with a role in mitosis, phosphorylation at Serine 87 is suppressed in S-phase and is mainly detected in the MUS81 molecules associated with EME1. Loss of CK2-dependent MUS81 phosphorylation contributes modestly to chromosome integrity, however, expression of the phosphomimic form induces DSBs accumulation in S-phase, because of unscheduled targeting of HJ-like DNA intermediates, and generates a wide chromosome instability phenotype. Collectively, our findings describe a novel regulatory mechanism controlling the MUS81 complex function in human cells. Furthermore, they indicate that, genome stability depends mainly on the ability of cells to counteract targeting of branched intermediates by the MUS81/EME1 complex in S-phase, rather than on a correct MUS81 function in mitosis.

Phosphorylation of Nephrin Triggers Ca2+ Signaling by Recruitment and Activation of Phospholipase C-{gamma}1.

PubMed

Harita, Yutaka; Kurihara, Hidetake; Kosako, Hidetaka; Tezuka, Tohru; Sekine, Takashi; Igarashi, Takashi; Ohsawa, Ikuroh; Ohta, Shigeo; Hattori, Seisuke

2009-03-27

A specialized intercellular junction between podocytes, known as the slit diaphragm (SD), forms the essential structural frame-work for glomerular filtration in the kidney. In addition, mounting evidence demonstrates that the SD also plays a crucial role as a signaling platform in physiological and pathological states. Nephrin, the major component of the SD, is tyrosine-phosphorylated by a Src family tyrosine kinase, Fyn, in developing or injured podocytes, recruiting Nck to Nephrin via its Src homology 2 domain to regulate dynamic actin remodeling. Dysregulated Ca(2+) homeostasis has also been implicated in podocyte damage, but the mechanism of how podocytes respond to injury is largely unknown. Here we have identified phospholipase C-gamma1 (PLC-gamma1) as a novel phospho-Nephrin-binding protein. When HEK293T cells expressing a chimeric protein consisting of CD8 and Nephrin cytoplasmic domain (CD) were treated with anti-CD8 and anti-mouse antibodies, clustering of Nephrin and phosphorylation of Nephrin-CD were induced. Upon this clustering, PLC-gamma1 was bound to phosphorylated Nephrin Tyr-1204, which induced translocation of PLC-gamma1 from cytoplasm to the CD8/Nephrin cluster on the plasma membrane. The recruitment of PLC-gamma1 to Nephrin activated PLC-gamma1, as detected by phosphorylation of PLC-gamma1 Tyr-783 and increase in inositol 1,4,5-trisphosphate level. We also found that Nephrin Tyr-1204 phosphorylation triggers the Ca(2+) response in a PLC-gamma1-dependent fashion. Furthermore, PLC-gamma1 is significantly phosphorylated in injured podocytes in vivo. Given the profound effect of PLC-gamma in diverse cellular functions, regulation of the Ca(2+) signaling by Nephrin may be important in modulating the glomerular filtration barrier function.

TALE factors poise promoters for activation by Hox proteins.

PubMed

Choe, Seong-Kyu; Ladam, Franck; Sagerström, Charles G

2014-01-27

Hox proteins form complexes with TALE cofactors from the Pbx and Prep/Meis families to control transcription, but it remains unclear how Hox:TALE complexes function. Examining a Hoxb1b:TALE complex that regulates zebrafish hoxb1a transcription, we find maternally deposited TALE proteins at the hoxb1a promoter already during blastula stages. These TALE factors recruit histone-modifying enzymes to promote an active chromatin profile at the hoxb1a promoter and also recruit RNA polymerase II (RNAPII) and P-TEFb. However, in the presence of TALE factors, RNAPII remains phosphorylated on serine 5 and hoxb1a transcription is inefficient. By gastrula stages, Hoxb1b binds together with TALE factors to the hoxb1a promoter. This triggers P-TEFb-mediated transitioning of RNAPII to the serine 2-phosphorylated form and efficient hoxb1a transcription. We conclude that TALE factors access promoters during early embryogenesis to poise them for activation but that Hox proteins are required to trigger efficient transcription. Copyright © 2014 Elsevier Inc. All rights reserved.

Activation of Akt, not connexin 43 protein ubiquitination, regulates gap junction stability.

PubMed

Dunn, Clarence A; Su, Vivian; Lau, Alan F; Lampe, Paul D

2012-01-20

The pore-forming gap junctional protein connexin 43 (Cx43) has a short (1-3 h) half-life in cells in tissue culture and in whole tissues. Although critical for cellular function in all tissues, the process of gap junction turnover is not well understood because treatment of cells with a proteasomal inhibitor results in larger gap junctions but little change in total Cx43 protein whereas lysosomal inhibitors increase total, mostly nonjunctional Cx43. To better understand turnover and identify potential sites of Cx43 ubiquitination, we prepared constructs of Cx43 with different lysines converted to arginines. However, when transfected into cells, a mutant version of Cx43 with all lysines converted to arginines behaved similarly to wild type in the presence of proteasomal and lysosomal inhibitors, indicating that ubiquitination of Cx43 did not appear to be playing a role in gap junction stability. Through the use of inhibitors and dominant negative constructs, we found that Akt (protein kinase B) activity controlled gap junction stability and was necessary to form larger stable gap junctions. Akt activation was increased upon proteasomal inhibition and resulted in phosphorylation of Cx43 at Akt phosphorylation consensus sites. Thus, we conclude that Cx43 ubiquitination is not necessary for the regulation of Cx43 turnover; rather, Akt activity, probably through direct phosphorylation of Cx43, controls gap junction stability. This linkage of a kinase involved in controlling cell survival and growth to gap junction stability may mechanistically explain how gap junctions and Akt play similar regulatory roles.

SnRK1 is differentially regulated in the cotyledon and embryo axe of bean (Phaseolus vulgaris L) seeds.

PubMed

Coello, Patricia; Martínez-Barajas, Eleazar

2014-07-01

SnRK1 activity is developmentally regulated in bean seeds and exhibits a transient increase with the highest value at 20 days after anthesis (DAA), which coincides with the beginning of protein and starch accumulation. The catalytic subunit of SnRK1 shows a consistent decrease throughout the seed development period. However, by 15 DAA a significant proportion of the catalytic subunit appears phosphorylated. The increase in activity and phosphorylation of the catalytic subunit coincides with a decrease in hexoses. However, SnRK1 activity is differentially regulated in the cotyledon and embryo axe, where a larger proportion of the catalytic subunit is phosphorylated. SnRK1 obtained from endosperm extract is inhibited by T6P and to a lesser extent by ADPG and UDPG, whereas the enzyme isolated from embryo is virtually insensitive to T6P but exhibits some inhibition by ADPG and UDPG. In cotyledon extracts, the effects of T6P and ADPG on SnRK1 activity are additive, whereas in embryo extract, T6P inhibits the enzyme only when ADPG is present. After fractionation on Sephacryl-S300, SnRK1 activity obtained from cotyledon extracts is detected as a single peak associated with a molecular weight of 250 kDa whereas that obtained form embryo axe extracts detected as 2 peaks associated with molecular weight of 250 and 180 kDa. In both cases, the catalytic subunit exhibits a wide distribution but is concentrated in the fractions with the highest activity. To analyse the composition of the complex, cotyledon and embryo extracts were treated with a reversible crosslinker (DSP). DSP induced the formation of complexes with molecular weights of 97 and 180 kDa in the cotyledon and embryo extracts, respectively. Since all the phosphorylated catalytic subunit is present in the complexes induced by DSP, it appears that the phosphorylation favors its interaction with other proteins. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Rewiring MAP kinases in Saccharomyces cerevisiae to regulate novel targets through ubiquitination.

PubMed

Groves, Benjamin; Khakhar, Arjun; Nadel, Cory M; Gardner, Richard G; Seelig, Georg

2016-08-15

Evolution has often copied and repurposed the mitogen-activated protein kinase (MAPK) signaling module. Understanding how connections form during evolution, in disease and across individuals requires knowledge of the basic tenets that govern kinase-substrate interactions. We identify criteria sufficient for establishing regulatory links between a MAPK and a non-native substrate. The yeast MAPK Fus3 and human MAPK ERK2 can be functionally redirected if only two conditions are met: the kinase and substrate contain matching interaction domains and the substrate includes a phospho-motif that can be phosphorylated by the kinase and recruit a downstream effector. We used a panel of interaction domains and phosphorylation-activated degradation motifs to demonstrate modular and scalable retargeting. We applied our approach to reshape the signaling behavior of an existing kinase pathway. Together, our results demonstrate that a MAPK can be largely defined by its interaction domains and compatible phospho-motifs and provide insight into how MAPK-substrate connections form.

HPr antagonizes the anti-σ70 activity of Rsd in Escherichia coli.

PubMed

Park, Young-Ha; Lee, Chang-Ro; Choe, Mangyu; Seok, Yeong-Jae

2013-12-24

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a multicomponent system that participates in a variety of physiological processes in addition to the phosphorylation-coupled transport of numerous sugars. In Escherichia coli and other enteric bacteria, enzyme IIA(Glc) (EIIA(Glc)) is known as the central processing unit of carbon metabolism and plays multiple roles, including regulation of adenylyl cyclase, the fermentation/respiration switch protein FrsA, glycerol kinase, and several non-PTS transporters, whereas the only known regulatory role of the E. coli histidine-containing phosphocarrier protein HPr is in the activation of glycogen phosphorylase. Because HPr is known to be more abundant than EIIA(Glc) in enteric bacteria, we assumed that there might be more regulatory mechanisms connected with HPr. The ligand fishing experiment in this study identified Rsd, an anti-sigma factor known to complex with σ(70) in stationary-phase cells, as an HPr-binding protein in E. coli. Only the dephosphorylated form of HPr formed a tight complex with Rsd and thereby inhibited complex formation between Rsd and σ(70). Dephosphorylated HPr, but not phosphorylated HPr, antagonized the inhibitory effect of Rsd on σ(70)-dependent transcriptions both in vivo and in vitro, and also influenced the competition between σ(70) and σ(S) for core RNA polymerase in the presence of Rsd. Based on these data, we propose that the anti-σ(70) activity of Rsd is regulated by the phosphorylation state-dependent interaction of HPr with Rsd.

HPr antagonizes the anti-σ70 activity of Rsd in Escherichia coli

PubMed Central

Park, Young-Ha; Lee, Chang-Ro; Choe, Mangyu; Seok, Yeong-Jae

2013-01-01

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a multicomponent system that participates in a variety of physiological processes in addition to the phosphorylation-coupled transport of numerous sugars. In Escherichia coli and other enteric bacteria, enzyme IIAGlc (EIIAGlc) is known as the central processing unit of carbon metabolism and plays multiple roles, including regulation of adenylyl cyclase, the fermentation/respiration switch protein FrsA, glycerol kinase, and several non-PTS transporters, whereas the only known regulatory role of the E. coli histidine-containing phosphocarrier protein HPr is in the activation of glycogen phosphorylase. Because HPr is known to be more abundant than EIIAGlc in enteric bacteria, we assumed that there might be more regulatory mechanisms connected with HPr. The ligand fishing experiment in this study identified Rsd, an anti-sigma factor known to complex with σ70 in stationary-phase cells, as an HPr-binding protein in E. coli. Only the dephosphorylated form of HPr formed a tight complex with Rsd and thereby inhibited complex formation between Rsd and σ70. Dephosphorylated HPr, but not phosphorylated HPr, antagonized the inhibitory effect of Rsd on σ70-dependent transcriptions both in vivo and in vitro, and also influenced the competition between σ70 and σS for core RNA polymerase in the presence of Rsd. Based on these data, we propose that the anti-σ70 activity of Rsd is regulated by the phosphorylation state-dependent interaction of HPr with Rsd. PMID:24324139

A Mass Spectrometry-Based Predictive Strategy Reveals ADAP1 is Phosphorylated at Tyrosine 364

DOE Office of Scientific and Technical Information (OSTI.GOV)

Littrell, BobbiJo R

The goal of this work was to identify phosphorylation sites within the amino acid sequence of human ADAP1. Using traditional mass spectrometry-based techniques we were unable to produce interpretable spectra demonstrating modification by phosphorylation. This prompted us to employ a strategy in which phosphorylated peptides were first predicted using peptide mapping followed by targeted MS/MS acquisition. ADAP1 was immunoprecipitated from extracts of HEK293 cells stably-transfected with ADAP1 cDNA. Immunoprecipitated ADAP1 was digested with proteolytic enzymes and analyzed by LC-MS in MS1 mode by high-resolution quadrupole time-of-flight mass spectrometry (QTOF-MS). Peptide molecular features were extracted using an untargeted data mining algorithm.more » Extracted peptide neutral masses were matched against the ADAP1 amino acid sequence with phosphorylation included as a predicted modification. Peptides with predicted phosphorylation sites were analyzed by targeted LC-MS2. Acquired MS2 spectra were then analyzed using database search engines to confirm phosphorylation. Spectra of phosphorylated peptides were validated by manual interpretation. Further confirmation was performed by manipulating phospho-peptide abundance using calf intestinal phosphatase (CIP) and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Of five predicted phosphopeptides, one, comprised of the sequence AVDRPMLPQEYAVEAHFK, was confirmed to be phosphorylated on a Tyrosine at position 364. Pre-treatment of cells with PMA prior to immunoprecipitation increased the ratio of phosphorylated to unphosphorylated peptide as determined by area counts of extracted ion chromatograms (EIC). Addition of CIP to immunoprecipitation reactions eliminated the phosphorylated form. A novel phosphorylation site was identified at Tyrosine 364. Phosphorylation at this site is increased by treatment with PMA. PMA promotes membrane translocation and activation of protein kinase C (PKC), indicating that Tyrosine 364 is phosphorylated by a PKC-dependent mechanism.« less

microRNA-21-induced dissociation of PDCD4 from rictor contributes to Akt-IKKβ-mTORC1 axis to regulate renal cancer cell invasion.

PubMed

Bera, Amit; Das, Falguni; Ghosh-Choudhury, Nandini; Kasinath, Balakuntalam S; Abboud, Hanna E; Choudhury, Goutam Ghosh

2014-10-15

Renal cancer metastasis may result from oncogenic forces that contribute to the primary tumor. We have recently identified microRNA-21 as an oncogenic driver of renal cancer cells. The mechanism by which miR-21 controls renal cancer cell invasion is poorly understood. We show that miR-21 directly downregulates the proapoptotic protein PDCD4 to increase migration and invasion of ACHN and 786-O renal cancer cells as a result of phosphorylation/activation of Akt and IKKβ, which activate NFκB-dependent transcription. Constitutively active (CA) Akt or CA IKKβ blocks PDCD4-mediated inhibition and restores renal cancer cell migration and invasion. PDCD4 inhibits mTORC1 activity, which was reversed by CA IKKβ. Moreover, CA mTORC1 restores cell migration and invasion inhibited by PDCD4 and dominant negative IKKβ. Moreover, PDCD4 negatively regulates mTORC2-dependent Akt phosphorylation upstream of this cascade. We show that PDCD4 forms a complex with rictor, an exclusive component of mTORC2, and that this complex formation is reduced in renal cancer cells due to increased miR-21 expression resulting in enhanced phosphorylation of Akt. Thus our results identify a previously unrecognized signaling node where high miR-21 levels reduce rictor-PDCD4 interaction to increase phosphorylation of Akt and contribute to metastatic fitness of renal cancer cells. Copyright © 2014 Elsevier Inc. All rights reserved.

microRNA-21-induced Dissociation of PDCD4 from Rictor Contributes to Akt-IKKβ-mTORC1 axis to Regulate Select Renal Cancer Cell Invasion

PubMed Central

Bera, Amit; Das, Falguni; Ghosh-Choudhury, Nandini; Kasinath, Balakuntalam S.; Abboud, Hanna E.; Choudhury, Goutam Ghosh

2014-01-01

Renal cancer metastasis may result from oncogenic forces that contribute to the primary tumor. We have recently identified microRNA-21 as an oncogenic driver of renal cancer cells. The mechanism by which miR-21 controls renal cancer cell invasion is poorly understood. We show that miR-21 directly downregulates the proapoptotic protein PDCD4 to increase migration and invasion of ACHN and 786-O renal cancer cells as a result of phosphorylation/activation of Akt and IKKβ, which activate NFκB-dependent transcription. Constitutively active (CA) Akt or CA IKKβ blocks PDCD4-mediated inhibition and restores renal cancer cell migration and invasion. PDCD4 inhibits mTORC1 activity, which was reversed by CA IKKβ. Moreover, CA mTORC1 restores cell migration and invasion inhibited by PDCD4- and dominant negative IKKβ. Moreover, PDCD4 negatively regulates mTORC2-dependent Akt phosphorylation upstream of this cascade. We show that PDCD4 forms a complex with rictor, an exclusive component of mTORC2, and that this complex formation is reduced in renal cancer cells due to increased miR-21 expression resulting in enhanced phosphorylation of Akt. Thus our results identify a previously unrecognized signaling node where high miR-21 levels reduce rictor-PDCD4 interaction to increase phosphorylation of Akt and contribute to metastatic fitness of renal cancer cells. PMID:25016284

Brain derived neurotrophic factor is involved in the regulation of glycogen synthase kinase 3β (GSK3β) signalling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gupta, Vivek, E-mail: vivek.gupta@mq.edu.au; Chitranshi, Nitin; You, Yuyi

2014-11-21

Highlights: • BDNF knockdown leads to activation of GSK3β in the neuronal cells. • BDNF knockdown can induce GSK3β activation beyond TrkB mediated effects. • BDNF impairment in vivo leads to age dependent activation of GSK3β in the retina. • Systemic treatment with TrkB agonist induces inhibition of retinal GSK3β. - Abstract: Glycogen synthase kinase 3β (GSK3β) is involved in several biochemical processes in neurons regulating cellular survival, gene expression, cell fate determination, metabolism and proliferation. GSK3β activity is inhibited through the phosphorylation of its Ser-9 residue. In this study we sought to investigate the role of BDNF/TrkB signalling inmore » the modulation of GSK3β activity. BDNF/TrkB signalling regulates the GSK3β activity both in vivo in the retinal tissue as well as in the neuronal cells under culture conditions. We report here for the first time that BDNF can also regulate GSK3β activity independent of its effects through the TrkB receptor signalling. Knockdown of BDNF lead to a decline in GSK3β phosphorylation without having a detectable effect on the TrkB activity or its downstream effectors Akt and Erk1/2. Treatment with TrkB receptor agonist had a stimulating effect on the GSK3β phosphorylation, but the effect was significantly less pronounced in the cells in which BDNF was knocked down. The use of TrkB receptor antagonist similarly, manifested itself in the form of downregulation of GSK3β phosphorylation, but a combined TrkB inhibition and BDNF knockdown exhibited a much stronger negative effect. In vivo, we observed reduced levels of GSK3β phosphorylation in the retinal tissues of the BDNF{sup +/−} animals implicating critical role of BDNF in the regulation of the GSK3β activity. Concluding, BDNF/TrkB axis strongly regulates the GSK3β activity and BDNF also exhibits GSK3β regulatory effect independent of its actions through the TrkB receptor signalling.« less

Short communication: High incubation temperature in bovine mammary epithelial cells reduced the activity of the mTOR signaling pathway.

PubMed

Kaufman, J D; Kassube, K R; Almeida, R A; Ríus, A G

2018-05-02

Hyperthermia alters utilization of AA in protein synthesis and cell-signaling activity in bovine mammary cells. Essential AA and insulin regulate translation of proteins by controlling the activity of mammalian target of rapamycin (mTOR) signaling pathway. The objectives of this study were to evaluate (1) the effects of incubation temperature on the mTOR signaling pathway and transcription of AA transporters in a bovine mammary alveolar cell line (MAC-T) and (2) the combined effects of incubation temperature and insulin on the mTOR signaling pathway in this cell line. Cells were cultured in medium with 10% fetal bovine serum at 37°C and 5% CO 2 . In experiment 1, cells were subjected to 37°C (control) or 41.5°C (high incubation temperature; HT) for 12 h. In experiment 2, cells were assigned to 1 of 4 treatments as a 2 × 2 factorial arrangement, including 2 cell culture temperatures (control and HT) and absence or presence of 1.0 μg/mL of insulin. Proteins were harvested and separated by gel electrophoresis. In experiment 1, gene expression of AA transporters (SLC1A1, SLC1A5, SLC3A2, SLC7A1, SLC7A5, and SLC36A1) were evaluated, and changes of ≥2 fold were deemed significantly different. In experiments 1 and 2, immunoblotting was used to identify total and site-specific phosphorylated forms of protein kinase B (Akt1; Ser473), p70 S6 kinase (S6K1; Thr389), ribosomal protein S6 (rpS6; Ser235/236), and eukaryotic elongation factor 2 (eEF2; Thr56). Phosphorylated and total forms of Akt1, S6K1, rpS6, and eEF2 were quantified and expressed as the ratio of phosphorylated to total protein. In experiment 1, HT resulted in a ≥2-fold increase expression of SLC1A1 and SLC3A2. High incubation temperature reduced the phosphorylated to total ratio of Akt1 and rpS6 and increased the phosphorylated to total ratio of eEF2. In experiment 2, we found no temperature by insulin interactions on phosphorylation state of the protein factors of interest. High incubation temperature reduced the phosphorylated to total ratio of Akt1. The addition of insulin increased the phosphorylated to total ratio of Akt1, S6K1, and rpS6. In summary, HT reduced the activity of the mTOR signaling pathway and increased the expression of AA transporters. High incubation temperature possibly reduced protein translation by reducing the mTOR signaling pathway activity in an effort to adapt to thermal stress. These results may help explain the direct effect of elevated temperature on AA metabolism and protein translation in heat-stressed animals. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

mTOR-dependent activation of the transcription factor TIF-IA links rRNA synthesis to nutrient availability.

PubMed

Mayer, Christine; Zhao, Jian; Yuan, Xuejun; Grummt, Ingrid

2004-02-15

In cycling cells, transcription of ribosomal RNA genes by RNA polymerase I (Pol I) is tightly coordinated with cell growth. Here, we show that the mammalian target of rapamycin (mTOR) regulates Pol I transcription by modulating the activity of TIF-IA, a regulatory factor that senses nutrient and growth-factor availability. Inhibition of mTOR signaling by rapamycin inactivates TIF-IA and impairs transcription-initiation complex formation. Moreover, rapamycin treatment leads to translocation of TIF-IA into the cytoplasm. Rapamycin-mediated inactivation of TIF-IA is caused by hypophosphorylation of Se 44 (S44) and hyperphosphorylation of Se 199 (S199). Phosphorylation at these sites affects TIF-IA activity in opposite ways, for example, phosphorylation of S44 activates and S199 inactivates TIF-IA. The results identify a new target formTOR-signaling pathways and elucidate the molecular mechanism underlying mTOR-dependent regulation of RNA synthesis.

Assay Methods for ACS Activity and ACS Phosphorylation by MAP Kinases In Vitro and In Vivo.

PubMed

Han, Xiaomin; Li, Guojing; Zhang, Shuqun

2017-01-01

Ethylene, a gaseous phytohormone, has profound effects on plant growth, development, and adaptation to the environment. Ethylene-regulated processes begin with the induction of ethylene biosynthesis. There are two key steps in ethylene biosynthesis. The first is the biosynthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) from S-Adenosyl-Methionine (SAM), a common precursor in many metabolic pathways, which is catalyzed by ACC synthase (ACS). The second is the oxidative cleavage of ACC to form ethylene under the action of ACC oxidase (ACO). ACC biosynthesis is the committing and generally the rate-limiting step in ethylene biosynthesis. As a result, characterizing the cellular ACS activity and understanding its regulation are important. In this chapter, we detail the methods used to measure, (1) the enzymatic activity of both recombinant and native ACS proteins, and (2) the phosphorylation of ACS protein by mitogen-activated protein kinases (MAPKs) in vivo and in vitro.

Immediate and Delayed Drug Therapy Effects on Low Dose Sarin Exposed Mice Myocardial Performance

DTIC Science & Technology

2011-03-01

to phosphorylate the serine hydroxyl residue in the active pocket of AChE, forming either a phosphoric or phosphonic acid ester which is extremely...London 2006). Brain natriuretic peptide exerts its 15 natriuretic, diuretic, and vasorelaxant effects through activation of its common receptor...exposed to asymptomatic levels would show no issues during normal activities , such garrison or non-field operations. However, this would quickly change

A protein-tyrosine phosphatase with sequence similarity to the SH2 domain of the protein-tyrosine kinases.

PubMed

Shen, S H; Bastien, L; Posner, B I; Chrétien, P

1991-08-22

The phosphorylation of proteins at tyrosine residues is critical in cellular signal transduction, neoplastic transformation and control of the mitotic cycle. These mechanisms are regulated by the activities of both protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPases). As in the PTKs, there are two classes of PTPases: membrane associated, receptor-like enzymes and soluble proteins. Here we report the isolation of a complementary DNA clone encoding a new form of soluble PTPase, PTP1C. The enzyme possesses a large noncatalytic region at the N terminus which unexpectedly contains two adjacent copies of the Src homology region 2 (the SH2 domain) found in various nonreceptor PTKs and other cytoplasmic signalling proteins. As with other SH2 sequences, the SH2 domains of PTP1C formed high-affinity complexes with the activated epidermal growth factor receptor and other phosphotyrosine-containing proteins. These results suggest that the SH2 regions in PTP1C may interact with other cellular components to modulate its own phosphatase activity against interacting substrates. PTPase activity may thus directly link growth factor receptors and other signalling proteins through protein-tyrosine phosphorylation.

Subetta increases phosphorylation of insulin receptor β-subunit alone and in the presence of insulin

PubMed Central

Gorbunov, E A; Nicoll, J; Kachaeva, E V; Tarasov, S A; Epstein, O I

2015-01-01

It has been previously shown that Subetta (a drug containing released-active forms of antibodies to the insulin receptor β-subunit and antibodies to endothelial nitric oxide synthase) stimulated insulin-induced adiponectin production by mature human adipocytes in the absence of insulin. Therefore, it was assumed that Subetta could activate the insulin receptor. To confirm this hypothesis, the capacity of Subetta to activate the insulin receptor in mature human adipocytes in the absence or presence of the insulin was investigated. Cells were incubated either with Subetta or with vehicle, or with basal medium for 3 days. Then, adipocytes were treated with water or insulin (100 nm) for 15 min. Following treatment, lysates were prepared and phosphorylation of insulin receptor β-subunits was analyzed by western blot analysis. It was shown that Subetta significantly increased (P<0.001) the ‘phosphorylated-insulin receptor β-subunit/total insulin receptor β-subunit' ratios in both the presence and the absence of insulin. These results support previously published data and indicate that Subetta could activate the insulin receptor through the effect on its β-subunits, whose conformational state is essential for insulin receptor activation. This action might serve as one of the primary mechanisms of the drug's antidiabetic effect. PMID:26148148

Phosphorylated (pT371)TRF1 is recruited to sites of DNA damage to facilitate homologous recombination and checkpoint activation

PubMed Central

McKerlie, Megan; Walker, John R.; Mitchell, Taylor R. H.; Wilson, Florence R.; Zhu, Xu-Dong

2013-01-01

TRF1, a duplex telomeric DNA-binding protein, plays an important role in telomere metabolism. We have previously reported that a fraction of endogenous TRF1 can stably exist free of telomere chromatin when it is phosphorylated at T371 by Cdk1; however, the role of this telomere-free (pT371)TRF1 has yet to be fully characterized. Here we show that phosphorylated (pT371)TRF1 is recruited to sites of DNA damage, forming damage-induced foci in response to ionizing radiation (IR), etoposide and camptothecin. We find that IR-induced (pT371)TRF1 foci formation is dependent on the ATM- and Mre11/Rad50/Nbs1-mediated DNA damage response. While loss of functional BRCA1 impairs the formation of IR-induced (pT371)TRF1 foci, depletion of either 53BP1 or Rif1 stimulates IR-induced (pT371)TRF1 foci formation. In addition, we show that TRF1 depletion or the lack of its phosphorylation at T371 impairs DNA end resection and repair of nontelomeric DNA double-strand breaks by homologous recombination. The lack of TRF1 phosphorylation at T371 also hampers the activation of the G2/M checkpoint and sensitizes cells to PARP inhibition, IR and camptothecin. Collectively, these results reveal a novel but important function of phosphorylated (pT371)TRF1 in facilitating DNA double-strand break repair and the maintenance of genome integrity. PMID:23997120

How quantitative measures unravel design principles in multi-stage phosphorylation cascades.

PubMed

Frey, Simone; Millat, Thomas; Hohmann, Stefan; Wolkenhauer, Olaf

2008-09-07

We investigate design principles of linear multi-stage phosphorylation cascades by using quantitative measures for signaling time, signal duration and signal amplitude. We compare alternative pathway structures by varying the number of phosphorylations and the length of the cascade. We show that a model for a weakly activated pathway does not reflect the biological context well, unless it is restricted to certain parameter combinations. Focusing therefore on a more general model, we compare alternative structures with respect to a multivariate optimization criterion. We test the hypothesis that the structure of a linear multi-stage phosphorylation cascade is the result of an optimization process aiming for a fast response, defined by the minimum of the product of signaling time and signal duration. It is then shown that certain pathway structures minimize this criterion. Several popular models of MAPK cascades form the basis of our study. These models represent different levels of approximation, which we compare and discuss with respect to the quantitative measures.

Evidence for two distinct phosphorylation pathways activated by high affinity immunoglobulin E receptors.

PubMed

Adamczewski, M; Paolini, R; Kinet, J P

1992-09-05

The high affinity receptor for immunoglobulin (Ig) E on mast cells, along with the antigen receptors on T and B cells and Fc receptors for IgG, belongs to a class of receptors which lack intrinsic kinase activity, but activate non-receptor tyrosine and serine/threonine kinases. Receptor engagement triggers a chain of signaling events leading from protein phosphorylation to activation of phosphatidylinositol-specific phospholipase C, an increase in intracellular calcium levels, and ultimately the activation of more specialized functions. IgE receptor disengagement leads to reversal of phosphorylation by undefined phosphatases and to inhibition of activation pathways. Here we show that phenylarsine oxide, a chemical which reacts with thiol groups and has been reported to inhibit tyrosine phosphatases, uncouples the IgE receptor-mediated phosphorylation signal from activation of phosphatidyl inositol metabolism, the increase in intracellular calcium levels, and serotonin release. Phenylarsine oxide inhibits neither the kinases (tyrosine and serine/threonine) phosphorylating the receptor and various cellular substrates nor, unexpectedly, the phosphatases responsible for the dephosphorylation following receptor disengagement. By contrast, it abolishes the receptor-mediated phosphorylation of phospholipase C-gamma 1, but not phospholipase C activity in vitro. Therefore the phosphorylation and activation of phospholipase C likely requires a phenylarsine oxide-sensitive element. Receptor aggregation thus activates at least two distinct phosphorylation pathways: a phenylarsine oxide-insensitive pathway leading to phosphorylation/dephosphorylation of the receptor and of various substrates and a sensitive pathway leading to phospholipase C-gamma 1 phosphorylation.

Structure and signalling functions of C3 receptors on human B cells.

PubMed

Frade, R

1990-03-01

CR1 (C3b receptor) and CR2 (C3d/EBV receptor) are two C3 receptors expressed on B lymphocytes. CR1 and CR2 have structural similarities and their cross-linking at the B cell surface by antibodies or specific ligands in multimeric forms induce B cell activation. However, activation of human B cells through cell surface interactions or by intracellular protein kinase C activators leads to phosphorylation of CR2 but not CR1. CR2 is phosphorylated on serine and tyrosine residues. Analysis of post-membrane events associated with CR2 revealed intracellular interactions of CR2 with p53, a plasma membrane anti-oncogene-encoded phosphoprotein, and with p120, a nuclear phosphoribonucleoprotein. These intracellular interactions probably represent important steps in the signalling functions of CR2.

Selective elimination of neuroblastoma cells by synergistic effect of Akt kinase inhibitor and tetrathiomolybdate.

PubMed

Navrátilová, Jarmila; Karasová, Martina; Kohutková Lánová, Martina; Jiráková, Ludmila; Budková, Zuzana; Pacherník, Jiří; Šmarda, Jan; Beneš, Petr

2017-09-01

Neuroblastoma is the most common extracranial solid tumour of infancy. Pathological activation of glucose consumption, glycolysis and glycolysis-activating Akt kinase occur frequently in neuroblastoma cells, and these changes correlate with poor prognosis of patients. Therefore, several inhibitors of glucose utilization and the Akt kinase activity are in preclinical trials as potential anti-cancer drugs. However, metabolic plasticity of cancer cells might undermine efficacy of this approach. In this work, we identified oxidative phosphorylation as compensatory mechanism preserving viability of neuroblastoma cells with inhibited glucose uptake/Akt kinase. It was oxidative phosphorylation that maintained intracellular level of ATP and proliferative capacity of these cells. The oxidative phosphorylation inhibitors (rotenone, tetrathiomolybdate) synergized with inhibitor of the Akt kinase/glucose uptake in down-regulation of both viability of neuroblastoma cells and clonogenic potential of cells forming neuroblastoma spheroids. Interestingly, tetrathiomolybdate acted as highly specific inhibitor of oxygen consumption and activator of lactate production in neuroblastoma cells, but not in normal fibroblasts and neuronal cells. Moreover, the reducing effect of tetrathiomolybdate on cell viability and the level of ATP in the cells with inhibited Akt kinase/glucose uptake was also selective for neuroblastoma cells. Therefore, efficient elimination of neuroblastoma cells requires inhibition of both glucose uptake/Akt kinase and oxidative phosphorylation activities. The use of tetrathiomolybdate as a mitochondrial inhibitor contributes to selectivity of this combined treatment, preferentially targeting neuroblastoma cells. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Navigating the conformational landscape of G protein-coupled receptor kinases during allosteric activation.

PubMed

Yao, Xin-Qiu; Cato, M Claire; Labudde, Emily; Beyett, Tyler S; Tesmer, John J G; Grant, Barry J

2017-09-29

G protein-coupled receptors (GPCRs) are essential for transferring extracellular signals into carefully choreographed intracellular responses controlling diverse aspects of cell physiology. The duration of GPCR-mediated signaling is primarily regulated via GPCR kinase (GRK)-mediated phosphorylation of activated receptors. Although many GRK structures have been reported, the mechanisms underlying GRK activation are not well-understood, in part because it is unknown how these structures map to the conformational landscape available to this enzyme family. Unlike most other AGC kinases, GRKs rely on their interaction with GPCRs for activation and not phosphorylation. Here, we used principal component analysis of available GRK and protein kinase A crystal structures to identify their dominant domain motions and to provide a framework that helps evaluate how close each GRK structure is to being a catalytically competent state. Our results indicated that disruption of an interface formed between the large lobe of the kinase domain and the regulator of G protein signaling homology domain (RHD) is highly correlated with establishment of the active conformation. By introducing point mutations in the GRK5 RHD-kinase domain interface, we show with both in silico and in vitro experiments that perturbation of this interface leads to higher phosphorylation activity. Navigation of the conformational landscape defined by this bioinformatics-based study is likely common to all GPCR-activated GRKs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Inhibitory effects of 1-O-methyl-fructofuranose from Schisandra chinensis fruit on melanogenesis in B16F0 melanoma cells.

PubMed

Oh, Eun Young; Jang, Ji Yeon; Choi, Yung Hyun; Choi, Young Whan; Choi, Byung Tae

2010-10-28

1-O-methyl-fructofuranose (1-O-MFF) from the fruit of Schisandra chinensis is a traditional Korean medicinal herb that has a variety of beneficial properties. The effect of purified 1-O-MFF on melanogenesis including the activation of related signaling pathways was investigated. The inhibitory activities of 1-O-MFF were examined by melanin synthesis, tyrosinase activity assay, Western blot and flow cytometric analyses in B16F0 mouse melanoma cells. 1-O-MFF significantly inhibited both melanin synthesis and tyrosinase activity in a concentration-dependent manner, and reduced the expression of melanogenic proteins including microphthalmia-associated transcription factor (MITF), tyrosinase and tyrosinase-related protein-1. 1-O-MFF phosphorylated and activated melanogenesis inhibitory proteins such as mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and Akt. Flow cytometry confirmed that 1-O-MFF phosphorylated ERK and Akt proteins and recovered partially phosphorylated forms in cells treated with the MEK/ERK inhibitor compound PD98059 and the phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor compound LY294002. The suppressive effects of 1-O-MFF on melanogenesis may involve down-regulation of MITF and its downstream signal pathway via the activation of MEK/ERK or PI3K/Akt. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Activated PLC-γ1 is Catalytically Induced at LAT but Activated PLC-γ1 is Localized at both LAT- and TCR-Containing Complexes

PubMed Central

Cruz-Orcutt, Noemi; Vacaflores, Aldo; Connolly, Sean F.; Bunnell, Stephen C.; Houtman, Jon C.D.

2014-01-01

Phospholipase C-γ1 (PLC-γ1) is a key regulator of T cell receptor (TCR)-induced signaling. Activation of the TCR enhances PLC-γ1 enzymatic function, resulting in calcium influx and the activation of PKC family members and RasGRP. The current model is that phosphorylation of LAT tyrosine 132 facilitates the recruitment of PLC-γ1, leading to its activation and function at the LAT complex. In this study, we examined the phosphorylation kinetics of LAT and PLC-γ1 and the cellular localization of activated PLC-γ1. We observed that commencement of the phosphorylation of LAT tyrosine 132 and PLC-γ1 tyrosine 783 occurred simultaneously, supporting the current model. However, once begun, PLC-γ1 activation occurred more rapidly than LAT tyrosine 132. The association of LAT and PLC-γ1 was more transient than the interaction of LAT and Grb2 and a pool of activated PLC-γ1 translocated away from LAT to cellular structures containing the TCR. These studies demonstrate that LAT and PLC-γ1 form transient interactions that catalyze the activation of PLC-γ1, but that activated PLC-γ1 resides in both LAT and TCR clusters. Together, this work highlights that our current model is incomplete and the activation and function of PLC-γ1 in T cells is highly complex. PMID:24412752

Dephosphorylation of the Core Clock Protein KaiC in the Cyanobacterial KaiABC Circadian Oscillator Proceeds via an ATP Synthase Mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Egli, Martin; Mori, Tetsuya; Pattanayek, Rekha

The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro from three proteins, KaiA, KaiB, and KaiC in the presence of ATP, to tick in a temperature-compensated manner. KaiC, the central cog of this oscillator, forms a homohexamer with 12 ATP molecules bound between its N- and C-terminal domains and exhibits unusual properties. Both the N-terminal (CI) and C-terminal (CII) domains harbor ATPase activity, and the subunit interfaces between CII domains are the sites of autokinase and autophosphatase activities. Hydrolysis of ATP correlates with phosphorylation at threonine and serine sites across subunits in an orchestrated manner, suchmore » that first T432 and then S431 are phosphorylated, followed by dephosphorylation of these residues in the same order. Although structural work has provided insight into the mechanisms of ATPase and kinase, the location and mechanism of the phosphatase have remained enigmatic. From the available experimental data based on a range of approaches, including KaiC crystal structures and small-angle X-ray scattering models, metal ion dependence, site-directed mutagenesis (i.e., E318, the general base), and measurements of the associated clock periods, phosphorylation patterns, and dephosphorylation courses as well as a lack of sequence motifs in KaiC that are typically associated with known phosphatases, we hypothesized that KaiCII makes use of the same active site for phosphorylation and dephosphorlyation. We observed that wild-type KaiC (wt-KaiC) exhibits an ATP synthase activity that is significantly reduced in the T432A/S431A mutant. We interpret the first observation as evidence that KaiCII is a phosphotransferase instead of a phosphatase and the second that the enzyme is capable of generating ATP, both from ADP and P{sub i} (in a reversal of the ATPase reaction) and from ADP and P-T432/P-S431 (dephosphorylation). This new concept regarding the mechanism of dephosphorylation is also supported by the strikingly similar makeups of the active sites at the interfaces between {alpha}/{beta} heterodimers of F1-ATPase and between monomeric subunits in the KaiCII hexamer. Several KaiCII residues play a critical role in the relative activities of kinase and ATP synthase, among them R385, which stabilizes the compact form and helps kinase action reach a plateau, and T426, a short-lived phosphorylation site that promotes and affects the order of dephosphorylation.« less

Casein kinase 1delta activates human recombinant deoxycytidine kinase by Ser-74 phosphorylation, but is not involved in the in vivo regulation of its activity.

PubMed

Smal, Caroline; Vertommen, Didier; Amsailale, Rachid; Arts, Angélique; Degand, Hervé; Morsomme, Pierre; Rider, Mark H; Neste, Eric Van Den; Bontemps, Françoise

2010-10-01

Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxynucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We recently showed that dCK was activated in vivo by phosphorylation of Ser-74. However, the protein kinase responsible was not identified. Ser-74 is located downstream a Glu-rich region, presenting similarity with the consensus phosphorylation motif of casein kinase 1 (CKI), and particularly of CKI delta. We showed that recombinant CKI delta phosphorylated several residues of bacterially overexpressed dCK: Ser-74, but also Ser-11, Ser-15, and Thr-72. Phosphorylation of dCK by CKI delta correlated with increased activity reaching at least 4-fold. Site-directed mutagenesis demonstrated that only Ser-74 phosphorylation was involved in dCK activation by CKI delta, strengthening the key role of this residue in the control of dCK activity. However, neither CKI delta inhibitors nor CKI delta siRNA-mediated knock-down modified Ser-74 phosphorylation or dCK activity in cultured cells. Moreover, these approaches did not prevent dCK activation induced by treatments enhancing Ser-74 phosphorylation. Taken together, the data preclude a role of CKI delta in the regulation of dCK activity in vivo. Nevertheless, phosphorylation of dCK by CKI delta could be a useful tool for elucidating the influence of Ser-74 phosphorylation on the structure-activity relationships in the enzyme. Copyright 2010 Elsevier Inc. All rights reserved.

Differential phosphorylation signals control endocytosis of GPR15

PubMed Central

Okamoto, Yukari; Shikano, Sojin

2017-01-01

GPR15 is an orphan G protein–coupled receptor (GPCR) that serves for an HIV coreceptor and was also recently found as a novel homing receptor for T-cells implicated in colitis. We show that GPR15 undergoes a constitutive endocytosis in the absence of ligand. The endocytosis was clathrin dependent and partially dependent on β-arrestin in HEK293 cells, and nearly half of the internalized GPR15 receptors were recycled to the plasma membrane. An Ala mutation of the distal C-terminal Arg-354 or Ser-357, which forms a consensus phosphorylation site for basophilic kinases, markedly reduced the endocytosis, whereas phosphomimetic mutation of Ser-357 to Asp did not. Ser-357 was phosphorylated in vitro by multiple kinases, including PKA and PKC, and pharmacological activation of these kinases enhanced both phosphorylation of Ser-357 and endocytosis of GPR15. These results suggested that Ser-357 phosphorylation critically controls the ligand-independent endocytosis of GPR15. The functional role of Ser-357 in endocytosis was distinct from that of a conserved Ser/Thr cluster in the more proximal C-terminus, which was responsible for the β-arrestin– and GPCR kinase–dependent endocytosis of GPR15. Thus phosphorylation signals may differentially control cell surface density of GPR15 through endocytosis. PMID:28615320

Protein Phosphorylation Profiling Using an In Situ Proximity Ligation Assay: Phosphorylation of AURKA-Elicited EGFR-Thr654 and EGFR-Ser1046 in Lung Cancer Cells

PubMed Central

Chen, Tzu-Chi; Liu, Yu-Wen; Huang, Yei-Hsuan; Yeh, Yi-Chen; Chou, Teh-Ying; Wu, Yu-Chung; Wu, Chun-Chi; Chen, Yi-Rong; Cheng, Hui-Chuan; Lu, Pei-Jung; Lai, Jin-Mei; Huang, Chi-Ying F.

2013-01-01

The epidermal growth factor receptor (EGFR), which is up-regulated in lung cancer, involves the activation of mitogenic signals and triggers multiple signaling cascades. To dissect these EGFR cascades, we used 14 different phospho-EGFR antibodies to quantify protein phosphorylation using an in situ proximity ligation assay (in situ PLA). Phosphorylation at EGFR-Thr654 and -Ser1046 was EGF-dependent in the wild-type (WT) receptor but EGF-independent in a cell line carrying the EGFR-L858R mutation. Using a ProtoAarray™ containing ∼5000 recombinant proteins on the protein chip, we found that AURKA interacted with the EGFR-L861Q mutant. Moreover, overexpression of EGFR could form a complex with AURKA, and the inhibitors of AURKA and EGFR decreased EGFR-Thr654 and -Ser1046 phosphorylation. Immunohistochemical staining of stage I lung adenocarcinoma tissues demonstrated a positive correlation between AURKA expression and phosphorylation of EGFR at Thr654 and Ser1046 in EGFR-mutant specimens, but not in EGFR-WT specimens. The interplay between EGFR and AURKA provides an explanation for the difference in EGF dependency between EGFR-WT and EGFR-mutant cells and may provide a new therapeutic strategy for lung cancer patients carrying EGFR mutations. PMID:23520446

An increase or a decrease in myosin II phosphorylation inhibits macrophage motility

PubMed Central

1991-01-01

Myosin II purified from mammalian non-muscle cells is phosphorylated on the 20-kD light chain subunit (MLC20) by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK). The importance of MLC20 phosphorylation in regulating cell motility was investigated by introducing either antibodies to MLCK (MK-Ab) or a Ca2+/calmodulin- independent, constitutively active form of MLCK (MK-) into macrophages. The effects of these proteins on cell motility were then determined using a quantitative chemotaxis assay. Chemotaxis is significantly diminished in macrophages containing MK-Ab compared to macrophages containing control antibodies. Moreover, there is an inverse relationship between the number of cells that migrate and the amount of MK-Ab introduced into cells. Interestingly, there is also an inverse relationship between the number of cells that migrate and the amount of MK- introduced into cells. Other experiments demonstrated that MK-Ab decreased intracellular MLC20 phosphorylation while MK- increased MLC20 phosphorylation. MK- also increased the amount of myosin associated with the cytoskeleton. These data demonstrate that the regulation of MLCK is an important aspect of cell motility and suggest that MLC20 phosphorylation must be maintained within narrow limits during translational motility by mammalian cells. PMID:2071674

Comprehensive Characterization of AMP-activated Protein Kinase Catalytic Domain by Top-down Mass Spectrometry

PubMed Central

Yu, Deyang; Peng, Ying; Ayaz-Guner, Serife; Gregorich, Zachery R.; Ge, Ying

2015-01-01

AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is essential in regulating energy metabolism in all eukaryotic cells. It is a heterotrimeric protein complex composed of a catalytic subunit (α) and two regulatory subunits (β and γ. C-terminal truncation of AMPKα at residue 312 yielded a protein that is active upon phosphorylation of Thr172 in the absence of β and γ subunits, which is refered to as the AMPK catalytic domain and commonly used to substitute for the AMPK heterotrimeric complex in in vitro kinase assays. However, a comprehensive characterization of the AMPK catalytic domain is lacking. Herein, we expressed a His-tagged human AMPK catalytic domin (denoted as AMPKΔ) in E. coli, comprehensively characterized AMPKΔ in its basal state and after in vitro phosphorylation using top-down mass spectrometry (MS), and assessed how phosphorylation of AMPKΔ affects its activity. Unexpectedly, we found that bacterially-expressed AMPKΔ was basally phosphorylated and localized the phosphorylation site to the His-tag. We found that AMPKΔ has noticeable basal activity and was capable of phosphorylating itself and its substrates without activating phosphorylation at Thr172. Moreover, our data suggested that Thr172 is the only site phosphorylated by its upstream kinase, liver kinase B1, and that this phosphorylation dramatically increases the kinase activity of AMPKΔ. Importantly, we demonstrated that top-down MS in conjunction with in vitro phosphorylation assay is a powerful approach for monitoring phosphorylation reaction and determining sequential order of phosphorylation events in kinase-substrate systems. PMID:26489410

Cross-talk between Rho-associated kinase and cyclic nucleotide-dependent kinase signaling pathways in the regulation of smooth muscle myosin light chain phosphatase.

PubMed

Grassie, Michael E; Sutherland, Cindy; Ulke-Lemée, Annegret; Chappellaz, Mona; Kiss, Enikö; Walsh, Michael P; MacDonald, Justin A

2012-10-19

Ca(2+) sensitization of smooth muscle contraction depends upon the activities of protein kinases, including Rho-associated kinase, that phosphorylate the myosin phosphatase targeting subunit (MYPT1) at Thr(697) and/or Thr(855) (rat sequence numbering) to inhibit phosphatase activity and increase contractile force. Both Thr residues are preceded by the sequence RRS, and it has been suggested that phosphorylation at Ser(696) prevents phosphorylation at Thr(697). However, the effects of Ser(854) and dual Ser(696)-Thr(697) and Ser(854)-Thr(855) phosphorylations on myosin phosphatase activity and contraction are unknown. We characterized a suite of MYPT1 proteins and phosphospecific antibodies for specificity toward monophosphorylation events (Ser(696), Thr(697), Ser(854), and Thr(855)), Ser phosphorylation events (Ser(696)/Ser(854)) and dual Ser/Thr phosphorylation events (Ser(696)-Thr(697) and Ser(854)-Thr(855)). Dual phosphorylation at Ser(696)-Thr(697) and Ser(854)-Thr(855) by cyclic nucleotide-dependent protein kinases had no effect on myosin phosphatase activity, whereas phosphorylation at Thr(697) and Thr(855) by Rho-associated kinase inhibited phosphatase activity and prevented phosphorylation by cAMP-dependent protein kinase at the neighboring Ser residues. Forskolin induced phosphorylation at Ser(696), Thr(697), Ser(854), and Thr(855) in rat caudal artery, whereas U46619 induced Thr(697) and Thr(855) phosphorylation and prevented the Ser phosphorylation induced by forskolin. Furthermore, pretreatment with forskolin prevented U46619-induced Thr phosphorylations. We conclude that cross-talk between cyclic nucleotide and RhoA signaling pathways dictates the phosphorylation status of the Ser(696)-Thr(697) and Ser(854)-Thr(855) inhibitory regions of MYPT1 in situ, thereby regulating the activity of myosin phosphatase and contraction.

Mucin1 shifts Smad3 signaling from the tumor-suppressive pSmad3C/p21(WAF1) pathway to the oncogenic pSmad3L/c-Myc pathway by activating JNK in human hepatocellular carcinoma cells.

PubMed

Li, Qiongshu; Liu, Guomu; Yuan, Hongyan; Wang, Juan; Guo, Yingying; Chen, Tanxiu; Zhai, Ruiping; Shao, Dan; Ni, Weihua; Tai, Guixiang

2015-02-28

Mucin1 (MUC1) is a transmembrane glycoprotein that acts as an oncogene in human hepatic tumorigenesis. Hepatocellular carcinoma (HCC) cells often gain advantage by reducing the tumor-suppressive activity of transforming growth factor beta (TGF-β) together with stimulation of its oncogenic activity as in MUC1 expressing HCC cells; however, molecular mechanisms remain largely unknown. Type I TGF-β receptor (TβRI) and c-Jun NH2-terminal kinase (JNK) differentially phosphorylate Smad3 mediator to create 2 phosphorylated forms: COOH-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). Here, we report that MUC1 overexpression in HCC cell lines suppresses TβRI-mediated pSmad3C signaling which involves growth inhibition by up-regulating p21(WAF1). Instead, MUC1 directly activates JNK to stimulate oncogenic pSmad3L signaling, which fosters cell proliferation by up-regulating c-Myc. Conversely, MUC1 gene silencing in MUC1 expressing HCC cells results in preserved tumor-suppressive function via pSmad3C, while eliminating pSmad3L-mediated oncogenic activity both in vitro and in vivo. In addition, high correlation between MUC1 and pSmad3L/c-Myc but not pSmad3C/p21(WAF1) expression was observed in HCC tissues from patients. Collectively, these results indicate that MUC1 shifts Smad3 signaling from a tumor-suppressive pSmad3C/p21(WAF1) to an oncogenic pSmad3L/c-Myc pathway by directly activating JNK in HCC cells, suggesting that MUC1 is an important target for HCC therapy.

Efficacy of glycogen synthase kinase-3β targeting against osteosarcoma via activation of β-catenin

PubMed Central

Yamamoto, Norio; Nishida, Hideji; Hayashi, Katsuhiro; Kimura, Hiroaki; Takeuchi, Akihiko; Miwa, Shinji; Igarashi, Kentaro; Kato, Takashi; Aoki, Yu; Higuchi, Takashi; Hirose, Mayumi; Hoffman, Robert M; Minamoto, Toshinari; Tsuchiya, Hiroyuki

2016-01-01

Development of innovative more effective therapy is required for refractory osteosarcoma patients. We previously established that glycogen synthase kinase-3β (GSK- 3β) is a therapeutic target in various cancer types. In the present study, we explored the therapeutic efficacy of GSK-3β inhibition against osteosarcoma and the underlying molecular mechanisms in an orthotopic mouse model. Expression and phosphorylation of GSK-3β in osteosarcoma and normal osteoblast cell lines was examined, together with efficacy of GSK-3β inhibition on cell survival, proliferation and apoptosis and on the growth of orthotopically-transplanted human osteosarcoma in nude mice. We also investigated changes in expression, phosphorylation and co-transcriptional activity of β-catenin in osteosarcoma cells following GSK-3β inhibition. Expression of the active form of GSK- 3β (tyrosine 216-phosphorylated) was higher in osteosarcoma than osteoblast cells. Inhibition of GSK-3β activity by pharmacological inhibitors or of its expression by RNA interference suppressed proliferation of osteosarcoma cells and induced apoptosis. Treatment with GSK-3β-specific inhibitors attenuated the growth of orthotopic osteosaroma in mice. Inhibition of GSK-3β reduced phosphorylation at GSK- 3β-phospho-acceptor sites in β-catenin and increased β-catenin expression, nuclear localization and co-transcriptional activity. These results suggest the efficacy of GSK-3β inhibitors is associated with activation of β-catenin, a putative tumor suppressor in bone and soft tissue sarcoma and an important component of osteogenesis. Our study thereby demonstrates a critical role for GSK-3β in sustaining survival and proliferation of osteosarcoma cells, and identifies this kinase as a potential therapeutic target against osteosarcoma. PMID:27780915

Involvement of distinct arrestin-1 elements in binding to different functional forms of rhodopsin

PubMed Central

Zhuang, Tiandi; Chen, Qiuyan; Cho, Min-Kyu; Vishnivetskiy, Sergey A.; Iverson, Tina M.; Gurevich, Vsevolod V.; Sanders, Charles R.

2013-01-01

Solution NMR spectroscopy of labeled arrestin-1 was used to explore its interactions with dark-state phosphorylated rhodopsin (P-Rh), phosphorylated opsin (P-opsin), unphosphorylated light-activated rhodopsin (Rh*), and phosphorylated light-activated rhodopsin (P-Rh*). Distinct sets of arrestin-1 elements were seen to be engaged by Rh* and inactive P-Rh, which induced conformational changes that differed from those triggered by binding of P-Rh*. Although arrestin-1 affinity for Rh* was seen to be low (KD > 150 μM), its affinity for P-Rh (KD ∼80 μM) was comparable to the concentration of active monomeric arrestin-1 in the outer segment, suggesting that P-Rh generated by high-gain phosphorylation is occupied by arrestin-1 under physiological conditions and will not signal upon photo-activation. Arrestin-1 was seen to bind P-Rh* and P-opsin with fairly high affinity (KD of ∼50 and 800 nM, respectively), implying that arrestin-1 dissociation is triggered only upon P-opsin regeneration with 11-cis-retinal, precluding noise generated by opsin activity. Based on their observed affinity for arrestin-1, P-opsin and inactive P-Rh very likely affect the physiological monomer-dimer-tetramer equilibrium of arrestin-1, and should therefore be taken into account when modeling photoreceptor function. The data also suggested that complex formation with either P-Rh* or P-opsin results in a global transition in the conformation of arrestin-1, possibly to a dynamic molten globule-like structure. We hypothesize that this transition contributes to the mechanism that triggers preferential interactions of several signaling proteins with receptor-activated arrestins. PMID:23277586

Involvement of distinct arrestin-1 elements in binding to different functional forms of rhodopsin.

PubMed

Zhuang, Tiandi; Chen, Qiuyan; Cho, Min-Kyu; Vishnivetskiy, Sergey A; Iverson, Tina M; Gurevich, Vsevolod V; Sanders, Charles R

2013-01-15

Solution NMR spectroscopy of labeled arrestin-1 was used to explore its interactions with dark-state phosphorylated rhodopsin (P-Rh), phosphorylated opsin (P-opsin), unphosphorylated light-activated rhodopsin (Rh*), and phosphorylated light-activated rhodopsin (P-Rh*). Distinct sets of arrestin-1 elements were seen to be engaged by Rh* and inactive P-Rh, which induced conformational changes that differed from those triggered by binding of P-Rh*. Although arrestin-1 affinity for Rh* was seen to be low (K(D) > 150 μM), its affinity for P-Rh (K(D) ~80 μM) was comparable to the concentration of active monomeric arrestin-1 in the outer segment, suggesting that P-Rh generated by high-gain phosphorylation is occupied by arrestin-1 under physiological conditions and will not signal upon photo-activation. Arrestin-1 was seen to bind P-Rh* and P-opsin with fairly high affinity (K(D) of~50 and 800 nM, respectively), implying that arrestin-1 dissociation is triggered only upon P-opsin regeneration with 11-cis-retinal, precluding noise generated by opsin activity. Based on their observed affinity for arrestin-1, P-opsin and inactive P-Rh very likely affect the physiological monomer-dimer-tetramer equilibrium of arrestin-1, and should therefore be taken into account when modeling photoreceptor function. The data also suggested that complex formation with either P-Rh* or P-opsin results in a global transition in the conformation of arrestin-1, possibly to a dynamic molten globule-like structure. We hypothesize that this transition contributes to the mechanism that triggers preferential interactions of several signaling proteins with receptor-activated arrestins.

Autoregulatory Characteristics of a Bacillus anthracis Serine/Threonine Kinase▿

PubMed Central

Bryant-Hudson, Katie M.; Shakir, Salika M.; Ballard, Jimmy D.

2011-01-01

BA-Stk1 is a serine/threonine kinase (STK) expressed by Bacillus anthracis. In previous studies, we found that BA-Stk1 activity is modulated through dephosphorylation by a partner phosphatase, BA-Stp1. In this study, we identified critical phosphorylation regions of BA-Stk1 and determined the contributions of these phosphodomains to autophosphorylation and substrate phosphorylation. The data indicate that BA-Stk1 undergoes trans-autophosphorylation within a regulatory domain, referred to as the activation loop, which carries eight putative regulatory serine and threonine residues. We identified activation loop mutants that impacted kinase activity in three different manners: regulation of autophosphorylation (T162), regulation of substrate phosphorylation (T159 and S169), and regulation of overall kinase activity (T163). Tandem mass spectrometry (MS/MS) analysis of the phosphorylation profile of each mutant revealed a second site of phosphorylation on the kinase that was influenced by the phosphorylation status of the activation loop. This second region of the kinase contained a single phosphorylation residue, S214. Previous work has shown S214 to be necessary for downstream substrate phosphorylation, and we have shown that this residue is subject to dephosphorylation by BA-Stp1. These findings indicate a connection between the phosphorylation status of the activation loop and phosphorylation of S214, and this suggests a previously undescribed model for how a bacterial STK shifts from a state of autophosphorylation to targeting downstream substrates. PMID:21296958

SH2-B promotes insulin receptor substrate 1 (IRS1)- and IRS2-mediated activation of the phosphatidylinositol 3-kinase pathway in response to leptin.

PubMed

Duan, Chaojun; Li, Minghua; Rui, Liangyou

2004-10-15

Leptin regulates energy homeostasis primarily by binding and activating its long form receptor (LRb). Deficiency of either leptin or LRb causes morbid obesity. Leptin stimulates LRb-associated JAK2, thus initiating multiple pathways including the Stat3 and phosphatidylinositol (PI) 3-kinase pathways that mediate leptin biological actions. Here we report that SH2-B, a JAK2-interacting protein, promotes activation of the PI 3-kinase pathway by recruiting insulin receptor substrate 1 (IRS1) and IRS2 in response to leptin. SH2-B directly bound, via its PH and SH2 domain, to both IRS1 and IRS2 both in vitro and in intact cells and mediated formation of a JAK2/SH2-B/IRS1 or IRS2 tertiary complex. Consequently, SH2-B dramatically enhanced leptin-stimulated tyrosine phosphorylation of IRS1 and IRS2 in HEK293 cells stably expressing LRb, thus promoting association of IRS1 and IRS2 with the p85 regulatory subunit of PI 3-kinase and phosphorylation and activation of Akt. SH2-B mutants with lower affinity for IRS1 and IRS2 exhibited reduced ability to promote association of JAK2 with IRS1, tyrosine phosphorylation of IRS1, and association of IRS1 with p85 in response to leptin. Moreover, deletion of the SH2-B gene impaired leptin-stimulated tyrosine phosphorylation of endogenous IRS1 in mouse embryonic fibroblasts (MEF), which was reversed by reintroduction of SH2-B. Similarly, SH2-B promoted growth hormone-stimulated tyrosine phosphorylation of IRS1 in both HEK293 and MEF cells. Our data suggest that SH2-B is a novel mediator of the PI 3-kinase pathway in response to leptin or other hormones and cytokines that activate JAK2.

Identification of Tyrosine 972 as a Novel Site of Jak2 Tyrosine Kinase Phosphorylation and its Role in Jak2 Activation

PubMed Central

McDoom, Issam; Ma, Xianyue; Kirabo, Annet; Lee, Kuang-Yung; Ostrov, David A.; Sayeski, Peter P.

2013-01-01

Jak2 is a 130 kDa tyrosine kinase that is important in a number of cellular signaling pathways. Its function is intrinsically regulated by the phosphorylation of a handful of its 49 tyrosines. Here, we report that tyrosine 972 (Y972) is a novel site of Jak2 phosphorylation, and hence auto-regulation. Specifically, we found that Y972 is phosphorylated and confirmed that this residue resides on the surface of the protein. Using expression plasmids that expressed either wild type Jak2 or a full length Jak2 cDNA containing a single Y972F substitution mutation, we investigated the consequences of losing Y972 phosphorylation on Jak2 function. We determined that the loss of Y972 phosphorylation significantly reduced both Jak2 total tyrosine phosphorylation and phosphorylation of Y1007/Y1008. Additionally, Y972 phosphorylation was shown to be important for maximal kinase function. Interestingly, in response to classical cytokine activation, the Jak2-Y972F mutant exhibited a moderately impaired level of activation when compared to wild type protein. However, when Jak2 was activated via a GPCR ligand, the ability of the Y972F mutant to activate was completely lost, therefore suggesting a differential role of Y972 in Jak2 activation. Finally, we found that phosphorylation of Y972 enhances Jak2 kinase function via a mechanism that appears to stabilize the active conformation of the protein. Collectively, our results suggest that Y972 is a novel site of Jak2 phosphorylation and plays an important differential role in ligand-dependent Jak2 activation via a mechanism that involves stabilization of the Jak2 active conformation. PMID:18636744

Regulation of NADH/CoQ oxidoreductase: do phosphorylation events affect activity?

PubMed

Maj, Mary C; Raha, Sandeep; Myint, Tomoko; Robinson, Brian H

2004-01-01

We had previously suggested that phosphorylation of proteins by mitochondrial kinases regulate the activity of NADH/CoQ oxidoreductase. Initial data showed that pyruvate dehydrogenase kinase (PDK) and cAMP-dependent protein kinase A (PKA) phosphorylate mitochondrial membrane proteins. Upon phosphorylation with crude PDK, mitochondria appeared to be deficient in NADH/cytochrome c reductase activity associated with increased superoxide production. Conversely, phosphorylation by PKA resulted in increased NADH/cytochrome c reductase activity and decreased superoxide formation. Current data confirms PKA involvement in regulating Complex I activity through phosphorylation of an 18 kDa subunit. Beef heart NADH/ cytochrome c reductase activity increases to 150% of control upon incubation with PKA and ATP-gamma-S. We have cloned the four human isoforms of PDK and purified beef heart Complex I. Incubation of mitochondria with PDK isoforms and ATP did not alter Complex I activity or superoxide production. Radiolabeling of mitochondria and purified Complex I with PDK failed to reveal phosphorylated proteins.

ABA-Induced Stomatal Closure Involves ALMT4, a Phosphorylation-Dependent Vacuolar Anion Channel of Arabidopsis[OPEN

PubMed Central

Baetz, Ulrike; Huck, Nicola V.; Zhang, Jingbo

2017-01-01

Stomatal pores are formed between a pair of guard cells and allow plant uptake of CO2 and water evaporation. Their aperture depends on changes in osmolyte concentration of guard cell vacuoles, specifically of K+ and Mal2−. Efflux of Mal2− from the vacuole is required for stomatal closure; however, it is not clear how the anion is released. Here, we report the identification of ALMT4 (ALUMINUM ACTIVATED MALATE TRANSPORTER4) as an Arabidopsis thaliana ion channel that can mediate Mal2− release from the vacuole and is required for stomatal closure in response to abscisic acid (ABA). Knockout mutants showed impaired stomatal closure in response to the drought stress hormone ABA and increased whole-plant wilting in response to drought and ABA. Electrophysiological data show that ALMT4 can mediate Mal2− efflux and that the channel activity is dependent on a phosphorylatable C-terminal serine. Dephosphomimetic mutants of ALMT4 S382 showed increased channel activity and Mal2− efflux. Reconstituting the active channel in almt4 mutants impaired growth and stomatal opening. Phosphomimetic mutants were electrically inactive and phenocopied the almt4 mutants. Surprisingly, S382 can be phosphorylated by mitogen-activated protein kinases in vitro. In brief, ALMT4 likely mediates Mal2− efflux during ABA-induced stomatal closure and its activity depends on phosphorylation. PMID:28874508

Synemin promotes AKT-dependent glioblastoma cell proliferation by antagonizing PP2A.

PubMed

Pitre, Aaron; Davis, Nathan; Paul, Madhumita; Orr, A Wayne; Skalli, Omar

2012-04-01

The intermediate filament protein synemin is present in astrocyte progenitors and glioblastoma cells but not in mature astrocytes. Here we demonstrate a role for synemin in enhancing glioblastoma cell proliferation and clonogenic survival, as synemin RNA interference decreased both behaviors by inducing G1 arrest along with Rb hypophosphorylation and increased protein levels of the G1/S inhibitors p21(Cip1) and p27(Kip1). Akt involvement was demonstrated by decreased phosphorylation of its substrate, p21(Cip1), and reduced Akt catalytic activity and phosphorylation at essential activation sites. Synemin silencing, however, did not affect the activities of PDPK1 and mTOR complex 2, which directly phosphorylate Akt activation sites, but instead enhanced the activity of the major regulator of Akt dephosphorylation, protein phosphatase type 2A (PP2A). This was accompanied by changes in PP2A subcellular distribution resulting in increased physical interactions between PP2A and Akt, as shown by proximity ligation assays (PLAs). PLAs and immunoprecipitation experiments further revealed that synemin and PP2A form a protein complex. In addition, treatment of synemin-silenced cells with the PP2A inhibitor cantharidic acid resulted in proliferation and pAkt and pRb levels similar to those of controls. Collectively these results indicate that synemin positively regulates glioblastoma cell proliferation by helping sequester PP2A away from Akt, thereby favoring Akt activation.

A truncated, activin-induced Smad3 isoform acts as a transcriptional repressor of FSHβ expression in mouse pituitary

PubMed Central

Kim, So-Youn; Zhu, Jie; Woodruff, Teresa K.

2011-01-01

The receptor-regulated protein Smad3 is key player in the signaling cascade stimulated by the binding of activin to its cell surface receptor. Upon phosphorylation, Smad3 forms a heterocomplex with Smad2 and Smad4, translocates to the nucleus and acts as a transcriptional co-activator. We have identified a unique isoform of Smad3 that is expressed in mature pituitary gonadotropes. 5' RACE revealed that this truncated Smad3 isoform is transcribed from an ATG site within exon 4 and consists of 7 exons encoding half of the linker region and the MH2 region. In pituitary cells, the truncated Smad3 isoform was phosphorylated upon activin treatment, in a manner that was temporally distinct from the phosphorylation of full-length Smad3. Activin-induced phosphorylation of Smad3 and the truncated Smad3 isoform was blocked by both follistatin and siRNA-mediated knockdown of Smad3. The truncated Smad3 isoform antagonized Smad3-mediated, activin-responsive promoter activity. We propose that the pituitary gonadotrope contains an ultra-short, activin-responsive feedback loop utilizing two different isoforms of Smad3, one which acts as an agonist (Smad3) and another that acts as an intracrine antagonist (truncated Smad3 isoform) to regulate FSHβ production. PMID:21664424

Nutrient-regulated Phosphorylation of ATG13 Inhibits Starvation-induced Autophagy*

PubMed Central

Puente, Cindy; Hendrickson, Ronald C.; Jiang, Xuejun

2016-01-01

Autophagy is a conserved catabolic process that utilizes a defined series of membrane trafficking events to generate a de novo double-membrane vesicle termed the autophagosome, which matures by fusing to the lysosome. Subsequently, the lysosome facilitates the degradation and recycling of the cytoplasmic cargo. In yeast, the upstream signals that regulate the induction of starvation-induced autophagy are clearly defined. The nutrient-sensing kinase Tor inhibits the activation of autophagy by regulating the formation of the Atg1-Atg13-Atg17 complex, through hyperphosphorylation of Atg13. However, in mammals, the ortholog complex ULK1-ATG13-FIP200 is constitutively formed. As such, the molecular mechanism by which mTOR regulates mammalian autophagy is unknown. Here we report the identification and characterization of novel nutrient-regulated phosphorylation sites on ATG13: Ser-224 and Ser-258. mTOR directly phosphorylates ATG13 on Ser-258 while Ser-224 is modulated by the AMPK pathway. In ATG13 knock-out cells reconstituted with an unphosphorylatable mutant of ATG13, ULK1 kinase activity is more potent, and amino acid starvation induced more rapid ATG13 and ULK1 translocation. These events culminated in a more rapid starvation-induced autophagy response. Therefore, ATG13 phosphorylation plays a crucial role in autophagy regulation. PMID:26801615

Receptor Type Protein Tyrosine Phosphatase ζ-Pleiotrophin Signaling Controls Endocytic Trafficking of DNER That Regulates Neuritogenesis▿ †

PubMed Central

Fukazawa, Nobuna; Yokoyama, Seisuke; Eiraku, Mototsugu; Kengaku, Mineko; Maeda, Nobuaki

2008-01-01

Protein tyrosine phosphatase ζ (PTPζ) is a receptor type protein tyrosine phosphatase that uses pleiotrophin as a ligand. Pleiotrophin inactivates the phosphatase activity of PTPζ, resulting in the increase of tyrosine phosphorylation levels of its substrates. We studied the functional interaction between PTPζ and DNER, a Notch-related transmembrane protein highly expressed in cerebellar Purkinje cells. PTPζ and DNER displayed patchy colocalization in the dendrites of Purkinje cells, and immunoprecipitation experiments indicated that these proteins formed complexes. Several tyrosine residues in and adjacent to the tyrosine-based and the second C-terminal sorting motifs of DNER were phosphorylated and were dephosphorylated by PTPζ, and phosphorylation of these tyrosine residues resulted in the accumulation of DNER on the plasma membrane. DNER mutants lacking sorting motifs accumulated on the plasma membrane of Purkinje cells and Neuro-2A cells and induced their process extension. While normal DNER was actively endocytosed and inhibited the retinoic-acid-induced neurite outgrowth of Neuro-2A cells, pleiotrophin stimulation increased the tyrosine phosphorylation level of DNER and suppressed the endocytosis of this protein, which led to the reversal of this inhibition, thus allowing neurite extension. These observations suggest that pleiotrophin-PTPζ signaling controls subcellular localization of DNER and thereby regulates neuritogenesis. PMID:18474614

sNASP inhibits TLR signaling to regulate immune response in sepsis.

PubMed

Yang, Feng-Ming; Zuo, Yong; Zhou, Wei; Xia, Chuan; Hahm, Bumsuk; Sullivan, Mark; Cheng, Jinke; Chang, Hui-Ming; Yeh, Edward Th

2018-06-01

Many Toll-like receptors (TLRs) signal through TNF receptor-associated factor 6 (TRAF6) to activate innate immune responses. Here, we show that somatic nuclear autoantigenic sperm protein (sNASP) binds to TRAF6 to prevent TRAF6 autoubiquitination in unstimulated macrophages. Following LPS stimulation, a complex consisting of sNASP, TRAF6, IRAK4, and casein kinase 2 (CK2) is formed. CK2 phosphorylates sNASP at serine 158, allowing sNASP to dissociate from TRAF6. Free TRAF6 is then autoubiquitinated, followed by activation of downstream signaling pathways. In sNasp S158A knockin (S158A-KI) mice, LPS-treated macrophages could not phosphorylate sNASP, which remained bound to TRAF6. S158A-KI mice were more susceptible to sepsis due to a marked reduction in IL-1β, TNF-α, and IFN-γ production accompanied by an inability to clear bacteria and recruit leukocytes. Furthermore, phosphorylation-regulated release of sNASP from TRAF6 is observed following activation of TLR-1, -2, -4, -5, and -6. Thus, sNASP is a negative regulator of TLR signaling to modulate the innate immune response.

The E3 ubiquitin ligase ZNRF2 is a substrate of mTORC1 and regulates its activation by amino acids

PubMed Central

Hoxhaj, Gerta; Caddye, Edward; Najafov, Ayaz; Houde, Vanessa P; Johnson, Catherine; Dissanayake, Kumara; Toth, Rachel; Campbell, David G; Prescott, Alan R; MacKintosh, Carol

2016-01-01

The mechanistic Target of Rapamycin complex 1 (mTORC1) senses intracellular amino acid levels through an intricate machinery, which includes the Rag GTPases, Ragulator and vacuolar ATPase (V-ATPase). The membrane-associated E3 ubiquitin ligase ZNRF2 is released into the cytosol upon its phosphorylation by Akt. In this study, we show that ZNRF2 interacts with mTOR on membranes, promoting the amino acid-stimulated translocation of mTORC1 to lysosomes and its activation in human cells. ZNRF2 also interacts with the V-ATPase and preserves lysosomal acidity. Moreover, knockdown of ZNRF2 decreases cell size and cell proliferation. Upon growth factor and amino acid stimulation, mTORC1 phosphorylates ZNRF2 on Ser145, and this phosphosite is dephosphorylated by protein phosphatase 6. Ser145 phosphorylation stimulates vesicle-to-cytosol translocation of ZNRF2 and forms a novel negative feedback on mTORC1. Our findings uncover ZNRF2 as a component of the amino acid sensing machinery that acts upstream of Rag-GTPases and the V-ATPase to activate mTORC1. DOI: http://dx.doi.org/10.7554/eLife.12278.001 PMID:27244671

Oncogenic Receptor Tyrosine Kinases Directly Phosphorylate Focal Adhesion Kinase (FAK) as a Resistance Mechanism to FAK-Kinase Inhibitors.

PubMed

Marlowe, Timothy A; Lenzo, Felicia L; Figel, Sheila A; Grapes, Abigail T; Cance, William G

2016-12-01

Focal adhesion kinase (FAK) is a major drug target in cancer and current inhibitors targeted to the ATP-binding pocket of the kinase domain have entered clinical trials. However, preliminary results have shown limited single-agent efficacy in patients. Despite these unfavorable data, the molecular mechanisms that drive intrinsic and acquired resistance to FAK-kinase inhibitors are largely unknown. We have demonstrated that receptor tyrosine kinases (RTK) can directly bypass FAK-kinase inhibition in cancer cells through phosphorylation of FAK's critical tyrosine 397 (Y397). We also showed that HER2 forms a direct protein-protein interaction with the FAK-FERM-F1 lobe, promoting direct phosphorylation of Y397. In addition, FAK-kinase inhibition induced two forms of compensatory RTK reprogramming: (i) the rapid phosphorylation and activation of RTK signaling pathways in RTK High cells and (ii) the long-term acquisition of RTKs novel to the parental cell line in RTK Low cells. Finally, HER2 +: cancer cells displayed resistance to FAK-kinase inhibition in 3D growth assays using a HER2 isogenic system and HER2 + cancer cell lines. Our data indicate a novel drug resistance mechanism to FAK-kinase inhibitors whereby HER2 and other RTKs can rescue and maintain FAK activation (pY397) even in the presence of FAK-kinase inhibition. These data may have important ramifications for existing clinical trials of FAK inhibitors and suggest that individual tumor stratification by RTK expression would be important to predict patient response to FAK-kinase inhibitors. Mol Cancer Ther; 15(12); 3028-39. ©2016 AACR. ©2016 American Association for Cancer Research.

Hydrophobic motif site-phosphorylated protein kinase CβII between mTORC2 and Akt regulates high glucose-induced mesangial cell hypertrophy.

PubMed

Das, Falguni; Ghosh-Choudhury, Nandini; Mariappan, Meenalakshmi M; Kasinath, Balakuntalam S; Choudhury, Goutam Ghosh

2016-04-01

PKCβII controls the pathologic features of diabetic nephropathy, including glomerular mesangial cell hypertrophy. PKCβII contains the COOH-terminal hydrophobic motif site Ser-660. Whether this hydrophobic motif phosphorylation contributes to high glucose-induced mesangial cell hypertrophy has not been determined. Here we show that, in mesangial cells, high glucose increased phosphorylation of PKCβII at Ser-660 in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. Using siRNAs to downregulate PKCβII, dominant negative PKCβII, and PKCβII hydrophobic motif phosphorylation-deficient mutant, we found that PKCβII regulates activation of mechanistic target of rapamycin complex 1 (mTORC1) and mesangial cell hypertrophy by high glucose. PKCβII via its phosphorylation at Ser-660 regulated phosphorylation of Akt at both catalytic loop and hydrophobic motif sites, resulting in phosphorylation and inactivation of its substrate PRAS40. Specific inhibition of mTORC2 increased mTORC1 activity and induced mesangial cell hypertrophy. In contrast, inhibition of mTORC2 decreased the phosphorylation of PKCβII and Akt, leading to inhibition of PRAS40 phosphorylation and mTORC1 activity and prevented mesangial cell hypertrophy in response to high glucose; expression of constitutively active Akt or mTORC1 restored mesangial cell hypertrophy. Moreover, constitutively active PKCβII reversed the inhibition of high glucose-stimulated Akt phosphorylation and mesangial cell hypertrophy induced by suppression of mTORC2. Finally, using renal cortexes from type 1 diabetic mice, we found that increased phosphorylation of PKCβII at Ser-660 was associated with enhanced Akt phosphorylation and mTORC1 activation. Collectively, our findings identify a signaling route connecting PI3-kinase to mTORC2 to phosphorylate PKCβII at the hydrophobic motif site necessary for Akt phosphorylation and mTORC1 activation, leading to mesangial cell hypertrophy.

Hydrophobic motif site-phosphorylated protein kinase CβII between mTORC2 and Akt regulates high glucose-induced mesangial cell hypertrophy

PubMed Central

Das, Falguni; Mariappan, Meenalakshmi M.; Kasinath, Balakuntalam S.; Choudhury, Goutam Ghosh

2016-01-01

PKCβII controls the pathologic features of diabetic nephropathy, including glomerular mesangial cell hypertrophy. PKCβII contains the COOH-terminal hydrophobic motif site Ser-660. Whether this hydrophobic motif phosphorylation contributes to high glucose-induced mesangial cell hypertrophy has not been determined. Here we show that, in mesangial cells, high glucose increased phosphorylation of PKCβII at Ser-660 in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. Using siRNAs to downregulate PKCβII, dominant negative PKCβII, and PKCβII hydrophobic motif phosphorylation-deficient mutant, we found that PKCβII regulates activation of mechanistic target of rapamycin complex 1 (mTORC1) and mesangial cell hypertrophy by high glucose. PKCβII via its phosphorylation at Ser-660 regulated phosphorylation of Akt at both catalytic loop and hydrophobic motif sites, resulting in phosphorylation and inactivation of its substrate PRAS40. Specific inhibition of mTORC2 increased mTORC1 activity and induced mesangial cell hypertrophy. In contrast, inhibition of mTORC2 decreased the phosphorylation of PKCβII and Akt, leading to inhibition of PRAS40 phosphorylation and mTORC1 activity and prevented mesangial cell hypertrophy in response to high glucose; expression of constitutively active Akt or mTORC1 restored mesangial cell hypertrophy. Moreover, constitutively active PKCβII reversed the inhibition of high glucose-stimulated Akt phosphorylation and mesangial cell hypertrophy induced by suppression of mTORC2. Finally, using renal cortexes from type 1 diabetic mice, we found that increased phosphorylation of PKCβII at Ser-660 was associated with enhanced Akt phosphorylation and mTORC1 activation. Collectively, our findings identify a signaling route connecting PI3-kinase to mTORC2 to phosphorylate PKCβII at the hydrophobic motif site necessary for Akt phosphorylation and mTORC1 activation, leading to mesangial cell hypertrophy. PMID:26739493

Mining protein phosphorylation information from biomedical literature using NLP parsing and Support Vector Machines.

PubMed

Raja, Kalpana; Natarajan, Jeyakumar

2018-07-01

Extraction of protein phosphorylation information from biomedical literature has gained much attention because of the importance in numerous biological processes. In this study, we propose a text mining methodology which consists of two phases, NLP parsing and SVM classification to extract phosphorylation information from literature. First, using NLP parsing we divide the data into three base-forms depending on the biomedical entities related to phosphorylation and further classify into ten sub-forms based on their distribution with phosphorylation keyword. Next, we extract the phosphorylation entity singles/pairs/triplets and apply SVM to classify the extracted singles/pairs/triplets using a set of features applicable to each sub-form. The performance of our methodology was evaluated on three corpora namely PLC, iProLink and hPP corpus. We obtained promising results of >85% F-score on ten sub-forms of training datasets on cross validation test. Our system achieved overall F-score of 93.0% on iProLink and 96.3% on hPP corpus test datasets. Furthermore, our proposed system achieved best performance on cross corpus evaluation and outperformed the existing system with recall of 90.1%. The performance analysis of our unique system on three corpora reveals that it extracts protein phosphorylation information efficiently in both non-organism specific general datasets such as PLC and iProLink, and human specific dataset such as hPP corpus. Copyright © 2018 Elsevier B.V. All rights reserved.

Cdk5 phosphorylation of WAVE2 regulates oligodendrocyte precursor cell migration through nonreceptor tyrosine kinase Fyn.

PubMed

Miyamoto, Yuki; Yamauchi, Junji; Tanoue, Akito

2008-08-13

Myelin formation of the CNS is a complex and dynamic process. Before the onset of myelination, oligodendrocytes (OLs), the myelin-forming glia of the CNS, proliferate and migrate along axons. Little is known about the molecular mechanisms underlying the early myelination processes. Here, we show that platelet-derived growth factor (PDGF), the crucial physiological ligand in early OL development, controls the migration of oligodendrocyte precursor cells (OPCs) through cyclin-dependent kinase 5 (Cdk5). PDGF stimulates Cdk5 activity in a time-dependent manner, whereas suppression of Cdk5 by the specific inhibitor roscovitine or by the retrovirus encoding short-hairpin RNA for Cdk5 impairs PDGF-dependent OPC migration. The activation of Cdk5 by PDGF is mediated by the phosphorylation of the nonreceptor tyrosine kinase, Fyn, whose inhibition reduces PDGF-dependent OPC migration. Furthermore, Cdk5 regulates PDGF-dependent OPC migration through the direct phosphorylation of WASP (Wiskott-Aldrich syndrome protein)-family verprolin-homologous protein 2 (WAVE2). Cdk5 phosphorylates WAVE2 at Ser-137 in vitro. Infection of the WAVE2 construct harboring the Ser-137-to-Ala reduces PDGF-dependent migration. Together, PDGF regulates OPC migration through an as-yet-unidentified signaling cascade coupling Fyn kinase to Cdk5 phosphorylation of WAVE2. These results provide new insights into both the role of Cdk5 in glial cells and the molecular mechanisms controlling the early developmental stage of OLs.

Effect of phosphorylation on antioxidant activities of pumpkin (Cucurbita pepo, Lady godiva) polysaccharide.

PubMed

Song, Yi; Ni, Yuanying; Hu, Xiaosong; Li, Quanhong

2015-11-01

Phosphorylated derivatives of pumpkin polysaccharide with different degree of substitution were synthesized using POCl3 and pyridine. Antioxidant activities and cytoprotective effects of unmodified polysaccharide and phosphorylated derivatives were investigated employing various in vitro systems. Results showed that high ratio of POCl3/pyridine could increase the degree of substitution and no remarkable degradation occurred in the phosphorylation process. Characteristic absorption of phosphorylation appeared both in the IR and (31)P NMR spectrum. The df values between 2.27 and 2.55 indicated the relatively expanded conformation of the phosphorylated derivatives. All the phosphorylated polysaccharides exhibited higher antioxidant activities. H2O2-induced oxidative damages on rat thymic lymphocyte were also prevented by the derivatives. In general, phosphorylation could improve the antioxidant activities of pumpkin polysaccharide both in vitro and in a cell system. Copyright © 2015 Elsevier B.V. All rights reserved.

The serine 814 of TRPC6 is phosphorylated under unstimulated conditions.

PubMed

Bousquet, Simon M; Monet, Michael; Boulay, Guylain

2011-03-23

TRPC are nonselective cation channels involved in calcium entry. Their regulation by phosphorylation has been shown to modulate their routing and activity. TRPC6 activity increases following phosphorylation by Fyn, and is inhibited by protein kinase G and protein kinase C. A previous study by our group showed that TRPC6 is phosphorylated under unstimulated conditions in a human embryonic kidney cells line (HEK293). To investigate the mechanism responsible for this phosphorylation, we used a MS/MS approach combined with metabolic labeling and showed that the serine at position 814 is phosphorylated in unstimulated cells. The mutation of Ser(814) into Ala decreased basal phosphorylation but did not modify TRPC6 activity. Even though Ser(814) is within a consensus site for casein kinase II (CK2), we showed that CK2 is not involved in the phosphorylation of TRPC6 and does not modify its activity. In summary, we identified a new basal phosphorylation site (Ser(814)) on TRPC6 and showed that CK2 is not responsible for the phosphorylation of this site.

The Serine 814 of TRPC6 Is Phosphorylated under Unstimulated Conditions

PubMed Central

Bousquet, Simon M.; Monet, Michael; Boulay, Guylain

2011-01-01

TRPC are nonselective cation channels involved in calcium entry. Their regulation by phosphorylation has been shown to modulate their routing and activity. TRPC6 activity increases following phosphorylation by Fyn, and is inhibited by protein kinase G and protein kinase C. A previous study by our group showed that TRPC6 is phosphorylated under unstimulated conditions in a human embryonic kidney cells line (HEK293). To investigate the mechanism responsible for this phosphorylation, we used a MS/MS approach combined with metabolic labeling and showed that the serine at position 814 is phosphorylated in unstimulated cells. The mutation of Ser814 into Ala decreased basal phosphorylation but did not modify TRPC6 activity. Even though Ser814 is within a consensus site for casein kinase II (CK2), we showed that CK2 is not involved in the phosphorylation of TRPC6 and does not modify its activity. In summary, we identified a new basal phosphorylation site (Ser814) on TRPC6 and showed that CK2 is not responsible for the phosphorylation of this site. PMID:21448286

Identification of in vivo phosphorylation sites on human deoxycytidine kinase. Role of Ser-74 in the control of enzyme activity.

PubMed

Smal, Caroline; Vertommen, Didier; Bertrand, Luc; Ntamashimikiro, Sandrine; Rider, Mark H; Van Den Neste, Eric; Bontemps, Françoise

2006-02-24

Deoxycytidine kinase (dCK) catalyzes the rate-limiting step of the deoxyribonucleoside salvage pathway in mammalian cells and plays a key role in the activation of numerous nucleoside analogues used in anti-cancer and antiviral chemotherapy. Although compelling evidence indicated that dCK activity might be regulated by phosphorylation/dephosphorylation, direct demonstration was lacking. Here we showed that dCK overexpressed in HEK 293T cells was labeled after incubating the cells with [32P]orthophosphate. Sorbitol, which was reported to decrease dCK activity, also decreased the labeling of dCK. These results indicated that dCK may exist as a phosphoprotein in vivo and that its activity can be correlated with its phosphorylation level. After purification of 32P-labeled dCK, digestion by trypsin, and analysis of the radioactive peptides by tandem mass spectrometry, the following four in vivo phosphorylation sites were identified: Thr-3, Ser-11, Ser-15, and Ser-74, the latter being the major phosphorylation site. Site-directed mutagenesis and use of an anti-phospho-Ser-74 antibody demonstrated that Ser-74 phosphorylation was crucial for dCK activity in HEK 293T cells, whereas phosphorylation of other identified sites did not seem essential. Phosphorylation of Ser-74 was also detected on endogenous dCK in leukemic cells, in which the Ser-74 phosphorylation state was increased by agents that enhanced dCK activity. Our study provided direct evidence that dCK activity can be controlled by phosphorylation in intact cells and highlights the importance of Ser-74 for dCK activity.

Activation of Escherichia coli antiterminator BglG requires its phosphorylation

PubMed Central

Rothe, Fabian M.; Bahr, Thomas; Stülke, Jörg; Rak, Bodo; Görke, Boris

2012-01-01

Transcriptional antiterminator proteins of the BglG family control the expression of enzyme II (EII) carbohydrate transporters of the bacterial phosphotransferase system (PTS). In the PTS, phosphoryl groups are transferred from phosphoenolpyruvate (PEP) via the phosphotransferases enzyme I (EI) and HPr to the EIIs, which phosphorylate their substrates during transport. Activity of the antiterminators is negatively controlled by reversible phosphorylation catalyzed by the cognate EIIs in response to substrate availability and positively controlled by the PTS. For the Escherichia coli BglG antiterminator, two different mechanisms for activation by the PTS were proposed. According to the first model, BglG is activated by HPr-catalyzed phosphorylation at a site distinct from the EII-dependent phosphorylation site. According to the second model, BglG is not activated by phosphorylation, but solely through interaction with EI and HPr, which are localized at the cell pole. Subsequently BglG is released from the cell pole to the cytoplasm as an active dimer. Here we addressed this discrepancy and found that activation of BglG requires phosphorylatable HPr or the HPr homolog FruB in vivo. Further, we uniquely demonstrate that purified BglG protein becomes phosphorylated by FruB as well as by HPr in vitro. Histidine residue 208 in BglG is essential for this phosphorylation. These data suggest that BglG is in fact activated by phosphorylation and that there is no principal difference between the PTS-exerted mechanisms controlling the activities of BglG family proteins in Gram-positive and Gram-negative bacteria. PMID:22984181

Mammalian transcription factor LSF is a target of ERK signaling

PubMed Central

Pagon, Zrinka; Volker, Janet; Cooper, Geoffrey M.; Hansen, Ulla

2012-01-01

LSF is a mammalian transcription factor that is rapidly and quantitatively phosphorylated upon growth induction of resting, peripheral human T cells, as assayed by a reduction in its electrophoretic mobility. The DNA-binding activity of LSF in primary T cells is greatly increased after this phosphorylation event [Volker et al., 1997]. We demonstrate here that LSF is also rapidly and quantitatively phosphorylated upon growth induction in NIH 3T3 cells, although its DNA-binding activity is not significantly altered. Three lines of experimentation established that ERK is responsible for phosphorylating LSF upon growth induction in both cell types. First, phosphorylation of LSF by ERK is sufficient to cause the reduced electrophoretic mobility of LSF. Second, the amount of ERK activity correlates with the extent of LSF phosphorylation in both primary human T cells and NIH 3T3 cells. Finally, specific inhibitors of the Ras/Raf/MEK/ERK pathway inhibit LSF modification in vivo. This phosphorylation by ERK is not sufficient for activation of LSF DNA-binding activity, as evidenced both in vitro and in mouse fibroblasts. Nonetheless, activation of ERK is a prerequisite for the substantial increase in LSF DNA-binding activity upon activation of resting T cells, indicating that ERK phosphorylation is necessary but not sufficient for activation of LSF in this cell type. PMID:12858339

Phosphorylation of serine-515 activates the Mammalian maintenance methyltransferase Dnmt1.

PubMed

Goyal, Rachna; Rathert, Philipp; Laser, Heike; Gowher, Humaira; Jeltsch, Albert

2007-09-01

DNA methyltransferase 1 methylates hemi-methylated CG sites generated during DNA replication. Serine 515 of this enzyme has been shown to be phosphorylated. To explore the importance of S515 phosphorylation, we generated mutants of Dnmt1 which removed the phosphorylation potential (S515A) or mimic phosphoserine (S515E), purified the proteins from insect cells and analyzed their DNA methylation activity in vitro. The S515E mutant was found to be active, while S515A mutant had severe loss in activity when compared to the wild type protein. The loss of activity of the S515A variant was not due to loss of DNA binding capacity. Furthermore, we show that a phosphorylated peptide whose sequence mimics the surrounding of Ser515 (EKIYIS(P)KIVVE) inhibited the activity of wild type Dnmt1 ten-fold more than the non-phosphorylated peptide. The inhibition was specific for Dnmt1 and for the particular peptide sequence. Our data suggest that phosphorylation of Ser515 is important for an interaction between the N-terminal domain of Dnmt1 and its catalytic domain that is necessary for activity and that this interaction is specifically disrupted by the phosphorylated peptide. We conclude that phosphorylation of Dnmt1 at Ser515 could be an important regulator of Dnmt1 activity during cell cycle and after proliferative stimuli.

Identification of Phosphorylation Codes for Arrestin Recruitment by G Protein-Coupled Receptors.

PubMed

Zhou, X Edward; He, Yuanzheng; de Waal, Parker W; Gao, Xiang; Kang, Yanyong; Van Eps, Ned; Yin, Yanting; Pal, Kuntal; Goswami, Devrishi; White, Thomas A; Barty, Anton; Latorraca, Naomi R; Chapman, Henry N; Hubbell, Wayne L; Dror, Ron O; Stevens, Raymond C; Cherezov, Vadim; Gurevich, Vsevolod V; Griffin, Patrick R; Ernst, Oliver P; Melcher, Karsten; Xu, H Eric

2017-07-27

G protein-coupled receptors (GPCRs) mediate diverse signaling in part through interaction with arrestins, whose binding promotes receptor internalization and signaling through G protein-independent pathways. High-affinity arrestin binding requires receptor phosphorylation, often at the receptor's C-terminal tail. Here, we report an X-ray free electron laser (XFEL) crystal structure of the rhodopsin-arrestin complex, in which the phosphorylated C terminus of rhodopsin forms an extended intermolecular β sheet with the N-terminal β strands of arrestin. Phosphorylation was detected at rhodopsin C-terminal tail residues T336 and S338. These two phospho-residues, together with E341, form an extensive network of electrostatic interactions with three positively charged pockets in arrestin in a mode that resembles binding of the phosphorylated vasopressin-2 receptor tail to β-arrestin-1. Based on these observations, we derived and validated a set of phosphorylation codes that serve as a common mechanism for phosphorylation-dependent recruitment of arrestins by GPCRs. Copyright © 2017 Elsevier Inc. All rights reserved.

Retinoic acid signaling and mouse embryonic stem cell differentiation: Cross talk between genomic and non-genomic effects of RA.

PubMed

Rochette-Egly, Cécile

2015-01-01

Retinoic acid (RA), the active derivative of vitamin A, a fat-soluble vitamin, plays key roles in cell growth and differentiation by activating nuclear receptors, RARs (α, β and γ), which are ligand dependent regulators of transcription. The past years highlighted several novelties in the field that increased the complexity of RA effects. Indeed, in addition to its classical genomic effects, RA also has extranuclear and non-transcriptional effects. RA induces the rapid and transient activation of kinase cascades, which are integrated in the nucleus via the phosphorylation of RARs at a conserved serine residue located in the N-terminal domain and their coregulators. In order to investigate the relevance of RARs' phosphorylation in cell differentiation, mouse embryonic stem (mES) cells were used as a model. When treated with RA, these pluripotent cells give rise to neuronal cells. Cells invalidated for each RAR were generated as well as stable rescue lines expressing RARs mutated in phosphor acceptor sites. Such a strategy revealed that RA-induced neuronal differentiation involves the RARγ2 subtype and requires RARγ2 phosphorylation. Moreover, in gene expression profiling experiments, the phosphorylated form of RARγ2 was found to regulate a small subset of genes through binding a novel RA response element consisting of two direct repeats with a 7 base pair spacer. These new findings suggest an important role for RAR phosphorylation during cell differentiation, and pave the way for further investigations with other cell types and during embryonic development. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics. Copyright © 2014 Elsevier B.V. All rights reserved.

Crop milk protein is synthesised following activation of the IRS1/Akt/TOR signalling pathway in the domestic pigeon (Columba livia).

PubMed

Hu, X-C; Gao, C-Q; Wang, X-H; Yan, H-C; Chen, Z-S; Wang, X-Q

2016-12-01

The experiment was conducted to study whether insulin receptor substance 1 (IRS1) / Protein kinase B (Akt)/target of the rapamycin (TOR) signalling pathway activation stimulates crop milk protein synthesis in the domestic pigeon (Columba livia). Crop milk was collected from ten 1-d-old squabs and analysed for nutrient content. During the non-breeding period and the first day of lactation, blood samples were collected from 5 pairs of breeding pigeons and the levels of prolactin and insulin were determined. Crop samples were collected from 5 pairs of breeders at d 14 and 16 of the incubation period and d 1, 3 and 7 of the lactation period. Crop samples were evaluated for changes in crop weight and thickness and changes in the expression patterns of IRS1/Akt/TOR signalling pathway-related proteins. The results demonstrated that prolactin induces a gradual increase in the relative weight and thickness of the crop, with crops reaching a maximum size at the third day of lactation. Pigeon crop milk contains 64.1% crude protein and 29.7% crude fat based on dry weight. Serum prolactin and insulin levels in the lactation period were significantly higher than those in the non-breeding period. Compared with non-breeding pigeons, the expression of the phosphorylated IRS1 phosphorylated Akt, phosphorylated TOR, phosphorylated ribosomal protein S6 kinase, phosphorylated S6, phosphorylated eukaryotic initiation factor 4E binding protein 1 and eukaryotic initiation factor 4E were significantly up-regulated in the crop of pigeons in the lactation period. In conclusion, prolactin might induce changes in crop tissue and form the physiological structure for crop milk synthesis. Furthermore, the synthesis of crop milk protein is regulated by activation of the IRS1/Akt/TOR signalling pathway.

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: II. Mutational analysis.

PubMed

Kitani, T; Okuno, S; Fujisawa, H

2001-10-01

We previously reported that rat brain Ca(2+)/calmodulin-dependent protein kinase (CaM-kinase) IV is inactivated by cAMP-dependent protein kinase (PKA) [Kameshita, I. and Fujisawa, H. (1991) Biochem. Biophys. Res. Commun. 180, 191-196]. In the preceding paper, we demonstrated that changes in the activity of CaM-kinase IV by PKA results from the phosphorylation of CaM-kinase kinase alpha by PKA and identified six phosphorylation sites, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA. In the present study, a causal relationship between the phosphorylation and change in the activity toward PKIV peptide has been studied using mutant enzymes with amino acid substitutions at the six phosphorylation sites. The following conclusions can be drawn from the experimental results: (i) Phosphorylation of Ser74 and/or unidentified sites causes an increase in activity; (ii) phosphorylation of Thr(108) or Ser(458) causes a decrease in the activity; (iii) the inhibitory effect of the phosphorylation of Thr(108) is canceled by the stimulatory effect of the phosphorylation, but that of Ser(458) is not; and (iv) the inhibitory effects of Thr(108) and Ser(458) are synergistic. In contrast to the activity toward PKIV peptide, the activity toward CaM-kinase IV appears to be decreased by the phosphorylation of Thr(108), but not significantly affected by the phosphorylation of Ser(458).

In vivo roles for myosin phosphatase targeting subunit-1 phosphorylation sites T694 and T852 in bladder smooth muscle contraction

PubMed Central

Chen, Cai-Ping; Chen, Xin; Qiao, Yan-Ning; Wang, Pei; He, Wei-Qi; Zhang, Cheng-Hai; Zhao, Wei; Gao, Yun-Qian; Chen, Chen; Tao, Tao; Sun, Jie; Wang, Ye; Gao, Ning; Kamm, Kristine E; Stull, James T; Zhu, Min-Sheng

2015-01-01

Force production and maintenance in smooth muscle is largely controlled by different signalling modules that fine tune myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. To investigate the regulation of MLCP activity in vivo, we analysed the role of two phosphorylation sites on MYPT1 (regulatory subunit of MLCP) that biochemically inhibit MLCP activity in vitro. MYPT1 is constitutively phosphorylated at T694 by unidentified kinases in vivo, whereas the T852 site is phosphorylated by RhoA-associated protein kinase (ROCK). We established two mouse lines with alanine substitution of T694 or T852. Isolated bladder smooth muscle from T852A mice displayed no significant changes in RLC phosphorylation or force responses, but force was inhibited with a ROCK inhibitor. In contrast, smooth muscles containing the T694A mutation showed a significant reduction of force along with reduced RLC phosphorylation. The contractile responses of T694A mutant smooth muscle were also independent of ROCK activation. Thus, phosphorylation of MYPT1 T694, but not T852, is a primary mechanism contributing to inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. The constitutive phosphorylation of MYPT1 T694 may provide a mechanism for regulating force maintenance of smooth muscle. Key points Force production and maintenance in smooth muscle is largely controlled by myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro. Here we separately investigated the contribution of these two phosphorylation sites in bladder smooth muscles by establishing two single point mutation mouse lines, T694A and T852A, and found that phosphorylation of MYPT1 T694, but not T852, mediates force maintenance via inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. Our findings reveal the role of MYPT1 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction. PMID:25433069

In vivo roles for myosin phosphatase targeting subunit-1 phosphorylation sites T694 and T852 in bladder smooth muscle contraction.

PubMed

Chen, Cai-Ping; Chen, Xin; Qiao, Yan-Ning; Wang, Pei; He, Wei-Qi; Zhang, Cheng-Hai; Zhao, Wei; Gao, Yun-Qian; Chen, Chen; Tao, Tao; Sun, Jie; Wang, Ye; Gao, Ning; Kamm, Kristine E; Stull, James T; Zhu, Min-Sheng

2015-02-01

Force production and maintenance in smooth muscle is largely controlled by myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro. Here we separately investigated the contribution of these two phosphorylation sites in bladder smooth muscles by establishing two single point mutation mouse lines, T694A and T852A, and found that phosphorylation of MYPT1 T694, but not T852, mediates force maintenance via inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. Our findings reveal the role of MYPT1 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction. Force production and maintenance in smooth muscle is largely controlled by different signalling modules that fine tune myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. To investigate the regulation of MLCP activity in vivo, we analysed the role of two phosphorylation sites on MYPT1 (regulatory subunit of MLCP) that biochemically inhibit MLCP activity in vitro. MYPT1 is constitutively phosphorylated at T694 by unidentified kinases in vivo, whereas the T852 site is phosphorylated by RhoA-associated protein kinase (ROCK). We established two mouse lines with alanine substitution of T694 or T852. Isolated bladder smooth muscle from T852A mice displayed no significant changes in RLC phosphorylation or force responses, but force was inhibited with a ROCK inhibitor. In contrast, smooth muscles containing the T694A mutation showed a significant reduction of force along with reduced RLC phosphorylation. The contractile responses of T694A mutant smooth muscle were also independent of ROCK activation. Thus, phosphorylation of MYPT1 T694, but not T852, is a primary mechanism contributing to inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. The constitutive phosphorylation of MYPT1 T694 may provide a mechanism for regulating force maintenance of smooth muscle. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

Extending Thymidine Kinase Activity to the Catalytic Repertoire of Human Deoxycytidine Kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hazra, Saugata; Sabini, Eliszbetta; Ort, Stephan

Salvage of nucleosides in the cytosol of human cells is carried out by deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1). Whereas TK1 is only responsible for thymidine phosphorylation, dCK is capable of converting dC, dA, and dG into their monophosphate forms. Using structural data on dCK, we predicted that select mutations at the active site would, in addition to making the enzyme faster, expand the catalytic repertoire of dCK to include thymidine. Specifically, we hypothesized that steric repulsion between the methyl group of the thymine base and Arg104 is the main factor preventing the phosphorylation of thymidine by wild-typemore » dCK. Here we present kinetic data on several dCK variants where Arg104 has been replaced by select residues, all performed in combination with the mutation of Asp133 to an alanine. We show that several hydrophobic residues at position 104 endow dCK with thymidine kinase activity. Depending on the exact nature of the mutations, the enzyme's substrate preference is modified. The R104M-D133A double mutant is a pyrimidine-specific enzyme due to large K{sub m} values with purines. The crystal structure of the double mutant R104M-D133A in complex with the L-form of thymidine supplies a structural explanation for the ability of this variant to phosphorylate thymidine and thymidine analogs. The replacement of Arg104 by a smaller residue allows L-dT to bind deeper into the active site, making space for the C5-methyl group of the thymine base. The unique catalytic properties of several of the mutants make them good candidates for suicide-gene/protein-therapy applications.« less

Akt phosphorylation regulates the tumour-suppressor merlin through ubiquitination and degradation.

PubMed

Tang, Xiaoling; Jang, Sung-Wuk; Wang, Xuerong; Liu, Zhixue; Bahr, Scott M; Sun, Shi-Yong; Brat, Daniel; Gutmann, David H; Ye, Keqiang

2007-10-01

The neurofibromatosis-2 (NF2) tumour-suppressor gene encodes an intracellular membrane-associated protein, called merlin, whose growth-suppressive function is dependent on its ability to form interactions through its intramolecular amino-terminal domain (NTD) and carboxy-terminal domain (CTD). Merlin phosphorylation plays a critical part in dictating merlin NTD/CTD interactions as well as in controlling binding to its effector proteins. Merlin is partially regulated by phosphorylation of Ser 518, such that hyperphosphorylated merlin is inactive and fails to form productive intramolecular and intermolecular interactions. Here, we show that the protein kinase Akt directly binds to and phosphorylates merlin on residues Thr 230 and Ser 315, which abolishes merlin NTD/CTD interactions and binding to merlin's effector protein PIKE-L and other binding partners. Furthermore, Akt-mediated phosphorylation leads to merlin degradation by ubiquitination. These studies demonstrate that Akt-mediated merlin phosphorylation regulates the function of merlin in the absence of an inactivating mutation.

Serine phosphorylation by SYK is critical for nuclear localization and transcription factor function of Ikaros

PubMed Central

Uckun, Fatih M.; Ma, Hong; Zhang, Jian; Ozer, Zahide; Dovat, Sinisa; Mao, Cheney; Ishkhanian, Rita; Goodman, Patricia; Qazi, Sanjive

2012-01-01

Ikaros is a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis through transcriptional regulation of the earliest stages of lymphocyte ontogeny and differentiation. Functional deficiency of Ikaros has been implicated in the pathogenesis of acute lymphoblastic leukemia, the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros activity is considered of paramount importance, but the operative molecular mechanisms responsible for its regulation remain largely unknown. Here we provide multifaceted genetic and biochemical evidence for a previously unknown function of spleen tyrosine kinase (SYK) as a partner and posttranslational regulator of Ikaros. We demonstrate that SYK phoshorylates Ikaros at unique C-terminal serine phosphorylation sites S358 and S361, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Mechanistically, we establish that SYK-induced Ikaros activation is essential for its nuclear localization and optimal transcription factor function. PMID:23071339

Oxidative stress increases eukaryotic initiation factor 4E phosphorylation in vascular cells.

PubMed Central

Duncan, Roger F; Peterson, Hazel; Hagedorn, Curt H; Sevanian, Alex

2003-01-01

Dysregulated cell growth can be caused by increased activity of protein synthesis eukaryotic initiation factor (eIF) 4E. Dysregulated cell growth is also characteristic of atherosclerosis. It is postulated that exposure of vascular cells, such as endothelial cells, smooth muscle cells and monocytes/macrophages, to oxidants, such as oxidized low-density lipoprotein (oxLDL), leads to the elaboration of growth factors and cytokines, which in turn results in smooth muscle cell hyperproliferation. To investigate whether activation of eIF4E might play a role in this hyperproliferative response, vascular cells were treated with oxLDL, oxidized lipid components of oxLDL and several model oxidants, including H(2)O(2) and dimethyl naphthoquinone. Exposure to each of these compounds led to a dose- and time-dependent increase in eIF4E phosphorylation in all three types of vascular cells, correlated with a modest increase in overall translation rate. No changes in eIF4EBP, eIF2 or eIF4B modification state were observed. Increased eIF4E phosphorylation was paralleled by increased presence of eIF4E in high-molecular-mass protein complexes characteristic of its most active form. Anti-oxidants at concentrations typically employed to block oxidant-induced cell signalling likewise promoted eIF4E phosphorylation. The results of this study indicate that increased eIF4E activity may contribute to the pathophysiological events in early atherogenesis by increasing the expression of translationally inefficient mRNAs encoding growth-promoting proteins. PMID:12215171

Influence of phosphorylation of THR-3, SER-11, and SER-15 on deoxycytidine kinase activity and stability.

PubMed

Smal, C; Ntamashimikiro, S; Arts, A; Van Den Neste, E; Bontemps, F

2010-06-01

Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxyribonucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We have recently shown that dCK is a phosphoprotein. Four in vivo phosphorylation sites were identified: Thr-3, Ser-11, Ser-15, and Ser-74. Site-directed mutagenesis demonstrated that phosphorylation of Ser-74, the major phosphorylated residue, strongly influences dCK activity in eucaryotic cells. Here, we show that phosphorylation of the three other sites, located in the N-terminal extremity of the protein, does not significantly modify dCK activity, but phosphorylation of Thr-3 could promote dCK stability.

δ-Tocopherol inhibits receptor tyrosine kinase-induced AKT activation in prostate cancer cells.

PubMed

Wang, Hong; Hong, Jungil; Yang, Chung S

2016-11-01

The cancer preventive activity of vitamin E is suggested by epidemiological studies and supported by animal studies with vitamin E forms, γ-tocopherol and δ-tocopherol (δ-T). Several recent large-scale cancer prevention trials with high dose of α-tocopherol, however, yielded disappointing results. Whether vitamin E prevents or promotes cancer is a serious concern. A better understanding of the molecular mechanisms of action of the different forms of tocopherols would enhance our understanding of this topic. In this study, we demonstrated that δ-T was the most effective tocopherol form in inhibiting prostate cancer cell growth, by inducing cell cycle arrest and apoptosis. By profiling the effects of δ-T on the cell signaling using the phospho-kinase array, we found that the most inhibited target was the phosphorylation of AKT on T308. Further study on the activation of AKT by EGFR and IGFR revealed that δ-T attenuated the EGF/IGF-induced activation of AKT (via the phosphorylation of AKT on T308 induced by the activation of PIK3). Expression of dominant active PIK3 and AKT in prostate cancer cell line DU145 in which PIK3, AKT, and PTEN are wild type caused the cells to be reflectory to the inhibition of δ-T, supporting that δ-T inhibits the PIK3-mediated activation of AKT. Our data also suggest that δ-T interferes with the EGF-induced EGFR internalization, which leads to the inhibition of the receptor tyrosine kinase-dependent activation of AKT. In summary, our results revealed a novel mechanism of δ-T in inhibiting prostate cancer cell growth, supporting the cancer preventive activity δ-T. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

HDAC2 phosphorylation-dependent Klf5 deacetylation and RARα acetylation induced by RAR agonist switch the transcription regulatory programs of p21 in VSMCs

PubMed Central

Zheng, Bin; Han, Mei; Shu, Ya-nan; Li, Ying-jie; Miao, Sui-bing; Zhang, Xin-hua; Shi, Hui-jing; Zhang, Tian; Wen, Jin-kun

2011-01-01

Abnormal proliferation of vascular smooth muscle cells (VSMCs) occurs in hypertension, atherosclerosis and restenosis after angioplasty, leading to pathophysiological vascular remodeling. As an important growth arrest gene, p21 plays critical roles in vascular remodeling. Regulation of p21 expression by retinoic acid receptor (RAR) and its ligand has important implications for control of pathological vascular remodeling. Nevertheless, the mechanism of RAR-mediated p21 expression in VSMCs remains poorly understood. Here, we show that, under basal conditions, RARα forms a complex with histone deacetylase 2 (HDAC2) and Krüppel-like factor 5 (Klf5) at the p21 promoter to inhibit its expression. Upon RARα agonist stimulation, HDAC2 is phosphorylated by CK2α. Phosphorylation of HDAC2, on the one hand, promotes its dissociation from RARα, thus allowing the liganded-RARα to interact with co-activators; on the other hand, it increases its interaction with Klf5, thus leading to deacetylation of Klf5. Deacetylation of Klf5 facilitates its dissociation from the p21 promoter, relieving its repressive effect on the p21 promoter. Interference with HDAC2 phosphorylation by either CK2α knockdown or the use of phosphorylation-deficient mutant of HDAC2 prevents the dissociation of Klf5 from the p21 promoter and impairs RAR agonist-induced p21 activation. Our results reveal a novel mechanism involving a phosphorylation-deacetylation cascade that functions to remove the basal repression complex from the p21 promoter upon RAR agonist treatment, allowing for optimum agonist-induced p21 expression. PMID:21383775

Generic temperature compensation of biological clocks by autonomous regulation of catalyst concentration

PubMed Central

Hatakeyama, Tetsuhiro S.; Kaneko, Kunihiko

2012-01-01

Circadian clocks—ubiquitous in life forms ranging from bacteria to multicellular organisms—often exhibit intrinsic temperature compensation; the period of circadian oscillators is maintained constant over a range of physiological temperatures, despite the expected Arrhenius form for the reaction coefficient. Observations have shown that the amplitude of the oscillation depends on the temperature but the period does not; this suggests that although not every reaction step is temperature independent, the total system comprising several reactions still exhibits compensation. Here we present a general mechanism for such temperature compensation. Consider a system with multiple activation energy barriers for reactions, with a common enzyme shared across several reaction steps. The steps with the highest activation energy rate-limit the cycle when the temperature is not high. If the total abundance of the enzyme is limited, the amount of free enzyme available to catalyze a specific reaction decreases as more substrates bind to the common enzyme. We show that this change in free enzyme abundance compensates for the Arrhenius-type temperature dependence of the reaction coefficient. Taking the example of circadian clocks with cyanobacterial proteins KaiABC, consisting of several phosphorylation sites, we show that this temperature compensation mechanism is indeed valid. Specifically, if the activation energy for phosphorylation is larger than that for dephosphorylation, competition for KaiA shared among the phosphorylation reactions leads to temperature compensation. Moreover, taking a simpler model, we demonstrate the generality of the proposed compensation mechanism, suggesting relevance not only to circadian clocks but to other (bio)chemical oscillators as well. PMID:22566655

Generic temperature compensation of biological clocks by autonomous regulation of catalyst concentration.

PubMed

Hatakeyama, Tetsuhiro S; Kaneko, Kunihiko

2012-05-22

Circadian clocks--ubiquitous in life forms ranging from bacteria to multicellular organisms--often exhibit intrinsic temperature compensation; the period of circadian oscillators is maintained constant over a range of physiological temperatures, despite the expected Arrhenius form for the reaction coefficient. Observations have shown that the amplitude of the oscillation depends on the temperature but the period does not; this suggests that although not every reaction step is temperature independent, the total system comprising several reactions still exhibits compensation. Here we present a general mechanism for such temperature compensation. Consider a system with multiple activation energy barriers for reactions, with a common enzyme shared across several reaction steps. The steps with the highest activation energy rate-limit the cycle when the temperature is not high. If the total abundance of the enzyme is limited, the amount of free enzyme available to catalyze a specific reaction decreases as more substrates bind to the common enzyme. We show that this change in free enzyme abundance compensates for the Arrhenius-type temperature dependence of the reaction coefficient. Taking the example of circadian clocks with cyanobacterial proteins KaiABC, consisting of several phosphorylation sites, we show that this temperature compensation mechanism is indeed valid. Specifically, if the activation energy for phosphorylation is larger than that for dephosphorylation, competition for KaiA shared among the phosphorylation reactions leads to temperature compensation. Moreover, taking a simpler model, we demonstrate the generality of the proposed compensation mechanism, suggesting relevance not only to circadian clocks but to other (bio)chemical oscillators as well.

Heterochromatin protein 1 gamma and IκB kinase alpha interdependence during tumour necrosis factor gene transcription elongation in activated macrophages.

PubMed

Thorne, James L; Ouboussad, Lylia; Lefevre, Pascal F

2012-09-01

IκB kinase α (IKKα) is part of the cytoplasmic IKK complex regulating nuclear factor-κB (NF-κB) release and translocation into the nucleus in response to pro-inflammatory signals. IKKα can also be recruited directly to the promoter of NF-κB-dependent genes by NF-κB where it phosphorylates histone H3 at serine 10, triggering recruitment of the bromodomain-containing protein 4 and the positive transcription elongation factor b. Herein, we report that IKKα travels with the elongating form of ribonucleic acid polymerase II together with heterochromatin protein 1 gamma (HP1γ) at NF-κB-dependent genes in activated macrophages. IKKα binds to and phosphorylates HP1γ, which in turn controls IKKα binding to chromatin and phosphorylation of the histone variant H3.3 at serine 31 within transcribing regions. Downstream of transcription end sites, IKKα accumulates with its inhibitor the CUE-domain containing protein 2, suggesting a link between IKKα inactivation and transcription termination.

Oxidative phosphorylation in a thermophilic, facultative chemoautotroph, Hydrogenophilus thermoluteolus, living prevalently in geothermal niches.

PubMed

Wakai, Satoshi; Masanari, Misa; Ikeda, Takumi; Yamaguchi, Naho; Ueshima, Saori; Watanabe, Kaori; Nishihara, Hirofumi; Sambongi, Yoshihiro

2013-04-01

Hydrogenophilus is a thermophilic, facultative chemoautotroph, which lives prevalently in high temperature geothermal niches. Despite the environmental distribution, little is known about its oxidative phosphorylation. Here, we show that inverted membrane vesicles derived from Hydrogenophilus thermoluteolus cells autotrophically cultivated with H2 formed a proton gradient on the addition of succinate, dl-lactate, and NADH, and exhibited oxidation activity toward these three organic compounds. These indicate the capability of mixotrophic growth of this bacterium. Biochemical analysis demonstrated that the same vesicles contained an F-type ATP synthase. The F1 sector of the ATP synthase purified from H. thermoluteolus membranes exhibited optimal ATPase activity at 65°C. Transformed Escherichia coli membranes expressing H. thermoluteolus F-type ATP synthase exhibited the same temperature optimum for the ATPase. These findings shed light on H. thermoluteolus oxidative phosphorylation from the aspects of membrane bioenergetics and ATPase biochemistry, which must be fundamental and advantageous in the biogeochemical cycles occurred in the high temperature geothermal niches. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

The role of the PI3K/mTOR signaling pathway in Staphylococcus epidermidis small colony variants intracellular survival.

PubMed

Magryś, Agnieszka; Bogut, Agnieszka; Kiełbus, Michał; Olender, Alina

2018-04-01

The objective of this study was to analyze how Staphylococcus epidermidis SCV and WT strains manipulate the PI3K/Akt/mTOR signaling pathway. Six S. epidermidis strains with normal phenotype (WT) and six S. epidermidis strains with SCV phenotype were isolated in parallel from six patients with the prosthetic hip joint infections. THP-1 activated cells were incubated with or without PI3K inhibitor-wortmannin or with mTOR inhibitor-rapamycin. Next, macrophages were exposed to S. epidermidis WT and SCV strains. After 4 h incubation, bacterial survival inside macrophages as well as PI3K-mTOR activation was analyzed. SCV strains of S. epidermidis increased the level of Akt phosphorylation, compared to uninfected macrophages and to their parental WT forms. Wild type variants of S. epidermidis phosphorylated Akt at similar or lower levels as control uninfected cells. Next, the induction of mTOR target, phosphorylated ribosomal protein S6, was measured in bacteria-infected macrophages. The level of phosphorylation was significantly reduced when the cells were exposed to WT strains of S. epidermidis. In contrast, the SCV strains activated S6 protein mostly at a level comparable to the control cells. Rapamycin inhibited mTOR activation as the number of p-S6 positive cells decreased in the tested cases. To conclude, the SCV strains activate the PI3K-Akt signaling pathway in opposite to WT strains. This fact however did not influence the increase in the number of live SCV bacteria as compared to the WT strains. Knowing that the PI3K-Akt pathway is involved in proinflammatory cytokines suppression, SCVs seem to use this pathway to reduce the inflammatory response during the infection.

Elevated ERK/p90 ribosomal S6 kinase activity underlies audiogenic seizure susceptibility in fragile X mice.

PubMed

Sawicka, Kirsty; Pyronneau, Alexander; Chao, Miranda; Bennett, Michael V L; Zukin, R Suzanne

2016-10-11

Fragile X syndrome (FXS) is the most common heritable cause of intellectual disability and a leading genetic form of autism. The Fmr1 KO mouse, a model of FXS, exhibits elevated translation in the hippocampus and the cortex. ERK (extracellular signal-regulated kinase) and mTOR (mechanistic target of rapamycin) signaling regulate protein synthesis by activating downstream targets critical to translation initiation and elongation and are known to contribute to hippocampal defects in fragile X. Here we show that the effect of loss of fragile X mental retardation protein (FMRP) on these pathways is brain region specific. In contrast to the hippocampus, ERK (but not mTOR) signaling is elevated in the neocortex of fragile X mice. Phosphorylation of ribosomal protein S6, typically a downstream target of mTOR, is elevated in the neocortex, despite normal mTOR activity. This is significant in that S6 phosphorylation facilitates translation, correlates with neuronal activation, and is altered in neurodevelopmental disorders. We show that in fragile X mice, S6 is regulated by ERK via the "alternative" S6 kinase p90-ribosomal S6 kinase (RSK), as evidenced by the site of elevated phosphorylation and the finding that ERK inhibition corrects elevated RSK and S6 activity. These findings indicate that signaling networks are altered in the neocortex of fragile X mice such that S6 phosphorylation receives aberrant input from ERK/RSK. Importantly, an RSK inhibitor reduces susceptibility to audiogenic seizures in fragile X mice. Our findings identify RSK as a therapeutic target for fragile X and suggest the therapeutic potential of drugs for the treatment of FXS may vary in a brain-region-specific manner.

Differential regulation of the transcriptional activity of the glucocorticoid receptor through site-specific phosphorylation.

PubMed

Kumar, Raj; Calhoun, William J

2008-12-01

Post-translational modifications such as phosphorylation are known to play an important role in the gene regulation by the transcription factors including the nuclear hormone receptor superfamily of which the glucocorticoid receptor (GR) is a member. Protein phosphorylation often switches cellular activity from one state to another. Like many other transcription factors, the GR is a phosphoprotein, and phosphorylation plays an important role in the regulation of GR activity. Cell signaling pathways that regulate phosphorylation of the GR and its associated proteins are important determinants of GR function under various physiological conditions. While the role of many phosphorylation sites in the GR is still not fully understood, the role of others is clearer. Several aspects of transcription factor function, including DNA binding affinity, interaction of transactivation domains with the transcription initiation complex, and shuttling between the cytoplasmic compartments, have all been linked to site-specific phosphorylation. All major phosphorylation sites in the human GR are located in the N-terminal domain including the major transactivation domain, AF1. Available literature clearly indicates that many of these potential phosphorylation sites are substrates for multiple kinases, suggesting the potential for a very complex regulatory network. Phosphorylated GR interacts favorably with critical coregulatory proteins and subsequently enhances transcriptional activity. In addition, the activities and specificities of coregulators may be subject to similar regulation by phosphorylation. Regulation of the GR activity due to phosphorylation appears to be site-specific and dependent upon specific cell signaling cascade. Taken together, site-specific phosphorylation and related kinase pathways play an important role in the action of the GR, and more precise mechanistic information will lead to fuller understanding of the complex nature of gene regulation by the GR- and related transcription factors. This review provides currently available information regarding the role of GR phosphorylation in its action, and highlights the possible underlying mechanisms of action.

Phosphorylation of SNAP-23 at Ser95 causes a structural alteration and negatively regulates Fc receptor-mediated phagosome formation and maturation in macrophages.

PubMed

Sakurai, Chiye; Itakura, Makoto; Kinoshita, Daiki; Arai, Seisuke; Hashimoto, Hitoshi; Wada, Ikuo; Hatsuzawa, Kiyotaka

2018-05-17

SNAP-23 is a plasma membrane-localized SNARE protein involved in Fc receptor (FcR)-mediated phagocytosis. However, the regulatory mechanism underlying its function remains elusive. Using phosphorylation specific-antibodies, SNAP-23 was found to be phosphorylated at Ser95 in macrophages. To understand the role of this phosphorylation, we established macrophage lines overexpressing the non-phosphorylatable S95A or the phospho-mimicking S95D mutation. The efficiency of phagosome formation and maturation was severely reduced in SNAP-23-S95D-overexpressing cells. To examine whether phosphorylation at Ser95 affected SNAP-23 structure, we constructed intramolecular Förster resonance energy transfer (FRET) probes of SNAP-23 designed to evaluate the approximation of the N-termini of the two SNARE motifs. Interestingly, a high FRET efficiency was detected on the membrane when the S95D probe was used, indicating that phosphorylation at Ser95 caused a dynamic structural shift to the closed form. Co-expression of IκB kinase (IKK) 2 enhanced the FRET efficiency of the wild-type probe on the phagosome membrane. Furthermore, the enhanced phagosomal FRET signal in interferon-γ-activated macrophages was largely dependent on IKK2, and this kinase mediated a delay in phagosome-lysosome fusion. These results suggested that SNAP-23 phosphorylation at Ser95 played an important role in the regulation of SNARE-dependent membrane fusion during FcR-mediated phagocytosis.

NMDA receptor mediates proliferation and CREB phosphorylation in postnatal Müller glia-derived retinal progenitors

PubMed Central

Ramírez, Mónica

2009-01-01

Purpose Postnatal retinal Müller glia are considered to be retinal progenitors as they retain the ability to dedifferentiate, proliferate, and differentiate to new retinal glia and neurons after injury. The proliferation and differentiation processes are coordinated by several extrinsic factors and neurotransmitters, including glutamate. Thus, the appropriate numbers and proportions of the different cell types are generated to form a functional retina during development and during injury repair. Here we analyze the changes in the proliferation of postnatal Müller glia-derived progenitors after activation of the N-methyl-D-aspartate (NMDA) glutamate receptors. Methods Müller glia-derived progenitor cell cultures were characterized by immunocytochemistry with antibodies against the NR1 subunit of the NMDA receptor and the progenitor cell marker nestin. The effect of glutamate receptor agonists and antagonists on cell proliferation was analyzed by BrdU incorporation or Ki67 immunostaining, cell counting, and by immunolabeling of phosphorylated cAMP response element binding protein (P-CREB) transcription factor. The effect of NMDA receptor activation was analyzed in vivo by P-CREB immunohistochemistry in retinal sections of Long-Evans NMDA injected rats. Results We show that NMDA receptor activation significantly increases the proliferation rate of Müller-glia derived progenitor cells and that this increase can be blocked by NMDA receptor antagonists. Furthermore, we show that CREB phosphorylation is induced in NMDA-treated Müller-glia derived progenitor cells in culture and that specific pharmacological inhibition of CREB phosphorylation results in a decreased number of proliferating cells. We confirmed the relevance of these observations by the analysis of retinal sections after NMDA injection in vivo where immunoreactivity to phosphorylated CREB is also increased after treatment. Conclusions In the present study we show that NMDA receptor activation induces postnatal Müller glia-derived retinal cell progenitor proliferation and transcription factor CREB phosphorylation both in culture and in vivo. The identification of the molecular determinants of mature retinal progenitors such as transcription factor CREB and NMDA receptor-induced players should facilitate the control of growth and manipulation of progenitor cell cultures and the possible identification of the molecular mechanisms involved in progenitor self-renewal. PMID:19365572

ABA Renewal Involves Enhancements in Both GluA2-Lacking AMPA Receptor Activity and GluA1 Phosphorylation in the Lateral Amygdala

PubMed Central

Park, Sungmo; Kim, Jihye; An, Bobae; Lee, Hyun Woo; Lee, Seungbok; Kim, Hyun; Lee, Justin C.; Lee, Sukwon; Choi, Sukwoo

2014-01-01

Fear renewal, the context-specific relapse of fear following fear extinction, is a leading animal model of post-traumatic stress disorders (PTSD) and fear-related disorders. Although fear extinction can diminish fear responses, this effect is restricted to the context where the extinction is carried out, and the extinguished fear strongly relapses when assessed in the original acquisition context (ABA renewal) or in a context distinct from the conditioning and extinction contexts (ABC renewal). We have previously identified Ser831 phosphorylation of GluA1 subunit in the lateral amygdala (LA) as a key molecular mechanism for ABC renewal. However, molecular mechanisms underlying ABA renewal remain to be elucidated. Here, we found that both the excitatory synaptic efficacy and GluA2-lacking AMPAR activity at thalamic input synapses onto the LA (T-LA synapses) were enhanced upon ABA renewal. GluA2-lacking AMPAR activity was also increased during low-threshold potentiation, a potential cellular substrate of renewal, at T-LA synapses. The microinjection of 1-naphtylacetyl-spermine (NASPM), a selective blocker of GluA2-lacking AMPARs, into the LA attenuated ABA renewal, suggesting a critical role of GluA2-lacking AMPARs in ABA renewal. We also found that Ser831 phosphorylation of GluA1 in the LA was increased upon ABA renewal. We developed a short peptide mimicking the Ser831-containing C-tail region of GluA1, which can be phosphorylated upon renewal (GluA1S); thus, the phosphorylated GluA1S may compete with Ser831-phosphorylated GluA1. This GluA1S peptide blocked the low-threshold potentiation when dialyzed into a recorded neuron. The microinjection of a cell-permeable form of GluA1S peptide into the LA attenuated ABA renewal. In support of the GluA1S experiments, a GluA1D peptide (in which the serine at 831 is replaced with a phosphomimetic amino acid, aspartate) attenuated ABA renewal when microinjected into the LA. These findings suggest that enhancements in both the GluA2-lacking AMPAR activity and GluA1 phosphorylation at Ser831 are required for ABA renewal. PMID:24925360

ABA renewal involves enhancements in both GluA2-lacking AMPA receptor activity and GluA1 phosphorylation in the lateral amygdala.

PubMed

Park, Kyungjoon; Song, Beomjong; Kim, Jeongyeon; Hong, Ingie; Song, Sangho; Lee, Junuk; Park, Sungmo; Kim, Jihye; An, Bobae; Lee, Hyun Woo; Lee, Seungbok; Kim, Hyun; Lee, Justin C; Lee, Sukwon; Choi, Sukwoo

2014-01-01

Fear renewal, the context-specific relapse of fear following fear extinction, is a leading animal model of post-traumatic stress disorders (PTSD) and fear-related disorders. Although fear extinction can diminish fear responses, this effect is restricted to the context where the extinction is carried out, and the extinguished fear strongly relapses when assessed in the original acquisition context (ABA renewal) or in a context distinct from the conditioning and extinction contexts (ABC renewal). We have previously identified Ser831 phosphorylation of GluA1 subunit in the lateral amygdala (LA) as a key molecular mechanism for ABC renewal. However, molecular mechanisms underlying ABA renewal remain to be elucidated. Here, we found that both the excitatory synaptic efficacy and GluA2-lacking AMPAR activity at thalamic input synapses onto the LA (T-LA synapses) were enhanced upon ABA renewal. GluA2-lacking AMPAR activity was also increased during low-threshold potentiation, a potential cellular substrate of renewal, at T-LA synapses. The microinjection of 1-naphtylacetyl-spermine (NASPM), a selective blocker of GluA2-lacking AMPARs, into the LA attenuated ABA renewal, suggesting a critical role of GluA2-lacking AMPARs in ABA renewal. We also found that Ser831 phosphorylation of GluA1 in the LA was increased upon ABA renewal. We developed a short peptide mimicking the Ser831-containing C-tail region of GluA1, which can be phosphorylated upon renewal (GluA1S); thus, the phosphorylated GluA1S may compete with Ser831-phosphorylated GluA1. This GluA1S peptide blocked the low-threshold potentiation when dialyzed into a recorded neuron. The microinjection of a cell-permeable form of GluA1S peptide into the LA attenuated ABA renewal. In support of the GluA1S experiments, a GluA1D peptide (in which the serine at 831 is replaced with a phosphomimetic amino acid, aspartate) attenuated ABA renewal when microinjected into the LA. These findings suggest that enhancements in both the GluA2-lacking AMPAR activity and GluA1 phosphorylation at Ser831 are required for ABA renewal.

Effect of WAVE2 phosphorylation on activation of the Arp2/3 complex.

PubMed

Nakanishi, Osamu; Suetsugu, Shiro; Yamazaki, Daisuke; Takenawa, Tadaomi

2007-03-01

Members of the family of WASP-family Verprolin homologous proteins (WAVEs) activate the Arp2/3 complex to induce actin polymerization. The WAVE family comprises three proteins, namely, WAVE1, WAVE2 and WAVE3. Among them, WAVE2 is crucial for activation of the Arp2/3 complex for the formation of branched actin filaments in lamellipodia. Activation of mitogen-activated protein (MAP) kinase signalling results in the phosphorylation of the WAVE family proteins; however, which of the three WAVE proteins is phosphorylated is unclear. We found that in vitro WAVE2 is directly phosphorylated by a MAP kinase, i.e. extracellular signal-regulated kinase (ERK) 2. The proline-rich region and the verprolin, cofilin and acidic (VCA) region of WAVE2 were phosphorylated. Interestingly, the phosphorylated VCA region had a higher affinity for the Arp2/3 complex. However, the phosphorylation of the VCA region resulted in reduced induction of Arp2/3-mediated actin polymerization in vitro. The role of the phosphorylation of the proline-rich region was not determined.

Regulation of Catalytic and Non-catalytic Functions of the Drosophila Ste20 Kinase Slik by Activation Segment Phosphorylation.

PubMed

Panneton, Vincent; Nath, Apurba; Sader, Fadi; Delaunay, Nathalie; Pelletier, Ariane; Maier, Dominic; Oh, Karen; Hipfner, David R

2015-08-21

Protein kinases carry out important functions in cells both by phosphorylating substrates and by means of regulated non-catalytic activities. Such non-catalytic functions have been ascribed to many kinases, including some members of the Ste20 family. The Drosophila Ste20 kinase Slik phosphorylates and activates Moesin in developing epithelial tissues to promote epithelial tissue integrity. It also functions non-catalytically to promote epithelial cell proliferation and tissue growth. We carried out a structure-function analysis to determine how these two distinct activities of Slik are controlled. We find that the conserved C-terminal coiled-coil domain of Slik, which is necessary and sufficient for apical localization of the kinase in epithelial cells, is not required for Moesin phosphorylation but is critical for the growth-promoting function of Slik. Slik is auto- and trans-phosphorylated in vivo. Phosphorylation of at least two of three conserved sites in the activation segment is required for both efficient catalytic activity and non-catalytic signaling. Slik function is thus dependent upon proper localization of the kinase via the C-terminal coiled-coil domain and activation via activation segment phosphorylation, which enhances both phosphorylation of substrates like Moesin and engagement of effectors of its non-catalytic growth-promoting activity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulation of 5'-adenosine monophosphate deaminase in the freeze tolerant wood frog, Rana sylvatica.

PubMed

Dieni, Christopher A; Storey, Kenneth B

2008-04-22

The wood frog, Rana sylvatica, is one of a few vertebrate species that have developed natural freeze tolerance, surviving days or weeks with 65-70% of its total body water frozen in extracellular ice masses. Frozen frogs exhibit no vital signs and their organs must endure multiple stresses, particularly long term anoxia and ischemia. Maintenance of cellular energy supply is critical to viability in the frozen state and in skeletal muscle, AMP deaminase (AMPD) plays a key role in stabilizing cellular energetics. The present study investigated AMPD control in wood frog muscle. Wood frog AMPD was subject to multiple regulatory controls: binding to subcellular structures, protein phosphorylation, and effects of allosteric effectors, cryoprotectants and temperature. The percentage of bound AMPD activity increased from 20 to 35% with the transition to the frozen state. Bound AMPD showed altered kinetic parameters compared with the free enzyme (S0.5 AMP was reduced, Hill coefficient fell to approximately 1.0) and the transition to the frozen state led to a 3-fold increase in S0.5 AMP of the bound enzyme. AMPD was a target of protein phosphorylation. Bound AMPD from control frogs proved to be a low phosphate form with a low S0.5 AMP and was phosphorylated in incubations that stimulated PKA, PKC, CaMK, or AMPK. Bound AMPD from frozen frogs was a high phosphate form with a high S0.5 AMP that was reduced under incubation conditions that stimulated protein phosphatases. Frog muscle AMPD was activated by Mg.ATP and Mg.ADP and inhibited by Mg.GTP, KCl, NaCl and NH4Cl. The enzyme product, IMP, uniquely inhibited only the bound (phosphorylated) enzyme from muscle of frozen frogs. Activators and inhibitors differentially affected the free versus bound enzyme. S0.5 AMP of bound AMPD was also differentially affected by high versus low assay temperature (25 vs 5 degrees C) and by the presence/absence of the natural cryoprotectant (250 mM glucose) that accumulates during freezing. Maintenance of long term viability under the ischemic conditions in frozen muscle requires attention to the control of cellular energetics. Differential regulatory controls on AMPD by mechanisms including binding to muscle proteins, actions allosteric effectors, glucose and temperature effects and reversible phosphorylation adjust enzyme function for an optimal role in controlling cellular adenylate levels in ischemic frozen muscle. Stable modification of AMPD properties via freeze-responsive phosphorylation may contribute both to AMPD control and to coordinating AMPD function with other enzymes of energy metabolism in cold ischemic muscle.

L-type Ca2+ channels in the heart: structure and regulation.

PubMed

Treinys, Rimantas; Jurevicius, Jonas

2008-01-01

This review analyzes the structure and regulation mechanisms of voltage-dependent L-type Ca(2+) channel in the heart. L-type Ca(2+) channels in the heart are composed of four different polypeptide subunits, and the pore-forming subunit alpha(1) is the most important part of the channel. In cardiac myocytes, Ca(2+) enter cell cytoplasm from extracellular space mainly through L-type Ca(2+) channels; these channels are very important system in heart Ca(2+) uptake regulation. L-type Ca(2+) channels are responsible for the activation of sarcoplasmic reticulum channels (RyR2) and force of muscle contraction generation in heart; hence, activity of the heart depends on L-type Ca(2+) channels. Phosphorylation of channel-forming subunits by different kinases is one of the most important ways to change the activity of L-type Ca(2+) channel. Additionally, the activity of L-type Ca(2+) channels depends on Ca(2+) concentration in cytoplasm. Ca(2+) current in cardiac cells can facilitate, and this process is regulated by phosphorylation of L-type Ca(2+) channels and intracellular Ca(2+) concentration. Disturbances in cellular Ca(2+) transport and regulation of L-type Ca(2+) channels are directly related to heart diseases, life quality, and life span.

Calcium regulation of oxidative phosphorylation in rat skeletal muscle mitochondria.

PubMed

Kavanagh, N I; Ainscow, E K; Brand, M D

2000-02-24

Activation of oxidative phosphorylation by physiological levels of calcium in mitochondria from rat skeletal muscle was analysed using top-down elasticity and regulation analysis. Oxidative phosphorylation was conceptually divided into three subsystems (substrate oxidation, proton leak and phosphorylation) connected by the membrane potential or the protonmotive force. Calcium directly activated the phosphorylation subsystem and (with sub-saturating 2-oxoglutarate) the substrate oxidation subsystem but had no effect on the proton leak kinetics. The response of mitochondria respiring on 2-oxoglutarate at two physiological concentrations of free calcium was quantified using control and regulation analysis. The partial integrated response coefficients showed that direct stimulation of substrate oxidation contributed 86% of the effect of calcium on state 3 oxygen consumption, and direct activation of the phosphorylation reactions caused 37% of the increase in phosphorylation flux. Calcium directly activated phosphorylation more strongly than substrate oxidation (78% compared to 45%) to achieve homeostasis of mitochondrial membrane potential during large increases in flux.

Rictor forms a complex with Cullin-1 to promote SGK1 ubiquitination and destruction

PubMed Central

Gao, Daming; Wan, Lixin; Inuzuka, Hiroyuki; Berg, Anders H.; Tseng, Alan; Zhai, Bo; Shaik, Shavali; Bennett, Eric; Tron, Adriana E.; Gasser, Jessica A.; Lau, Alan; Gygi, Steven; Harper, J. Wade; DeCaprio, James A.; Toker, Alex; Wei, Wenyi

2010-01-01

Summary The Rictor/mTOR complex (also known as mTORC2) plays a critical role in cellular homeostasis by phosphorylating AGC kinases such as Akt and SGK at their hydrophobic motifs to activate downstream signaling. However, the regulation of mTORC2 and whether it has additional function(s), remains largely unknown. Here we report that Rictor associates with Cullin-1 to form a functional E3 ubiquitin ligase. Rictor, but not Raptor or mTOR alone promotes SGK1 ubiquitination. Loss of Rictor/Cullin-1-mediated ubiquitination leads to increased SGK1 protein levels as detected in Rictor null cells. Moreover, as part of a feedback mechanism, phosphorylation of Rictor at T1135 by multiple AGC kinases disrupts the interaction between Rictor and Cullin-1 to impair SGK1 ubiquitination. These findings indicate that the Rictor/Cullin-1 E3 ligase activity is regulated by a specific signal relay cascade and that misregulation of this mechanism may contribute to the frequent overexpression of SGK1 in various human cancers. PMID:20832730

Plasmonic ruler on field-effect devices for kinase drug discovery applications.

PubMed

Bhalla, Nikhil; Formisano, Nello; Miodek, Anna; Jain, Aditya; Di Lorenzo, Mirella; Pula, Giordano; Estrela, Pedro

2015-09-15

Protein kinases are cellular switches that mediate phosphorylation of proteins. Abnormal phosphorylation of proteins is associated with lethal diseases such as cancer. In the pharmaceutical industry, protein kinases have become an important class of drug targets. This study reports a versatile approach for the detection of protein phosphorylation. The change in charge of the myelin basic protein upon phosphorylation by the protein kinase C-alpha (PKC-α) in the presence of adenosine 5'-[γ-thio] triphosphate (ATP-S) was detected on gold metal-insulator-semiconductor (Au-MIS) capacitor structures. Gold nanoparticles (AuNPs) can then be attached to the thio-phosphorylated proteins, forming a Au-film/AuNP plasmonic couple. This was detected by a localized surface plasmon resonance (LSPR) technique alongside MIS capacitance. All reactions were validated using surface plasmon resonance technique and the interaction of AuNPs with the thio-phosphorylated proteins quantified by quartz crystal microbalance. The plasmonic coupling was also visualized by simulations using finite element analysis. The use of this approach in drug discovery applications was demonstrated by evaluating the response in the presence of a known inhibitor of PKC-α kinase. LSPR and MIS on a single platform act as a cross check mechanism for validating kinase activity and make the system robust to test novel inhibitors. Copyright © 2015 Elsevier B.V. All rights reserved.

Upstream kinases of plant SnRKs are involved in salt stress tolerance.

PubMed

Barajas-Lopez, Juan de Dios; Moreno, Jose Ramon; Gamez-Arjona, Francisco M; Pardo, Jose M; Punkkinen, Matleena; Zhu, Jian-Kang; Quintero, Francisco J; Fujii, Hiroaki

2018-01-01

Sucrose non-fermenting 1-related protein kinases (SnRKs) are important for plant growth and stress responses. This family has three clades: SnRK1, SnRK2 and SnRK3. Although plant SnRKs are thought to be activated by upstream kinases, the overall mechanism remains obscure. Geminivirus Rep-Interacting Kinase (GRIK)1 and GRIK2 phosphorylate SnRK1s, which are involved in sugar/energy sensing, and the grik1-1 grik2-1 double mutant shows growth retardation under regular growth conditions. In this study, we established another Arabidopsis mutant line harbouring a different allele of gene GRIK1 (grik1-2 grik2-1) that grows similarly to the wild-type, enabling us to evaluate the function of GRIKs under stress conditions. In the grik1-2 grik2-1 double mutant, phosphorylation of SnRK1.1 was reduced, but not eliminated, suggesting that the grik1-2 mutation is a weak allele. In addition to high sensitivity to glucose, the grik1-2 grik2-1 mutant was sensitive to high salt, indicating that GRIKs are also involved in salinity signalling pathways. Salt Overly Sensitive (SOS)2, a member of the SnRK3 subfamily, is a critical mediator of the response to salinity. GRIK1 phosphorylated SOS2 in vitro, resulting in elevated kinase activity of SOS2. The salt tolerance of sos2 was restored to normal levels by wild-type SOS2, but not by a mutated form of SOS2 lacking the T168 residue phosphorylated by GRIK1. Activation of SOS2 by GRIK1 was also demonstrated in a reconstituted system in yeast. Our results indicate that GRIKs phosphorylate and activate SnRK1 and other members of the SnRK3 family, and that they play important roles in multiple signalling pathways in vivo. © 2017 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

Analysis of Phosphorylation of the Receptor-Like Protein Kinase HAESA during Arabidopsis Floral Abscission

PubMed Central

Taylor, Isaiah; Wang, Ying; Seitz, Kati; Baer, John; Bennewitz, Stefan; Mooney, Brian P.; Walker, John C.

2016-01-01

Receptor-like protein kinases (RLKs) are the largest family of plant transmembrane signaling proteins. Here we present functional analysis of HAESA, an RLK that regulates floral organ abscission in Arabidopsis. Through in vitro and in vivo analysis of HAE phosphorylation, we provide evidence that a conserved phosphorylation site on a region of the HAE protein kinase domain known as the activation segment positively regulates HAE activity. Additional analysis has identified another putative activation segment phosphorylation site common to multiple RLKs that potentially modulates HAE activity. Comparative analysis suggests that phosphorylation of this second activation segment residue is an RLK specific adaptation that may regulate protein kinase activity and substrate specificity. A growing number of RLKs have been shown to exhibit biologically relevant dual specificity toward serine/threonine and tyrosine residues, but the mechanisms underlying dual specificity of RLKs are not well understood. We show that a phospho-mimetic mutant of both HAE activation segment residues exhibits enhanced tyrosine auto-phosphorylation in vitro, indicating phosphorylation of this residue may contribute to dual specificity of HAE. These results add to an emerging framework for understanding the mechanisms and evolution of regulation of RLK activity and substrate specificity. PMID:26784444

p38 MAPK mediates fibrogenic signal through Smad3 phosphorylation in rat myofibroblasts.

PubMed

Furukawa, Fukiko; Matsuzaki, Koichi; Mori, Shigeo; Tahashi, Yoshiya; Yoshida, Katsunori; Sugano, Yasushi; Yamagata, Hideo; Matsushita, Masanori; Seki, Toshihito; Inagaki, Yutaka; Nishizawa, Mikio; Fujisawa, Junichi; Inoue, Kyoichi

2003-10-01

Hepatic stellate cells (HSCs) spontaneously transdifferentiate into myofibroblast (MFB)-phenotype on plastic dishes. This response recapitulates the features of activation in vivo. Transforming growth factor beta (TGF-beta) plays a prominent role in stimulating liver fibrogenesis by MFBs. In quiescent HSCs, TGF-beta signaling involves TGF-beta type I receptor (TbetaRI)-mediated phosphorylation of serine residues within the conserved SSXS motif at the C-terminus of Smad2 and Smad3. The middle linker regions of Smad2 and Smad3 also are phosphorylated by mitogen-activated protein kinase (MAPK). This study elucidates the change of Smad3-mediated signals during the transdifferentiation process. By using antibodies highly specific to the phosphorylated C-terminal region and the phosphorylated linker region of Smad3, we found that TGF-beta-dependent Smad3 phosphorylation at the C-terminal region decreased, but that the phosphorylation at the linker region increased in the process of transdifferentiation. TGF-beta activated the p38 MAPK pathway, further leading to Smad3 phosphorylation at the linker region in the cultured MFBs, irrespective of Smad2. The phosphorylation promoted hetero-complex formation and nuclear translocation of Smad3 and Smad4. Once combined with TbetaRI-phosphorylated Smad2, the Smad3 and Smad4 complex bound to plasminogen activator inhibitor-type I promoter could enhance the transcription. In addition, Smad3 phosphorylation mediated by the activated TbetaRI was impaired severely in MFBs during chronic liver injury, whereas Smad3 phosphorylation at the linker region was remarkably induced by p38 MAPK pathway. In conclusion, p38 MAPK-dependent Smad3 phosphorylation promoted extracellular matrix production in MFBs both in vitro and in vivo.

Structural characterization by NMR of a double phosphorylated chimeric peptide vaccine for treatment of Alzheimer's disease.

PubMed

Ramírez-Gualito, Karla; Richter, Monique; Matzapetakis, Manolis; Singer, David; Berger, Stefan

2013-04-26

Rational design of peptide vaccines becomes important for the treatment of some diseases such as Alzheimer's disease (AD) and related disorders. In this study, as part of a larger effort to explore correlations of structure and activity, we attempt to characterize the doubly phosphorylated chimeric peptide vaccine targeting a hyperphosphorylated epitope of the Tau protein. The 28-mer linear chimeric peptide consists of the double phosphorylated B cell epitope Tau₂₂₉₋₂₃₇[pThr231/pSer235] and the immunomodulatory T cell epitope Ag85B₂₄₁₋₂₅₅ originating from the well-known antigen Ag85B of the Mycobacterium tuberculosis, linked by a four amino acid sequence -GPSL-. NMR chemical shift analysis of our construct demonstrated that the synthesized peptide is essentially unfolded with a tendency to form a β-turn due to the linker. In conclusion, the -GPSL- unit presumably connects the two parts of the vaccine without transferring any structural information from one part to the other. Therefore, the double phosphorylated epitope of the Tau peptide is flexible and accessible.

The end of a monolith: Deconstructing the Cnn-Polo interaction.

PubMed

Eisman, Robert C; Phelps, Melissa A S; Kaufman, Thomas C

2016-04-02

In Drosophila melanogaster a functional pericentriolar matrix (PCM) at mitotic centrosomes requires Centrosomin-Long Form (Cnn-LF) proteins. Moreover, tissue culture cells have shown that the centrosomal localization of both Cnn-LF and Polo kinase are co-dependent, suggesting a direct interaction. Our recent study found Cnn potentially binds to and is phosphorylated by Polo kinase at 2 residues encoded by Exon1A, the initiating exon of a subset of Cnn isoforms. These interactions are required for the centrosomal localization of Cnn-LF in syncytial embryos and a mutation of either phosphorylation site is sufficient to block localization of both mutant and wild-type Cnn when they are co-expressed. Immunoprecipitation experiments show that Cnn-LF interacts directly with mitotically activated Polo kinase and requires the 2 phosphorylation sites in Exon1A. These IP experiments also show that Cnn-LF proteins form multimers. Depending on the stoichiometry between functional and mutant peptides, heteromultimers exhibit dominant negative or positive trans-complementation (rescue) effects on mitosis. Additionally, following the completion of meiosis, Cnn-Short Form (Cnn-SF) proteins are required for polar body formation in embryos, a process previously shown to require Polo kinase. These findings, when combined with previous work, clearly demonstrate the complexity of cnn and show that a view of cnn as encoding a single peptide is too simplistic.

The end of a monolith: Deconstructing the Cnn-Polo interaction

PubMed Central

2016-01-01

ABSTRACT In Drosophila melanogaster a functional pericentriolar matrix (PCM) at mitotic centrosomes requires Centrosomin-Long Form (Cnn-LF) proteins. Moreover, tissue culture cells have shown that the centrosomal localization of both Cnn-LF and Polo kinase are co-dependent, suggesting a direct interaction. Our recent study found Cnn potentially binds to and is phosphorylated by Polo kinase at 2 residues encoded by Exon1A, the initiating exon of a subset of Cnn isoforms. These interactions are required for the centrosomal localization of Cnn-LF in syncytial embryos and a mutation of either phosphorylation site is sufficient to block localization of both mutant and wild-type Cnn when they are co-expressed. Immunoprecipitation experiments show that Cnn-LF interacts directly with mitotically activated Polo kinase and requires the 2 phosphorylation sites in Exon1A. These IP experiments also show that Cnn-LF proteins form multimers. Depending on the stoichiometry between functional and mutant peptides, heteromultimers exhibit dominant negative or positive trans-complementation (rescue) effects on mitosis. Additionally, following the completion of meiosis, Cnn-Short Form (Cnn-SF) proteins are required for polar body formation in embryos, a process previously shown to require Polo kinase. These findings, when combined with previous work, clearly demonstrate the complexity of cnn and show that a view of cnn as encoding a single peptide is too simplistic. PMID:27096551

Positive regulation of deoxycytidine kinase activity by phosphorylation of Ser-74 in B-cell chronic lymphocytic leukaemia lymphocytes.

PubMed

Smal, Caroline; Van Den Neste, Eric; Maerevoet, Marie; Poiré, Xavier; Théate, Ivan; Bontemps, Françoise

2007-08-08

Deoxycytidine kinase (dCK) activates several antileukaemic nucleoside analogues. We have recently reported that the activity of dCK, overexpressed in HEK 293T cells, correlates with its phosphorylation level on Ser-74. Here, we show that dCK from B-cell chronic lymphocytic leukaemia (B-CLL) lymphocytes can be detected by an anti-phospho-Ser-74 antibody and that interindividual variability in dCK activity is related to its phosphorylation level on Ser-74. Moreover, pharmacological intervention modified Ser-74 phosphorylation, in close parallel with changes in dCK activity. These results suggest that activation of dCK via phosphorylation of Ser-74 might constitute a new therapeutic strategy to enhance activation and efficacy of nucleoside analogues.

Attenuation of Phosphorylation-dependent Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Disease-causing Mutations at the Transmission Interface*

PubMed Central

Chin, Stephanie; Yang, Donghe; Miles, Andrew J.; Eckford, Paul D. W.; Molinski, Steven; Wallace, B. A.; Bear, Christine E.

2017-01-01

Cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein that functions as a phosphorylation-regulated anion channel. The interface between its two cytosolic nucleotide binding domains and coupling helices conferred by intracellular loops extending from the channel pore domains has been referred to as a transmission interface and is thought to be critical for the regulated channel activity of CFTR. Phosphorylation of the regulatory domain of CFTR by protein kinase A (PKA) is required for its channel activity. However, it was unclear if phosphorylation modifies the transmission interface. Here, we studied purified full-length CFTR protein using spectroscopic techniques to determine the consequences of PKA-mediated phosphorylation. Synchrotron radiation circular dichroism spectroscopy confirmed that purified full-length wild-type CFTR is folded and structurally responsive to phosphorylation. Intrinsic tryptophan fluorescence studies of CFTR showed that phosphorylation reduced iodide-mediated quenching, consistent with an effect of phosphorylation in burying tryptophans at the transmission interface. Importantly, the rate of phosphorylation-dependent channel activation was compromised by the introduction of disease-causing mutations in either of the two coupling helices predicted to interact with nucleotide binding domain 1 at the interface. Together, these results suggest that phosphorylation modifies the interface between the catalytic and pore domains of CFTR and that this modification facilitates CFTR channel activation. PMID:28003367

Structural basis of regulation and substrate specificity of protein kinase CK2 deduced from the modeling of protein-protein interactions

PubMed Central

Rekha, Nambudiry; Srinivasan, N

2003-01-01

Background Protein Kinase Casein Kinase 2 (PKCK2) is an ubiquitous Ser/Thr kinase expressed in all eukaryotes. It phosphorylates a number of proteins involved in various cellular processes. PKCK2 holoenzyme is catalytically active tetramer, composed of two homologous or identical and constitutively active catalytic (α) and two identical regulatory (β) subunits. The tetramer cannot phosphorylate some substrates that can be phosphorylated by PKCK2α in isolation. The present work explores the structural basis of this feature using computational analysis and modeling. Results We have initially built a model of PKCK2α bound to a substrate peptide with a conformation identical to that of the substrates in the available crystal structures of other kinases complexed with the substrates/ pseudosubstrates. In this model however, the fourth acidic residue in the consensus pattern of the substrate, S/T-X-X-D/E where S/T is the phosphorylation site, did not result in interaction with the active form of PKCK2α and is highly solvent exposed. Interaction of the acidic residue is observed if the substrate peptide adopts conformations as seen in β turn, α helix, or 310 helices. This type of conformation is observed and accommodated well by PKCK2α in calmodulin where the phosphorylation site is at the central helix. PP2A carries sequence patterns for PKCK2α phosphorylation. While the possibility of PP2A being phosphorylated by PKCK2 has been raised in the literature we use the model of PP2A to generate a model of PP2A-PKCK2α complex. PKCK2β undergoes phosphorylation by holoenzyme at the N-terminal region, and is accommodated very well in the limited space available at the substrate-binding site of the holoenzyme while the space is insufficient to accommodate the binding of PP2A or calmodulin in the holoenzyme. Conclusion Charge and shape complimentarity seems to play a role in substrate recognition and binding to PKCK2α, along with the consensus pattern. The detailed conformation of the substrate peptide binding to PKCK2 differs from the conformation of the substrate/pseudo substrate peptide that is bound to other kinases in the crystal structures reported. The ability of holoenzyme to phosphorylate substrate proteins seems to depend on the accessibility of the P-site in limited space available in holoenzyme. PMID:12740046

PsRBR1 encodes a pea retinoblastoma-related protein that is phosphorylated in axillary buds during dormancy-to-growth transition

PubMed Central

Shimizu-Sato, Sae; Ike, Yoko

2007-01-01

In intact plants, cells in axillary buds are arrested at the G1 phase of the cell cycle during dormancy. In mammalian cells, the cell cycle is suppressed at the G1 phase by the activities of retinoblastoma tumor suppressor gene (RB) family proteins, depending on their phosphorylation state. Here, we report the isolation of a pea cDNA clone encoding an RB-related protein (PsRBR1, Accession No. AB012024) with a high degree of amino acid conservation in comparison with RB family proteins. PsRBR1 protein was detected as two polypeptides using an anti-PsRBR1 antibody in dormant axillary buds, whereas it was detected as three polypeptides, which were the same two polypeptides and another larger polypeptide 2 h after terminal decapitation. Both in vitro-synthesized PsPRB1 protein and lambda protein phosphatase-treated PsRBR1 protein corresponded to the smallest polypeptide detected by anti-PsRBR1 antibody, suggesting that the three polypeptides correspond to non-phosphorylated form of PsRBR1 protein, and lower- and higher-molecular mass forms of phosphorylated PsRBR1 protein. Furthermore, in vivo labeling with [32P]-inorganic phosphate indicated that PsRBR1 protein was more phosphorylated before mRNA accumulation of cell cycle regulatory genes such as PCNA. Together these findings suggest that dormancy-to-growth transition in pea axillary buds is regulated by molecular mechanisms of cell cycle control similar to those in mammals, and that the PsRBR1 protein has an important role in suppressing the cell cycle during dormancy in axillary buds. PMID:18034314

Phosphorylation Dependence and Stoichiometry of the Complex Formed by Tyrosine Hydroxylase and 14-3-3γ*

PubMed Central

Kleppe, Rune; Rosati, Sara; Jorge-Finnigan, Ana; Alvira, Sara; Ghorbani, Sadaf; Haavik, Jan; Valpuesta, José María; Heck, Albert J. R.; Martinez, Aurora

2014-01-01

Phosphorylated tyrosine hydroxylase (TH) can form complexes with 14-3-3 proteins, resulting in enzyme activation and stabilization. Although TH was among the first binding partners identified for these ubiquitous regulatory proteins, the binding stoichiometry and the activation mechanism remain unknown. To address this, we performed native mass spectrometry analyses of human TH (nonphosphorylated or phosphorylated on Ser19 (TH-pS19), Ser40 (TH-pS40), or Ser19 and Ser40 (TH-pS19pS40)) alone and together with 14-3-3γ. Tetrameric TH-pS19 (224 kDa) bound 14-3-3γ (58.3 kDa) with high affinity (Kd = 3.2 nM), generating complexes containing either one (282.4 kDa) or two (340.8 kDa) dimers of 14-3-3. Electron microscopy also revealed one major population of an asymmetric complex, consistent with one TH tetramer and one 14-3-3 dimer, and a minor population of a symmetric complex of one TH tetramer with two 14-3-3 dimers. Lower phosphorylation stoichiometries (0.15–0.54 phosphate/monomer) produced moderate changes in binding kinetics, but native MS detected much less of the symmetric TH:14-3-3γ complex. Interestingly, dephosphorylation of [32P]-TH-pS19 was mono-exponential for low phosphorylation stoichiometries (0.18–0.52), and addition of phosphatase accelerated the dissociation of the TH-pS19:14-3-3γ complex 3- to 4-fold. All together this is consistent with a model in which the pS19 residues in the TH tetramer contribute differently in the association to 14-3-3γ. Complex formation between TH-pS40 and 14-3-3γ was not detected via native MS, and surface plasmon resonance showed that the interaction was very weak. Furthermore, TH-pS19pS40 behaved similarly to TH-pS19 in terms of binding stoichiometry and affinity (Kd = 2.1 nM). However, we found that 14-3-3γ inhibited the phosphorylation rate of TH-pS19 by PKA (3.5-fold) on Ser40. We therefore conclude that Ser40 does not significantly contribute to the binding of 14-3-3γ, and rather has reduced accessibility in the TH:14-3-3γ complex. This adds to our understanding of the fine-tuned physiological regulation of TH, including hierarchical phosphorylation at multiple sites. PMID:24947669

Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65

PubMed Central

Kazlauskaite, Agne; Kondapalli, Chandana; Gourlay, Robert; Campbell, David G.; Ritorto, Maria Stella; Hofmann, Kay; Alessi, Dario R.; Knebel, Axel; Trost, Matthias; Muqit, Miratul M. K.

2014-01-01

We have previously reported that the Parkinson's disease-associated kinase PINK1 (PTEN-induced putative kinase 1) is activated by mitochondrial depolarization and stimulates the Parkin E3 ligase by phosphorylating Ser65 within its Ubl (ubiquitin-like) domain. Using phosphoproteomic analysis, we identified a novel ubiquitin phosphopeptide phosphorylated at Ser65 that was enriched 14-fold in HEK (human embryonic kidney)-293 cells overexpressing wild-type PINK1 stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide m-chlorophenylhydrazone), to activate PINK1, compared with cells expressing kinase-inactive PINK1. Ser65 in ubiquitin lies in a similar motif to Ser65 in the Ubl domain of Parkin. Remarkably, PINK1 directly phosphorylates Ser65 of ubiquitin in vitro. We undertook a series of experiments that provide striking evidence that Ser65-phosphorylated ubiquitin (ubiquitinPhospho−Ser65) functions as a critical activator of Parkin. First, we demonstrate that a fragment of Parkin lacking the Ubl domain encompassing Ser65 (ΔUbl-Parkin) is robustly activated by ubiquitinPhospho−Ser65, but not by non-phosphorylated ubiquitin. Secondly, we find that the isolated Parkin Ubl domain phosphorylated at Ser65 (UblPhospho−Ser65) can also activate ΔUbl-Parkin similarly to ubiquitinPhospho−Ser65. Thirdly, we establish that ubiquitinPhospho−Ser65, but not non-phosphorylated ubiquitin or UblPhospho−Ser65, activates full-length wild-type Parkin as well as the non-phosphorylatable S65A Parkin mutant. Fourthly, we provide evidence that optimal activation of full-length Parkin E3 ligase is dependent on PINK1-mediated phosphorylation of both Parkin at Ser65 and ubiquitin at Ser65, since only mutation of both proteins at Ser65 completely abolishes Parkin activation. In conclusion, the findings of the present study reveal that PINK1 controls Parkin E3 ligase activity not only by phosphorylating Parkin at Ser65, but also by phosphorylating ubiquitin at Ser65. We propose that phosphorylation of Parkin at Ser65 serves to prime the E3 ligase enzyme for activation by ubiquitinPhospho−Ser65, suggesting that small molecules that mimic ubiquitinPhospho−Ser65 could hold promise as novel therapies for Parkinson's disease. PMID:24660806

Maize plasma membrane aquaporins belonging to the PIP1 and PIP2 subgroups are in vivo phosphorylated.

PubMed

Van Wilder, Valérie; Miecielica, Urszula; Degand, Hervé; Derua, Rita; Waelkens, Etienne; Chaumont, François

2008-09-01

Aquaporins are channel proteins that facilitate transmembrane water movement. In this study, we showed that plasma membrane intrinsic proteins (PIPs) from maize shoots are in vitro and in vivo phosphorylated on serine residues by a calcium-dependent kinase associated with the membrane fraction. Mass spectrometry identified phosphorylated peptides corresponding to the C-terminal region of (i) ZmPIP2;1, ZmPIP2;2 and/or ZmPIP2;7; (ii) ZmPIP2;3 and/or ZmPIP2;4; (iii) ZmPIP2;6; together with (iv) a phosphorylated peptide located in the N-terminal region of ZmPIP1;1, ZmPIP1;2, ZmPIP1;3 and/or ZmPIP1;4. The role of phosphorylation in the water channel activity of wild-type and mutant ZmPIP2;1 was studied in Xenopus laevis oocytes. Activation of endogenous protein kinase A increased the osmotic water permeability coefficient of ZmPIP2;1-expressing oocytes, suggesting that phosphorylation activates its channel activity. Mutation of S126 or S203, putative phosphorylated serine residues conserved in all plant PIPs, to alanine decreased ZmPIP2;1 activity by 30-50%, without affecting its targeting to the plasma membrane. Mutation of S285, which is phosphorylated in planta, to alanine or glutamate did not affect the water channel activity. These results indicate that, in oocytes, S126 and S203 play an important role in ZmPIP2;1 activity and that phosphorylation of S285 is not required for its activity.

Muscarinic agonists and phorbol esters increase tyrosine phosphorylation of a 40-kilodalton protein in hippocampal slices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stratton, K.R.; Worley, P.F.; Huganir, R.L.

The authors have used the hippocampal slice preparation to investigate the regulation of protein tyrosine phosphorylation in brain. After pharmacological treatment of intact slices, proteins were separated by electrophoresis, and levels of protein tyrosine phosphorylation were assessed by immunoblotting with specific anti-phosphotyrosine antibodies. Phorbol esters, activators of the serine- and threonine-phosphorylating enzyme protein kinase C, selectively increase tyrosine phosphorylation of a soluble protein with an apparent molecular mass of approximately 40 kilodaltons. Muscarinic agonists such as carbachol and oxotremorine M that strongly activate the inositol phospholipid system also increase tyrosine phosphorylation of this protein. Neurotransmitter activation of the inositol phospholipidmore » system and protein kinase C appears to trigger a cascade leading to increased tyrosine phosphorylation.« less

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

PubMed Central

Bastia, Deepak; Srivastava, Pankaj; Zaman, Shamsu; Choudhury, Malay; Mohanty, Bidyut K.; Bacal, Julien; Langston, Lance D.; Pasero, Philippe; O’Donnell, Michael E.

2016-01-01

Several important physiological transactions, including control of replicative life span (RLS), prevention of collision between replication and transcription, and cellular differentiation, require programmed replication fork arrest (PFA). However, a general mechanism of PFA has remained elusive. We previously showed that the Tof1–Csm3 fork protection complex is essential for PFA by antagonizing the Rrm3 helicase that displaces nonhistone protein barriers that impede fork progression. Here we show that mutations of Dbf4-dependent kinase (DDK) of Saccharomyces cerevisiae, but not other DNA replication factors, greatly reduced PFA at replication fork barriers in the spacer regions of the ribosomal DNA array. A key target of DDK is the mini chromosome maintenance (Mcm) 2–7 complex, which is known to require phosphorylation by DDK to form an active CMG [Cdc45 (cell division cycle gene 45), Mcm2–7, GINS (Go, Ichi, Ni, and San)] helicase. In vivo experiments showed that mutational inactivation of DDK caused release of Tof1 from the chromatin fractions. In vitro binding experiments confirmed that CMG and/or Mcm2–7 had to be phosphorylated for binding to phospho-Tof1–Csm3 but not to its dephosphorylated form. Suppressor mutations that bypass the requirement for Mcm2–7 phosphorylation by DDK restored PFA in the absence of the kinase. Retention of Tof1 in the chromatin fraction and PFA in vivo was promoted by the suppressor mcm5-bob1, which bypassed DDK requirement, indicating that under this condition a kinase other than DDK catalyzed the phosphorylation of Tof1. We propose that phosphorylation regulates the recruitment and retention of Tof1–Csm3 by the replisome and that this complex antagonizes the Rrm3 helicase, thereby promoting PFA, by preserving the integrity of the Fob1–Ter complex. PMID:27298353

Phosphorylated AKT preserves stallion sperm viability and motility by inhibiting caspases 3 and 7.

PubMed

Gallardo Bolaños, Juan M; Balao da Silva, Carolina M; Martín Muñoz, Patricia; Morillo Rodríguez, Antolín; Plaza Dávila, María; Rodríguez-Martínez, Heriberto; Aparicio, Inés M; Tapia, José A; Ortega Ferrusola, Cristina; Peña, Fernando J

2014-08-01

AKT, also referred to as protein kinase B (PKB or RAC), plays a critical role in controlling cell survival and apoptosis. To gain insights into the mechanisms regulating sperm survival after ejaculation, the role of AKT was investigated in stallion spermatozoa using a specific inhibitor and a phosphoflow approach. Stallion spermatozoa were washed and incubated in Biggers-Whitten-Whittingham medium, supplemented with 1% polyvinyl alcohol (PVA) in the presence of 0 (vehicle), 10, 20 or 30 μM SH5, an AKT inhibitor. SH5 treatment reduced the percentage of sperm displaying AKT phosphorylation, with inhibition reaching a maximum after 1 h of incubation. This decrease in phosphorylation was attributable to either dephosphorylation or suppression of the active phosphorylation pathway. Stallion spermatozoa spontaneously dephosphorylated during in vitro incubation, resulting in a lack of a difference in AKT phosphorylation between the SH5-treated sperm and the control after 4 h of incubation. AKT inhibition decreased the proportion of motile spermatozoa (total and progressive) and the sperm velocity. Similarly, AKT inhibition reduced membrane integrity, leading to increased membrane permeability and reduced the mitochondrial membrane potential concomitantly with activation of caspases 3 and 7. However, the percentage of spermatozoa exhibiting oxidative stress, the production of mitochondrial superoxide radicals, DNA oxidation and DNA fragmentation were not affected by AKT inhibition. It is concluded that AKT maintains the membrane integrity of ejaculated stallion spermatozoa, presumably by inhibiting caspases 3 and 7, which prevents the progression of spermatozoa to an incomplete form of apoptosis. © 2014 Society for Reproduction and Fertility.

NADPH oxidase activation in neutrophils: Role of the Phosphorylation of its subunits.

PubMed

Belambri, Sahra A; Rolas, Loïc; Raad, Houssam; Hurtado-Nedelec, Margarita; Dang, Pham My-Chan; El-Benna, Jamel

2018-05-14

Neutrophils are key cells of innate immunity and during inflammation. Upon activation, they produce large amounts of superoxide anion (O 2 -. ) and ensuing reactive oxygen species (ROS) to kill phagocytized microbes. The enzyme responsible for O 2 -. production is called the phagocyte NADPH oxidase. This is a multicomponent enzyme system that becomes active after assembly of four cytosolic proteins (p47 phox , p67 phox , p40 phox and Rac2) with the transmembrane proteins (p22 phox and gp91 phox , which form the cytochrome b 558 ). gp91 phox represents the catalytic subunit of the NADPH oxidase and is also called NOX2. NADPH oxidase-derived ROS are essential for microbial killing and innate immunity; however, excessive ROS production induces tissue injury and prolonged inflammatory reactions that contribute to inflammatory diseases. Thus, NADPH oxidase activation must be tightly regulated in time and space in order to limit ROS production. NADPH oxidase activation is regulated by several processes such as phosphorylation of its components, exchange of GDP/GTP on Rac2 and binding of p47 phox and p40 phox to phospholipids. This review aims to provide new insights into the role of the phosphorylation of the NADPH oxidase components, i.e., gp91 phox , p22 phox , p47 phox , p67 phox and p40 phox , in the activation of this enzyme. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor.

PubMed

Jouvin, M H; Adamczewski, M; Numerof, R; Letourneur, O; Vallé, A; Kinet, J P

1994-02-25

Nonreceptor tyrosine kinases such as the newly described 70-kDa (ZAP-70/Syk) and Src-related tyrosine kinases are coupled to a variety of receptors, including the antigen receptors on B- and T-cells and the Fc receptors for IgE (Fc epsilon RI) and IgG (Fc gamma RI, Fc gamma RIII/CD16). Various subunits of these receptors contain homologous activation motifs which appear capable of autonomously triggering cell activation. Two forms of this motif are present in the Fc epsilon RI multimeric complex: one in the beta chain and one in the gamma chain. Here we show that each of the two tyrosine kinases known to be involved in Fc epsilon RI signaling is controlled by a distinct motif-containing chain. Lyn associates with the nonactivated beta chain, whereas gamma promotes the activation of Syk. We also show that neither the beta nor the gamma motif alone can account for the full signaling capacity of the entire receptor. We propose that, upon triggering of the tetrameric receptor, Lyn already bound to beta becomes activated and phosphorylates beta and gamma; the phosphorylation of gamma induces the association of Syk with gamma and also the activation of Syk, resulting in the phosphorylation and activation of phospholipase C gamma 1. Cooperative recruitment of specific kinases by the various signaling chains found in this family of antigen receptors could represent a way to achieve the full signaling capacity of the multimeric complexes.

The assembly, activation, and substrate specificity of Cyclin D1/Cdk2 complexes

PubMed Central

Jahn, Stephan C.; Law, Mary E.; Corsino, Patrick E.; Rowe, Thomas C.; Davis, Bradley J.; Law, Brian K.

2013-01-01

Previous studies have shown conflicting data regarding Cyclin D1/Cdk2 complexes and, considering the widespread overexpression of Cyclin D1 in cancer, it is important to fully understand their relevance. While many have shown Cyclin D1/Cdk2 complexes to form active complexes, others have failed to show activity or association. Here, using a novel p21-PCNA fusion protein as well as p21 mutant proteins, we show that p21 is a required scaffolding protein, with Cyclin D1 and Cdk2 failing to complex in its absence. These p21/Cyclin D1/Cdk2 complexes are active and also bind the trimeric PCNA complex, with each trimer capable of independently binding distinct Cyclin/Cdk complexes. We also show that increased p21 levels due to treatment with chemotherapeutic agents result in increased formation and kinase activity of Cyclin D1/Cdk2 complexes, and that Cyclin D1/Cdk2 complexes are able to phosphorylate a number of substrates in addition to Rb. Nucleophosmin and Cdh1, two proteins important for centrosome replication and implicated in the chromosomal instability of cancer are shown to be phosphorylated by Cyclin D1/Cdk2 complexes. Additionally, PSF is identified as a novel Cdk2 substrate, being phosphorylated by Cdk2 complexed with either Cyclin E or Cyclin D1, and given the many functions of PSF, it could have important implications on cellular activity. PMID:23627734

Histidine phosphorylation relieves copper inhibition in the mammalian potassium channel KCa3.1

PubMed Central

Srivastava, Shekhar; Panda, Saswati; Li, Zhai; Fuhs, Stephen R; Hunter, Tony; Thiele, Dennis J; Hubbard, Stevan R; Skolnik, Edward Y

2016-01-01

KCa2.1, KCa2.2, KCa2.3 and KCa3.1 constitute a family of mammalian small- to intermediate-conductance potassium channels that are activated by calcium-calmodulin. KCa3.1 is unique among these four channels in that activation requires, in addition to calcium, phosphorylation of a single histidine residue (His358) in the cytoplasmic region, by nucleoside diphosphate kinase-B (NDPK-B). The mechanism by which KCa3.1 is activated by histidine phosphorylation is unknown. Histidine phosphorylation is well characterized in prokaryotes but poorly understood in eukaryotes. Here, we demonstrate that phosphorylation of His358 activates KCa3.1 by antagonizing copper-mediated inhibition of the channel. Furthermore, we show that activated CD4+ T cells deficient in intracellular copper exhibit increased KCa3.1 histidine phosphorylation and channel activity, leading to increased calcium flux and cytokine production. These findings reveal a novel regulatory mechanism for a mammalian potassium channel and for T-cell activation, and highlight a unique feature of histidine versus serine/threonine and tyrosine as a regulatory phosphorylation site. DOI: http://dx.doi.org/10.7554/eLife.16093.001 PMID:27542194

Transforming growth factor-beta and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent Smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury.

PubMed

Yoshida, Katsunori; Matsuzaki, Koichi; Mori, Shigeo; Tahashi, Yoshiya; Yamagata, Hideo; Furukawa, Fukiko; Seki, Toshihito; Nishizawa, Mikio; Fujisawa, Junichi; Okazaki, Kazuichi

2005-04-01

After liver injury, transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) regulate the activation of hepatic stellate cells (HSCs) and tissue remodeling. Mechanisms of PDGF signaling in the TGF-beta-triggered cascade are not completely understood. TGF-beta signaling involves phosphorylation of Smad2 and Smad3 at linker and C-terminal regions. Using antibodies to distinguish Smad2/3 phosphorylated at linker regions from those phosphorylated at C-terminal regions, we investigated Smad2/3-mediated signaling in rat liver injured by CCl(4) administration and in cultured HSCs. In acute liver injury, Smad2/3 were transiently phosphorylated at both regions. Although linker-phosphorylated Smad2 remained in the cytoplasm of alpha-smooth muscle actin-immunoreactive mesenchymal cells adjacent to necrotic hepatocytes in centrilobular areas, linker-phosphorylated Smad3 accumulated in the nuclei. c-Jun N-terminal kinase (JNK) in the activated HSCs directly phosphorylated Smad2/3 at linker regions. Co-treatment of primary cultured HSCs with TGF-beta and PDGF activated the JNK pathway, subsequently inducing endogenous linker phosphorylation of Smad2/3. The JNK pathway may be involved in migration of resident HSCs within the space of Disse to the sites of tissue damage because the JNK inhibitor SP600125 inhibited HSC migration induced by TGF-beta and PDGF signals. Moreover, treatment of HSCs with both TGF-beta and PDGF increased transcriptional activity of plasminogen activator inhibitor-1 through linker phosphorylation of Smad3. In conclusion, TGF-beta and PDGF activate HSCs by transmitting their signals through JNK-mediated Smad2/3 phosphorylation at linker regions, both in vivo and in vitro.

PI3K/Akt Signaling Pathway Activates the WNK-OSR1/SPAK-NCC Phosphorylation Cascade in Hyperinsulinemic db/db Mice

PubMed Central

Nishida, Hidenori; Sohara, Eisei; Nomura, Naohiro; Chiga, Motoko; Alessi, Dario R; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi

2013-01-01

Metabolic syndrome patients have insulin resistance, which causes hyperinsulinemia, which in turn causes aberrant increased renal sodium reabsorption. The precise mechanisms underlying this greater salt-sensitivity of hyperinsulinemic patients remain unclear. Abnormal activation of the recently-identified WNK kinase-OSR1/SPAK kinases-NCC transporter phosphorylation cascade results in the salt-sensitive hypertension of pseudohypoaldosteronism type II. Here, we report a study of renal WNK-OSR1/SPAK-NCC cascade activation in the db/db mouse model of hyperinsulinemic metabolic syndrome. Thiazide sensitivity was increased, suggesting greater activity of NCC in db/db mice. In fact, increased phosphorylation of OSR1/SPAK and NCC was observed. In both SpakT243A/+ and Osr1T185A/+ knock-in db/db mice, which carry mutations that disrupt the signal from WNK kinases, increased phosphorylation of NCC and elevated blood pressure were completely corrected, indicating that phosphorylation of SPAK and OSR1 by WNK kinases is required for the increased activation and phosphorylation of NCC in this model. Renal phosphorylated Akt was increased in db/db mice, suggesting that increased NCC phosphorylation is regulated by the PI3K/Akt signaling cascade in the kidney in response to hyperinsulinemia. A PI3K inhibitor (NVP-BEZ235) corrected the increased OSR1/SPAK-NCC phosphorylation. Another more specific PI3K inhibitor (GDC-0941) and an Akt inhibitor (MK-2206) also inhibited increased NCC phosphorylation. These results indicate that the PI3K/Akt signaling pathway activates the WNK-OSR1/SPAK-NCC phosphorylation cascade in db/db mice. This mechanism may play a role in the pathogenesis of salt-sensitive hypertension in human hyperinsulinemic conditions such as the metabolic syndrome. PMID:22949526

Natural and synthetic STAT3 inhibitors reduce hepcidin expression in differentiated mouse hepatocytes expressing the active phosphorylated STAT3 form.

PubMed

Fatih, Nadia; Camberlein, Emilie; Island, Marie Laure; Corlu, Anne; Abgueguen, Emmanuelle; Détivaud, Lénaïck; Leroyer, Patricia; Brissot, Pierre; Loréal, Olivier

2010-05-01

During the inflammatory process, hepcidin overexpression favours the development of anaemia of chronic diseases which represents the second most common form of anaemia worldwide. The identification of therapeutic agents decreasing hepcidin expression is therefore an important goal. The aim of this study was to target the STAT3 signalling involved in the development of increased hepcidin expression related to chronic inflammation. In a co-culture model associating mouse hepatocytes and rat liver epithelial cells, the mRNA levels of hepcidin1, albumin, aldolase B, Cyp3a4, Stat3, Smad4 and iron regulatory genes were measured by real-time PCR. STAT3 and phosphorylated SMAD1/5/8 proteins were analysed by Western blot. At variance of hepatocyte pure culture, co-culture provided high levels of hepcidin1 mRNA, reaching 400% of the freshly isolated hepatocyte values after 6 days of culture. Hepcidin expression was associated with the maintenance of hepatocyte phenotype, STAT3 phosphorylation and functional BMP/SMAD pathway. Stat3 siRNAs inhibited the hepcidin1 mRNA expression. STAT3 inhibitors, including curcumin, AG490 and a peptide (PpYLKTK), reduced hepcidin1 mRNA expression even when cells were additionally exposed to IL-6. Hepcidin1 mRNA was expressed at high levels by hepatocytes in the co-culture model, and STAT3 pathway activation was controlled through STAT3 inhibitors. Such inhibitors could be useful to prevent anaemia related to hepcidin overexpression during chronic inflammation.

Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex.

PubMed

Ganaie, Safder S; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve; Qiu, Jianming

2017-05-01

Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases.

A CONSTITUTIVELY ACTIVE FORM OF NEUROKININ 1 RECEPTOR AND NEUROKININ 1 RECEPTOR-MEDIATED APOPTOSIS IN GLIOBLASTOMAS

PubMed Central

Akazawa, Toshimasa; Kwatra, Shawn G.; Goldsmith, Laura E.; Richardson, Mark D.; Cox, Elizabeth A.; Sampson, John H.; Kwatra, Madan M.

2009-01-01

Previous studies have shown that neurokinin 1 receptor (NK1R) occurs naturally in human glioblastomas and its stimulation causes cell proliferation. In the present study we show that stimulation of NK1R in human U373 glioblastoma cells by substance P (SP) increases Akt phosphorylation by 2.5-fold, with an EC50 of 57 nM. Blockade of NK1R lowers basal phosphorylation of Akt, indicating the presence of a constitutively active form of NK1R; similar results are seen in U251 MG and DBTRG-05 glioblastoma cells. Linkage of NK1R to Akt implicates NK1R in apoptosis of glioblastoma cells. Indeed, treatment of serum-starved U373 cells with SP reduces apoptosis by 53 ± 1% (P < 0.05), and treatment with NK1R antagonist L-733,060 increases apoptosis by 64 ± 16 % (P < 0.01). Further, the blockade of NK1R in human glioblastoma cells with L-733,060 causes cleavage of Caspase-3 and proteolysis of poly (ADP-ribose) polymerase (PARP). Experiments designed to elucidate the mechanism of NK1R-mediated Akt phosphorylation revealed total involvement of non-receptor tyrosine kinase Src and PI-3-kinase, a partial involvement of epidermal growth factor receptor (EGFR), and no involvement of MEK. Taken together, the results of the present study indicate a key role for NK1R in glioblastoma apoptosis. PMID:19519779

Solution structure of the IIAChitobiose-IIBChitobiose complex of the N,N'-diacetylchitobiose branch of the Escherichia coli phosphotransferase system.

PubMed

Jung, Young-Sang; Cai, Mengli; Clore, G Marius

2010-02-05

The solution structure of the IIA-IIB complex of the N,N'-diacetylchitobiose (Chb) transporter of the Escherichia coli phosphotransferase system has been solved by NMR. The active site His-89 of IIA(Chb) was mutated to Glu to mimic the phosphorylated state and the active site Cys-10 of IIB(Chb) was substituted by serine to prevent intermolecular disulfide bond formation. Binding is weak with a K(D) of approximately 1.3 mm. The two complementary interaction surfaces are largely hydrophobic, with the protruding active site loop (residues 9-16) of IIB(Chb) buried deep within the active site cleft formed at the interface of two adjacent subunits of the IIA(Chb) trimer. The central hydrophobic portion of the interface is surrounded by a ring of polar and charged residues that provide a relatively small number of electrostatic intermolecular interactions that serve to correctly align the two proteins. The conformation of the active site loop in unphosphorylated IIB(Chb) is inconsistent with the formation of a phosphoryl transition state intermediate because of steric hindrance, especially from the methyl group of Ala-12 of IIB(Chb). Phosphorylation of IIB(Chb) is accompanied by a conformational change within the active site loop such that its path from residues 11-13 follows a mirror-like image relative to that in the unphosphorylated state. This involves a transition of the phi/psi angles of Gly-13 from the right to left alpha-helical region, as well as smaller changes in the backbone torsion angles of Ala-12 and Met-14. The resulting active site conformation is fully compatible with the formation of the His-89-P-Cys-10 phosphoryl transition state without necessitating any change in relative translation or orientation of the two proteins within the complex.

Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling.

PubMed

Choi, Jun-Hyuk; Lindsey-Boltz, Laura A; Kemp, Michael; Mason, Aaron C; Wold, Marc S; Sancar, Aziz

2010-08-03

ATR kinase is a critical upstream regulator of the checkpoint response to various forms of DNA damage. Previous studies have shown that ATR is recruited via its binding partner ATR-interacting protein (ATRIP) to replication protein A (RPA)-covered single-stranded DNA (RPA-ssDNA) generated at sites of DNA damage where ATR is then activated by TopBP1 to phosphorylate downstream targets including the Chk1 signal transducing kinase. However, this critical feature of the human ATR-initiated DNA damage checkpoint signaling has not been demonstrated in a defined system. Here we describe an in vitro checkpoint system in which RPA-ssDNA and TopBP1 are essential for phosphorylation of Chk1 by the purified ATR-ATRIP complex. Checkpoint defective RPA mutants fail to activate ATR kinase in this system, supporting the conclusion that this system is a faithful representation of the in vivo reaction. Interestingly, we find that an alternative form of RPA (aRPA), which does not support DNA replication, can substitute for the checkpoint function of RPA in vitro, thus revealing a potential role for aRPA in the activation of ATR kinase. We also find that TopBP1 is recruited to RPA-ssDNA in a manner dependent on ATRIP and that the N terminus of TopBP1 is required for efficient recruitment and activation of ATR kinase.

A truncated, activin-induced Smad3 isoform acts as a transcriptional repressor of FSHβ expression in mouse pituitary.

PubMed

Kim, So-Youn; Zhu, Jie; Woodruff, Teresa K

2011-08-06

The receptor-regulated protein Smad3 is key player in the signaling cascade stimulated by the binding of activin to its cell surface receptor. Upon phosphorylation, Smad3 forms a heterocomplex with Smad2 and Smad4, translocates to the nucleus and acts as a transcriptional co-activator. We have identified a unique isoform of Smad3 that is expressed in mature pituitary gonadotropes. 5' RACE revealed that this truncated Smad3 isoform is transcribed from an ATG site within exon 4 and consists of 7 exons encoding half of the linker region and the MH2 region. In pituitary cells, the truncated Smad3 isoform was phosphorylated upon activin treatment, in a manner that was temporally distinct from the phosphorylation of full-length Smad3. Activin-induced phosphorylation of Smad3 and the truncated Smad3 isoform was blocked by both follistatin and siRNA-mediated knockdown of Smad3. The truncated Smad3 isoform antagonized Smad3-mediated, activin-responsive promoter activity. We propose that the pituitary gonadotrope contains an ultra-short, activin-responsive feedback loop utilizing two different isoforms of Smad3, one which acts as an agonist (Smad3) and another that acts as an intracrine antagonist (truncated Smad3 isoform) to regulate FSHβ production. Copyright © 2011 Elsevier Ltd. All rights reserved.

AMP-activated protein kinase-mediated feedback phosphorylation controls the Ca2+/calmodulin (CaM) dependence of Ca2+/CaM-dependent protein kinase kinase β.

PubMed

Nakanishi, Akihiro; Hatano, Naoya; Fujiwara, Yuya; Sha'ri, Arian; Takabatake, Shota; Akano, Hiroki; Kanayama, Naoki; Magari, Masaki; Nozaki, Naohito; Tokumitsu, Hiroshi

2017-12-01

The Ca 2+ /calmodulin-dependent protein kinase kinase β (CaMKKβ)/5'-AMP-activated protein kinase (AMPK) phosphorylation cascade affects various Ca 2+ -dependent metabolic pathways and cancer growth. Unlike recombinant CaMKKβ that exhibits higher basal activity (autonomous activity), activation of the CaMKKβ/AMPK signaling pathway requires increased intracellular Ca 2+ concentrations. Moreover, the Ca 2+ /CaM dependence of CaMKKβ appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKKβ activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKKβ at multiple residues by CaMKKβ-activated AMPK in addition to autophosphorylation in vitro , leading to reduced autonomous, but not Ca 2+ /CaM-activated, CaMKKβ activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKKβ indicated that Thr 144 phosphorylation by activated AMPK converts CaMKKβ into a Ca 2+ /CaM-dependent enzyme as shown by completely Ca 2+ /CaM-dependent CaMKK activity of a phosphomimetic T144E CaMKKβ mutant. CaMKKβ mutant analysis indicated that the C-terminal domain (residues 471-587), including the autoinhibitory region, plays an important role in stabilizing an inactive conformation in a Thr 144 phosphorylation-dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr 144 antibody revealed phosphorylation of Thr 144 in CaMKKβ in transfected COS-7 cells that was further enhanced by exogenous expression of AMPKα. These results indicate that AMPK-mediated feedback phosphorylation of CaMKKβ regulates the CaMKKβ/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca 2+ -dependent AMPK activation by CaMKKβ. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Discovery of cellular substrates for protein kinase A using a peptide array screening protocol.

PubMed

Smith, F Donelson; Samelson, Bret K; Scott, John D

2011-08-15

Post-translational modification of proteins is a universal form of cellular regulation. Phosphorylation on serine, threonine, tyrosine or histidine residues by protein kinases is the most widespread and versatile form of covalent modification. Resultant changes in activity, localization or stability of phosphoproteins drives cellular events. MS and bioinformatic analyses estimate that ~30% of intracellular proteins are phosphorylated at any given time. Multiple approaches have been developed to systematically define targets of protein kinases; however, it is likely that we have yet to catalogue the full complement of the phosphoproteome. The amino acids that surround a phosphoacceptor site are substrate determinants for protein kinases. For example, basophilic enzymes such as PKA (protein kinase A), protein kinase C and calmodulin-dependent kinases recognize basic side chains preceding the target serine or threonine residues. In the present paper we describe a strategy using peptide arrays and motif-specific antibodies to identify and characterize previously unrecognized substrate sequences for protein kinase A. We found that the protein kinases PKD (protein kinase D) and MARK3 [MAP (microtubule-associated protein)-regulating kinase 3] can both be phosphorylated by PKA. Furthermore, we show that the adapter protein RIL [a product of PDLIM4 (PDZ and LIM domain protein 4)] is a PKA substrate that is phosphorylated on Ser(119) inside cells and that this mode of regulation may control its ability to affect cell growth. © The Authors Journal compilation © 2011 Biochemical Society

Internalisation and multiple phosphorylation of γ-Conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Capraro, Jessica; Magni, Chiara, E-mail: chiara.magni@unimi.it; Faoro, Franco

Highlights: •A glycaemia-reducing lupin seed protein is internalized by HepG2 cells. •The protein accumulates in the cytosol in an intact form. •The internalized protein is multiply phosphorylated. -- Abstract: Lupin seed γ-Conglutin is a protein capable of reducing glycaemia in mammalians and increasing glucose uptake by model cells. This work investigated whether γ-Conglutin is internalised into the target cells and undergoes any covalent change during the process, as a first step to understanding its mechanism of action. To this purpose, γ-Conglutin-treated and untreated HepG2 cells were submitted to confocal and transmission electron microscopy. Immune-revelation of γ-Conglutin at various intervals revealedmore » its accumulation inside the cytosol. In parallel, 2D-electrophoresis of the cell lysates and antibody reaction of the blotted maps showed the presence of the protein intact subunits inside the treated cells, whilest no trace of the protein was found in the control cells. However, γ-Conglutin-related spots with an unexpectedly low pI were also observed in the maps. These spots were excised, trypsin-treated and submitted to MS/MS spectrometric analysis. The presence of phosphorylated amino acids was detected. These findings, by showing that γ-Conglutin is internalised by HepG2 cells in an intact form and is modified by multiple phosphorylation, open the way to the understanding of the lupin γ-Conglutin insulin-mimetic activity.« less

Ezrin and moesin expression in canine and feline osteosarcoma.

PubMed

Hlavaty, Juraj; Wolfesberger, Birgitt; Hauck, Marlene; Obermayer-Pietsch, Barbara; Fuchs-Baumgartinger, Andrea; Miller, Ingrid; Walter, Ingrid

2017-08-01

Biological features of canine osteosarcomas (OS) differ markedly from those found in feline and resemble more human osteosarcomas, in particular for their high rate of metastasis and poor prognosis. Ezrin, radixin and moesin are members of the ERM protein family and link the actin cytoskeleton with the cell membrane. Ezrin and moesin have been shown to be of prognostic significance in tumor progression due to their role in the metastatic process. The objective of this study was to analyze ezrin and moesin protein expression in a series of dog (n = 16) and cat (n = 8) osteosarcoma samples using immunohistochemistry and western blot techniques. We found that cat OS have a higher moesin expression compared to dog OS, however, the active phosphorylated forms of moesin and ezrin Tyr353 were more abundant in the dog samples. A statistically significant difference was found for the low and high immunohistochemical scores of ezrin and pan-phospho-ERM proteins between cat and dog. Although phospho-ezrin Thr567 was higher in feline OS, the membranous localization in dog OS samples indicates the presence of the biologically active form. Therefore, the observed differences in phosphorylated forms of ezrin and moesin status should be further studied to demonstrate if they are relevant for different biological behavior between dog and cat OS.

Antiapoptotic and Trophic Effects of Dominant-Negative Forms of Dual Leucine Zipper Kinase in Dopamine Neurons of the Substantia Nigra In Vivo

PubMed Central

Chen, Xiqun; Rzhetskaya, Margarita; Kareva, Tatyana; Bland, Ross; During, Matthew J.; Tank, A. William; Kholodilov, Nikolai; Burke, Robert E.

2009-01-01

There is extensive evidence that the mitogen-activated protein kinase (MAPK) signaling cascade mediates programmed cell death in neurons. However, current evidence that the mixed linage kinases (MLKs), upstream in this cascade, mediate cell death is based, in the in vivo context, entirely on pharmacological approaches. The compounds used in these studies have neither complete specificity nor selectivity among these kinases. Therefore, to better address the molecular specificity of the MLKs in mediating neuron death, we used dominant-negative constructs delivered by AAV (adenoassociated virus) vector transfer. We assessed effects in a neurotoxin model of parkinsonism, in which neuroprotection by pharmacologic MLK inhibition has been reported. We find that two dominant-negative forms of dual leucine zipper kinase (DLK) inhibit apoptosis and enhance long-term survival of dopamine neurons, but a dominant negative of MLK3 does not. Interestingly, the kinase-dead form of DLK not only blocks apoptosis but also has trophic effects on dopamine neurons. Although the MAPK cascade activates a number of downstream cell death mediators, we find that inhibition of DLK correlates closely with blockade of phosphorylation of c-jun and prevention of cell death. We conclude that DLK acts primarily through c-jun phosphorylation to mediate cell death in this model. PMID:18199767

MenaINV dysregulates cortactin phosphorylation to promote invadopodium maturation

PubMed Central

Weidmann, Maxwell D.; Surve, Chinmay R.; Eddy, Robert J.; Chen, Xiaoming; Gertler, Frank B.; Sharma, Ved P.; Condeelis, John S.

2016-01-01

Invadopodia, actin-based protrusions of invasive carcinoma cells that focally activate extracellular matrix-degrading proteases, are essential for the migration and intravasation of tumor cells during dissemination from the primary tumor. We have previously shown that cortactin phosphorylation at tyrosine residues, in particular tyrosine 421, promotes actin polymerization at newly-forming invadopodia, promoting their maturation to matrix-degrading structures. However, the mechanism by which cells regulate the cortactin tyrosine phosphorylation-dephosphorylation cycle at invadopodia is unknown. Mena, an actin barbed-end capping protein antagonist, is expressed as various splice-isoforms. The MenaINV isoform is upregulated in migratory and invasive sub-populations of breast carcinoma cells, and is involved in tumor cell intravasation. Here we show that forced MenaINV expression increases invadopodium maturation to a far greater extent than equivalent expression of other Mena isoforms. MenaINV is recruited to invadopodium precursors just after their initial assembly at the plasma membrane, and promotes the phosphorylation of cortactin tyrosine 421 at invadopodia. In addition, we show that cortactin phosphorylation at tyrosine 421 is suppressed by the phosphatase PTP1B, and that PTP1B localization to the invadopodium is reduced by MenaINV expression. We conclude that MenaINV promotes invadopodium maturation by inhibiting normal dephosphorylation of cortactin at tyrosine 421 by the phosphatase PTP1B. PMID:27824079

Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation

NASA Astrophysics Data System (ADS)

Gone, Swapna; Alfonso-Prieto, Mercedes; Paudyal, Samridhdi; Nicholson, Allen W.

2016-05-01

Ribonuclease III (RNase III) is a conserved, gene-regulatory bacterial endonuclease that cleaves double-helical structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control, reflective of its global regulatory functions. Escherichia coli (Ec) RNase III catalytic activity is known to increase during bacteriophage T7 infection, reflecting the expression of the phage-encoded protein kinase, T7PK. However, the mechanism of catalytic enhancement is unknown. This study shows that Ec-RNase III is phosphorylated on serine in vitro by purified T7PK, and identifies the targets as Ser33 and Ser34 in the N-terminal catalytic domain. Kinetic experiments reveal a 5-fold increase in kcat and a 1.4-fold decrease in Km following phosphorylation, providing a 7.4-fold increase in catalytic efficiency. Phosphorylation does not change the rate of substrate cleavage under single-turnover conditions, indicating that phosphorylation enhances product release, which also is the rate-limiting step in the steady-state. Molecular dynamics simulations provide a mechanism for facilitated product release, in which the Ser33 phosphomonoester forms a salt bridge with the Arg95 guanidinium group, thereby weakening RNase III engagement of product. The simulations also show why glutamic acid substitution at either serine does not confer enhancement, thus underscoring the specific requirement for a phosphomonoester.

Antisense protein tyrosine phosphatase 1B reverses activation of p38 mitogen-activated protein kinase in liver of ob/ob mice.

PubMed

Gum, Rebecca J; Gaede, Lori L; Heindel, Matthew A; Waring, Jeffrey F; Trevillyan, James M; Zinker, Bradley A; Stark, Margery E; Wilcox, Denise; Jirousek, Michael R; Rondinone, Cristina M; Ulrich, Roger G

2003-06-01

Phosphorylation of stress-activated kinase p38, a MAPK family member, was increased in liver of ob/ob diabetic mice relative to lean littermates. Treatment of ob/ob mice with protein tyrosine phosphatase 1B (PTP1B) antisense oligonucleotides (ASO) reduced phosphorylation of p38 in liver-to below lean littermate levels-and normalized plasma glucose while reducing plasma insulin. Phosphorylation of ERK, but not JNK, was also decreased in ASO-treated mice. PTP1B ASO decreased TNFalpha protein levels and phosphorylation of the transcription factor cAMP response element binding protein (CREB) in liver, both of which can occur through decreased phosphorylation of p38 and both of which have been implicated in insulin resistance or hyperglycemia. Decreased p38 phosphorylation was not directly due to decreased phosphorylation of the kinases that normally phosphorylate p38-MKK3 and MKK6. Additionally, p38 phosphorylation was not enhanced in liver upon insulin stimulation of ASO-treated ob/ob mice (despite increased activation of other signaling molecules) corroborating that p38 is not directly affected via the insulin receptor. Instead, decreased phosphorylation of p38 may be due to increased expression of MAPK phosphatases, particularly the p38/ERK phosphatase PAC1 (phosphatase of activated cells). This study demonstrates that reduction of PTP1B protein using ASO reduces activation of p38 and its substrates TNFalpha and CREB in liver of diabetic mice, which correlates with decreased hyperglycemia and hyperinsulinemia.

Heat-shock protein-25/27 phosphorylation by the delta isoform of protein kinase C.

PubMed Central

Maizels, E T; Peters, C A; Kline, M; Cutler, R E; Shanmugam, M; Hunzicker-Dunn, M

1998-01-01

Small heat-shock proteins (sHSPs) are widely expressed 25-28 kDa proteins whose functions are dynamically regulated by phosphorylation. While recent efforts have clearly delineated a stress-responsive p38 mitogen-activated protein-kinase (MAPK)-dependent kinase pathway culminating in activation of the heat-shock (HSP)-kinases, mitogen-activated protein-kinase-activated protein kinase-2 and -3, not all sHSP phosphorylation events can be explained by the p38 MAPK-dependent pathway. The contribution of protein kinase C (PKC) to sHSP phosphorylation was suggested by early studies but later questioned on the basis of the reported poor ability of purified PKC to phosphorylate sHSP in vitro. The current study re-evaluates the role of PKC in sHSP phosphorylation in the light of the isoform complexity of the PKC family. We evaluated the sHSP phosphorylation status in rat corpora lutea obtained from two stages of pregnancy, mid-pregnancy and late-pregnancy, which express different levels of the novel PKC isoform, PKC-delta. Two-dimensional Western blot analysis showed that HSP-27 was more highly phosphorylated in vivo in corpora lutea of late pregnancy, corresponding to the developmental stage in which PKC-delta is abundant and active. Late-pregnant luteal extracts contained a lipid-sensitive HSP-kinase activity which exactly co-purified with PKC-delta using hydroxyapatite and S-Sepharose column chromatography. To determine whether there might be preferential phosphorylation of sHSP by a particular PKC isoform, purified recombinant PKC isoforms corresponding to those PKC isoforms detected in rat corpora lutea were evaluated for HSP-kinase activity in vitro. Recombinant PKC-delta effectively catalysed the phosphorylation of sHSP in vitro, and PKC-alpha was 30-50% as effective as an HSP-kinase; other PKCs tested (beta1, beta2, epsilon and zeta) were poor HSP-kinases. These results show that select PKC family members can function as direct HSP-kinases in vitro. Moreover, the observation of enhanced luteal HSP-27 phosphorylation in vivo, in late pregnancy, when PKC-delta is abundant and active, suggests that select PKC family members contribute to sHSP phosphorylation events in vivo. PMID:9620873

UVC-induced apoptosis in Dubca cells is independent of JNK activation and p53{sup Ser-15} phosphorylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chathoth, Shahanas; Thayyullathil, Faisal; Hago, Abdulkader

2009-06-12

Ultraviolet C (UVC) irradiation in mammalian cell lines activates a complex signaling network that leads to apoptosis. By using Dubca cells as a model system, we report the presence of a UVC-induced apoptotic pathway that is independent of c-Jun N-terminal kinases (JNKs) activation and p53 phosphorylation at Ser{sup 15}. Irradiation of Dubca cells with UVC results in a rapid JNK activation and phosphorylation of its downstream target c-Jun, as well as, phosphorylation of activating transcription factor 2 (ATF2). Pre-treatment with JNK inhibitor, SP600125, inhibited UVC-induced c-Jun phosphorylation without preventing UVC-induced apoptosis. Similarly, inhibition of UVC-induced p53 phosphorylation did not preventmore » Dubca cell apoptosis, suggesting that p53{sup Ser-15} phosphorylation is not associated with UVC-induced apoptosis signaling. The pan-caspase inhibitor z-VAD-fmk inhibited UVC-induced PARP cleavage, DNA fragmentation, and ultimately apoptosis of Dubca cells. Altogether, our study clearly indicates that UVC-induced apoptosis is independent of JNK and p53 activation in Dubca cells, rather, it is mediated through a caspase dependent pathway. Our findings are not in line with the ascribed critical role for JNKs activation, and downstream phosphorylation of targets such as c-Jun and ATF2 in UVC-induced apoptosis.« less

In vivo phosphorylation of WRKY transcription factor by MAPK.

PubMed

Ishihama, Nobuaki; Adachi, Hiroaki; Yoshioka, Miki; Yoshioka, Hirofumi

2014-01-01

Plants activate signaling networks in response to diverse pathogen-derived signals, facilitating transcriptional reprogramming through mitogen-activated protein kinase (MAPK) cascades. Identification of phosphorylation targets of MAPK and in vivo detection of the phosphorylated substrates are important processes to elucidate the signaling pathway in plant immune responses. We have identified a WRKY transcription factor, which is phosphorylated by defense-related MAPKs, SIPK and WIPK. Recent evidence demonstrated that some group I WRKY transcription factors, which contain a conserved motif in the N-terminal region, are activated by MAPK-dependent phosphorylation. In this chapter, we describe protocols for preparation of anti-phosphopeptide antibodies, detection of activated MAPKs using anti-phospho-MAPK antibody, and activated WRKY using anti-phospho-WRKY antibody, respectively.

The insulin receptor substrate (IRS)-1 pleckstrin homology domain functions in downstream signaling.

PubMed

Vainshtein, I; Kovacina, K S; Roth, R A

2001-03-16

The pleckstrin homology (PH) domain of the insulin receptor substrate-1 (IRS-1) plays a role in directing this molecule to the insulin receptor, thereby regulating its tyrosine phosphorylation. In this work, the role of the PH domain in subsequent signaling was studied by constructing constitutively active forms of IRS-1 in which the inter-SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase was fused to portions of the IRS-1 molecule. Chimeric molecules containing the PH domain were found to activate the downstream response of stimulating the Ser/Thr kinase Akt. A chimera containing point mutations in the PH domain that abolished the ability of this domain to bind phosphatidylinositol 4,5-bisphosphate prevented these molecules from activating Akt. These mutations also decreased by about 70% the amount of the constructs present in a particulate fraction of the cells. These results indicate that the PH domain of IRS-1, in addition to directing this protein to the receptor for tyrosine phosphorylation, functions in the ability of this molecule to stimulate subsequent responses. Thus, compromising the function of the PH domain, e.g. in insulin-resistant states, could decrease both the ability of IRS-1 to be tyrosine phosphorylated by the insulin receptor and to link to subsequent downstream targets.

Purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae.

PubMed

Elbing, Karin; McCartney, Rhonda R; Schmidt, Martin C

2006-02-01

Members of the Snf1/AMPK family of protein kinases are activated by distinct upstream kinases that phosphorylate a conserved threonine residue in the Snf1/AMPK activation loop. Recently, the identities of the Snf1- and AMPK-activating kinases have been determined. Here we describe the purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae. The identities of proteins associated with the Snf1-activating kinases were determined by peptide mass fingerprinting. These kinases, Sak1, Tos3 and Elm2 do not appear to require the presence of additional subunits for activity. Sak1 and Snf1 co-purify and co-elute in size exclusion chromatography, demonstrating that these two proteins form a stable complex. The Snf1-activating kinases phosphorylate the activation loop threonine of Snf1 in vitro with great specificity and are able to do so in the absence of beta and gamma subunits of the Snf1 heterotrimer. Finally, we showed that the Snf1 kinase domain isolated from bacteria as a GST fusion protein can be activated in vitro and shows substrate specificity in the absence of its beta and gamma subunits.

Purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae

PubMed Central

2005-01-01

Members of the Snf1/AMPK family of protein kinases are activated by distinct upstream kinases that phosphorylate a conserved threonine residue in the Snf1/AMPK activation loop. Recently, the identities of the Snf1- and AMPK-activating kinases have been determined. Here we describe the purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae. The identities of proteins associated with the Snf1-activating kinases were determined by peptide mass fingerprinting. These kinases, Sak1, Tos3 and Elm2 do not appear to require the presence of additional subunits for activity. Sak1 and Snf1 co-purify and co-elute in size exclusion chromatography, demonstrating that these two proteins form a stable complex. The Snf1-activating kinases phosphorylate the activation loop threonine of Snf1 in vitro with great specificity and are able to do so in the absence of β and γ subunits of the Snf1 heterotrimer. Finally, we showed that the Snf1 kinase domain isolated from bacteria as a GST fusion protein can be activated in vitro and shows substrate specificity in the absence of its β and γ subunits. PMID:16201971

PHOSPHORYLATION BY EXTRACTS OF NITROSOMONAS EUROPAEA

PubMed Central

Burge, W. D.; Malavolta, E.; Delwiche, C. C.

1963-01-01

Burge, W. D. (University of California, Berkeley), E. Malavolta, and C. C. Delwiche. Phosphorylation by extracts of Nitrosomonas europaea. J. Bacteriol. 85:106–110. 1963.—Cellfree preparations of Nitrosomonas europaea are capable of oxidizing hydroxylamine, but not ammonium ion, to nitrite. The quantity of nitrite formed by our preparations was, at most, equivalent to only 70% of the hydroxylamine added. Although the preparations had a strong phosphatase activity, resulting in a net loss of organic phosphate during the experimental period, P32-labeled inorganic phosphate was found to be incorporated into the organic fraction, including adenosine triphosphate (ATP) and adenosine diphosphate (ADP). The provision of hydroxylamine as substrate resulted in the formation of nitrite and an increased incorporation of P32 into the organic fraction. It is concluded that the chemosynthetic autotroph Nitrosomonas, in common with certain other autotrophic organisms and heterotrophs, is capable of converting energy released in the oxidation of its inorganic substrate into high-energy phosphate units (ATP and ADP) for the mediation of other energy-requiring reactions. The simultaneous formation of ATP and ADP is interpreted as evidence for an adenylate kinase activity. The preparations used exhibited a considerable endogenous incorporation of P32 into organic phosphate in the absence of added hydroxylamine. Cyanide inhibited both phosphorylation and the oxidation of hydroxylamine. Both the supernatant and particulate fractions of a Nitrosomonas extract subjected to centrifugal fields of 100,000 × g were active in phosphorylation and nitrite formation, but these activities appeared to be uncoupled in the particulate fraction and only partially coupled in the supernatant solution. This most likely reflects a significant endogenous respiration, and not a real lack of coupling between the two reactions. PMID:14016952

Identification and subsequent phosphorylation of sequestered partially processed caseins in the lactating guinea-pig mammary gland.

PubMed Central

Boulton, A P; Pascall, J C; Craig, R K

1984-01-01

Golgi and endoplasmic-reticulum fractions were prepared from the lactating guinea-pig mammary gland. The endoplasmic-reticulum fraction was highly active in the processing and sequestration of milk-protein primary translation products. Explants from the lactating gland in organ culture were used to identify milk-protein intermediates present in the secretory pathway, and the timing of the events leading to their post-translational modification. With [35S]methionine, the milk proteins labelled after a short pulse (3 min) were represented by the partially processed (but not phosphorylated) caseins and alpha-lactalbumin sequestered within membrane-bound vesicles. After a 30 min labelling period, higher-Mr caseins with electrophoretic mobilities identical with those of the phosphorylated caseins isolated from milk were identified in the incubation medium, and sequestered within membrane-bound vesicles. Pulse-chase experiments established a precursor-product relationship between these forms. Secretion is apparent approx. 30 min after sequestration. Caseins are highly phosphorylated; removal of the phosphate residues with acid phosphatase results in proteins with increased electrophoretic mobility, similar to those of the partially processed early casein intermediates found sequestered in explants after a 3 min pulse with [35S]methionine, and those sequestered within microsomal membranes after mRNA-directed cell-free protein synthesis. A comparison of the proteins labelled during both short (5 min) and long (30 min) pulses with [35S]methionine and [32P]Pi shows that, in contrast with the 35S-labelled caseins, those labelled with [32P]Pi exhibit only electrophoretic mobilities identical with those of the mature caseins isolated from milk and those identified after long labelling periods with [35S]methionine. No phosphorylated early intermediate forms of caseins were identified. We conclude that the synthesis and post-translational modification of guinea-pig caseins occurs in two stages, (i) an early event involving synthesis and sequestration within the endoplasmic reticulum, an event that involves signal-peptide removal, followed (ii) 10-20 min later by phosphorylation at a different point in the secretory pathway, probably in the Golgi complex. Secretion of the phosphorylated caseins occurs 10-20 min later. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:6477529

Shp2 Associates with and Enhances Nephrin Tyrosine Phosphorylation and Is Necessary for Foot Process Spreading in Mouse Models of Podocyte Injury.

PubMed

Verma, Rakesh; Venkatareddy, Madhusudan; Kalinowski, Anne; Patel, Sanjeevkumar R; Salant, David J; Garg, Puneet

2016-02-15

In most forms of glomerular diseases, loss of size selectivity by the kidney filtration barrier is associated with changes in the morphology of podocytes. The kidney filtration barrier is comprised of the endothelial lining, the glomerular basement membrane, and the podocyte intercellular junction, or slit diaphragm. The cell adhesion proteins nephrin and neph1 localize to the slit diaphragm and transduce signals in a Src family kinase Fyn-mediated tyrosine phosphorylation-dependent manner. Studies in cell culture suggest nephrin phosphorylation-dependent signaling events are primarily involved in regulation of actin dynamics and lamellipodium formation. Nephrin phosphorylation is a proximal event that occurs both during development and following podocyte injury. We hypothesized that abrogation of nephrin phosphorylation following injury would prevent nephrin-dependent actin remodeling and foot process morphological changes. Utilizing a biased screening approach, we found nonreceptor Src homology 2 (sh2) domain-containing phosphatase Shp2 to be associated with phosphorylated nephrin. We observed an increase in nephrin tyrosine phosphorylation in the presence of Shp2 in cell culture studies. In the human glomerulopathies minimal-change nephrosis and membranous nephropathy, there is an increase in Shp2 phosphorylation, a marker of increased Shp2 activity. Mouse podocytes lacking Shp2 do not develop foot process spreading when subjected to podocyte injury in vivo using protamine sulfate or nephrotoxic serum (NTS). In the NTS model, we observed a lack of foot process spreading in mouse podocytes with Shp2 deleted and smaller amounts of proteinuria. Taken together, these results suggest that Shp2-dependent signaling events are necessary for changes in foot process structure and function following injury. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Shp2 Associates with and Enhances Nephrin Tyrosine Phosphorylation and Is Necessary for Foot Process Spreading in Mouse Models of Podocyte Injury

PubMed Central

Verma, Rakesh; Venkatareddy, Madhusudan; Kalinowski, Anne; Patel, Sanjeevkumar R.; Salant, David J.

2015-01-01

In most forms of glomerular diseases, loss of size selectivity by the kidney filtration barrier is associated with changes in the morphology of podocytes. The kidney filtration barrier is comprised of the endothelial lining, the glomerular basement membrane, and the podocyte intercellular junction, or slit diaphragm. The cell adhesion proteins nephrin and neph1 localize to the slit diaphragm and transduce signals in a Src family kinase Fyn-mediated tyrosine phosphorylation-dependent manner. Studies in cell culture suggest nephrin phosphorylation-dependent signaling events are primarily involved in regulation of actin dynamics and lamellipodium formation. Nephrin phosphorylation is a proximal event that occurs both during development and following podocyte injury. We hypothesized that abrogation of nephrin phosphorylation following injury would prevent nephrin-dependent actin remodeling and foot process morphological changes. Utilizing a biased screening approach, we found nonreceptor Src homology 2 (sh2) domain-containing phosphatase Shp2 to be associated with phosphorylated nephrin. We observed an increase in nephrin tyrosine phosphorylation in the presence of Shp2 in cell culture studies. In the human glomerulopathies minimal-change nephrosis and membranous nephropathy, there is an increase in Shp2 phosphorylation, a marker of increased Shp2 activity. Mouse podocytes lacking Shp2 do not develop foot process spreading when subjected to podocyte injury in vivo using protamine sulfate or nephrotoxic serum (NTS). In the NTS model, we observed a lack of foot process spreading in mouse podocytes with Shp2 deleted and smaller amounts of proteinuria. Taken together, these results suggest that Shp2-dependent signaling events are necessary for changes in foot process structure and function following injury. PMID:26644409

Regulation of N-formyl peptide-mediated degranulation by receptor phosphorylation.

PubMed

Vines, Charlotte M; Xue, Mei; Maestas, Diane C; Cimino, Daniel F; Prossnitz, Eric R

2002-12-15

One of the major functions of the N-formyl peptide receptor (FPR) is to mediate leukocyte degranulation. Phosphorylation of the C-terminal domain of the FPR is required for receptor internalization and desensitization. Although arrestins mediate phosphorylation-dependent desensitization, internalization, and initiation of novel signaling cascades for a number of G protein-coupled receptors, their roles in FPR regulation and signaling remain unclear. CXCR1-mediated degranulation of RBL-2H3 cells is promoted by arrestin binding. To determine whether receptor phosphorylation or arrestin binding is required to promote FPR-mediated degranulation, we used RBL-2H3 cells stably transfected with either the wild-type FPR or a mutant form, DeltaST, which is incapable of undergoing ligand-stimulated phosphorylation. We observed that stimulation of wild-type FPR resulted in very low levels of degranulation compared with that mediated by cross-linking of the Fc(epsilon)RI receptor. Stimulation of the DeltaST mutant, however, resulted in levels of degranulation comparable to those of the Fc(epsilon)RI receptor, demonstrating that neither receptor phosphorylation nor arrestin binding was necessary to initiate FPR-mediated degranulation. Degranulation initiated by the DeltaST mutant was proportional to the level of active cell surface receptor, suggesting that either receptor internalization or desensitization may be responsible for terminating degranulation of the wild-type FPR. To distinguish between these possibilities, we used a partially phosphorylation-deficient mutant of the FPR that can undergo internalization, but not desensitization. Degranulation by this mutant FPR was indistinguishable from that of the DeltaST mutant, indicating that FPR phosphorylation or binding of arrestin but not internalization terminates the degranulation response.

Modes of caldesmon binding to actin: sites of caldesmon contact and modulation of interactions by phosphorylation.

PubMed

Foster, D Brian; Huang, Renjian; Hatch, Victoria; Craig, Roger; Graceffa, Philip; Lehman, William; Wang, C-L Albert

2004-12-17

Smooth muscle caldesmon binds actin and inhibits actomyosin ATPase activity. Phosphorylation of caldesmon by extracellular signal-regulated kinase (ERK) reverses this inhibitory effect and weakens actin binding. To better understand this function, we have examined the phosphorylation-dependent contact sites of caldesmon on actin by low dose electron microscopy and three-dimensional reconstruction of actin filaments decorated with a C-terminal fragment, hH32K, of human caldesmon containing the principal actin-binding domains. Helical reconstruction of negatively stained filaments demonstrated that hH32K is located on the inner portion of actin subdomain 1, traversing its upper surface toward the C-terminal segment of actin, and forms a bridge to the neighboring actin monomer of the adjacent long pitch helical strand by connecting to its subdomain 3. Such lateral binding was supported by cross-linking experiments using a mutant isoform, which was capable of cross-linking actin subunits. Upon ERK phosphorylation, however, the mutant no longer cross-linked actin to polymers. Three-dimensional reconstruction of ERK-phosphorylated hH32K indeed indicated loss of the interstrand connectivity. These results, together with fluorescence quenching data, are consistent with a phosphorylation-dependent conformational change that moves the C-terminal end segment of caldesmon near the phosphorylation site but not the upstream region around Cys(595), away from F-actin, thus neutralizing its inhibitory effect on actomyosin interactions. The binding pattern of hH32K suggests a mechanism by which unphosphorylated, but not ERK-phosphorylated, caldesmon could stabilize actin filaments and resist F-actin severing or depolymerization in both smooth muscle and nonmuscle cells.

Non-Aggregating Tau Phosphorylation by Cyclin-Dependent Kinase 5 Contributes to Motor Neuron Degeneration in Spinal Muscular Atrophy

PubMed Central

Miller, Nimrod; Feng, Zhihua; Edens, Brittany M.; Yang, Ben; Shi, Han; Sze, Christie C.; Hong, Benjamin Taige; Su, Susan C.; Cantu, Jorge A.; Topczewski, Jacek; Crawford, Thomas O.; Ko, Chien-Ping; Sumner, Charlotte J.; Ma, Long

2015-01-01

Mechanisms underlying motor neuron degeneration in spinal muscular atrophy (SMA), the leading inherited cause of infant mortality, remain largely unknown. Many studies have established the importance of hyperphosphorylation of the microtubule-associated protein tau in various neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. However, tau phosphorylation in SMA pathogenesis has yet to be investigated. Here we show that tau phosphorylation on serine 202 (S202) and threonine 205 (T205) is increased significantly in SMA motor neurons using two SMA mouse models and human SMA patient spinal cord samples. Interestingly, phosphorylated tau does not form aggregates in motor neurons or neuromuscular junctions (NMJs), even at late stages of SMA disease, distinguishing it from other tauopathies. Hyperphosphorylation of tau on S202 and T205 is mediated by cyclin-dependent kinase 5 (Cdk5) in SMA disease condition, because tau phosphorylation at these sites is significantly reduced in Cdk5 knock-out mice; genetic knock-out of Cdk5 activating subunit p35 in an SMA mouse model also leads to reduced tau phosphorylation on S202 and T205 in the SMA;p35−/− compound mutant mice. In addition, expression of the phosphorylation-deficient tauS202A,T205A mutant alleviates motor neuron defects in a zebrafish SMA model in vivo and mouse motor neuron degeneration in culture, whereas expression of phosphorylation-mimetic tauS202E,T205E promotes motor neuron defects. More importantly, genetic knock-out of tau in SMA mice rescues synapse stripping on motor neurons, NMJ denervation, and motor neuron degeneration in vivo. Altogether, our findings suggest a novel mechanism for SMA pathogenesis in which hyperphosphorylation of non-aggregating tau by Cdk5 contributes to motor neuron degeneration. PMID:25878277

Differential recognition of syk-binding sites by each of the two phosphotyrosine-binding pockets of the Vav SH2 domain.

PubMed

Chen, Chih-Hong; Piraner, Dan; Gorenstein, Nina M; Geahlen, Robert L; Beth Post, Carol

2013-11-01

The association of spleen tyrosine kinase (Syk), a central tyrosine kinase in B cell signaling, with Vav SH2 domain is controlled by phosphorylation of two closely spaced tyrosines in Syk linker B: Y342 and Y346. Previous studies established both singly phosphorylated and doubly phosphorylated forms play a role in signaling. The structure of the doubly phosphorylated form identified a new recognition of phosphotyrosine whereby two phosphotyrosines bind simultaneously to the Vav SH2 domain, one in the canonical pTyr pocket and one in the specificity pocket on the opposite side of the central β-sheet. It is unknown if the specificity pocket can bind phosphotyrosine independent of phosphotyrosine binding the pTyr pocket. To address this gap in knowledge, we determined the structure of the complex between Vav1 SH2 and a peptide (SykLB-YpY) modeling the singly phosphorylated-Y346 form of Syk with unphosphorylated Y342. The nuclear magnetic resonance (NMR) data conclusively establish that recognition of phosphotyrosine is swapped between the two pockets; phosphorylated pY346 binds the specificity pocket of Vav1 SH2, and unphosphorylated Y342 occupies what is normally the pTyr binding pocket. Nearly identical changes in chemical shifts occurred upon binding all three forms of singly and doubly phosphorylated peptides; however, somewhat smaller shift perturbations for SykLB-YpY from residues in regions of high internal mobility suggest that internal motions are coupled to binding affinity. The differential recognition that includes this swapped binding of phosphotyrosine to the specificity pocket of Vav SH2 increases the repertoire of possible phosphotyrosine binding by SH2 domains in regulating protein-protein interactions in cellular signaling. Copyright © 2013 Wiley Periodicals, Inc.

Phosphorylation of Trihelix Transcriptional Repressor ASR3 by MAP KINASE4 Negatively Regulates Arabidopsis Immunity

PubMed Central

Li, Bo; Jiang, Shan; Yu, Xiao; Cheng, Cheng; Chen, Sixue; Cheng, Yanbing; Yuan, Joshua S.; Jiang, Daohong; He, Ping; Shan, Libo

2015-01-01

Proper control of immune-related gene expression is crucial for the host to launch an effective defense response. Perception of microbe-associated molecular patterns (MAMPs) induces rapid and profound transcriptional reprogramming via unclear mechanisms. Here, we show that ASR3 (ARABIDOPSIS SH4-RELATED3) functions as a transcriptional repressor and plays a negative role in regulating pattern-triggered immunity (PTI) in Arabidopsis thaliana. ASR3 belongs to a plant-specific trihelix transcription factor family for which functional studies are lacking. MAMP treatments induce rapid phosphorylation of ASR3 at threonine 189 via MPK4, a mitogen-activated protein kinase that negatively regulates PTI responses downstream of multiple MAMP receptors. ASR3 possesses transcriptional repressor activity via its ERF-associated amphiphilic repression motifs and negatively regulates a large subset of flg22-induced genes. Phosphorylation of ASR3 by MPK4 enhances its DNA binding activity to suppress gene expression. Importantly, the asr3 mutant shows enhanced disease resistance to virulent bacterial pathogen infection, whereas transgenic plants overexpressing the wild-type or phospho-mimetic form of ASR3 exhibit compromised PTI responses. Our studies reveal a function of the trihelix transcription factors in plant innate immunity and provide evidence that ASR3 functions as a transcriptional repressor regulated by MAMP-activated MPK4 to fine-tune plant immune gene expression. PMID:25770109

Adiponectin Receptors Form Homomers and Heteromers Exhibiting Distinct Ligand Binding and Intracellular Signaling Properties*

PubMed Central

Almabouada, Farid; Diaz-Ruiz, Alberto; Rabanal-Ruiz, Yoana; Peinado, Juan R.; Vazquez-Martinez, Rafael; Malagon, Maria M.

2013-01-01

Adiponectin binds to two widely expressed receptors (AdipoR1 and AdipoR2) that contain seven transmembrane domains but, unlike G-protein coupled receptors, present an extracellular C terminus and a cytosolic N terminus. Recently, AdipoR1 was found to associate in high order complexes. However, it is still unknown whether AdipoR2 may also form homomers or heteromers with AdipoR1 or if such interactions may be functionally relevant. Herein, we have analyzed the oligomerization pattern of AdipoRs by FRET and immunoprecipitation and evaluated both the internalization of AdipoRs in response to various adiponectin isoforms and the effect of adiponectin binding to different AdipoR combinations on AMP-activated protein kinase phosphorylation and peroxisome proliferator-activated receptor α activation. Transfection of HEK293AD cells with AdipoR1 and AdipoR2 showed that both receptors colocalize at both the plasma membrane and the endoplasmic reticulum. Co-transfection with the different AdipoR pairs yielded high FRET efficiencies in non-stimulated cells, which indicates that AdipoR1 and AdipoR2 form homo- and heteromeric complexes under resting conditions. Live FRET imaging suggested that both homo- and heteromeric AdipoR complexes dissociate in response to adiponectin, but heteromers separate faster than homomers. Finally, phosphorylation of AMP-activated protein kinase in response to adiponectin was delayed in cells wherein heteromer formation was favored. In sum, our findings indicate that AdipoR1 and AdipoR2 form homo- and heteromers that present unique interaction behaviors and signaling properties. This raises the possibility that the pleiotropic, tissue-dependent functions of adiponectin depend on the expression levels of AdipoR1 and AdipoR2 and, therefore, on the steady-state proportion of homo- and heteromeric complexes. PMID:23255609

Adiponectin receptors form homomers and heteromers exhibiting distinct ligand binding and intracellular signaling properties.

PubMed

Almabouada, Farid; Diaz-Ruiz, Alberto; Rabanal-Ruiz, Yoana; Peinado, Juan R; Vazquez-Martinez, Rafael; Malagon, Maria M

2013-02-01

Adiponectin binds to two widely expressed receptors (AdipoR1 and AdipoR2) that contain seven transmembrane domains but, unlike G-protein coupled receptors, present an extracellular C terminus and a cytosolic N terminus. Recently, AdipoR1 was found to associate in high order complexes. However, it is still unknown whether AdipoR2 may also form homomers or heteromers with AdipoR1 or if such interactions may be functionally relevant. Herein, we have analyzed the oligomerization pattern of AdipoRs by FRET and immunoprecipitation and evaluated both the internalization of AdipoRs in response to various adiponectin isoforms and the effect of adiponectin binding to different AdipoR combinations on AMP-activated protein kinase phosphorylation and peroxisome proliferator-activated receptor α activation. Transfection of HEK293AD cells with AdipoR1 and AdipoR2 showed that both receptors colocalize at both the plasma membrane and the endoplasmic reticulum. Co-transfection with the different AdipoR pairs yielded high FRET efficiencies in non-stimulated cells, which indicates that AdipoR1 and AdipoR2 form homo- and heteromeric complexes under resting conditions. Live FRET imaging suggested that both homo- and heteromeric AdipoR complexes dissociate in response to adiponectin, but heteromers separate faster than homomers. Finally, phosphorylation of AMP-activated protein kinase in response to adiponectin was delayed in cells wherein heteromer formation was favored. In sum, our findings indicate that AdipoR1 and AdipoR2 form homo- and heteromers that present unique interaction behaviors and signaling properties. This raises the possibility that the pleiotropic, tissue-dependent functions of adiponectin depend on the expression levels of AdipoR1 and AdipoR2 and, therefore, on the steady-state proportion of homo- and heteromeric complexes.

Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation

PubMed Central

Kazlauskaite, Agne; Martínez-Torres, R Julio; Wilkie, Scott; Kumar, Atul; Peltier, Julien; Gonzalez, Alba; Johnson, Clare; Zhang, Jinwei; Hope, Anthony G; Peggie, Mark; Trost, Matthias; van Aalten, Daan MF; Alessi, Dario R; Prescott, Alan R; Knebel, Axel; Walden, Helen; Muqit, Miratul MK

2015-01-01

Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser65)—which lies within its ubiquitin-like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser65. How Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitinPhospho-Ser65 binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser65 by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitinPhospho-Ser65, thereby promoting Parkin Ser65 phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser65 phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitinPhospho-Ser65 to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser65. Finally, purified Parkin maximally phosphorylated at Ser65 in vitro cannot be further activated by the addition of ubiquitinPhospho-Ser65. Our results thus suggest that a major role of ubiquitinPhospho-Ser65 is to promote PINK1-mediated phosphorylation of Parkin at Ser65, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser65-binding pocket on the surface of Parkin that is critical for the ubiquitinPhospho-Ser65 interaction. This study provides new mechanistic insights into Parkin activation by ubiquitinPhospho-Ser65, which could aid in the development of Parkin activators that mimic the effect of ubiquitinPhospho-Ser65. PMID:26116755

Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation.

PubMed

Kazlauskaite, Agne; Martínez-Torres, R Julio; Wilkie, Scott; Kumar, Atul; Peltier, Julien; Gonzalez, Alba; Johnson, Clare; Zhang, Jinwei; Hope, Anthony G; Peggie, Mark; Trost, Matthias; van Aalten, Daan M F; Alessi, Dario R; Prescott, Alan R; Knebel, Axel; Walden, Helen; Muqit, Miratul M K

2015-08-01

Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser(65))--which lies within its ubiquitin-like domain (Ubl)--and indirectly through phosphorylation of ubiquitin at Ser(65). How Ser(65)-phosphorylated ubiquitin (ubiquitin(Phospho-Ser65)) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitin(Phospho-Ser65) binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser(65) by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitin(Phospho-Ser65), thereby promoting Parkin Ser(65) phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser(65) phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitin(Phospho-Ser65) to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser(65). Finally, purified Parkin maximally phosphorylated at Ser(65) in vitro cannot be further activated by the addition of ubiquitin(Phospho-Ser65). Our results thus suggest that a major role of ubiquitin(Phospho-Ser65) is to promote PINK1-mediated phosphorylation of Parkin at Ser(65), leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser(65)-binding pocket on the surface of Parkin that is critical for the ubiquitin(Phospho-Ser65) interaction. This study provides new mechanistic insights into Parkin activation by ubiquitin(Phospho-Ser65), which could aid in the development of Parkin activators that mimic the effect of ubiquitin(Phospho-Ser65). © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM)-mediated Inhibitory Signaling is Regulated by Sequential Phosphorylation Mediated by Distinct Nonreceptor Tyrosine Kinases: A Case Study Involving PECAM-1

PubMed Central

Tourdot, Benjamin E.; Brenner, Michelle K.; Keough, Kathleen C.; Holyst, Trudy; Newman, Peter J.; Newman, Debra K.

2013-01-01

The activation state of many blood and vascular cells is tightly controlled by a delicate balance between receptors that contain immunoreceptor tyrosine-based activation motifs (ITAMs) and those that contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Precisely how the timing of cellular activation by ITAM-coupled receptors is regulated by ITIM-containing receptors is, however, poorly understood. Using platelet endothelial cell adhesion molecule 1 (PECAM-1) as a prototypical ITIM-bearing receptor, we demonstrate that initiation of inhibitory signaling occurs via a novel, sequential process in which Src family kinases phosphorylate the C-terminal ITIM, thereby enabling phosphorylation of the N-terminal ITIM of PECAM-1 by other Src homology 2 domain-containing non-receptor tyrosine kinases (NRTKs). NRTKs capable of mediating the second phosphorylation event include C-terminal Src kinase (Csk) and Bruton’s tyrosine kinase (Btk). Btk and Csk function downstream of phosphatidylinositol 3-kinase (PI3K) activation during ITAM-dependent platelet activation. In ITAM-activated platelets that were treated with a PI3K inhibitor, PECAM-1 was phosphorylated but did not bind the tandem SH2 domain-containing tyrosine phosphatase SHP-2, indicating that it was not phosphorylated on its N-terminal ITIM. Csk bound to and phosphorylated PECAM-1 more efficiently than did Btk, and required its SH2 domain to perform these functions. Additionally, the phosphorylation of the N-terminal ITIM of Siglec-9 by Csk is enhanced by the prior phosphorylation of its C-terminal ITIM, providing evidence that the ITIMs of other dual ITIM-containing receptors are also sequentially phosphorylated. On the basis of these findings, we propose that sequential ITIM phosphorylation provides a general mechanism for precise temporal control over the recruitment and activation of tandem SH2 domain-containing tyrosine phosphatases that dampen ITAM-dependent signals. PMID:23418871

The oligomerization state determines regulatory properties and inhibitor sensitivity of type 4 cAMP-specific phosphodiesterases.

PubMed

Richter, Wito; Conti, Marco

2004-07-16

PDE4 splice variants are classified into long and short forms depending on the presence or absence of two unique N-terminal domains termed upstream conserved regions 1 and 2 (UCR1 and -2). We have shown previously that the UCR module mediates dimerization of PDE4 long forms, whereas short forms, which lack UCR1, behave as monomers. In the present study, we demonstrate that dimerization is an essential structural element that determines the regulatory properties and inhibitor sensitivities of PDE4 enzymes. Comparing the properties of the dimeric wild type PDE4D3 with several monomeric mutant PDE4D3 constructs revealed that disruption of dimerization ablates the activation of PDE4 long forms by either protein kinase A phosphorylation or phosphatidic acid binding. Moreover, the analysis of heterodimers consisting of a catalytically active and a catalytically inactive PDE4D3 subunit indicates that protein kinase A phosphorylation of both subunits is essential to fully activate PDE4 enzymes. In addition to affecting enzyme regulation, disruption of dimerization reduces the sensitivity of the enzymes toward the prototypical PDE4 inhibitor rolipram. Parallel binding assays indicated that this shift in rolipram sensitivity is likely mediated by a decrease in the number of inhibitor binding sites in the high affinity rolipram binding state. Thus, although dimerization is not a requirement for high affinity rolipram binding, it functions to stabilize PDE4 long forms in their high affinity rolipram binding conformation. Taken together, our data indicate that dimerization defines the properties of PDE4 enzymes and suggest a common structural and functional organization for all PDEs.

Novel Autophosphorylation Sites of Src Family Kinases Regulate Kinase Activity and SH2 Domain Binding Capacity

PubMed Central

Weir, Marion E.; Mann, Jacqueline E.; Corwin, Thomas; Fulton, Zachary W.; Hao, Jennifer M.; Maniscalco, Jeanine F.; Kenney, Marie C.; Roque, Kristal M. Roman; Chapdelaine, Elizabeth F.; Stelzl, Ulrich; Deming, Paula B.; Ballif, Bryan A.; Hinkle, Karen L.

2016-01-01

Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly-regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly-phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the site C-terminal to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. PMID:27001024

Characterization of STAT5B phosphorylation correlating with expression of cytokine-inducible SH2-containing protein (CIS).

PubMed

Cooper, John C; Boustead, Jared N; Yu, Chao-Lan

2006-06-01

Cytokine-inducible SH2-containing protein (CIS) is the first identified member of genes encoding for the suppressor of cytokine signaling (SOCS). CIS is also a well-known target gene of signal transducer and activator of transcription 5 (STAT5) pathways, providing normal negative feedback control of signaling by cytokines and growth factors. Three other SOCS genes, SOCS1, SOCS2, and SOCS3, can be silenced by DNA hypermethylation in human cancers, suggesting a potential mechanism for constitutive STAT activation. However, it is not known whether CIS expression is similarly perturbed in tumor cells. We report here the absence of CIS expression in T lymphoma LSTRA that overexpresses the Lck protein tyrosine kinase and exhibits elevated STAT5 activity. Pervanadate-induced CIS expression and STAT5 binding to the CIS promoter in vivo over a short time course implies that mechanisms other than DNA hypermethylation may contribute to defective CIS expression in LSTRA cells. Comparison with cytokine-dependent BaF3 cells stimulated with interleukin-3 (IL-3) further reveals that CIS induction correlates with specific STAT5b post-translational modifications. It exhibits as the slowest migrating form through SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. This distinctly modified STAT5b is the predominant form that binds to the consensus STAT5 sites in the CIS promoter and accumulates in the nucleus. In vitro phosphatase assays and phosphoamino acid analysis suggest the involvement of phosphorylation on residues other than the highly conserved tyrosine and serine sites in this distinct STAT5b mobility shift. All together, our results provide a novel link between incomplete STAT5b phosphorylation and defective SOCS gene expression in cancer cells.

CUB domain-containing protein 1 and the epidermal growth factor receptor cooperate to induce cell detachment.

PubMed

Law, Mary E; Ferreira, Renan B; Davis, Bradley J; Higgins, Paul J; Kim, Jae-Sung; Castellano, Ronald K; Chen, Sixue; Luesch, Hendrik; Law, Brian K

2016-08-05

While localized malignancies often respond to available therapies, most disseminated cancers are refractory. Novel approaches, therefore, are needed for the treatment of metastatic disease. CUB domain-containing protein1 (CDCP1) plays an important role in metastasis and drug resistance; the mechanism however, is poorly understood. Breast cancer cell lines were engineered to stably express EGFR, CDCP1 or phosphorylation site mutants of CDCP1. These cell lines were used for immunoblot analysis or affinity purification followed by immunoblot analysis to assess protein phosphorylation and/or protein complex formation with CDCP1. Kinase activity was evaluated using phosphorylation site-specific antibodies and immunoblot analysis in in vitro kinase assays. Protein band excision and mass spectrometry was utilized to further identify proteins complexed with CDCP1 or ΔCDCP1, which is a mimetic of the cleaved form of CDCP1. Cell detachment was assessed using cell counting. This paper reports that CDCP1 forms ternary protein complexes with Src and EGFR, facilitating Src activation and Src-dependent EGFR transactivation. Importantly, we have discovered that a class of compounds termed Disulfide bond Disrupting Agents (DDAs) blocks CDCP1/EGFR/Src ternary complex formation and downstream signaling. CDCP1 and EGFR cooperate to induce detachment of breast cancer cells from the substratum and to disrupt adherens junctions. Analysis of CDCP1-containing complexes using proteomics techniques reveals that CDCP1 associates with several proteins involved in cell adhesion, including adherens junction and desmosomal cadherins, and cytoskeletal elements. Together, these results suggest that CDCP1 may facilitate loss of adhesion by promoting activation of EGFR and Src at sites of cell-cell and cell-substratum contact.

Lessons from a tarantula: new insights into muscle thick filament and myosin interacting-heads motif structure and function.

PubMed

Alamo, Lorenzo; Koubassova, Natalia; Pinto, Antonio; Gillilan, Richard; Tsaturyan, Andrey; Padrón, Raúl

2017-10-01

The tarantula skeletal muscle X-ray diffraction pattern suggested that the myosin heads were helically arranged on the thick filaments. Electron microscopy (EM) of negatively stained relaxed tarantula thick filaments revealed four helices of heads allowing a helical 3D reconstruction. Due to its low resolution (5.0 nm), the unambiguous interpretation of densities of both heads was not possible. A resolution increase up to 2.5 nm, achieved by cryo-EM of frozen-hydrated relaxed thick filaments and an iterative helical real space reconstruction, allowed the resolving of both heads. The two heads, "free" and "blocked", formed an asymmetric structure named the "interacting-heads motif" (IHM) which explained relaxation by self-inhibition of both heads ATPases. This finding made tarantula an exemplar system for thick filament structure and function studies. Heads were shown to be released and disordered by Ca 2+ -activation through myosin regulatory light chain phosphorylation, leading to EM, small angle X-ray diffraction and scattering, and spectroscopic and biochemical studies of the IHM structure and function. The results from these studies have consequent implications for understanding and explaining myosin super-relaxed state and thick filament activation and regulation. A cooperative phosphorylation mechanism for activation in tarantula skeletal muscle, involving swaying constitutively Ser35 mono-phosphorylated free heads, explains super-relaxation, force potentiation and post-tetanic potentiation through Ser45 mono-phosphorylated blocked heads. Based on this mechanism, we propose a swaying-swinging, tilting crossbridge-sliding filament for tarantula muscle contraction.

Transcriptional and translational control of ornithine decarboxylase during Ras transformation.

PubMed Central

Shantz, Lisa M

2004-01-01

ODC (ornithine decarboxylase) activity is induced following ras activation. However, the Ras effector pathways responsible are unknown. These experiments used NIH-3T3 cells expressing partial-loss-of-function Ras mutants to activate selectively pathways downstream of Ras and examined the contribution of each pathway to ODC induction. Overexpression of Ras12V, a constitutively active mutant, resulted in ODC activities up to 20-fold higher than controls. Stable transfections of Ras partial-loss-of-function mutants and constitutively active forms of MEK (MAPK kinase) and Akt indicated that activation of more than one Ras effector pathway is necessary for the complete induction of ODC activity. The increase in ODC activity in Ras12V-transformed cells is not owing to a substantial change in ODC protein half-life, which increased by <2-fold. Northern-blot analysis and reporter assays suggested that the mechanism of ODC induction involves both a modest increase in the transcription of ODC mRNA and a much more considerable increase in the translation of mRNA into protein. ODC transcription was controlled through a pathway dependent on Raf/MEK/ERK (where ERK stands for extracellular-signal-regulated kinase) activation, whereas activation of the phosphoinositide 3-kinase and the Raf/MEK/ERK pathways were necessary for translational regulation of ODC. The increase in ODC synthesis was accompanied by changes in phosphorylation of eukaryotic initiation factor 4E and its binding protein 4E-BP1. Results show that the phosphoinositide 3-kinase pathway regulates phosphorylation of both proteins, whereas the Raf/MEK/ERK pathway affects only the eukaryotic initiation factor 4E phosphorylation. PMID:14519103

The role of STATs in transcriptional control and their impact on cellular function.

PubMed

Bromberg, J; Darnell, J E

2000-05-15

The STAT proteins (Signal Transducers and Activators of Transcription), were identified in the last decade as transcription factors which were critical in mediating virtually all cytokine driven signaling. These proteins are latent in the cytoplasm and become activated through tyrosine phosphorylation which typically occurs through cytokine receptor associated kinases (JAKs) or growth factor receptor tyrosine kinases. Recently a number of non-receptor tyrosine kinases (for example src and abl) have been found to cause STAT phosphorylation. Phosphorylated STATs form homo- or hetero-dimers, enter the nucleus and working coordinately with other transcriptional co-activators or transcription factors lead to increased transcriptional initiation. In normal cells and in animals, ligand dependent activation of the STATs is a transient process, lasting for several minutes to several hours. In contrast, in many cancerous cell lines and tumors, where growth factor dysregulation is frequently at the heart of cellular transformation, the STAT proteins (in particular Stats 1, 3 and 5) are persistently tyrosine phosphorylated or activated. The importance of STAT activation to growth control in experiments using anti-sense molecules or dominant negative STAT protein encoding constructs performed in cell lines or studies in animals lacking specific STATs strongly indicate that STATs play an important role in controlling cell cycle progression and apoptosis. Stat1 plays an important role in growth arrest, in promoting apoptosis and is implicated as a tumor suppressor; while Stats 3 and 5 are involved in promoting cell cycle progression and cellular transformation and preventing apoptosis. Many questions remain including: (1) a better understanding of how the STAT proteins through association with other factors increase transcription initiation; (2) a more complete definition of the sets of genes which are activated by different STATs and (3) how these sets of activated genes differ as a function of cell type. Finally, in the context of many cancers, where STATs are frequently persistently activated, an understanding of the mechanisms leading to their constitutive activation and defining the potential importance of persistent STAT activation in human tumorigenesis remains. Oncogene (2000).

Stimulation of dihydroxyacetone and glycerol kinase activity in Streptococcus faecalis by phosphoenolpyruvate-dependent phosphorylation catalyzed by enzyme I and HPr of the phosphotransferase systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deutscher, J.; Sauerwald, H.

1986-06-01

Recently a report was given of the phosphoenolpyruvate (PEP)-dependent phosphorylation of a 55-kilodalton protein of Streptococus faecalis catalyzed by enzyme I and histidine-containing protein (HPr) of the phosphotransferase system. The purified 55-kilodalton protein was found to exhibit dihydroxyacetone kinase activity. Glycerol was six times more slowly phosphorylated than dihydroxyacetone. The K/sub m/s were found to 0.7 mM for ATP, 0.45 mM for dihydroxyacetone, and 0.9 MM for glycerol. PEP-dependent phosphorylation of dihydroxyacetone kinase stimulated phosphorylation of both substrates about 10-fold. Fructose 1,6-diphosphate at concentrations higher than 2 mM inhibited the activity of phosphorylated and unphosphorylated dihydroxyacetone kinase in a noncompetitivemore » manner. The rate of PEP-dependent phosphorylation of dihydroxyacetone kinase was about 200-fold slower than the phosphorylation rate of III proteins (also called enzyme III or factor III), which so far have been considered the only phosphoryl acceptors of histidyl-phosphorylated HPr. P-Dihydroxyacetone kinase was found to be able to transfer its phosphoryl group in a backward reaction to HPr. Following (/sup 32/P)PEP-dependent phosphorylation and tryptic digestion of dihydroxyacetone kinase, the authors isolated a labeled peptide composed of 37 amino acids, as determined by amino acid analysis. The single histidyl residue of this peptide most likely carries the phosphoryl group in phosphorylated dihydroxyacetone kinase.« less

Phosphorylation of sucrose synthase at serine 170: occurrence and possible role as a signal for proteolysis.

PubMed

Hardin, Shane C; Tang, Guo-Qing; Scholz, Anke; Holtgraewe, Daniela; Winter, Heike; Huber, Steven C

2003-09-01

Sequence analysis identified serine 170 (S170) of the maize (Zea mays L.) SUS1 sucrose synthase (SUS) protein as a possible, second phosphorylation site. Maize leaves contained two calcium-dependent protein kinase activities and a calcium-independent kinase activity with characteristics of an sucrose non-fermenting 1 (SNF1)-related protein kinase. Phosphorylation of the novel S170 and the known serine 15 (S15) site by these protein kinases was determined in peptide substrates and detected in SUS1 protein substrates utilizing sequence- and phosphorylation-specific antibodies. We demonstrate phosphorylation of S170 in vitro and in vivo. The calcium-dependent protein kinases phosphorylated both S170 and S15, whereas SNF1-related protein kinase activity was restricted to S15. Calcium-dependent protein-kinase-mediated S170 and S15 phosphorylation kinetics were determined in wild-type and mutant SUS1 substrates. These analyses revealed that kinase specificity for S170 was threefold lower than that for S15, and that phosphorylation of S170 was stimulated by prior phosphorylation at the S15 site. The SUS-binding peptides encoded by early nodulin 40 (ENOD40) specifically antagonized S170 phosphorylation in vitro. A model wherein S170 phosphorylation functions as part of a mechanism targeting SUS for proteasome-mediated degradation is supported by the observations that SUS proteolytic fragments: (i) were detected and possessed relatively high phosphorylated-S170 (pS170) stoichiometry; (ii) were spatially coincident with proteasome activity within developing leaves; and (iii) co-sedimented with proteasome activity. In addition, full-length pS170-SUS protein was less stable than S170-SUS in cultured leaf segments and was stabilized by proteasome inhibition. Post-translational control of SUS protein level through pS170-promoted proteolysis may explain the specific and significant decrease in SUS abundance that accompanies the sink-to-source transition in developing maize leaves.

Syntheses and insulin-like activity of phosphorylated galactose derivatives.

PubMed

Caro, H N; Martín-Lomas, M; Bernabé, M

1993-02-24

The syntheses of the poly-phosphorylated galactosides 6, 8, 10, 13, 16, and 20, isolated as sodium salts, have been performed. The non-phosphorylated disaccharide 17 and trisaccharide 21 have been prepared via glycosylation of the 2-(trimethylsilyl)ethyl galactosides 3 and 2, respectively, and subsequent complete deprotection. Preliminary insulin-like activity of the phosphorylated derivatives is reported.

In Vivo Overexpression of Tissue-Nonspecific Alkaline Phosphatase Increases Skeletal Mineralization and Affects the Phosphorylation Status of Osteopontin

PubMed Central

Narisawa, Sonoko; Yadav, Manisha C.; Millán, José Luis

2013-01-01

Functional ablation of tissue-nonspecific alkaline phosphatase (TNAP) (Alpl−/− mice) leads to hypophosphatasia, characterized by rickets/osteomalacia attributable to elevated levels of extracellular inorganic pyrophosphate, a potent mineralization inhibitor. Osteopontin (OPN) is also elevated in the plasma and skeleton of Alpl−/− mice. Phosphorylated OPN is known to inhibit mineralization, however, the phosphorylation status of the increased OPN found in Alpl−/− mice is unknown. Here, we generated a transgenic mouse line expressing human TNAP under control of an osteoblast-specific Col1a1 promoter (Col1a1-Tnap). The transgene is expressed in osteoblasts, periosteum, and cortical bones, and plasma levels of TNAP in mice expressing Col1a1-Tnap are 10-20 times higher than those of wild-type mice. The Col1a1-Tnap animals are healthy and exhibit increased bone mineralization by microCT analysis. Crossbreeding of Col1a1-Tnap transgenic mice to Alpl−/− mice rescues the lethal hypophosphatasia phenotype characteristic of this disease model. Osteoblasts from [Col1a1-Tnap] mice mineralize better than non-transgenic controls and osteoblasts from [Col1a1-Tnap+/−; Alpl−/−] mice are able to mineralize to the level of Alpl+/− heterozygous osteoblasts, while Alpl−/− osteoblasts show no mineralization. We found that the increased levels of OPN in bone tissue of Alpl−/− mice are comprised of phosphorylated forms of OPN while WT and [Col1a1-Tnap+/−; Alpl−/−] mice had both phosphorylated and dephosphorylated forms of OPN. OPN from [Col1a1-Tnap] osteoblasts were more phosphorylated than non-transgenic control cells. Titanium dioxide-liquid chromatography and tandem mass spectrometry analysis revealed that OPN peptides derived from Alpl−/− bone and osteoblasts yielded a higher proportion of phosphorylated peptides than samples from WT mice, and at least two phosphopeptides, p(S174FQVS178DEQY182PDAT186DEDLT191)SHMK and FRIp(S299HELES304S305S306S307)EVN, with one non-localized site each, appear to be preferred sites of TNAP action on OPN. Our data suggest that the pro-mineralization role of TNAP may be related not only to its accepted pyrophosphatase activity but also to its ability to modify the phosphorylation status of OPN. PMID:23427088

Molecular mechanisms underlying PINK1 and Parkin catalyzed ubiquitylation of substrates on damaged mitochondria.

PubMed

Koyano, Fumika; Matsuda, Noriyuki

2015-10-01

PINK1 and Parkin are gene products that cause genetic recessive Parkinsonism. PINK1 is a protein kinase and Parkin is a ubiquitin ligase (E3) that links ubiquitin to a substrate. Importantly, under steady state conditions, the enzymatic activity of Parkin is completely suppressed, but is activated when mitochondria become abnormal. In 2013 and 2014, biochemical and structure-function analyses revealed a number of critical mechanistic insights. First, Parkin is a self-inhibitory E3 that suppresses its E3 activity via intramolecular interactions. Second, in response to a decrease in mitochondrial membrane potential, PINK1 phosphorylates Ser65 in both the Parkin ubiquitin-like domain and ubiquitin itself. These phosphorylation events cooperate to relieve the Parkin autoinhibition. Third, activated Parkin forms a ubiquitin-thioester bond at Cys431 to produce a reaction intermediate that catalyzes ubiquitylation of substrates on damaged mitochondria. While the molecular mechanism regulating Parkin enzymatic activity has largely eluded clarification, a complete picture is now emerging. Copyright © 2015 Elsevier B.V. All rights reserved.

Mechanisms underlying the inhibitory effects of arsenic compounds on protein tyrosine phosphatase (PTP)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rehman, Kanwal; Chen, Zhe; Wang, Wen Wen

2012-09-15

Arsenic binding to biomolecules is considered one of the major toxic mechanisms, which may also be related to the carcinogenic risks of arsenic in humans. At the same time, arsenic is also known to activate the phosphorylation-dependent signaling pathways including the epidermal growth factor receptor, the mitogen-activated protein kinase and insulin/insulin-like growth factor-1 pathways. These signaling pathways originate at the level of receptor tyrosine kinases whose phosphorylation status is regulated by opposing protein tyrosine phosphatase (PTP) activity. Reversible tyrosine phosphorylation, which is governed by the balanced action of protein tyrosine kinases and phosphatases, regulates important signaling pathways that are involvedmore » in the control of cell proliferation, adhesion and migration. In the present study, we have focused on the interaction of cellular PTPs with toxic trivalent arsenite (iAs{sup III}) and its intermediate metabolites such as monomethylarsonous acid (MMA{sup III}) and dimethylarsinous acid (DMA{sup III}) in vitro, and then determined the arsenic binding site in PTP by the use of recombinant PTPs (e.g., PTP1B and CD45). Interestingly, the activities of PTP1B (cytoplasm-form) or CD45 (receptor-linked form) were observed to be strongly inhibited by both methylated metabolites (i.e., MMA{sup III} and DMA{sup III}) but not by iAs{sup III}. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has clearly confirmed that the organic intermediate, DMA{sup III} directly bound to the active site cysteine residue of PTP1B (e.g., Cys215), resulting in inhibition of enzyme activity. These results suggest that arsenic exposure may disturb the cellular signaling pathways through PTP inactivation. Highlights: ► This study focused on the interaction of PTPs with trivalent arsenicals in vitro. ► We for the first time confirmed that DMA{sup III} strongly inhibited activity of PTP1B. ► DMA{sup III} directly bound to PTP1B, resulting in inhibition of enzyme activity.« less

HIPK2 restricts SIRT1 activity upon severe DNA damage by a phosphorylation-controlled mechanism

PubMed Central

Conrad, E; Polonio-Vallon, T; Meister, M; Matt, S; Bitomsky, N; Herbel, C; Liebl, M; Greiner, V; Kriznik, B; Schumacher, S; Krieghoff-Henning, E; Hofmann, T G

2016-01-01

Upon severe DNA damage a cellular signalling network initiates a cell death response through activating tumour suppressor p53 in association with promyelocytic leukaemia (PML) nuclear bodies. The deacetylase Sirtuin 1 (SIRT1) suppresses cell death after DNA damage by antagonizing p53 acetylation. To facilitate efficient p53 acetylation, SIRT1 function needs to be restricted. How SIRT1 activity is regulated under these conditions remains largely unclear. Here we provide evidence that SIRT1 activity is limited upon severe DNA damage through phosphorylation by the DNA damage-responsive kinase HIPK2. We found that DNA damage provokes interaction of SIRT1 and HIPK2, which phosphorylates SIRT1 at Serine 682 upon lethal damage. Furthermore, upon DNA damage SIRT1 and HIPK2 colocalize at PML nuclear bodies, and PML depletion abrogates DNA damage-induced SIRT1 Ser682 phosphorylation. We show that Ser682 phosphorylation inhibits SIRT1 activity and impacts on p53 acetylation, apoptotic p53 target gene expression and cell death. Mechanistically, we found that DNA damage-induced SIRT1 Ser682 phosphorylation provokes disruption of the complex between SIRT1 and its activator AROS. Our findings indicate that phosphorylation-dependent restriction of SIRT1 activity by HIPK2 shapes the p53 response. PMID:26113041

Magnetic Electrochemical Sensing Platform for Biomonitoring of Exposure to Organophosphorus Pesticides and Nerve Agents Based on Simultaneous Measurement of Total Enzyme Amount and Enzyme Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Dan; Wang, Jun; Wang, Limin

We report a new approach for electrochemical quantification of enzymatic inhibition and phosphorylation for biomonitoring of exposure to organophosphorus (OP) pesticides and nerve agents based on a magnetic beads (MBs) immunosensing platform. The principle of this approach is based on the combination of MBs immuno-capture based enzyme activity assay and competitive immunoassay of total amount of enzyme for simultaneous detection of enzyme inhibition and phosphorylation in biological fluids. Butyrylcholinesterase (BChE) was chosen as a model enzyme. In competitive immunoassay, the target total BChE in a sample (mixture of OP-inhibited BChE and active BChE) competes with the BChE modified on themore » MBs to bind to the limited anti-BChE antibody labeled with quantum dots (QDs-anti-BChE), and followed by electrochemical stripping analysis of the bound QDs conjugate on the MBs. This assay shows a linear response over the total BChE concentration range of 0.1~20 nM. Simultaneously, real time BChE activity was measured on an electrochemical carbon nanotube-based sensor coupled with microflow injection system after immuno-capture by MBs-anti-BChE conjugate. Therefore, the formed phosphorylated adduct (OP-BChE) can be estimated by the difference values of the total amount BChE (including active and OP-inhibited) and active BChE from established calibration curves. This approach not only eliminates the difficulty in screening of low-dose OP exposure (less than 20% inhibition of BChE) because of individual variation of BChE values, but also avoids the drawback of the scarce availability of OP-BChE antibody. It is sensitive enough to detect 0.5 nM OP-BChE, which is less than 2% BChE inhibition. This method offers a new method for rapid, accurate, selective and inexpensive quantification of phosphorylated adducts and enzyme inhibition for biomonitoring of OP and nerve agent exposures.« less

Induction of c-fos expression through JNK-mediated TCF/Elk-1 phosphorylation.

PubMed Central

Cavigelli, M; Dolfi, F; Claret, F X; Karin, M

1995-01-01

Growth factors induce c-fos transcription by stimulating phosphorylation of transcription factor TCF/Elk-1, which binds to the serum response element (SRE). Under such conditions Elk-1 could be phosphorylated by the mitogen-activated protein kinases (MAPKs) ERK1 and ERK2. However, c-fos transcription and SRE activity are also induced by stimuli, such as UV irradiation and activation of the protein kinase MEKK1, that cause only an insignificant increase in ERK1/2 activity. However, both of these stimuli strongly activate two other MAPKs, JNK1 and JNK2, and stimulate Elk-1 transcriptional activity and phosphorylation. We find that the JNKs are the predominant Elk-1 activation domain kinases in extracts of UV-irradiated cells and that immunopurified JNK1/2 phosphorylate Elk-1 on the same major sites recognized by ERK1/2, that potentiate its transcriptional activity. Finally, we show that UV irradiation, but not serum or phorbol esters, stimulate translocation of JNK1 to the nucleus. As Elk-1 is most likely phosphorylated while bound to the c-fos promoter, these results suggest that UV irradiation and MEKK1 activation stimulate TCF/Elk-1 activity through JNK activation, while growth factors induce c-fos through ERK activation. Images PMID:8846788

Activation of the Low Molecular Weight Protein Tyrosine Phosphatase in Keratinocytes Exposed to Hyperosmotic Stress

PubMed Central

Cavalheiro, Renan P.; Machado, Daisy; Cruz, Bread L. G.; Paredes-Gamero, Edgar J.; Gomes-Marcondes, Maria C. C.; Zambuzzi, Willian F.; Vasques, Luciana; Nader, Helena B.; Souza, Ana Carolina S.; Justo, Giselle Z.

2015-01-01

Herein, we provide new contribution to the mechanisms involved in keratinocytes response to hyperosmotic shock showing, for the first time, the participation of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP) activity in this event. We reported that sorbitol-induced osmotic stress mediates alterations in the phosphorylation of pivotal cytoskeletal proteins, particularly Src and cofilin. Furthermore, an increase in the expression of the phosphorylated form of LMWPTP, which was followed by an augment in its catalytic activity, was observed. Of particular importance, these responses occurred in an intracellular milieu characterized by elevated levels of reduced glutathione (GSH) and increased expression of the antioxidant enzymes glutathione peroxidase and glutathione reductase. Altogether, our results suggest that hyperosmostic stress provides a favorable cellular environment to the activation of LMWPTP, which is associated with increased expression of antioxidant enzymes, high levels of GSH and inhibition of Src kinase. Finally, the real contribution of LMWPTP in the hyperosmotic stress response of keratinocytes was demonstrated through analysis of the effects of ACP1 gene knockdown in stressed and non-stressed cells. LMWPTP knockdown attenuates the effects of sorbitol induced-stress in HaCaT cells, mainly in the status of Src kinase, Rac and STAT5 phosphorylation and activity. These results describe for the first time the participation of LMWPTP in the dynamics of cytoskeleton rearrangement during exposure of human keratinocytes to hyperosmotic shock, which may contribute to cell death. PMID:25781955

Phenylbutyrate Therapy for Pyruvate Dehydrogenase Complex Deficiency and Lactic Acidosis

PubMed Central

Ferriero, Rosa; Manco, Giuseppe; Lamantea, Eleonora; Nusco, Edoardo; Ferrante, Mariella I.; Sordino, Paolo; Stacpoole, Peter W.; Lee, Brendan; Zeviani, Massimo; Brunetti-Pierri, Nicola

2014-01-01

Lactic acidosis is a build-up of lactic acid in the blood and tissues, which can be due to several inborn errors of metabolism as well as nongenetic conditions. Deficiency of pyruvate dehydrogenase complex (PDHC) is the most common genetic disorder leading to lactic acidosis. Phosphorylation of specific serine residues of the E1α subunit of PDHC by pyruvate dehydrogenase kinase (PDK) inactivates the enzyme, whereas dephosphorylation restores PDHC activity. We found that phenylbutyrate enhances PDHC enzymatic activity in vitro and in vivo by increasing the proportion of unphosphorylated enzyme through inhibition of PDK. Phenylbutyrate given to C57B6/L wild-type mice results in a significant increase in PDHC enzyme activity and a reduction of phosphorylated E1α in brain, muscle, and liver compared to saline-treated mice. By means of recombinant enzymes, we showed that phenylbutyrate prevents phosphorylation of E1α through binding and inhibition of PDK, providing a molecular explanation for the effect of phenylbutyrate on PDHC activity. Phenylbutyrate increases PDHC activity in fibroblasts from PDHC-deficient patients harboring various molecular defects and corrects the morphological, locomotor, and biochemical abnormalities in the noam631 zebrafish model of PDHC deficiency. In mice, phenylbutyrate prevents systemic lactic acidosis induced by partial hepatectomy. Because phenylbutyrate is already approved for human use in other diseases, the findings of this study have the potential to be rapidly translated for treatment of patients with PDHC deficiency and other forms of primary and secondary lactic acidosis. PMID:23467562

Phosphorylation of 1-Aminocyclopropane-1-Carboxylic Acid Synthase by MPK6, a Stress-Responsive Mitogen-Activated Protein Kinase, Induces Ethylene Biosynthesis in ArabidopsisW⃞

PubMed Central

Liu, Yidong; Zhang, Shuqun

2004-01-01

Mitogen-activated protein kinases (MAPKs) are implicated in regulating plant growth, development, and response to the environment. However, the underlying mechanisms are unknown because of the lack of information about their substrates. Using a conditional gain-of-function transgenic system, we demonstrated that the activation of SIPK, a tobacco (Nicotiana tabacum) stress-responsive MAPK, induces the biosynthesis of ethylene. Here, we report that MPK6, the Arabidopsis thaliana ortholog of tobacco SIPK, is required for ethylene induction in this transgenic system. Furthermore, we found that selected isoforms of 1-aminocyclopropane-1-carboxylic acid synthase (ACS), the rate-limiting enzyme of ethylene biosynthesis, are substrates of MPK6. Phosphorylation of ACS2 and ACS6 by MPK6 leads to the accumulation of ACS protein and, thus, elevated levels of cellular ACS activity and ethylene production. Expression of ACS6DDD, a gain-of-function ACS6 mutant that mimics the phosphorylated form of ACS6, confers constitutive ethylene production and ethylene-induced phenotypes. Increasing numbers of stress stimuli have been shown to activate Arabidopsis MPK6 or its orthologs in other plant species. The identification of the first plant MAPK substrate in this report reveals one mechanism by which MPK6/SIPK regulates plant stress responses. Equally important, this study uncovers a signaling pathway that modulates the biosynthesis of ethylene, an important plant hormone, in plants under stress. PMID:15539472

TGF-beta and HGF transmit the signals through JNK-dependent Smad2/3 phosphorylation at the linker regions.

PubMed

Mori, Shigeo; Matsuzaki, Koichi; Yoshida, Katsunori; Furukawa, Fukiko; Tahashi, Yoshiya; Yamagata, Hideo; Sekimoto, Go; Seki, Toshihito; Matsui, Hirofumi; Nishizawa, Mikio; Fujisawa, Jun-ichi; Okazaki, Kazuichi

2004-09-23

Although hepatocyte growth factor (HGF) can act synergistically or antagonistically with transforming growth factor-beta (TGF-beta) signaling, molecular mechanism of their crosstalk remains unknown. Using antibodies which selectively distinguished receptor-regulated Smads (R-Smads) phosphorylated at linker regions from those at C-terminal regions, we herein showed that either HGF or TGF-beta treatment of normal stomach-origin cells activated the JNK pathway, thereafter inducing endogenous R-Smads phosphorylation at linker regions. However, the phosphorylation at their C-terminal regions was not induced by HGF treatment. The activated JNK could directly phosphorylate R-Smads in vitro at the same sites that were phosphorylated in response to TGF-beta or HGF in vivo. Thus, the linker regions of R-Smads were the common phosphorylation sites for HGF and TGF-beta signaling pathways. The phosphorylation induced by simultaneous treatment with HGF and TGF-beta allowed R-Smads to associate with Smad4 and to translocate into the nucleus. JNK pathway involved HGF and TGF-beta-mediated infiltration potency since a JNK inhibitor SP600125 caused the reduction of invasive capacity induced by HGF and TGF-beta signals. Moreover, a combined treatment with HGF and TGF-beta led to a potent increase in plasminogen activator inhibitor type 1 transcriptional activity through Smad3 phosphorylation at the linker region. In contrast, HGF treatment reduced TGF-beta-dependent activation of p15INK4B promoter, in which Smad3 phosphorylation at the C-terminal region was involved. In conclusion, HGF and TGF-beta transmit the signals through JNK-mediated R-Smads phosphorylation at linker regions.

Cdk5-dependent phosphorylation of liprinα1 mediates neuronal activity-dependent synapse development

PubMed Central

Huang, Huiqian; Lin, Xiaochen; Liang, Zhuoyi; Zhao, Teng; Du, Shengwang; Loy, Michael M. T.; Lai, Kwok-On; Fu, Amy K. Y.

2017-01-01

The experience-dependent modulation of brain circuitry depends on dynamic changes in synaptic connections that are guided by neuronal activity. In particular, postsynaptic maturation requires changes in dendritic spine morphology, the targeting of postsynaptic proteins, and the insertion of synaptic neurotransmitter receptors. Thus, it is critical to understand how neuronal activity controls postsynaptic maturation. Here we report that the scaffold protein liprinα1 and its phosphorylation by cyclin-dependent kinase 5 (Cdk5) are critical for the maturation of excitatory synapses through regulation of the synaptic localization of the major postsynaptic organizer postsynaptic density (PSD)-95. Whereas Cdk5 phosphorylates liprinα1 at Thr701, this phosphorylation decreases in neurons in response to neuronal activity. Blockade of liprinα1 phosphorylation enhances the structural and functional maturation of excitatory synapses. Nanoscale superresolution imaging reveals that inhibition of liprinα1 phosphorylation increases the colocalization of liprinα1 with PSD-95. Furthermore, disruption of liprinα1 phosphorylation by a small interfering peptide, siLIP, promotes the synaptic localization of PSD-95 and enhances synaptic strength in vivo. Our findings collectively demonstrate that the Cdk5-dependent phosphorylation of liprinα1 is important for the postsynaptic organization during activity-dependent synapse development. PMID:28760951

Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner.

PubMed

Newton, Phillip T; Vuppalapati, Karuna K; Bouderlique, Thibault; Chagin, Andrei S

2015-01-01

Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H(+)-ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A1 or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p < 0.018). This activation of MTORC1 was further confirmed in bone organ culture and promoted potent stimulation of longitudinal growth (p < 0.001). Importantly, the same effect was observed in ATG5 (autophagy-related 5)-deficient bones suggesting a macroautophagy-independent mechanism of MTORC1 inhibition by lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity.

PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon

2015-04-17

Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Alamore » (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression.« less

Conformationally constrained peptides target the allosteric kinase dimer interface and inhibit EGFR activation.

PubMed

Fulton, Melody D; Hanold, Laura E; Ruan, Zheng; Patel, Sneha; Beedle, Aaron M; Kannan, Natarajan; Kennedy, Eileen J

2018-03-15

Although EGFR is a highly sought-after drug target, inhibitor resistance remains a challenge. As an alternative strategy for kinase inhibition, we sought to explore whether allosteric activation mechanisms could effectively be disrupted. The kinase domain of EGFR forms an atypical asymmetric dimer via head-to-tail interactions and serves as a requisite for kinase activation. The kinase dimer interface is primarily formed by the H-helix derived from one kinase monomer and the small lobe of the second monomer. We hypothesized that a peptide designed to resemble the binding surface of the H-helix may serve as an effective disruptor of EGFR dimerization and activation. A library of constrained peptides was designed to mimic the H-helix of the kinase domain and interface side chains were optimized using molecular modeling. Peptides were constrained using peptide "stapling" to structurally reinforce an alpha-helical conformation. Peptide stapling was demonstrated to notably enhance cell permeation of an H-helix derived peptide termed EHBI2. Using cell-based assays, EHBI2 was further shown to significantly reduce EGFR activity as measured by EGFR phosphorylation and phosphorylation of the downstream signaling substrate Akt. To our knowledge, this is the first H-helix-based compound targeting the asymmetric interface of the kinase domain that can successfully inhibit EGFR activation and signaling. This study presents a novel, alternative targeting site for allosteric inhibition of EGFR. Copyright © 2017 Elsevier Ltd. All rights reserved.

Phosphorylation of αB-crystallin supports reactive astrogliosis in demyelination

PubMed Central

Yoon, Jane; van Horssen, Jack; Han, May H.; Bollyky, Paul L.; Palmer, Theo D.; Steinman, Lawrence

2017-01-01

The small heat shock protein αB-crystallin (CRYAB) has been implicated in multiple sclerosis (MS) pathogenesis. Earlier studies have indicated that CRYAB inhibits inflammation and attenuates clinical disease when administered in the experimental autoimmune encephalomyelitis model of MS. In this study, we evaluated the role of CRYAB in primary demyelinating events. Using the cuprizone model of demyelination, a noninflammatory model that allows the analysis of glial responses in MS, we show that endogenous CRYAB expression is associated with increased severity of demyelination. Moreover, we demonstrate a strong correlation between the expression of CRYAB and the extent of reactive astrogliosis in demyelinating areas and in in vitro assays. In addition, we reveal that CRYAB is differentially phosphorylated in astrocytes in active demyelinating MS lesions, as well as in cuprizone-induced lesions, and that this phosphorylation is required for the reactive astrocyte response associated with demyelination. Furthermore, taking a proteomics approach to identify proteins that are bound by the phosphorylated forms of CRYAB in primary cultured astrocytes, we show that there is clear differential binding of protein targets due to the specific phosphorylation of CRYAB. Subsequent Ingenuity Pathway Analysis of these targets reveals implications for intracellular pathways and biological processes that could be affected by these modifications. Together, these findings demonstrate that astrocytes play a pivotal role in demyelination, making them a potential target for therapeutic intervention, and that phosphorylation of CRYAB is a key factor supporting the pathogenic response of astrocytes to oligodendrocyte injury. PMID:28196893

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fouassier, Laura; Nichols, Matthew T.; Gidey, Elizabeth

Ezrin-Radixin-Moesin (ERM) binding phosphoprotein 50 (EBP50, a.k.a. NHERF-1) is a scaffold protein essential for the localization and coordinated activity of apical transporters, enzymes and receptors in epithelial cells. EBP50 acts via multiple protein binding interactions, including oligomerization through interactions of its PSD95-Dlg-ZO1 (PDZ) domains. EBP50 can be phosphorylated on multiple sites and phosphorylation of specific sites modulates the extent of oligomerization. The aim of the present study was to test the capacity of protein kinase C (PKC) to phosphorylate EBP50 and to regulate its oligomerization. In vitro experiments showed that the catalytic subunit of PKC directly phosphorylates EBP50. In HEK-293more » cells transfected with rat EBP50 cDNA, a treatment with 12 myristate 13-acetate (PMA) induced a translocation of PKC{alpha} and {beta} isoforms to the membrane and increased {sup 32}P incorporation into EBP50. In co-transfection/co-precipitation studies, PMA treatment stimulated EBP50 oligomerization. Mass spectrometry analysis of full-length EBP50 and phosphorylation analyses of specific domains, and of mutated or truncated forms of EBP50, indicated that PKC-induced phosphorylation of EBP50 occurred on the Ser{sup 337}/Ser{sup 338} residue within the carboxyl-tail domain of the protein. Truncation of Ser{sup 337}/Ser{sup 338} also diminished PKC-induced oligomerization of EBP50. These results suggest the PKC signaling pathway can impact EBP50-dependent cellular functions by regulating EBP50 oligomerization.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bin, Geng; Bo, Zhang; Jing, Wang

TNF-α is known to induce osteoblasts apoptosis, whereas mechanical stimulation has been shown to enhance osteoblast survival. In the present study, we found that mechanical stimulation in the form of fluid shear stress (FSS) suppresses TNF-α induced apoptosis in MC3T3-E1 cells. Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family that has been implicated in cell survival. We also demonstrated that FSS imposed by flow chamber in vitro leads to a markedly activation of ERK5, which was shown to be protective against TNF-α-induced apoptosis, whereas the transfection of siRNA against ERK5 (ERK5-siRNA) reversed themore » FSS-medicated anti-apoptotic effects. An initial FSS-mediated activation of ERK5 that phosphorylates AKT to increase its activity, and a following forkhead box O 3a (FoxO3a) was phosphorylated by activated AKT. Phosphorylated FoxO3a is sequestered in the cytoplasm, and prevents it from translocating to nucleus where it can increase the expression of FasL and Bim. The inhibition of AKT-FoxO3a signalings by a PI3K (PI3-kinase)/AKT inhibitor (LY294002) or the transfection of ERK5-siRNA led to the nuclear translocation of non-phosphorylated FoxO3a, and increased the protein expression of FasL and Bim. In addition, the activation of caspase-3 by TNF-α was significantly inhibited by aforementioned FSS-medicated mechanisms. In brief, the activation of ERK5-AKT-FoxO3a signaling pathways by FSS resulted in a decreased expression of FasL and Bim and an inhibition of caspase-3 activation, which exerts a protective effect that prevents osteoblasts from apoptosis. - Highlights: • Fluid shear stress inhibits osteoblast apoptosis induced by TNF-α. • Inhibition of ERK5 activity by transfection of ERK5 siRNA blocks FSS-mediated anti-apoptotic effect in osteoblast. • Activated ERK5-AKT-FoxO3a-Bim/FasL signaling pathways by FSS is required to protect osteoblast from apoptosis.« less

Suspension survival mediated by PP2A-STAT3-Col XVII determines tumour initiation and metastasis in cancer stem cells

PubMed Central

Liu, Chen-Chi; Lin, Shih-Pei; Hsu, Han-Shui; Yang, Shung-Haur; Lin, Chiu-Hua; Yang, Muh-Hwa; Hung, Mien-Chie; Hung, Shih-Chieh

2016-01-01

Targeting tumour-initiating cells (TICs) would lead to new therapies to cure cancer. We previously demonstrated that TICs have the capacity to survive under suspension conditions, while other cells undergo anoikis. Here we show that TICs exhibit increased phosphorylation levels of S727STAT3 because of PP2A inactivation. Collagen 17 gene expression is upregulated in a STAT3-dependent manner, which also stabilizes laminin 5 and engages cells to form hemidesmosome-like junctions in response. Blocking the PP2A-S727STAT3-collagen 17 pathway inhibits the suspension survival of TICs and their ability to form tumours in mice, while activation of the same pathway increases the suspension survival and tumour-initiation capacities of bulk cancer cells. The S727STAT3 phosphorylation levels correlate with collagen 17 expression in colon tumour samples, and correlate inversely with survival. Finally, this signalling axis enhances the ability of TIC to form tumours in mouse models of malignant lung cancer pleural effusion and spontaneous colon cancer metastasis. PMID:27306323

Suspension survival mediated by PP2A-STAT3-Col XVII determines tumour initiation and metastasis in cancer stem cells.

PubMed

Liu, Chen-Chi; Lin, Shih-Pei; Hsu, Han-Shui; Yang, Shung-Haur; Lin, Chiu-Hua; Yang, Muh-Hwa; Hung, Mien-Chie; Hung, Shih-Chieh

2016-06-16

Targeting tumour-initiating cells (TICs) would lead to new therapies to cure cancer. We previously demonstrated that TICs have the capacity to survive under suspension conditions, while other cells undergo anoikis. Here we show that TICs exhibit increased phosphorylation levels of S727STAT3 because of PP2A inactivation. Collagen 17 gene expression is upregulated in a STAT3-dependent manner, which also stabilizes laminin 5 and engages cells to form hemidesmosome-like junctions in response. Blocking the PP2A-S727STAT3-collagen 17 pathway inhibits the suspension survival of TICs and their ability to form tumours in mice, while activation of the same pathway increases the suspension survival and tumour-initiation capacities of bulk cancer cells. The S727STAT3 phosphorylation levels correlate with collagen 17 expression in colon tumour samples, and correlate inversely with survival. Finally, this signalling axis enhances the ability of TIC to form tumours in mouse models of malignant lung cancer pleural effusion and spontaneous colon cancer metastasis.

[Changes of protein tyrosine phosphorylation in erythrocyte band 3 glucose-6-phosphate dehydrogenase deficiency].

PubMed

Yu, Guoyu; Li, Jialin; Tian, Xingya; Lin, Hong; Wang, Xiaoying

2002-11-01

To explore the hemolytic mechanism of glucose-6-phosphate dehydrogenase (G6PD) deficient erythrocytes in the view of phosphorylation of membrane protein. The alternation of membrane protein phosphorylation and the effect of dithiothreitol (DTT) on protein phosphorylation were analysed by Western blot technique. The activity of phosphotyrosine phosphatase (PTPs) was determined by using p-nitrophenyl phosphate as substrate. Tyrosine phosphorylation of band 3 protein was obviously enhanced in G6PD-deficient erythrocytes. The activity of PTPs was low compared to the normal erythrocytes. The level of phosphotyrosine in G6PD-deficient erythrocytes incubated with DTT was almost the same as in those without DTT. The results were consistent with the activity of PTPs. PTPs activity reduction and tyrosine phosphorylation enhancement induced by oxidation in G6PD deficiency play an important role in erythrocytes hemolysis. However, the alternation of thiol group is not the only factor affecting the activity of PTPs in G6PD-deficient erythrocytes.

Stretch-induced ERK2 phosphorylation requires PLA2 activity in skeletal myotubes.

PubMed

Burkholder, Thomas J

2009-08-14

Mechanical stretch rapidly activates multiple signaling cascades, including phospholipases and kinases, to stimulate protein synthesis and growth. The purpose of this study was to determine whether PLA2 activation contributes to stretch-induced phosphorylation of ERK2 in skeletal muscle myotubes. Myotubes derived from neonatal C57 mice were cultured on silicone membranes and subjected to brief cyclic stretch. Inhibition of PLA2 prevented ERK2 phosphorylation, while inhibition of prostaglandin or leukotriene synthesis did not. ERK2 phosphorylation was also blocked by genistein and PD98059, implicating the canonical raf-MEK-ERK cassette. It appears that PLA2, but not further metabolism of arachidonic acid, is required for stretch-induced activation of ERK2. Exposure to exogenous arachidonic acid had no effect on ERK2 phosphorylation, but exposure to lysophosphatidylcholine, the other metabolite of PLA2, caused a dose-dependent increase in ERK2 phosphorylation. These results suggest that stretch-induced activation of ERK2 may result from an interaction between PLA2 derived lysophosphatidylcholine and membrane receptors.

Stretch-induced ERK2 phosphorylation requires PLA2 activity in skeletal myotubes

PubMed Central

Burkholder, Thomas J.

2009-01-01

Mechanical stretch rapidly activates multiple signaling cascades, including phospholipases and kinases, to stimulate protein synthesis and growth. The purpose of this study was to determine whether PLA2 activation contributes to stretch-induced phosphorylation of ERK2 in skeletal muscle myotubes. Myotubes derived from neonatal C57 mice were cultured on silicone membranes and subjected to brief cyclic stretch. Inhibition of PLA2 prevented ERK2 phosphorylation, while inhibition of prostaglandin or leukotriene synthesis did not. ERK2 phosphorylation was also blocked by genistein and PD98059, implicating the canonical raf-MEK-ERK cassette. It appears that PLA2, but not further metabolism of arachidonic acid, is required for stretch-induced activation of ERK2. Exposure to exogenous arachidonic acid had no effect on ERK2 phosphorylation, but exposure to lysophosphatidylcholine, the other metabolite of PLA2, caused a dose-dependent increase in ERK2 phosphorylation. These results suggest that stretch-induced activation of ERK2 may result from an interaction between PLA2 derived lysophosphatidylcholine and membrane receptors. PMID:19524551

Neither Aurora B activity nor histone H3 phosphorylation is essential for chromosome condensation during meiotic maturation of porcine oocytes.

PubMed

Jelínková, Lucie; Kubelka, Michal

2006-05-01

Aurora kinase B (AURKB) is a chromosomal passenger protein that is essential for a number of processes during mitosis. Its activity is regulated by association with two other passenger proteins, INCENP and Survivin, and by phosphorylation on Thr 232. In this study, we examine expression and phosphorylation on Thr-232 of AURKB during meiotic maturation of pig oocytes in correlation with histone H3 phosphorylation and chromosome condensation. We show that histone H3 phosphorylation on Ser-10, but not on Ser-28, correlates with progressive chromosome condensation during oocyte maturation; Ser-10 phosphorylation starts around the time of the breakdown of the nuclear envelope, with the maximal activity in metaphase I, whereas Ser-28 phosphorylation does not significantly change in maturing oocytes. Treatment of oocytes with 50 microM butyrolactone I (BL-I), an inhibitor of cyclin-dependent kinases, or cycloheximide (10 microg/ml), inhibitor of proteosynthesis, results in a block of oocytes in the germinal vesicle stage, when nuclear membrane remains intact; however, condensed chromosome fibers or highly condensed chromosome bivalents can be seen in the nucleoplasm of BL-I- or cycloheximide-treated oocytes, respectively. In these treated oocytes, no or only very weak AURKB activity and phosphorylation of histone H3 on Ser-10 can be detected after 27 h of treatment, whereas phosphorylation on Ser-28 is not influenced. These results suggest that AURKB activity and Ser-10 phosphorylation of histone H3 are not required for chromosome condensation in pig oocytes, but might be required for further processing of chromosomes during meiosis.

Revisiting Frank-Starling: regulatory light chain phosphorylation alters the rate of force redevelopment (ktr ) in a length-dependent fashion.

PubMed

Toepfer, Christopher N; West, Timothy G; Ferenczi, Michael A

2016-09-15

Regulatory light chain (RLC) phosphorylation has been shown to alter the ability of muscle to produce force and power during shortening and to alter the rate of force redevelopment (ktr ) at submaximal [Ca(2+) ]. Increasing RLC phosphorylation ∼50% from the in vivo level in maximally [Ca(2+) ]-activated cardiac trabecula accelerates ktr . Decreasing RLC phosphorylation to ∼70% of the in vivo control level slows ktr and reduces force generation. ktr is dependent on sarcomere length in the physiological range 1.85-1.94 μm and RLC phosphorylation modulates this response. We demonstrate that Frank-Starling is evident at maximal [Ca(2+) ] activation and therefore does not necessarily require length-dependent change in [Ca(2+) ]-sensitivity of thin filament activation. The stretch response is modulated by changes in RLC phosphorylation, pinpointing RLC phosphorylation as a modulator of the Frank-Starling law in the heart. These data provide an explanation for slowed systolic function in the intact heart in response to RLC phosphorylation reduction. Force and power in cardiac muscle have a known dependence on phosphorylation of the myosin-associated regulatory light chain (RLC). We explore the effect of RLC phosphorylation on the ability of cardiac preparations to redevelop force (ktr ) in maximally activating [Ca(2+) ]. Activation was achieved by rapidly increasing the temperature (temperature-jump of 0.5-20ºC) of permeabilized trabeculae over a physiological range of sarcomere lengths (1.85-1.94 μm). The trabeculae were subjected to shortening ramps over a range of velocities and the extent of RLC phosphorylation was varied. The latter was achieved using an RLC-exchange technique, which avoids changes in the phosphorylation level of other proteins. The results show that increasing RLC phosphorylation by 50% accelerates ktr by ∼50%, irrespective of the sarcomere length, whereas decreasing phosphorylation by 30% slows ktr by ∼50%, relative to the ktr obtained for in vivo phosphorylation. Clearly, phosphorylation affects the magnitude of ktr following step shortening or ramp shortening. Using a two-state model, we explore the effect of RLC phosphorylation on the kinetics of force development, which proposes that phosphorylation affects the kinetics of both attachment and detachment of cross-bridges. In summary, RLC phosphorylation affects the rate and extent of force redevelopment. These findings were obtained in maximally activated muscle at saturating [Ca(2+) ] and are not explained by changes in the Ca(2+) -sensitivity of acto-myosin interactions. The length-dependence of the rate of force redevelopment, together with the modulation by the state of RLC phosphorylation, suggests that these effects play a role in the Frank-Starling law of the heart. © 2016 Wellcome Trust The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Phenylbutyrate therapy for maple syrup urine disease

PubMed Central

Brunetti-Pierri, Nicola; Lanpher, Brendan; Erez, Ayelet; Ananieva, Elitsa A.; Islam, Mohammad; Marini, Juan C.; Sun, Qin; Yu, Chunli; Hegde, Madhuri; Li, Jun; Wynn, R. Max; Chuang, David T.; Hutson, Susan; Lee, Brendan

2011-01-01

Therapy with sodium phenylacetate/benzoate or sodium phenylbutyrate in urea cycle disorder patients has been associated with a selective reduction in branched-chain amino acids (BCAA) in spite of adequate dietary protein intake. Based on this clinical observation, we investigated the potential of phenylbutyrate treatment to lower BCAA and their corresponding α-keto acids (BCKA) in patients with classic and variant late-onset forms of maple syrup urine disease (MSUD). We also performed in vitro and in vivo experiments to elucidate the mechanism for this effect. We found that BCAA and BCKA are both significantly reduced following phenylbutyrate therapy in control subjects and in patients with late-onset, intermediate MSUD. In vitro treatment with phenylbutyrate of control fibroblasts and lymphoblasts resulted in an increase in the residual enzyme activity, while treatment of MSUD cells resulted in the variable response which did not simply predict the biochemical response in the patients. In vivo phenylbutyrate increases the proportion of active hepatic enzyme and unphosphorylated form over the inactive phosphorylated form of the E1α subunit of the branched-chain α-keto acid dehydrogenase complex (BCKDC). Using recombinant enzymes, we show that phenylbutyrate prevents phosphorylation of E1α by inhibition of the BCKDC kinase to activate BCKDC overall activity, providing a molecular explanation for the effect of phenylbutyrate in a subset of MSUD patients. Phenylbutyrate treatment may be a valuable treatment for reducing the plasma levels of neurotoxic BCAA and their corresponding BCKA in a subset of MSUD patients and studies of its long-term efficacy are indicated. PMID:21098507

Phenylbutyrate therapy for maple syrup urine disease.

PubMed

Brunetti-Pierri, Nicola; Lanpher, Brendan; Erez, Ayelet; Ananieva, Elitsa A; Islam, Mohammad; Marini, Juan C; Sun, Qin; Yu, Chunli; Hegde, Madhuri; Li, Jun; Wynn, R Max; Chuang, David T; Hutson, Susan; Lee, Brendan

2011-02-15

Therapy with sodium phenylacetate/benzoate or sodium phenylbutyrate in urea cycle disorder patients has been associated with a selective reduction in branched-chain amino acids (BCAA) in spite of adequate dietary protein intake. Based on this clinical observation, we investigated the potential of phenylbutyrate treatment to lower BCAA and their corresponding α-keto acids (BCKA) in patients with classic and variant late-onset forms of maple syrup urine disease (MSUD). We also performed in vitro and in vivo experiments to elucidate the mechanism for this effect. We found that BCAA and BCKA are both significantly reduced following phenylbutyrate therapy in control subjects and in patients with late-onset, intermediate MSUD. In vitro treatment with phenylbutyrate of control fibroblasts and lymphoblasts resulted in an increase in the residual enzyme activity, while treatment of MSUD cells resulted in the variable response which did not simply predict the biochemical response in the patients. In vivo phenylbutyrate increases the proportion of active hepatic enzyme and unphosphorylated form over the inactive phosphorylated form of the E1α subunit of the branched-chain α-keto acid dehydrogenase complex (BCKDC). Using recombinant enzymes, we show that phenylbutyrate prevents phosphorylation of E1α by inhibition of the BCKDC kinase to activate BCKDC overall activity, providing a molecular explanation for the effect of phenylbutyrate in a subset of MSUD patients. Phenylbutyrate treatment may be a valuable treatment for reducing the plasma levels of neurotoxic BCAA and their corresponding BCKA in a subset of MSUD patients and studies of its long-term efficacy are indicated.

Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain-binding capacity.

PubMed

Weir, Marion E; Mann, Jacqueline E; Corwin, Thomas; Fulton, Zachary W; Hao, Jennifer M; Maniscalco, Jeanine F; Kenney, Marie C; Roman Roque, Kristal M; Chapdelaine, Elizabeth F; Stelzl, Ulrich; Deming, Paula B; Ballif, Bryan A; Hinkle, Karen L

2016-04-01

Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the C-terminal site to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. © 2016 Federation of European Biochemical Societies.

gp140, the EBV/C3d receptor (CR2) of human B lymphocytes, is involved in cell-free phosphorylation of p120, a nuclear ribonucleoprotein.

PubMed

Delcayre, A X; Fiandino, A; Barel, M; Frade, R

1987-12-01

gp140, the EB/C3d receptor (EBV/C3dR; CR2), is a membrane site involved in human B cell regulation. Cross-linking of this receptor on the cell surface by its specific ligands led to the enhancement of B cell proliferation in synergy with T cell factors. In vitro activation of human peripheral B lymphocytes by cross-linking membrane immunoglobulins with anti-mu antibody induced EBV/C3dR phosphorylation. These studies were pursued by analyzing cell-free phosphorylation of EBV/C3dR isolated from Raji cell fractions, and immobilized on OKB7, a monoclonal anti-EBV/C3dR antibody. Three EBV/C3dR-related antigens which could be cell-free phosphorylated were detected: gp140, the EBV/C3dR, p130 and p120. gp140, the mature form of EBV/C3dR, was isolated from plasma membrane and from purified nuclei. p130 was identified as an intracellular intermediate of EBV/C3dR glycosylation, localized in low-density microsomes. Phosphoamino acid analysis of EBV/C3dR allowed the detection of phosphotyrosine and phosphoserine residues. These data suggest that EBV/C3dR could carry an autophosphorylation activity and could be associated to serine kinases. Using polyclonal anti-p120 antibody and anti-120 kDa nuclear ribonucleoprotein monoclonal antibody (mAb), p120 was identified as a nuclear ribonucleoprotein antigenically not related to EBV/C3dR. Detection of p120 on EBV/C3dR, immobilized on OKB7, was due to interactions between both antigens, instead of anti-EBV/C3dR mAb cross-reactivity with p120. Cell-free phosphorylation of p120 was under the control of EBV/C3dR. However, it is not yet established whether other nuclear or membrane components were involved in the control of p120 cell-free phosphorylation by EBV/C3dR. From the data presented herein, we propose that phosphorylation of a 120-kDa nuclear ribonucleoprotein by EBV/C3dR-associated kinases could represent a crucial step in in vivo regulation of human B cell activation.

Protein kinase A catalytic subunit primed for action: Time-lapse crystallography of Michaelis complex formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Das, Amit; Gerlits, Oksana O.; Parks, Jerry M.

The catalytic subunit of the cyclic AMP-dependent protein kinase A (PKAc) catalyzes the transfer of the γ-phosphate of bound Mg 2ATP to a serine or threonine residue of a protein substrate. Here, time-lapse X-ray crystallography was used to capture a series of complexes of PKAc with an oligopeptide substrate and unreacted Mg 2ATP, including the Michaelis complex, that reveal important geometric rearrangements in and near the active site preceding the phosphoryl transfer reaction. Contrary to the prevailing view, Mg 2+ binds first to the M1 site as a complex with ATP and is followed by Mg 2+ binding to themore » M2 site. Furthermore, the target serine hydroxyl of the peptide substrate rotates away from the active site toward the bulk solvent, which breaks the hydrogen bond with D166. In conclusion, the serine hydroxyl of the substrate rotates back toward D166 to form the Michaelis complex with the active site primed for phosphoryl transfer.« less

Protein kinase A catalytic subunit primed for action: Time-lapse crystallography of Michaelis complex formation

DOE PAGES

Das, Amit; Gerlits, Oksana O.; Parks, Jerry M.; ...

2015-11-12

The catalytic subunit of the cyclic AMP-dependent protein kinase A (PKAc) catalyzes the transfer of the γ-phosphate of bound Mg 2ATP to a serine or threonine residue of a protein substrate. Here, time-lapse X-ray crystallography was used to capture a series of complexes of PKAc with an oligopeptide substrate and unreacted Mg 2ATP, including the Michaelis complex, that reveal important geometric rearrangements in and near the active site preceding the phosphoryl transfer reaction. Contrary to the prevailing view, Mg 2+ binds first to the M1 site as a complex with ATP and is followed by Mg 2+ binding to themore » M2 site. Furthermore, the target serine hydroxyl of the peptide substrate rotates away from the active site toward the bulk solvent, which breaks the hydrogen bond with D166. In conclusion, the serine hydroxyl of the substrate rotates back toward D166 to form the Michaelis complex with the active site primed for phosphoryl transfer.« less

A Functional Approach Reveals a Genetic and Physical Interaction between Ribonucleotide Reductase and CHK1 in Mammalian Cells

PubMed Central

Taricani, Lorena; Shanahan, Frances; Malinao, Maria-Christina; Beaumont, Maribel; Parry, David

2014-01-01

Ribonucleotide reductase (RNR) enzyme is composed of the homodimeric RRM1 and RRM2 subunits, which together form a heterotetramic active enzyme that catalyzes the de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes. In this study, we show that ablation of RRM1 and RRM2 by siRNA induces G1/S phase arrest, phosphorylation of Chk1 on Ser345 and phosphorylation of γ-H2AX on S139. Combinatorial ablation of RRM1 or RRM2 and Chk1 causes a dramatic accumulation of γ-H2AX, a marker of double-strand DNA breaks, suggesting that activation of Chk1 in this context is essential for suppression of DNA damage. Significantly, we demonstrate for the first time that Chk1 and RNR subunits co-immunoprecipitate from native cell extracts. These functional genomic studies suggest that RNR is a critical mediator of replication checkpoint activation. PMID:25375241

Immunohistochemical Evaluation of AKT Protein Activation in Canine Mast Cell Tumours

PubMed Central

Rodriguez, S.; Fadlalla, K.; Graham, T.; Tameru, B.; Fermin, C. D.; Samuel, T.

2011-01-01

Summary The pathogenesis of canine mast cell tumour (MCT) remains unknown. Moreover, therapeutic options are limited and resistance to targeted drugs and recurrences are common, necessitating the identification of additional cellular targets for therapy. In this study we investigated the expression of phosphorylated AKT protein in 25 archival canine MCT samples by immunohistochemistry and examined the correlation between the immunohistochemical scores and histopathological tumour grades. AKT protein was detected in all of the samples and 24 of the 25 samples expressed the phosphorylated form of the protein, albeit with variable intensity. However, when the immunohistochemical scores of weak, intermediate and strong labelling were compared with the histopathological grades of I to III, there was no strong correlation. This study suggests that canine MCT cells have activated AKT and indicates the need for further research on the role of the AKT protein and the possibility of targeting the AKT signalling pathway in MCTs. PMID:22289273

Phosphatidylinositol 3-kinase/Akt signaling pathway activates the WNK-OSR1/SPAK-NCC phosphorylation cascade in hyperinsulinemic db/db mice.

PubMed

Nishida, Hidenori; Sohara, Eisei; Nomura, Naohiro; Chiga, Motoko; Alessi, Dario R; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi

2012-10-01

Metabolic syndrome patients have insulin resistance, which causes hyperinsulinemia, which in turn causes aberrant increased renal sodium reabsorption. The precise mechanisms underlying this greater salt sensitivity of hyperinsulinemic patients remain unclear. Abnormal activation of the recently identified with-no-lysine kinase (WNK)-oxidative stress-responsive kinase 1 (OSR1)/STE20/SPS1-related proline/alanine-rich kinase (SPAK)-NaCl cotransporter (NCC) phosphorylation cascade results in the salt-sensitive hypertension of pseudohypoaldosteronism type II. Here, we report a study of renal WNK-OSR1/SPAK-NCC cascade activation in the db/db mouse model of hyperinsulinemic metabolic syndrome. Thiazide sensitivity was increased, suggesting greater activity of NCC in db/db mice. In fact, increased phosphorylation of OSR1/SPAK and NCC was observed. In both SpakT243A/+ and Osr1T185A/+ knock-in db/db mice, which carry mutations that disrupt the signal from WNK kinases, increased phosphorylation of NCC and elevated blood pressure were completely corrected, indicating that phosphorylation of SPAK and OSR1 by WNK kinases is required for the increased activation and phosphorylation of NCC in this model. Renal phosphorylated Akt was increased in db/db mice, suggesting that increased NCC phosphorylation is regulated by the phosphatidylinositol 3-kinase/Akt signaling cascade in the kidney in response to hyperinsulinemia. A phosphatidylinositol 3-kinase inhibitor (NVP-BEZ235) corrected the increased OSR1/SPAK-NCC phosphorylation. Another more specific phosphatidylinositol 3-kinase inhibitor (GDC-0941) and an Akt inhibitor (MK-2206) also inhibited increased NCC phosphorylation. These results indicate that the phosphatidylinositol 3-kinase/Akt signaling pathway activates the WNK-OSR1/SPAK-NCC phosphorylation cascade in db/db mice. This mechanism may play a role in the pathogenesis of salt-sensitive hypertension in human hyperinsulinemic conditions, such as the metabolic syndrome.

Activation of AMPK reduces the co-transporter activity of NKCC1.

PubMed

Fraser, Scott A; Davies, Matthew; Katerelos, Marina; Gleich, Kurt; Choy, Suet-Wan; Steel, Rohan; Galic, Sandra; Mount, Peter F; Kemp, Bruce E; Power, David A

2014-01-01

The co-transporter activity of Na(+)-K(+)-2Cl(-) 1 (NKCC1) is dependent on phosphorylation. In this study we show the energy-sensing kinase AMPK inhibits NKCC1 activity. Three separate AMPK activators (AICAR, Phenformin and A-769662) inhibited NKCC1 flux in a variety of nucleated cells. Treatment with A-769662 resulted in a reduction of NKCC1(T212/T217) phosphorylation, and this was reversed by treatment with the non-selective AMPK inhibitor Compound C. AMPK dependence was confirmed by treatment of AMPK null mouse embryonic fibroblasts, where A-769662 had no effect on NKCC1 mediated transport. AMPK was found to directly phosphorylate a recombinant human-NKCC1 N-terminal fragment (1-293) with the phosphorylated site identified as S77. Mutation of Serine 77 to Alanine partially prevented the inhibitory effect of A-769662 on NKCC1 activity. In conclusion, AMPK can act to reduce NKCC1-mediated transport. While the exact mechanism is still unclear there is evidence for both a direct effect on phosphorylation of S77 and reduced phosphorylation of T212/217.

Phospho-mimicking Atf1 mutants bypass the transcription activating function of the MAP kinase Sty1 of fission yeast.

PubMed

Sánchez-Mir, Laura; Salat-Canela, Clàudia; Paulo, Esther; Carmona, Mercè; Ayté, José; Oliva, Baldo; Hidalgo, Elena

2018-02-01

Stress-dependent activation of signaling cascades is often mediated by phosphorylation events, but the exact nature and role of these phosphorelays are frequently poorly understood. Here, we review which are the consequences of the stress-dependent phosphorylation of a transcription factor on gene activation. In fission yeast, the MAP kinase Sty1 is activated upon several environmental hazards and promotes cell adaptation and survival, greatly through activation of a gene program mediated by the transcription factor Atf1. Although described decades ago, the role of the phosphorylation of Atf1 by Sty1 is still a matter of debate. We present here a brief review of recent data, obtained through the characterization of several phosphorylation mutant derivatives of Atf1, demonstrating that Atf1 phosphorylation does not stabilize the factor nor stimulates its binding to DNA. Rather, it provides a structural platform of interaction with the transcriptional machinery. Based on these findings, future work will establish how this phosphorylated trans-activation domain promotes the massive gene expression shift allowing cellular adaptation to stress.

AMPK is activated early in cerebellar granule cells undergoing apoptosis and influences VADC1 phosphorylation status and activity.

PubMed

Bobba, A; Casalino, E; Amadoro, G; Petragallo, V A; Atlante, A

2017-09-01

The neurodegeneration of cerebellar granule cells, after low potassium induced apoptosis, is known to be temporally divided into an early and a late phase. Voltage-dependent anion channel-1 (VDAC1) protein, changing from the closed inactive state to the active open state, is central to the switch between the early and late phase. It is also known that: (i) VDAC1 can undergo phosphorylation events and (ii) AMP-activated protein kinase (AMPK), the sensor of cellular stress, may have a role in neuronal homeostasis. In the view of this, the involvement of AMPK activation and its correlation with VDAC1 status and activity has been investigated in the course of cerebellar granule cells apoptosis. The results reported in this study show that an increased level of the phosphorylated, active, isoform of AMPK occurs in the early phase, peaks at 3 h and guarantees an increase in the phosphorylation status of VDCA1, resulting in a reduced activity of this latter. However this situation is transient in nature, since, in the late phase, AMPK activation decreases as well as the level of phosphorylated VDAC1. In a less phosphorylated status, VDAC1 fully recovers its gating activity and drives cells along the death route.

The Zds proteins control entry into mitosis and target protein phosphatase 2A to the Cdc25 phosphatase

PubMed Central

Wicky, Sidonie; Tjandra, Hendri; Schieltz, David; Yates, John; Kellogg, Douglas R.

2011-01-01

The Wee1 kinase restrains entry into mitosis by phosphorylating and inhibiting cyclin-dependent kinase 1 (Cdk1). The Cdc25 phosphatase promotes entry into mitosis by removing Cdk1 inhibitory phosphorylation. Experiments in diverse systems have established that Wee1 and Cdc25 are regulated by protein phosphatase 2A (PP2A), but a full understanding of the function and regulation of PP2A in entry into mitosis has remained elusive. In budding yeast, entry into mitosis is controlled by a specific form of PP2A that is associated with the Cdc55 regulatory subunit (PP2ACdc55). We show here that related proteins called Zds1 and Zds2 form a tight stoichiometric complex with PP2ACdc55 and target its activity to Cdc25 but not to Wee1. Conditional inactivation of the Zds proteins revealed that their function is required primarily at entry into mitosis. In addition, Zds1 undergoes cell cycle–dependent changes in phosphorylation. Together, these observations define a role for the Zds proteins in controlling specific functions of PP2ACdc55 and suggest that upstream signals that regulate PP2ACdc55 may play an important role in controlling entry into mitosis. PMID:21119008

The Zds proteins control entry into mitosis and target protein phosphatase 2A to the Cdc25 phosphatase.

PubMed

Wicky, Sidonie; Tjandra, Hendri; Schieltz, David; Yates, John; Kellogg, Douglas R

2011-01-01

The Wee1 kinase restrains entry into mitosis by phosphorylating and inhibiting cyclin-dependent kinase 1 (Cdk1). The Cdc25 phosphatase promotes entry into mitosis by removing Cdk1 inhibitory phosphorylation. Experiments in diverse systems have established that Wee1 and Cdc25 are regulated by protein phosphatase 2A (PP2A), but a full understanding of the function and regulation of PP2A in entry into mitosis has remained elusive. In budding yeast, entry into mitosis is controlled by a specific form of PP2A that is associated with the Cdc55 regulatory subunit (PP2A(Cdc55)). We show here that related proteins called Zds1 and Zds2 form a tight stoichiometric complex with PP2A(Cdc55) and target its activity to Cdc25 but not to Wee1. Conditional inactivation of the Zds proteins revealed that their function is required primarily at entry into mitosis. In addition, Zds1 undergoes cell cycle-dependent changes in phosphorylation. Together, these observations define a role for the Zds proteins in controlling specific functions of PP2A(Cdc55) and suggest that upstream signals that regulate PP2A(Cdc55) may play an important role in controlling entry into mitosis.

Procarcinogenic effects of cyclosporine A are mediated through the activation of TAK1/TAB1 signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Jianmin; Walsh, Stephanie B.; Verney, Zoe M.

Research highlights: {yields} Organ transplant recipients are highly susceptible to early skin cancer development. {yields} CsA-mediated TGFB1-dependent TAK1/TAB1 signaling augments invasive tumor growth. {yields} CsA enhances accumulation of upstream kinases, ZMP, AMPK and IRAK to activate TAK1. {yields} TAK1 mediates enhanced proliferation and reduced apoptosis via CsA-dependent NF{kappa}B. -- Abstract: Cyclosporine A (CsA) is an immunosuppressive drug commonly used for maintaining chronic immune suppression in organ transplant recipients. It is known that patients receiving CsA manifest increased growth of aggressive non-melanoma skin cancers. However, the underlying mechanism by which CsA augments tumor growth is not fully understood. Here, we showmore » that CsA augments the growth of A431 epidermoid carcinoma xenograft tumors by activating tumor growth factor {beta}-activated kinase1 (TAK1). The activation of TAK1 by CsA occurs at multiple levels by kinases ZMP, AMPK and IRAK. TAK1 forms heterodimeric complexes with TAK binding protein 1 and 2 (TAB1/TAB2) which in term activate nuclear factor {kappa}B (NF{kappa}B) and p38 MAP kinase. Transcriptional activation of NF{kappa}B is evidenced by IKK{beta}-mediated phosphorylation-dependent degradation of I{kappa}B and consequent nuclear translocation of p65. This also leads to enhancement in the expression of its transcriptional target genes cyclin D1, Bcl2 and COX-2. Similarly, activation of p38 leads to enhanced inflammation-related signaling shown by increased phosphorylation of MAPKAPK2 and which in turn phosphorylates its substrate HSP27. Activation of both NF{kappa}B and p38 MAP kinase provide mitogenic stimuli to augment the growth of SCCs.« less

Vasopressin activates Akt/mTOR pathway in smooth muscle cells cultured in high glucose concentration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Montes, Daniela K.; Brenet, Marianne; Muñoz, Vanessa C.

Highlights: •AVP induces mTOR phosphorylation in A-10 cells cultured in high glucose concentration. •The mTOR phosphorylation is mediated by the PI3K/Akt pathway activation. •The AVP-induced mTOR phosphorylation inhibited autophagy and stimulated cell proliferation. -- Abstract: Mammalian target of rapamycin (mTOR) complex is a key regulator of autophagy, cell growth and proliferation. Here, we studied the effects of arginine vasopressin (AVP) on mTOR activation in vascular smooth muscle cells cultured in high glucose concentration. AVP induced the mTOR phosphorylation in A-10 cells grown in high glucose, in contrast to cells cultured in normal glucose; wherein, only basal phosphorylation was observed. Themore » AVP-induced mTOR phosphorylation was inhibited by a PI3K inhibitor. Moreover, the AVP-induced mTOR activation inhibited autophagy and increased thymidine incorporation in cells grown in high glucose. This increase was abolished by rapamycin which inhibits the mTORC1 complex formation. Our results suggest that AVP stimulates mTOR phosphorylation by activating the PI3K/Akt signaling pathway and, subsequently, inhibits autophagy and raises cell proliferation in A-10 cells maintained in high glucose concentration.« less

Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression

PubMed Central

Landry, Benjamin D; Mapa, Claudine E; Arsenault, Heather E; Poti, Kristin E; Benanti, Jennifer A

2014-01-01

To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division. PMID:24714560

Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression.

PubMed

Landry, Benjamin D; Mapa, Claudine E; Arsenault, Heather E; Poti, Kristin E; Benanti, Jennifer A

2014-05-02

To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division.

Tyrosine kinase activity of EphA2 promotes its S897 phosphorylation and glioblastoma cell proliferation.

PubMed

Hamaoka, Yuho; Negishi, Manabu; Katoh, Hironori

2018-05-23

EphA2, a member of the Eph family of receptor tyrosine kinases, has been reported to promote tumor malignancy through phosphorylation of serine 897 (S897). Here, we found that overexpression of wild-type EphA2 induced S897 phosphorylation through ERK activation without growth factors or cytokines and promoted glioblastoma cell proliferation. However, overexpression of a kinase-inactive mutant of EphA2 failed to induce ERK activation, S897 phosphorylation, and promotion of glioblastoma cell proliferation. These data suggest that when overexpressed, EphA2 induces ERK activation through its tyrosine kinase activity, leading to S897 phosphorylation and promotion of glioblastoma cell proliferation. Our findings provide a new insight into how EphA2 mediates glioblastoma progression. Copyright © 2018 Elsevier Inc. All rights reserved.

The PreS2 activator MHBst of hepatitis B virus activates c-raf-1/Erk2 signaling in transgenic mice

PubMed Central

Hildt, Eberhard; Munz, Barbara; Saher, Gesine; Reifenberg, Kurt; Hofschneider, Peter Hans

2002-01-01

The large hepatitis B virus (HBV) surface protein (LHBs) and C-terminally truncated middle size surface proteins (MHBst) form the family of the PreS2 activator proteins of HBV. Their transcriptional activator function is based on the cytoplasmic orientation of the PreS2 domain. MHBst activators are paradigmatic for this class of activators. Here we report that MHBst is protein kinase C (PKC)-dependently phosphorylated at Ser28. The integrity of the phosphorylation site is essential for the activator function. MHBst triggers PKC-dependent activation of c-Raf-1/Erk2 signaling that is a prerequisite for MHBst-dependent activation of AP-1 and NF-κB. To analyze the pathophysiological relevance of these data in vivo, transgenic mice were established that produce the PreS2 activator MHBst specifically in the liver. In these mice, a permanent PreS2-dependent specific activation of c-Raf-1/Erk2 signaling was observed, resulting in an increased hepatocyte proliferation rate. In transgenics older than 15 months, an increased incidence of liver tumors occurs. These data suggest that PreS2 activators LHBs and MHBst exert a tumor promoter-like function by activation of key enzymes of proliferation control. PMID:11847101

The PreS2 activator MHBs(t) of hepatitis B virus activates c-raf-1/Erk2 signaling in transgenic mice.

PubMed

Hildt, Eberhard; Munz, Barbara; Saher, Gesine; Reifenberg, Kurt; Hofschneider, Peter Hans

2002-02-15

The large hepatitis B virus (HBV) surface protein (LHBs) and C-terminally truncated middle size surface proteins (MHBs(t)) form the family of the PreS2 activator proteins of HBV. Their transcriptional activator function is based on the cytoplasmic orientation of the PreS2 domain. MHBs(t) activators are paradigmatic for this class of activators. Here we report that MHBs(t) is protein kinase C (PKC)-dependently phosphorylated at Ser28. The integrity of the phosphorylation site is essential for the activator function. MHBs(t) triggers PKC-dependent activation of c-Raf-1/Erk2 signaling that is a prerequisite for MHBs(t)-dependent activation of AP-1 and NF-kappaB. To analyze the pathophysiological relevance of these data in vivo, transgenic mice were established that produce the PreS2 activator MHBs(t) specifically in the liver. In these mice, a permanent PreS2-dependent specific activation of c-Raf-1/Erk2 signaling was observed, resulting in an increased hepatocyte proliferation rate. In transgenics older than 15 months, an increased incidence of liver tumors occurs. These data suggest that PreS2 activators LHBs and MHBs(t) exert a tumor promoter-like function by activation of key enzymes of proliferation control.

Phosphorylation and transport in the Na-K-2Cl cotransporters, NKCC1 and NKCC2A, compared in HEK-293 cells.

PubMed

Hannemann, Anke; Flatman, Peter W

2011-03-25

Na-K-2Cl cotransporters help determine cell composition and volume. NKCC1 is widely distributed whilst NKCC2 is only found in the kidney where it plays a vital role reabsorbing 20% of filtered NaCl. NKCC2 regulation is poorly understood because of its restricted distribution and difficulties with its expression in mammalian cell cultures. Here we compare phosphorylation of the N-termini of the cotransporters, measured with phospho-specific antibodies, with bumetanide-sensitive transport of K(+) ((86)Rb(+)) (activity) in HEK-293 cells stably expressing fNKCC1 or fNKCC2A which were cloned from ferret kidney. Activities of transfected transporters were distinguished from those of endogenous ones by working at 37 °C. fNKCC1 and fNKCC2A activities were highest after pre-incubation of cells in hypotonic low-[Cl(-)] media to reduce cell [Cl(-)] and volume during flux measurement. Phosphorylation of both transporters more than doubled. Pre-incubation with ouabain also strongly stimulated fNKCC1 and fNKCC2A and substantially increased phosphorylation, whereas pre-incubation in Na(+)-free media maximally stimulated fNKCC1 and doubled its phosphorylation, but inhibited fNKCC2A, with a small increase in its phosphorylation. Kinase inhibitors halved phosphorylation and activity of both transporters whereas inhibition of phosphatases with calyculin A strongly increased phosphorylation of both transporters but only slightly stimulated fNKCC1 and inhibited fNCCC2A. Thus kinase inhibition reduced phosphorylation and transport, and transport stimulation was only seen when phosphorylation increased, but transport did not always increase with phosphorylation. This suggests phosphorylation of the N-termini determines the transporters' potential capacity to move ions, but final activity also depends on other factors. Transport cannot be reliably inferred solely using phospho-specific antibodies on whole-cell lysates.

Phosphorylation of Glutathione S-Transferase P1 (GSTP1) by Epidermal Growth Factor Receptor (EGFR) Promotes Formation of the GSTP1-c-Jun N-terminal kinase (JNK) Complex and Suppresses JNK Downstream Signaling and Apoptosis in Brain Tumor Cells*

PubMed Central

Okamura, Tatsunori; Antoun, Gamil; Keir, Stephen T.; Friedman, Henry; Bigner, Darell D.; Ali-Osman, Francis

2015-01-01

Under normal physiologic conditions, the glutathione S-transferase P1 (GSTP1) protein exists intracellularly as a dimer in reversible equilibrium with its monomeric subunits. In the latter form, GSTP1 binds to the mitogen-activated protein kinase, JNK, and inhibits JNK downstream signaling. In tumor cells, which frequently are characterized by constitutively high GSTP1 expression, GSTP1 undergoes phosphorylation by epidermal growth factor receptor (EGFR) at tyrosine residues 3, 7, and 198. Here we report on the effect of this EGFR-dependent GSTP1 tyrosine phosphorylation on the interaction of GSTP1 with JNK, on the regulation of JNK downstream signaling by GSTP1, and on tumor cell survival. Using in vitro and in vivo growing human brain tumors, we show that tyrosine phosphorylation shifts the GSTP1 dimer-monomer equilibrium to the monomeric state and facilitates the formation of the GSTP1-JNK complex, in which JNK is functionally inhibited. Targeted mutagenesis and functional analysis demonstrated that the increased GSTP1 binding to JNK results from phosphorylation of the GSTP1 C-terminal Tyr-198 by EGFR and is associated with a >2.5-fold decrease in JNK downstream signaling and a significant suppression of both spontaneous and drug-induced apoptosis in the tumor cells. The findings define a novel mechanism of regulatory control of JNK signaling that is mediated by the EGFR/GSTP1 cross-talk and provides a survival advantage for tumors with activated EGFR and high GSTP1 expression. The results lay the foundation for a novel strategy of dual EGFR/GSTP1 for treating EGFR+ve, GSTP1 expressing GBMs. PMID:26429914

Human Parturition Involves Phosphorylation of Progesterone Receptor-A at Serine-345 in Myometrial Cells.

PubMed

Amini, Peyvand; Michniuk, Daniel; Kuo, Kelly; Yi, Lijuan; Skomorovska-Prokvolit, Yelenna; Peters, Gregory A; Tan, Huiqing; Wang, Junye; Malemud, Charles J; Mesiano, Sam

2016-11-01

The hypothesis that phosphorylation of progesterone receptor (PR) isoforms, PR-A and PR-B, in myometrial cells affects progesterone action in the context of human parturition was tested. Immunodetection of phosphoserine (pSer) PR forms in term myometrium revealed that the onset of labor is associated with increased phosphorylation of PR-A at serine-345 (pSer345-PRA) and that pSer345-PRA localized to the nucleus of myometrial cells. In explant cultures of term myometrium generation of pSer345-PRA was induced by interleukin-1β and dependent on progesterone, suggesting that pSer345-PRA generation is induced by a proinflammatory stimulus. In the hTERT-HM A/B human myometrial cell line, abundance of pSer345-PRA was induced by progesterone in a dose- (EC 50 ∼1 nM) and time-dependent manner. Prevention of pSer345 (by site-directed mutagenesis) abolished the capacity for PR-A to inhibit anti-inflammatory actions of progesterone mediated by PR-B but had no effect on the transrepressive activity of PR-A at a canonical progesterone response element. Taken together, the data show that human parturition involves the phosphorylation of PR-A at serine-345 in myometrial cells and that this process is ligand dependent and induced by a proinflammatory stimulus. We also found that in myometrial cells, pSer345 activates the capacity for PR-A to inhibit antiinflammatory actions of progesterone mediated by PR-B. Phosphorylation of PR-A at serine-345 may be an important functional link between tissue-level inflammation and PR-A-mediated functional progesterone withdrawal to trigger parturition.

Dopamine D2 receptor signaling modulates mutant ataxin-1 S776 phosphorylation and aggregation.

PubMed

Hearst, Scoty M; Lopez, Mariper E; Shao, Qingmei; Liu, Yong; Vig, Parminder J S

2010-08-01

Spinocerebellar ataxia 1 (SCA1) is a dominantly inherited neurodegenerative disease associated with progressive ataxia resulting from the loss of cerebellar Purkinje cells (PCs) and neurons in the brainstem. In PCs of SCA1 transgenic mice, the disease causing ataxin-1 protein mediates the formation of S100B containing cytoplasmic vacuoles and further self-aggregates to form intranuclear inclusions. The exact function of the ataxin-1 protein is not fully understood. However, the aggregation and neurotoxicity of the mutant ataxin-1 protein is dependent on the phosphorylation at serine 776 (S776). Although protein kinase A (PKA) has been implicated as the S776 kinase, the mechanism of PKA/ataxin-1 regulation in SCA1 is still not clear. We propose that a dopamine D(2) receptor (D2R)/S100B pathway may be involved in modulating PKA activity in PCs. Using a D2R/S100B HEK stable cell line transiently transfected with GFP-ataxin-1[82Q], we demonstrate that stimulation of the D2R/S100B pathway caused a reduction in mutant ataxin-1 S776 phosphorylation and ataxin-1 aggregation. Activation of PKA by forskolin resulted in an enhanced S776 phosphorylation and increased ataxin-1 nuclear aggregation, which was suppressed by treatment with D2R agonist bromocriptine and PKA inhibitor H89. Furthermore, treating SCA1 transgenic PC slice cultures with forskolin induced neurodegenerative morphological abnormalities in PC dendrites consistent with those observed in vivo. Taken together our data support a mechanism where PKA dependent mutant ataxin-1 phosphorylation and aggregation can be regulated by D2R/S100B signaling.

Dopamine D2 Receptor Signaling Modulates Mutant Ataxin-1 S776 Phosphorylation and Aggregation

PubMed Central

Hearst, SM; Lopez, ME; Shao, Q; Liu, Y; Vig, PJS

2010-01-01

Spinocerebellar ataxia 1 (SCA1) is a dominantly inherited neurodegenerative disease associated with progressive ataxia resulting from the loss of cerebellar Purkinje cells (PCs) and neurons in the brainstem. In PCs of SCA1 transgenic (Tg) mice, the disease causing ataxin-1 protein mediates the formation of S100B containing cytoplasmic vacuoles and further self-aggregates to form intranuclear inclusions. The exact function of the ataxin-1 protein is not fully understood. However, the aggregation and neurotoxicity of the mutant ataxin-1 protein is dependent on the phosphorylation at serine 776 (S776). Although protein kinase A (PKA) has been implicated as the S776 kinase, the mechanism of PKA/ataxin-1 regulation in SCA1 is still not clear. We propose that a dopamine D2 receptor (D2R)/S100B pathway may be involved in modulating PKA activity in PCs. Using a D2R/S100B HEK stable cell line transiently transfected with GFP-ataxin-1[82Q], we demonstrate that stimulation of the D2R/S100B pathway caused a reduction in mutant ataxin-1 S776 phosphorylation and ataxin-1 aggregation. Activation of PKA by forskolin resulted in an enhanced S776 phosphorylation and increased ataxin-1 nuclear aggregation, which was suppressed by treatment with D2R agonist bromocriptine and PKA inhibitor H89. Furthermore, treating SCA1 Tg PC slice cultures with forskolin induced neurodegenerative morphological abnormalities in PC dendrites consistent with those observed in vivo. Taken together our data support a mechanism where PKA dependent mutant ataxin-1 phosphorylation and aggregation can be regulated by D2R/S100B signaling. PMID:20477910

Human Parturition Involves Phosphorylation of Progesterone Receptor-A at Serine-345 in Myometrial Cells

PubMed Central

Amini, Peyvand; Michniuk, Daniel; Kuo, Kelly; Yi, Lijuan; Skomorovska-Prokvolit, Yelenna; Peters, Gregory A.; Tan, Huiqing; Wang, Junye; Malemud, Charles J.

2016-01-01

The hypothesis that phosphorylation of progesterone receptor (PR) isoforms, PR-A and PR-B, in myometrial cells affects progesterone action in the context of human parturition was tested. Immunodetection of phosphoserine (pSer) PR forms in term myometrium revealed that the onset of labor is associated with increased phosphorylation of PR-A at serine-345 (pSer345-PRA) and that pSer345-PRA localized to the nucleus of myometrial cells. In explant cultures of term myometrium generation of pSer345-PRA was induced by interleukin-1β and dependent on progesterone, suggesting that pSer345-PRA generation is induced by a proinflammatory stimulus. In the hTERT-HMA/B human myometrial cell line, abundance of pSer345-PRA was induced by progesterone in a dose- (EC50 ∼1 nM) and time-dependent manner. Prevention of pSer345 (by site-directed mutagenesis) abolished the capacity for PR-A to inhibit anti-inflammatory actions of progesterone mediated by PR-B but had no effect on the transrepressive activity of PR-A at a canonical progesterone response element. Taken together, the data show that human parturition involves the phosphorylation of PR-A at serine-345 in myometrial cells and that this process is ligand dependent and induced by a proinflammatory stimulus. We also found that in myometrial cells, pSer345 activates the capacity for PR-A to inhibit antiinflammatory actions of progesterone mediated by PR-B. Phosphorylation of PR-A at serine-345 may be an important functional link between tissue-level inflammation and PR-A-mediated functional progesterone withdrawal to trigger parturition. PMID:27653036

Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling.

PubMed Central

Denhardt, D T

1996-01-01

The features of three distinct protein phosphorylation cascades in mammalian cells are becoming clear. These signalling pathways link receptor-mediated events at the cell surface or intracellular perturbations such as DNA damage to changes in cytoskeletal structure, vesicle transport and altered transcription factor activity. The best known pathway, the Ras-->Raf-->MEK-->ERK cascade [where ERK is extracellular-signal-regulated kinase and MEK is mitogen-activated protein (MAP) kinase/ERK kinase], is typically stimulated strongly by mitogens and growth factors. The other two pathways, stimulated primarily by assorted cytokines, hormones and various forms of stress, predominantly utilize p21 proteins of the Rho family (Rho, Rac and CDC42), although Ras can also participate. Diagnostic of each pathway is the MAP kinase component, which is phosphorylated by a unique dual-specificity kinase on both tyrosine and threonine in one of three motifs (Thr-Glu-Tyr, Thr-Phe-Tyr or Thr-Gly-Tyr), depending upon the pathway. In addition to activating one or more protein phosphorylation cascades, the initiating stimulus may also mobilize a variety of other signalling molecules (e.g. protein kinase C isoforms, phospholipid kinases, G-protein alpha and beta gamma subunits, phospholipases, intracellular Ca2+). These various signals impact to a greater or lesser extent on multiple downstream effectors. Important concepts are that signal transmission often entails the targeted relocation of specific proteins in the cell, and the reversible formation of protein complexes by means of regulated protein phosphorylation. The signalling circuits may be completed by the phosphorylation of upstream effectors by downstream kinases, resulting in a modulation of the signal. Signalling is terminated and the components returned to the ground state largely by dephosphorylation. There is an indeterminant amount of cross-talk among the pathways, and many of the proteins in the pathways belong to families of closely related proteins. The potential for more than one signal to be conveyed down a pathway simultaneously (multiplex signalling) is discussed. The net effect of a given stimulus on the cell is the result of a complex intracellular integration of the intensity and duration of activation of the individual pathways. The specific outcome depends on the particular signalling molecules expressed by the target cells and on the dynamic balance among the pathways. PMID:8836113

Bi-functional, substrate mimicking RNA inhibits MSK1-mediated cAMP-response element-binding protein phosphorylation and reveals magnesium ion-dependent conformational changes of the kinase.

PubMed

Hamm, Jorg; Alessi, Dario R; Biondi, Ricardo M

2002-11-29

The design of specific inhibitors for protein kinases is an important step toward elucidation of intracellular signal transduction pathways and to guide drug discovery programs. We devised a model approach to generate specific, competitive kinase inhibitors by isolating substrate mimics containing two independent binding sites with an anti-idiotype strategy from combinatorial RNA libraries. As a general test for the ability to generate highly specific kinase inhibitors, we selected the transcription factor cAMP-response element-binding protein (CREB) that is phosphorylated on the same serine residue by the protein kinase MSK1 as well as by RSK1. The sequences and structures of these kinases are very similar, about 60% of their amino acids are identical. Nevertheless, we can demonstrate that the selected RNA inhibitors inhibit specifically CREB phosphorylation by MSK1 but do not affect CREB phosphorylation by RSK1. The inhibitors interact preferentially with the inactive form of MSK1. Furthermore, we demonstrate that RNA ligands can be conformation-specific probes, and this feature allowed us to describe magnesium ion-dependent conformational changes of MSK1 upon activation.

Regulation of renal fibrosis by Smad3 Thr388 phosphorylation.

PubMed

Qu, Xinli; Li, Xueling; Zheng, Yaowu; Ren, Yi; Puelles, Victor G; Caruana, Georgina; Nikolic-Paterson, David J; Li, Jinhua

2014-04-01

Transforming growth factor-β (TGF-β) promotes tissue fibrosis via receptor-mediated phosphorylation of the receptor-activated Smad2/3, together with Smad4. Of these, Smad3 plays a major profibrotic role in mouse models of tissue fibrosis. Transcriptional activity of the Smad3 protein is regulated by phosphorylation of residues in the C-terminal domain and the linker region. Herein, we examined the role of a novel phosphorylation site within the MH2 domain (T388) in the regulation of Smad3 activity. Confocal microscopy using an Smad3 phosphorylated T388-specific antibody identified phosphorylation of Smad3 T388 in myofibroblasts and tubular epithelial cells in human focal and segmental glomerulosclerosis and mouse models of unilateral ureteric obstruction and diabetic nephropathy, whereas phosphorylated T388 was largely absent in normal kidney. In vitro, TGF-β1 induced phosphorylation of Smad3 T388 in a biphasic pattern. A point mutation of T388/V in an Smad3 construct demonstrated that phosphorylation of T388 promotes Smad3 binding to Smad4 and CDK8, but was not necessary for nuclear translocation. Furthermore, T388 phosphorylation was required for TGF-β-induced collagen I gene promoter activity and extracellular matrix production in cultured fibroblasts. In conclusion, our study identifies phosphorylation of T388 in the Smad3 MH2 domain as an important mechanism that regulates the profibrotic TGF-β/Smad3 signaling pathway, which has direct relevance to human and experimental fibrotic kidney disease. Copyright © 2014. Published by Elsevier Inc.

Activation of p38 in C2C12 myotubes following ATP depletion depends on extracellular glucose.

PubMed

Hsu, Chia George; Burkholder, Thomas J

2015-06-01

Muscle cells adjust their glucose metabolism in response to myriad stimuli, and particular attention has been paid to glucose metabolism after contraction, ATP depletion, and insulin stimulation. Each of these requires translocation of GLUT4 to the cell membrane, and may require activation of glucose transporters by p38. In contrast, AICAR stimulates glucose transport without activation of p38, suggesting that p38 activation may be an indirect consequence of accelerated glucose transport or metabolism. This study was designed to investigate the contribution of AMPK and p38 to ATP homeostasis and glucose metabolism to test the hypothesis that p38 reflects glycolytic activity rather than controls glucose uptake. Treating mature myotubes with rotenone caused transient ATP depletion in 15 min with recovery by 120 min, associated with increased lactate production. Both ACC and p38 were rapidly phosphorylated, but ACC remained phosphorylated while p38 phosphorylation declined as ATP recovered. AMPK inhibition blocked ATP recovery, lactate production, and phosphorylation of p38 and ACC. Inhibition of p38 had little effect. AICAR induced ACC phosphorylation, but not lactate production or p38 phosphorylation. Finally, removing extracellular glucose potentiated rotenone-induced AMPK activation, but reduced lactate generation, ATP recovery and p38 activation. Thus, glucose metabolism is highly sensitive to ATP homeostasis via AMPK activity, but p38 activity is dispensable. Although p38 is strongly phosphorylated during ATP depletion, this appears to be an indirect consequence of accelerated glycolysis.

Cool-associated Tyrosine-phosphorylated Protein 1 Is Required for the Anchorage-independent Growth of Cervical Carcinoma Cells by Binding Paxillin and Promoting AKT Activation*

PubMed Central

Yoo, Sungsoo M.; Latifkar, Arash; Cerione, Richard A.; Antonyak, Marc A.

2017-01-01

Cool-associated tyrosine-phosphorylated protein 1 (Cat-1) is a signaling scaffold as well as an ADP-ribosylation factor-GTPase-activating protein. Although best known for its role in cell migration, we recently showed that the ability of Cat-1 to bind paxillin, a major constituent of focal complexes, is also essential for the anchorage-independent growth of HeLa cervical carcinoma cells. Here we set out to learn more about the underlying mechanism by which Cat-paxillin interactions mediate this effect. We show that knocking down paxillin expression in HeLa cells promotes their ability to form colonies in soft agar, whereas ectopically expressing paxillin in these cells inhibits this transformed growth phenotype. Although knocking down Cat-1 prevents HeLa cells from forming colonies in soft agar, when paxillin is knocked down together with Cat-1, the cells are again able to undergo anchorage-independent growth. These results suggest that the requirement of Cat-1 for this hallmark of cellular transformation is coupled to its ability to bind paxillin and abrogate its actions as a negative regulator of anchorage-independent growth. We further show that knocking down Cat-1 expression in HeLa cells leads to a reduction in Akt activation, which can be reversed by knocking down paxillin. Moreover, expression of constitutively active forms of Akt1 and Akt2 restores the anchorage-independent growth capability of HeLa cells depleted of Cat-1 expression. Together, these findings highlight a novel mechanism whereby interactions between Cat-1 and its binding partner paxillin are necessary to ensure sufficient Akt activation so that cancer cells are able to grow under anchorage-independent conditions. PMID:28100775

Cool-associated Tyrosine-phosphorylated Protein 1 Is Required for the Anchorage-independent Growth of Cervical Carcinoma Cells by Binding Paxillin and Promoting AKT Activation.

PubMed

Yoo, Sungsoo M; Latifkar, Arash; Cerione, Richard A; Antonyak, Marc A

2017-03-03

Cool-associated tyrosine-phosphorylated protein 1 (Cat-1) is a signaling scaffold as well as an ADP-ribosylation factor-GTPase-activating protein. Although best known for its role in cell migration, we recently showed that the ability of Cat-1 to bind paxillin, a major constituent of focal complexes, is also essential for the anchorage-independent growth of HeLa cervical carcinoma cells. Here we set out to learn more about the underlying mechanism by which Cat-paxillin interactions mediate this effect. We show that knocking down paxillin expression in HeLa cells promotes their ability to form colonies in soft agar, whereas ectopically expressing paxillin in these cells inhibits this transformed growth phenotype. Although knocking down Cat-1 prevents HeLa cells from forming colonies in soft agar, when paxillin is knocked down together with Cat-1, the cells are again able to undergo anchorage-independent growth. These results suggest that the requirement of Cat-1 for this hallmark of cellular transformation is coupled to its ability to bind paxillin and abrogate its actions as a negative regulator of anchorage-independent growth. We further show that knocking down Cat-1 expression in HeLa cells leads to a reduction in Akt activation, which can be reversed by knocking down paxillin. Moreover, expression of constitutively active forms of Akt1 and Akt2 restores the anchorage-independent growth capability of HeLa cells depleted of Cat-1 expression. Together, these findings highlight a novel mechanism whereby interactions between Cat-1 and its binding partner paxillin are necessary to ensure sufficient Akt activation so that cancer cells are able to grow under anchorage-independent conditions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Human Cystathionine-β-Synthase Phosphorylation on Serine227 Modulates Hydrogen Sulfide Production in Human Urothelium.

PubMed

d'Emmanuele di Villa Bianca, Roberta; Mitidieri, Emma; Esposito, Davide; Donnarumma, Erminia; Donnarumm, Erminia; Russo, Annapina; Fusco, Ferdinando; Ianaro, Angela; Mirone, Vincenzo; Cirino, Giuseppe; Russo, Giulia; Sorrentino, Raffaella

2015-01-01

Urothelium, the epithelial lining the inner surface of human bladder, plays a key role in bladder physiology and pathology. It responds to chemical, mechanical and thermal stimuli by releasing several factors and mediators. Recently it has been shown that hydrogen sulfide contributes to human bladder homeostasis. Hydrogen sulfide is mainly produced in human bladder by the action of cystathionine-β-synthase. Here, we demonstrate that human cystathionine-β-synthase activity is regulated in a cGMP/PKG-dependent manner through phosphorylation at serine 227. Incubation of human urothelium or T24 cell line with 8-Bromo-cyclic-guanosine monophosphate (8-Br-cGMP) but not dibutyryl-cyclic-adenosine monophosphate (d-cAMP) causes an increase in hydrogen sulfide production. This result is congruous with the finding that PKG is robustly expressed but PKA only weakly present in human urothelium as well as in T24 cells. The cGMP/PKG-dependent phosphorylation elicited by 8-Br-cGMP is selectively reverted by KT5823, a specific PKG inhibitor. Moreover, the silencing of cystathionine-β-synthase in T24 cells leads to a marked decrease in hydrogen sulfide production either in basal condition or following 8-Br-cGMP challenge. In order to identify the phosphorylation site, recombinant mutant proteins of cystathionine-β-synthase in which Ser32, Ser227 or Ser525 was mutated in Ala were generated. The Ser227Ala mutant cystathionine-β-synthase shows a notable reduction in basal biosynthesis of hydrogen sulfide becoming unresponsive to the 8-Br-cGMP challenge. A specific antibody that recognizes the phosphorylated form of cystathionine-β-synthase has been produced and validated by using T24 cells and human urothelium. In conclusion, human cystathionine-β-synthase can be phosphorylated in a PKG-dependent manner at Ser227 leading to an increased catalytic activity.

Human Cystathionine-β-Synthase Phosphorylation on Serine227 Modulates Hydrogen Sulfide Production in Human Urothelium

PubMed Central

d’Emmanuele di Villa Bianca, Roberta; Donnarumm, Erminia; Russo, Annapina; Fusco, Ferdinando; Ianaro, Angela; Mirone, Vincenzo; Cirino, Giuseppe; Russo, Giulia; Sorrentino, Raffaella

2015-01-01

Urothelium, the epithelial lining the inner surface of human bladder, plays a key role in bladder physiology and pathology. It responds to chemical, mechanical and thermal stimuli by releasing several factors and mediators. Recently it has been shown that hydrogen sulfide contributes to human bladder homeostasis. Hydrogen sulfide is mainly produced in human bladder by the action of cystathionine-β-synthase. Here, we demonstrate that human cystathionine-β-synthase activity is regulated in a cGMP/PKG-dependent manner through phosphorylation at serine 227. Incubation of human urothelium or T24 cell line with 8-Bromo-cyclic-guanosine monophosphate (8-Br-cGMP) but not dibutyryl-cyclic-adenosine monophosphate (d-cAMP) causes an increase in hydrogen sulfide production. This result is congruous with the finding that PKG is robustly expressed but PKA only weakly present in human urothelium as well as in T24 cells. The cGMP/PKG-dependent phosphorylation elicited by 8-Br-cGMP is selectively reverted by KT5823, a specific PKG inhibitor. Moreover, the silencing of cystathionine-β-synthase in T24 cells leads to a marked decrease in hydrogen sulfide production either in basal condition or following 8-Br-cGMP challenge. In order to identify the phosphorylation site, recombinant mutant proteins of cystathionine-β-synthase in which Ser32, Ser227 or Ser525 was mutated in Ala were generated. The Ser227Ala mutant cystathionine-β-synthase shows a notable reduction in basal biosynthesis of hydrogen sulfide becoming unresponsive to the 8-Br-cGMP challenge. A specific antibody that recognizes the phosphorylated form of cystathionine-β-synthase has been produced and validated by using T24 cells and human urothelium. In conclusion, human cystathionine-β-synthase can be phosphorylated in a PKG-dependent manner at Ser227 leading to an increased catalytic activity. PMID:26368121

AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells.

PubMed

Lorenzato, Annalisa; Biolatti, Marta; Delogu, Giuseppe; Capobianco, Giampiero; Farace, Cristiano; Dessole, Salvatore; Cossu, Antonio; Tanda, Francesco; Madeddu, Roberto; Olivero, Martina; Di Renzo, Maria Flavia

2013-10-15

The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. © 2013 Elsevier Inc. All rights reserved.

Gab1 Acts as an Adapter Molecule Linking the Cytokine Receptor gp130 to ERK Mitogen-Activated Protein Kinase

PubMed Central

Takahashi-Tezuka, Mariko; Yoshida, Yuichi; Fukada, Toshiyuki; Ohtani, Takuya; Yamanaka, Yojiro; Nishida, Keigo; Nakajima, Koichi; Hibi, Masahiko; Hirano, Toshio

1998-01-01

Gab1 has structural similarities with Drosophila DOS (daughter of sevenless), which is a substrate of the protein tyrosine phosphatase Corkscrew. Both Gab1 and DOS have a pleckstrin homology domain and tyrosine residues, potential binding sites for various SH2 domain-containing adapter molecules when they are phosphorylated. We found that Gab1 was tyrosine phosphorylated in response to various cytokines, such as interleukin-6 (IL-6), IL-3, alpha interferon (IFN-α), and IFN-γ. Upon the stimulation of IL-6 or IL-3, Gab1 was found to form a complex with phosphatidylinositol (PI)-3 kinase and SHP-2, a homolog of Corkscrew. Mutational analysis of gp130, the common subunit of IL-6 family cytokine receptors, revealed that neither tyrosine residues of gp130 nor its carboxy terminus was required for tyrosine phosphorylation of Gab1. Expression of Gab1 enhanced gp130-dependent mitogen-activated protein (MAP) kinase ERK2 activation. A mutation of tyrosine 759, the SHP-2 binding site of gp130, abrogated the interactions of Gab1 with SHP-2 and PI-3 kinase as well as ERK2 activation. Furthermore, ERK2 activation was inhibited by a dominant negative p85 PI-3 kinase, wortmannin, or a dominant negative Ras. These observations suggest that Gab1 acts as an adapter molecule in transmitting signals to ERK MAP kinase for the cytokine receptor gp130 and that SHP-2, PI-3 kinase, and Ras are involved in Gab1-mediated ERK activation. PMID:9632795

Estradiol-induced object recognition memory consolidation is dependent on activation of mTOR signaling in the dorsal hippocampus

PubMed Central

Fortress, Ashley M.; Fan, Lu; Orr, Patrick T.; Zhao, Zaorui; Frick, Karyn M.

2013-01-01

The mammalian target of rapamycin (mTOR) signaling pathway is an important regulator of protein synthesis and is essential for various forms of hippocampal memory. Here, we asked whether the enhancement of object recognition memory consolidation produced by dorsal hippocampal infusion of 17β-estradiol (E2) is dependent on mTOR signaling in the dorsal hippocampus, and whether E2-induced mTOR signaling is dependent on dorsal hippocampal phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) activation. We first demonstrated that the enhancement of object recognition induced by E2 was blocked by dorsal hippocampal inhibition of ERK, PI3K, or mTOR activation. We then showed that an increase in dorsal hippocampal ERK phosphorylation 5 min after intracerebroventricular (ICV) E2 infusion was also blocked by dorsal hippocampal infusion of the three cell signaling inhibitors. Next, we found that ICV infusion of E2 increased phosphorylation of the downstream mTOR targets S6K (Thr-421) and 4E-BP1 in the dorsal hippocampus 5 min after infusion, and that this phosphorylation was blocked by dorsal hippocampal infusion of inhibitors of ERK, PI3K, and mTOR. Collectively, these data demonstrate for the first time that activation of the dorsal hippocampal mTOR signaling pathway is necessary for E2 to enhance object recognition memory consolidation and that E2-induced mTOR activation is dependent on upstream activation of ERK and PI3K signaling. PMID:23422279

Synaptic Activation of Ribosomal Protein S6 Phosphorylation Occurs Locally in Activated Dendritic Domains

ERIC Educational Resources Information Center

Pirbhoy, Patricia Salgado; Farris, Shannon; Steward, Oswald

2016-01-01

Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6)…

Tuning stochastic transition rates in a bistable genetic network.

NASA Astrophysics Data System (ADS)

Chickarmane, Vijay; Peterson, Carsten

2009-03-01

We investigate the stochastic dynamics of a simple genetic network, a toggle switch, in which the system makes transitions between the two alternative states. Our interest is in exploring whether such stochastic transitions, which occur due to the intrinsic noise such as transcriptional and degradation events, can be slowed down/speeded up, without changing the mean expression levels of the two genes, which comprise the toggle network. Such tuning is achieved by linking a signaling network to the toggle switch. The signaling network comprises of a protein, which can exist either in an active (phosphorylated) or inactive (dephosphorylated) form, and where its state is determined by one of the genetic network components. The active form of the protein in turn feeds back on the dynamics of the genetic network. We find that the rate of stochastic transitions from one state to the other, is determined essentially by the speed of phosphorylation, and hence the rate can be modulated by varying the phosphatase levels. We hypothesize that such a network architecture can be implemented as a general mechanism for controlling transition rates and discuss applications in population studies of two differentiated cell lineages, ex: the myeloid/erythroid lineage in hematopoiesis.

RACK1 forms a complex with FGFR1 and PKM2, and stimulates the growth and migration of squamous lung cancer cells.

PubMed

Zhou, Chengzhi; Chen, Tao; Xie, Zhanhong; Qin, Yinyin; Ou, Yangming; Zhang, Jiexia; Li, Shiyue; Chen, Rongchang; Zhong, Nanshan

2017-11-01

Phosphorylation of Pyruvate Kinase M2 (PKM2) on Tyr105 by fibroblast growth factor receptor 1 (FGFR1) has been shown to promote its nuclear localization as well as cell growth in lung cancer. Better understanding the regulation of this process would benefit the clinical treatment for lung cancer. Here, it has been found that the adaptor protein receptor for activated PKC kinase (RACK1) formed a complex with FGFR1 and PKM2, and activated the FGFR1/PKM2 signaling. Knocking down the expression of RACK1 impaired the phosphorylation on Tyr105 of PKM2 and inhibited the growth and migration of lung cancer cells, while over-expression of RACK1 in lung cancer cells led to the resistance to Erdafitinib. Moreover, knocking down the expression of RACK1 impaired the tumorigenesis of lung cancer driven by LKB loss and mutated Ras (KrasG12D). Taken together, our study demonstrated the pivotal roles of RACK1 in FGFR1/PKM2 signaling, suggesting FGFR1/RACK1/PKM2 might be a therapeutic target for lung cancer treatment. © 2017 Wiley Periodicals, Inc.

Engineering Visual Arrestin-1 with Special Functional Characteristics*

PubMed Central

Vishnivetskiy, Sergey A.; Chen, Qiuyan; Palazzo, Maria C.; Brooks, Evan K.; Altenbach, Christian; Iverson, Tina M.; Hubbell, Wayne L.; Gurevich, Vsevolod V.

2013-01-01

Arrestin-1 preferentially binds active phosphorylated rhodopsin. Previously, a mutant with enhanced binding to unphosphorylated active rhodopsin (Rh*) was shown to partially compensate for lack of rhodopsin phosphorylation in vivo. Here we showed that reengineering of the receptor binding surface of arrestin-1 further improves the binding to Rh* while preserving protein stability. In mammals, arrestin-1 readily self-associates at physiological concentrations. The biological role of this phenomenon can only be elucidated by replacing wild type arrestin-1 in living animals with a non-oligomerizing mutant retaining all other functions. We demonstrate that constitutively monomeric forms of arrestin-1 are sufficiently stable for in vivo expression. We also tested the idea that individual functions of arrestin-1 can be independently manipulated to generate mutants with the desired combinations of functional characteristics. Here we showed that this approach is feasible; stable forms of arrestin-1 with high Rh* binding can be generated with or without the ability to self-associate. These novel molecular tools open the possibility of testing of the biological role of arrestin-1 self-association and pave the way to elucidation of full potential of compensational approach to gene therapy of gain-of-function receptor mutations. PMID:23250748

Regulation of tyrosine hydroxylase activity and phosphorylation at Ser(19) and Ser(40) via activation of glutamate NMDA receptors in rat striatum.

PubMed

Lindgren, N; Xu, Z Q; Lindskog, M; Herrera-Marschitz, M; Goiny, M; Haycock, J; Goldstein, M; Hökfelt, T; Fisone, G

2000-06-01

The activity of tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by phosphorylation. In this study, we examined the effects of activation of NMDA receptors on the state of phosphorylation and activity of tyrosine hydroxylase in rat striatal slices. NMDA produced a time-and concentration-dependent increase in the levels of phospho-Ser(19)-tyrosine hydroxylase in nigrostriatal nerve terminals. This increase was not associated with any changes in the basal activity of tyrosine hydroxylase, measured as DOPA accumulation. Forskolin, an activator of adenylyl cyclase, stimulated tyrosine hydroxylase phosphorylation at Ser(40) and caused a significant increase in DOPA accumulation. NMDA reduced forskolin-mediated increases in both Ser(40) phosphorylation and DOPA accumulation. In addition, NMDA reduced the increase in phospho-Ser(40)-tyrosine hydroxylase produced by okadaic acid, an inhibitor of protein phosphatase 1 and 2A, but not by a cyclic AMP analogue, 8-bromo-cyclic AMP. These results indicate that, in the striatum, glutamate decreases tyrosine hydroxylase phosphorylation at Ser(40) via activation of NMDA receptors by reducing cyclic AMP production. They also provide a mechanism for the demonstrated ability of NMDA to decrease tyrosine hydroxylase activity and dopamine synthesis.

Tyrosine hydroxylase is activated and phosphorylated at different sites in rat pheochromocytoma PC 12 cells treated with phorbol ester and forskolin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tachikawa, E.; Tank, A.W.; Weiner, D.H.

1986-03-01

The effects of phorbol ester (4..beta..-phorbol, 12..beta..-myristate, 13..cap alpha..-acetate; TPA), an activator of Ca/sup + +//phospholipid-dependent protein kinase (PK-C), and forskolin, which stimulates adenylate cyclase and cyclic AMP-dependent protein kinase (cAMP-PK), on the activation and phosphorylation of tyrosine hydroxylase (TH) in rat pheochromocytoma (PC 12) cells were examined. Incubation of the cells with TPA (0.01-1 ..mu..M) or forskolin (0.01-0.1 ..mu..M) produces increases in activation and phosphorylation of TH in a concentration-dependent manner. The stimulatory effects of TPA are dependent on extracellular Ca/sup + +/ and are inhibited by pretreatment of the cells with trifluoperazine (TFP). The effects of forskolin aremore » independent of Ca/sup + +/ and are not inhibited by TFP. In cells treated with forskolin, the time course of the increase in cAMP correlates with the increases in TH activity and phosphorylation. cAMP levels do not increase in cells treated with TPA. There is an increase in the phosphorylation of only one tryptic phosphopeptide derived from TH in cells treated with either forskolin or TPA. The peptide phosphorylated in TPA-treated cells exhibits different elution characteristics on HPLC from that in forskolin-treated cells. The authors conclude that TH in PC 12 cells is phosphorylated on different sites by cAMP-PK and PK-C. Phosphorylation of either of these sites is associated with enzyme activation.« less

Phosphatidylinositol kinase. A component of the chromaffin-granule membrane

PubMed Central

Phillips, John H.

1973-01-01

Phosphorylation of bovine chromaffin granules by ATP leads to the formation of diphosphoinositide in the granule membrane. Both phosphatidylinositol kinase and its substrate are components of this membrane, and triphosphoinositide is not formed under the conditions of the assay. The reaction is Mg2+-dependent and is stimulated by Mn2+ and F− ions. The initial reaction is rapid, with a broad pH profile and a `transition' temperature for its activation energy at 27°C. The apparent Km for ATP is 5μm. ATP, N-ethylmaleimide, Cu2+ ions and NaIO4 are inhibitory. The phospholipids of chromaffin-granule membranes have been analysed: 6.8% of the lipid P is found in phosphatidylinositol, and only 2–3% in phosphatidylserine. Comparison of the rate of phosphorylation of intact and lysed granules suggests that the sites for phosphorylation are on the outer (cytoplasmic) surface of the granules, and diphosphoinositide may therefore make an important contribution to the charge of the chromaffin granule in vivo. PMID:4360713

Structural studies of ROK fructokinase YdhR from Bacillus subtilis : insights into substrate binding and fructose specificity.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nocek, B.; Stein, A.; Jedrzejczak, R.

2011-02-18

The main pathway of bacterial sugar phosphorylation utilizes specific phosphoenolpyruvate phosphotransferase system (PTS) enzymes. In addition to the classic PTS system, a PTS-independent secondary system has been described in which nucleotide-dependent sugar kinases are used for monosaccharide phosphorylation. Fructokinase (FK), which phosphorylates d-fructose with ATP as a cofactor, has been shown to be a member of this secondary system. Bioinformatic analysis has shown that FK is a member of the 'ROK' (bacterial Repressors, uncharacterized Open reading frames, and sugar Kinases) sequence family. In this study, we report the crystal structures of ROK FK from Bacillus subtilis (YdhR) (a) apo andmore » in the presence of (b) ADP and (c) ADP/d-fructose. All structures show that YdhR is a homodimer with a monomer composed of two similar {alpha}/{beta} domains forming a large cleft between domains that bind ADP and d-fructose. Enzymatic activity assays support YdhR function as an ATP-dependent fructose kinase.« less

Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex

PubMed Central

Ganaie, Safder S.; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve

2017-01-01

Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases. PMID:28459842

Protein phosphorylation as a mechanism for osmotic-stress activation of sucrose-phosphate synthase in spinach leaves.

PubMed

Toroser, D; Huber, S C

1997-07-01

Experiments were performed to investigated the mechanism of sucrose-phosphate synthase (SPS) activation by osmotic stress in darkened spinach (Spinacia oleracea L.) leaves. The activation was stable through immunopurification and was not the result of an increased SPS protein level. The previously described Ca(2+)-independent peak III kinase, obtained by ion-exchange chromatography, is confirmed to be the predominant enzyme catalyzing phosphorylation and inactivation of dephosphoserine-158-SPS. A new, Ca(2+)-dependent SPS-protein kinase activity (peak IV kinase) was also resolved and shown to phosphorylate and activate phosphoserine-158-SPS in vitro. The peak IV kinase also phosphorylated a synthetic peptide (SP29) based on the amino acid sequence surrounding serine-424, which also contains the motif described for the serine-158 regulatory phosphorylation site; i.e. basic residues at P-3 and P-6 and a hydrophobic residue at P-5. Peak IV kinase had a native molecular weight of approximately 150,000 as shown by gel filtration. The SP29 peptide was not phosphorylated by the inactivating peak III kinase. Osmotically stressed leaves showed increased peak IV kinase activity with the SP29 peptide as a substrate. Tryptic 32P-phosphopeptide analysis of SPS from excised spinach leaves fed [32P]inorganic P showed increased phosphorylation of the tryptic peptide containing serine-424. Therefore, at least part of the osmotic stress activation of SPS in dark leaves results from phosphorylation of serine-424 catalyzed by a Ca(2+)-dependent, 150-kD protein kinase.

Molecular Imaging of Phosphorylation Events for Drug Development

PubMed Central

Chan, C. T.; Paulmurugan, R.; Reeves, R. E.; Solow-Cordero, D.; Gambhir, S. S.

2014-01-01

Purpose Protein phosphorylation mediated by protein kinases controls numerous cellular processes. A genetically encoded, generalizable split firefly luciferase (FL)-assisted complementation system was developed for noninvasive monitoring phosphorylation events and efficacies of kinase inhibitors in cell culture and in small living subjects by optical bioluminescence imaging. Procedures An Akt sensor (AST) was constructed to monitor Akt phosphorylation and the effect of different PI-3K and Akt inhibitors. Specificity of AST was determined using a non-phosphorylable mutant sensor containing an alanine substitution (ASA). Results The PI-3K inhibitor LY294002 and Akt kinase inhibitor perifosine led to temporal- and dose-dependent increases in complemented FL activities in 293T human kidney cancer cells stably expressing AST (293T/AST) but not in 293T/ASA cells. Inhibition of endogenous Akt phosphorylation and kinase activities by perifosine also correlated with increase in complemented FL activities in 293T/AST cells but not in 293T/ASA cells. Treatment of nude mice bearing 293T/AST xenografts with perifosine led to a 2-fold increase in complemented FL activities compared to that of 293T/ASA xenografts. Our system was used to screen a small chemical library for novel modulators of Akt kinase activity. Conclusion This generalizable approach for noninvasive monitoring of phosphorylation events will accelerate the discovery and validation of novel kinase inhibitors and modulators of phosphorylation events. PMID:19048345

Study of phosphorylation of translation elongation factor 2 (EF-2) from wheat germ.

PubMed

Smailov, S K; Lee, A V; Iskakov, B K

1993-04-26

Phosphorylation of elongation factor 2 (EF-2) by specific Ca2+/calmodulin-dependent kinase is considered as a possible mechanism of regulation of protein biosynthesis in animal cells at the level of polypeptide chain elongation. In this report we show that wheat germ EF-2 can be intensively phosphorylated by the rabbit reticulocyte EF-2 kinase. Phosphorylation results in inhibition of the activity of plant EF-2 in poly(U)-dependent cell-free translation system. Thus, the activity of EF-2 in plant cells can be potentially regulated by phosphorylation. However, we could not detect endogenous EF-2 kinase activity in wheat germ either in vitro or in vivo. Furthermore, EF-2 kinase activity is not displayed in different organs of wheat and other higher plants.

Regulation of Neurite Outgrowth in N1E-115 Cells through PDZ-Mediated Recruitment of Diacylglycerol Kinase ζ

PubMed Central

Yakubchyk, Yury; Abramovici, Hanan; Maillet, Jean-Christian; Daher, Elias; Obagi, Christopher; Parks, Robin J.; Topham, Matthew K.; Gee, Stephen H.

2005-01-01

Syntrophins are scaffold proteins that regulate the subcellular localization of diacylglycerol kinase ζ (DGK-ζ), an enzyme that phosphorylates the lipid second-messenger diacylglycerol to yield phosphatidic acid. DGK-ζ and syntrophins are abundantly expressed in neurons of the developing and adult brain, but their function is unclear. Here, we show that they are present in cell bodies, neurites, and growth cones of cultured cortical neurons and differentiated N1E-115 neuroblastoma cells. Overexpression of DGK-ζ in N1E-115 cells induced neurite formation in the presence of serum, which normally prevents neurite outgrowth. This effect was independent of DGK-ζ kinase activity but dependent on a functional C-terminal PDZ-binding motif, which specifically interacts with syntrophin PDZ domains. DGK-ζ mutants with a blocked C terminus acted as dominant-negative inhibitors of outgrowth from serum-deprived N1E-115 cells and cortical neurons. Several lines of evidence suggest DGK-ζ promotes neurite outgrowth through association with the GTPase Rac1. DGK-ζ colocalized with Rac1 in neuronal processes and DGK-ζ-induced outgrowth was inhibited by dominant-negative Rac1. Moreover, DGK-ζ directly interacts with Rac1 through a binding site located within its C1 domains. Together with syntrophin, these proteins form a tertiary complex in N1E-115 cells. A DGK-ζ mutant that mimics phosphorylation of the MARCKS domain was unable to bind an activated Rac1 mutant (Rac1V12) and phorbol myristate acetate-induced protein kinase C activation inhibited the interaction of DGK-ζ with Rac1V12, suggesting protein kinase C-mediated phosphorylation of the MARCKS domain negatively regulates DGK-ζ binding to active Rac1. Collectively, these findings suggest DGK-ζ, syntrophin, and Rac1 form a regulated signaling complex that controls polarized outgrowth in neuronal cells. PMID:16055737

Platelet-Derived Growth Factor Receptor Activation Promotes the Prodestructive Invadosome-Forming Phenotype of Synoviocytes from Patients with Rheumatoid Arthritis.

PubMed

Charbonneau, Martine; Lavoie, Roxane R; Lauzier, Annie; Harper, Kelly; McDonald, Patrick P; Dubois, Claire M

2016-04-15

Fibroblast-like synoviocytes (FLS) play a major role in invasive joint destruction in rheumatoid arthritis (RA). This prodestructive phenotype has been shown to involve autocrine TGF-β that triggers formation of matrix-degrading invadosomes through molecular mechanisms that are not fully elucidated. The platelet-derived growth factor (PDGF) receptor (PDGFR) family of receptor tyrosine kinases (RTK) has been shown to cooperate with TGF-β in various pathological conditions. We therefore sought to determine whether RTK activity played a role in invadosome biogenesis. We demonstrated that, among the common RTKs, PDGFR-αβ was specifically phosphorylated in FLS from RA patients. Phosphorylation of PDGFR-αβ was also elevated in RA synovial tissues. Interference with PDGFR activation or PDGF neutralization inhibited invadosome formation in RA synoviocytes, indicating the presence of an autocrine PDGFR activation loop that involved endogenous PDGF. Among the PDGF-A-D isoforms, only PDGF-B was found both significantly elevated in FLS lines from RA patients, and related to high-invadosome forming cells. Addition of TGF-β upregulated invadosome formation, PDGF-B mRNA expression, and phosphorylation of PDGFR. All of these functions were efficiently suppressed by TGF-β neutralization or interference with the Smad/TβR1or PI3K/Akt pathway. Among the class 1 PI3K family proteins known to be expressed in RA synoviocytes, PI3Kα was selectively involved in PDGF-B expression, whereas both PI3Kα and PI3Kδ participated in invadosome formation. Our findings demonstrate that PDGFR is a critical RTK required for the prodestructive phenotype of RA synovial cells. They also provide evidence for an association between autocrine TGF-β and PDGFR-mediated invadosome formation in RA synoviocytes that involves the production of PDGF-B induced by TGF-β. Copyright © 2016 by The American Association of Immunologists, Inc.

Phosphorylation acts positively and negatively to regulate MRTF-A subcellular localisation and activity

PubMed Central

Panayiotou, Richard; Miralles, Francesc; Pawlowski, Rafal; Diring, Jessica; Flynn, Helen R; Skehel, Mark; Treisman, Richard

2016-01-01

The myocardin-related transcription factors (MRTF-A and MRTF-B) regulate cytoskeletal genes through their partner transcription factor SRF. The MRTFs bind G-actin, and signal-regulated changes in cellular G-actin concentration control their nuclear accumulation. The MRTFs also undergo Rho- and ERK-dependent phosphorylation, but the function of MRTF phosphorylation, and the elements and signals involved in MRTF-A nuclear export are largely unexplored. We show that Rho-dependent MRTF-A phosphorylation reflects relief from an inhibitory function of nuclear actin. We map multiple sites of serum-induced phosphorylation, most of which are S/T-P motifs and show that S/T-P phosphorylation is required for transcriptional activation. ERK-mediated S98 phosphorylation inhibits assembly of G-actin complexes on the MRTF-A regulatory RPEL domain, promoting nuclear import. In contrast, S33 phosphorylation potentiates the activity of an autonomous Crm1-dependent N-terminal NES, which cooperates with five other NES elements to exclude MRTF-A from the nucleus. Phosphorylation thus plays positive and negative roles in the regulation of MRTF-A. DOI: http://dx.doi.org/10.7554/eLife.15460.001 PMID:27304076

Triggering sporulation in Bacillus subtilis with artificial two-component systems reveals the importance of proper Spo0A activation dynamics.

PubMed

Vishnoi, Monika; Narula, Jatin; Devi, Seram Nganbiton; Dao, Hoang-Anh; Igoshin, Oleg A; Fujita, Masaya

2013-10-01

Sporulation initiation in Bacillus subtilis is controlled by the phosphorylated form of the master regulator Spo0A which controls transcription of a multitude of sporulation genes. In this study, we investigated the importance of temporal dynamics of phosphorylated Spo0A (Spo0A∼P) accumulation by rewiring the network controlling its phosphorylation. We showed that simultaneous induction of KinC, a kinase that can directly phosphorylate Spo0A, and Spo0A itself from separately controlled inducible promoters can efficiently trigger sporulation even under nutrient rich conditions. However, the sporulation efficiency in this artificial two-component system was significantly impaired when KinC and/or Spo0A induction was too high. Using mathematical modelling, we showed that gradual accumulation of Spo0A∼P is essential for the proper temporal order of the Spo0A regulon expression, and that reduction in sporulation efficiency results from the reversal of that order. These insights led us to identify premature repression of DivIVA as one possible explanation for the adverse effects of accelerated accumulation of Spo0A∼P on sporulation. Moreover, we found that positive feedback resulting from autoregulation of the native spo0A promoter leads to robust control of Spo0A∼P accumulation kinetics. Thus we propose that a major function of the conserved architecture of the sporulation network is controlling Spo0A activation dynamics. © 2013 John Wiley & Sons Ltd.

Pr-specific phytochrome phosphorylation in vitro by a protein kinase present in anti-phytochrome maize immunoprecipitates

NASA Technical Reports Server (NTRS)

Biermann, B. J.; Pao, L. I.; Feldman, L. J.

1994-01-01

Protein kinase activity has repeatedly been found to co-purify with the plant photoreceptor phytochrome, suggesting that light signals received by phytochrome may be transduced or modulated through protein phosphorylation. In this study immunoprecipitation techniques were used to characterize protein kinase activity associated with phytochrome from maize (Zea mays L.). A protein kinase that specifically phosphorylated phytochrome was present in washed anti-phytochrome immunoprecipitates of etiolated coleoptile proteins. No other substrate tested was phosphorylated by this kinase. Adding salts or detergents to disrupt low-affinity protein interactions reduced background phosphorylation in immunoprecipitates without affecting phytochrome phosphorylation, indicating that the protein kinase catalytic activity is either intrinsic to the phytochrome molecule or associated with it by high-affinity interactions. Red irradiation (of coleoptiles or extracts) sufficient to approach photoconversion saturation reduced phosphorylation of immunoprecipitated phytochrome. Subsequent far-red irradiation reversed the red-light effect. Phytochrome phosphorylation was stimulated about 10-fold by a co-immunoprecipitated factor. The stimulatory factor was highest in immunoprecipitates when Mg2+ was present in immunoprecipitation reactions but remained in the supernatant in the absence of Mg2+. These observations provide strong support for the hypothesis that phytochrome-associated protein kinase modulates light responses in vivo. Since only phytochrome was found to be phosphorylated, the co-immunoprecipitated protein kinase may function to regulate receptor activity.

CULLIN-3 Controls TIMELESS Oscillations in the Drosophila Circadian Clock

PubMed Central

Lamouroux, Annie; Chélot, Elisabeth; Rouyer, François

2012-01-01

Eukaryotic circadian clocks rely on transcriptional feedback loops. In Drosophila, the PERIOD (PER) and TIMELESS (TIM) proteins accumulate during the night, inhibit the activity of the CLOCK (CLK)/CYCLE (CYC) transcriptional complex, and are degraded in the early morning. The control of PER and TIM oscillations largely depends on post-translational mechanisms. They involve both light-dependent and light-independent pathways that rely on the phosphorylation, ubiquitination, and proteasomal degradation of the clock proteins. SLMB, which is part of a CULLIN-1-based E3 ubiquitin ligase complex, is required for the circadian degradation of phosphorylated PER. We show here that CULLIN-3 (CUL-3) is required for the circadian control of PER and TIM oscillations. Expression of either Cul-3 RNAi or dominant negative forms of CUL-3 in the clock neurons alters locomotor behavior and dampens PER and TIM oscillations in light-dark cycles. In constant conditions, CUL-3 deregulation induces behavioral arrhythmicity and rapidly abolishes TIM cycling, with slower effects on PER. CUL-3 affects TIM accumulation more strongly in the absence of PER and forms protein complexes with hypo-phosphorylated TIM. In contrast, SLMB affects TIM more strongly in the presence of PER and preferentially associates with phosphorylated TIM. CUL-3 and SLMB show additive effects on TIM and PER, suggesting different roles for the two ubiquitination complexes on PER and TIM cycling. This work thus shows that CUL-3 is a new component of the Drosophila clock, which plays an important role in the control of TIM oscillations. PMID:22879814

Phospholipase C-gamma 1 binding to intracellular receptors for activated protein kinase C.

PubMed

Disatnik, M H; Hernandez-Sotomayor, S M; Jones, G; Carpenter, G; Mochly-Rosen, D

1994-01-18

Phospholipase C-gamma 1 (PLC-gamma 1; EC 3.1.4.11) hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate and is activated in response to growth factor stimulation and tyrosine phosphorylation. Concomitantly, the enzyme translocates from the cytosol to the particulate cell fraction. A similar process of activation-induced translocation from the cytosol to the cell particulate fraction has also been described for protein kinase C (PKC). We have previously shown that activated PKC binds to specific receptor proteins, receptors for activated C kinase, or RACKs, of approximately 30 kDa. Here, we show that PLC-gamma 1 bound to these RACKs and inhibited subsequent PKC binding to RACKs. However, unlike PKC, the binding of PLC-gamma 1 to RACKs did not require phospholipids and calcium. After epidermal growth factor treatment of intact A-431 cells, the binding of PLC-gamma 1 to RACKs increased as compared with PLC-gamma 1 from control cells. This increase in PLC-gamma 1 binding to RACKs was due to the phosphorylation of PLC-gamma 1. Additional data indicated that PLC-gamma 1 binds to RACKs in solution; epidermal growth factor receptor-dependent PLC-gamma 1 phosphorylation and activation decreased in the presence of RACKs. It is possible that, in vivo, PLC-gamma 1 associates with RACKs or with other PLC-gamma 1-specific anchoring proteins in the particulate cell fraction. Since a PKC C2 homologous region is present in PLC-gamma 1, the C2 region may mediate the activation-induced translocation of the enzyme to the cell particulate fraction and the anchoring protein-PLC-gamma 1 complex may be the active translocated form of PLC-gamma 1.

Na/K-ATPase regulates bovine sperm capacitation through raft- and non-raft-mediated signaling mechanisms.

PubMed

Rajamanickam, Gayathri D; Kastelic, John P; Thundathil, Jacob C

2017-11-01

Highly dynamic lipid microdomains (rafts) in the sperm plasma membrane contain several signaling proteins that regulate sperm capacitation. Na/K-ATPase isoforms (testis-specific isoform ATP1A4 and ubiquitous isoform ATP1A1) are abundant in bovine sperm plasma membrane. We previously reported that incubation of bovine sperm with ouabain, a specific Na/K-ATPase ligand, induced tyrosine phosphorylation of several sperm proteins during capacitation. The objective of this study was to investigate the roles of lipid rafts and non-rafts in Na/K-ATPase enzyme activity and signaling during bovine sperm capacitation. Content of ATP1A4 and, to a lesser extent, ATP1A1 was increased in raft and non-raft fractions of capacitated sperm, although non-raft enzyme activities of both isoforms were higher than the corresponding activities in rafts from capacitated sperm. Yet, ATP1A4 was the predominant isoform responsible for total Na/K-ATPase activity in both rafts and non-rafts. A comparative increase in phosphorylation of signaling molecules was observed in both raft (CAV1) and non-raft (EGFR and ERK1/2) membrane fractions during capacitation. Although SRC was phosphorylated in both membrane fractions, the non-raft fraction possessed more of this activated form. We also inferred, by immunoprecipitation, that ATP1A4 interacted with CAV1 and EGFR in the raft fraction, whereas interactions of ATP1A4 with SRC, EGFR, and ERK1/2 occurred in the non-raft fraction of ouabain-capacitated sperm; conversely, ATP1A1 interacted only with CAV1 in both fractions of uncapacitated and capacitated sperm. In conclusion, both raft and non-raft cohorts of Na/K-ATPase isoforms contributed to phosphorylation of signaling molecules during bovine sperm capacitation. © 2017 Wiley Periodicals, Inc.

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: I. Biochemical analysis.

PubMed

Okuno, S; Kitani, T; Fujisawa, H

2001-10-01

Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) I and IV are activated upon phosphorylation of their Thr(177) and Thr(196), respectively, by the upstream Ca(2+)/calmodulin-dependent protein kinases CaM-kinase kinase alpha and beta, and deactivated upon dephosphorylation by protein phosphatases such as CaM-kinase phosphatase. Recent studies demonstrated that the activity of CaM-kinase kinase alpha is decreased upon phosphorylation by cAMP-dependent protein kinase (PKA), and the relationship between the inhibition and phosphorylation of CaM-kinase kinase alpha by PKA has been studied. In the present study, we demonstrate that the activity of CaM-kinase kinase alpha toward PKIV peptide, which contains the sequence surrounding Thr(196) of CaM-kinase IV, is increased by incubation with PKA in the presence of Ca(2+)/calmodulin but decreased in its absence, while the activity toward CaM-kinase IV is decreased by incubation with PKA in both the presence and absence of Ca(2+)/calmodulin. Six phosphorylation sites on CaM-kinase kinase alpha, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA, were identified by amino acid sequence analysis of the phosphopeptides purified from the tryptic digest of the phosphorylated enzymes. The presence of Ca(2+)/calmodulin suppresses phosphorylation on Ser(52), Ser(74), Thr(108), and Ser(458) by PKA, but accelerates phosphorylation on Ser(475). The changes in the activity of the enzyme upon phosphorylation appear to occur as a result of conformational changes induced by phosphorylation on several sites.

A small peptide promotes EphA2 kinase-dependent signaling by stabilizing EphA2 dimers.

PubMed

Singh, Deo R; Pasquale, Elena B; Hristova, Kalina

2016-09-01

The EphA2 receptor tyrosine kinase is known to promote cancer cell malignancy in the absence of activation by ephrin ligands. This behavior depends on high EphA2 phosphorylation on Ser897 and low tyrosine phosphorylation, resulting in increased cell migration and invasiveness. We have previously shown that EphA2 forms dimers in the absence of ephrin ligand binding, and that dimerization of unliganded EphA2 can decrease EphA2 Ser897 phosphorylation. We have also identified a small peptide called YSA, which binds EphA2 and competes with the naturally occurring ephrin ligands. Here, we investigate the effect of YSA on EphA2 dimer stability and EphA2 function using quantitative FRET techniques, Western blotting, and cell motility assays. We find that the YSA peptide stabilizes the EphA2 dimer, increases EphA2 Tyr phosphorylation, and decreases both Ser897 phosphorylation and cell migration. The experiments demonstrate that the small peptide ligand YSA reduces EphA2 Ser897 pro-tumorigenic signaling by stabilizing the EphA2 dimer. This work is a proof-of-principle demonstration that EphA2 homointeractions in the plasma membrane can be pharmacologically modulated to decrease the pro-tumorigenic signaling of the receptor. Copyright © 2016 Elsevier B.V. All rights reserved.

Tyrosine phosphorylation switching of a G protein.

PubMed

Li, Bo; Tunc-Ozdemir, Meral; Urano, Daisuke; Jia, Haiyan; Werth, Emily G; Mowrey, David D; Hicks, Leslie M; Dokholyan, Nikolay V; Torres, Matthew P; Jones, Alan M

2018-03-30

Heterotrimeric G protein complexes are molecular switches relaying extracellular signals sensed by G protein-coupled receptors (GPCRs) to downstream targets in the cytoplasm, which effect cellular responses. In the plant heterotrimeric GTPase cycle, GTP hydrolysis, rather than nucleotide exchange, is the rate-limiting reaction and is accelerated by a receptor-like regulator of G signaling (RGS) protein. We hypothesized that posttranslational modification of the Gα subunit in the G protein complex regulates the RGS-dependent GTPase cycle. Our structural analyses identified an invariant phosphorylated tyrosine residue (Tyr 166 in the Arabidopsis Gα subunit AtGPA1) located in the intramolecular domain interface where nucleotide binding and hydrolysis occur. We also identified a receptor-like kinase that phosphorylates AtGPA1 in a Tyr 166 -dependent manner. Discrete molecular dynamics simulations predicted that phosphorylated Tyr 166 forms a salt bridge in this interface and potentially affects the RGS protein-accelerated GTPase cycle. Using a Tyr 166 phosphomimetic substitution, we found that the cognate RGS protein binds more tightly to the GDP-bound Gα substrate, consequently reducing its ability to accelerate GTPase activity. In conclusion, we propose that phosphorylation of Tyr 166 in AtGPA1 changes the binding pattern with AtRGS1 and thereby attenuates the steady-state rate of the GTPase cycle. We coin this newly identified mechanism "substrate phosphoswitching." © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

The Akt1-eNOS axis illustrates the specificity of kinase-substrate relationships in vivo.

PubMed

Schleicher, Michael; Yu, Jun; Murata, Takahisa; Derakhshan, Berhad; Atochin, Dimitriy; Qian, Li; Kashiwagi, Satoshi; Di Lorenzo, Annarita; Harrison, Kenneth D; Huang, Paul L; Sessa, William C

2009-08-04

Akt1 is critical for many in vivo functions; however, the cell-specific substrates responsible remain to be defined. Here, we examine the importance of endothelial nitric oxide synthase (eNOS) as an Akt1 substrate by generating Akt1-deficient mice (Akt1(-/-) mice) carrying knock-in mutations (serine to aspartate or serine to alanine substitutions) of the critical Akt1 phosphorylation site on eNOS (serine 1176) that render the enzyme "constitutively active" or "less active." The eNOS mutations did not influence several phenotypes in Akt1(-/-) mice; however, the defective postnatal angiogenesis characteristic of Akt1(-/-) mice was rescued by crossing the Akt1(-/-) mice with mice carrying the constitutively active form of eNOS, but not by crossing with mice carrying the less active eNOS mutant. This genetic rescue resulted in the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha) and increased production of HIF-1alpha-responsive genes in vivo and in vitro. Thus, Akt1 regulates angiogenesis largely through phosphorylation of eNOS and NO-dependent signaling.

SRC activates TAZ for intestinal tumorigenesis and regeneration.

PubMed

Byun, Mi Ran; Hwang, Jun-Ha; Kim, A Rum; Kim, Kyung Min; Park, Jung Il; Oh, Ho Taek; Hwang, Eun Sook; Hong, Jeong-Ho

2017-12-01

Proto-oncogene tyrosine-protein kinase Src (cSRC) is involved in colorectal cancer (CRC) development and damage-induced intestinal regeneration, although the cellular mechanisms involved are poorly understood. Here, we report that transcriptional coactivator with PDZ binding domain (TAZ) is activated by cSRC, regulating CRC cell proliferation and tumor formation, where cSRC overexpression increases TAZ expression in CRC cells. In contrast, knockdown of cSRC decreases TAZ expression. Additionally, direct phosphorylation of TAZ at Tyr316 by cSRC stimulates nuclear localization and facilitates transcriptional enhancer factor TEF-3 (TEAD4)-mediated transcription. However, a TAZ phosphorylation mutant significantly decreased cell proliferation, wound healing, colony forming, and tumor formation. In a CRC mouse model, Apc Min/+ , activated SRC expression was associated with increased TAZ expression in polyps and TAZ depletion decreased polyp formation. Moreover, intestinal TAZ knockout mice had intestinal regeneration defects following γ-irradiation. Finally, significant correspondence between SRC activation and TAZ overexpression was observed in CRC patients. These results suggest that TAZ is a critical factor for SRC-mediated intestinal tumor formation and regeneration. Copyright © 2017 Elsevier B.V. All rights reserved.

The active site of oxidative phosphorylation and the origin of hyperhomocysteinemia in aging and dementia.

PubMed

McCully, Kilmer S

2015-01-01

The active site of oxidative phosphorylation and adenosine triphosphate (ATP) synthesis in mitochondria is proposed to consist of two molecules of thioretinamide bound to cobalamin, forming thioretinaco, complexed with ozone, oxygen, nicotinamide adenine dinucleotide. and inorganic phosphate, TR2CoO3O2NAD(+)H2PO4(-). Reduction of the pyridinium nitrogen of the nicotinamide group by an electron from electron transport complexes initiates polymerization of phosphate with adenosine diphosphate, yielding nicotinamide riboside and ATP bound to thioretinaco ozonide oxygen. A second electron reduces oxygen to hydroperoxyl radical, releasing ATP from the active site. A proton gradient is created within F1F0 ATPase complexes of mitochondria by reaction of protons with reduced nicotinamide riboside and with hydroperoxyl radical, yielding reduced nicotinamide riboside and hydroperoxide. The hyperhomocysteinemia of aging and dementia is attributed to decreased synthesis of adenosyl methionine by thioretinaco ozonide and ATP, causing decreased allosteric activation of cystathionine synthase and decreased allosteric inhibition of methylenetetrahydrofolate reductase and resulting in dysregulation of methionine metabolism. © 2015 by the Association of Clinical Scientists, Inc.

8-C-(E-phenylethenyl)quercetin from onion/beef soup induces autophagic cell death in colon cancer cells through ERK activation.

PubMed

Zhao, Yueliang; Fan, Daming; Zheng, Zong-Ping; Li, Edmund T S; Chen, Feng; Cheng, Ka-Wing; Wang, Mingfu

2017-02-01

Quercetin, a flavonoid, widely distributed in edible fruits and vegetables, was reported to effectively inhibit 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP) formation in a food model (roast beef patties) with itself being converted into a novel compound 8-C-(E-phenylethenyl)quercetin (8-CEPQ). Here we investigated whether 8-CEPQ could be formed in a real food system, and tested its anticancer activity in human colon cancer cell lines. LC-MS was applied for the determination of 8-CEPQ formation in onion/beef soup. Anticancer activity of 8-CEPQ was evaluated by using cell viability assay and flow cytometry. Results showed that 8-CEPQ suppressed proliferation and caused G 2 phase arrest in colon cancer cells. Based on immunofluorescent staining assay, western blot assay, and RNA knockdown data, we found that 8-CEPQ did not cause apoptotic cell death. Instead, it induced autophagic cell death. Moreover, treatment with 8-CEPQ induced phosphorylation of extracellular signal-regulated kinase (ERK). Inhibition of ERK phosphorylation by the mitogen-activated protein kinase kinase (MEK)/ERK inhibitor U0126 attenuated 8-CEPQ-induced autophagy and reversed 8-CEPQ-mediated cell growth inhibition. Our results demonstrate that 8-CEPQ, a novel quercetin derivative, could be formed in onion/beef soup. 8-CEPQ inhibited colon cancer cell growth by inducing autophagic cell death through ERK activation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Roles of cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1.

PubMed

Shinohara, M; Kodama, A; Matozaki, T; Fukuhara, A; Tachibana, K; Nakanishi, H; Takai, Y

2001-06-01

Gab-1 is a multiple docking protein that is tyrosine phosphorylated by receptor tyrosine kinases such as c-Met, hepatocyte growth factor/scatter factor receptor, and epidermal growth factor receptor. We have now demonstrated that cell-cell adhesion also induces marked tyrosine phosphorylation of Gab-1 and that disruption of cell-cell adhesion results in its dephosphorylation. An anti-E-cadherin antibody decreased cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas the expression of E-cadherin specifically induced tyrosine phosphorylation of Gab-1. A relatively selective inhibitor of Src family kinases reduced cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas expression of a dominant-negative mutant of Csk increased it. Disruption of cell-cell adhesion, which reduced tyrosine phosphorylation of Gab-1, also reduced the activation of mitogen-activated protein kinase and Akt in response to cell-cell adhesion. These results indicate that E-cadherin-mediated cell-cell adhesion induces tyrosine phosphorylation by a Src family kinase of Gab-1, thereby regulating the activation of Ras/MAP kinase and phosphatidylinositol 3-kinase/Akt cascades.

Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation.

PubMed

Madero-Pérez, Jesús; Fdez, Elena; Fernández, Belén; Lara Ordóñez, Antonio J; Blanca Ramírez, Marian; Gómez-Suaga, Patricia; Waschbüsch, Dieter; Lobbestael, Evy; Baekelandt, Veerle; Nairn, Angus C; Ruiz-Martínez, Javier; Aiastui, Ana; López de Munain, Adolfo; Lis, Pawel; Comptdaer, Thomas; Taymans, Jean-Marc; Chartier-Harlin, Marie-Christine; Beilina, Alexandria; Gonnelli, Adriano; Cookson, Mark R; Greggio, Elisa; Hilfiker, Sabine

2018-01-23

Mutations in LRRK2 are a common genetic cause of Parkinson's disease (PD). LRRK2 interacts with and phosphorylates a subset of Rab proteins including Rab8a, a protein which has been implicated in various centrosome-related events. However, the cellular consequences of such phosphorylation remain elusive. Human neuroblastoma SH-SY5Y cells stably expressing wildtype or pathogenic LRRK2 were used to test for polarity defects in the context of centrosomal positioning. Centrosomal cohesion deficits were analyzed from transiently transfected HEK293T cells, as well as from two distinct peripheral cell types derived from LRRK2-PD patients. Kinase assays, coimmunoprecipitation and GTP binding/retention assays were used to address Rab8a phosphorylation by LRRK2 and its effects in vitro. Transient transfections and siRNA experiments were performed to probe for the implication of Rab8a and its phosphorylated form in the centrosomal deficits caused by pathogenic LRRK2. Here, we show that pathogenic LRRK2 causes deficits in centrosomal positioning with effects on neurite outgrowth, cell polarization and directed migration. Pathogenic LRRK2 also causes deficits in centrosome cohesion which can be detected in peripheral cells derived from LRRK2-PD patients as compared to healthy controls, and which are reversed upon LRRK2 kinase inhibition. The centrosomal cohesion and polarity deficits can be mimicked when co-expressing wildtype LRRK2 with wildtype but not phospho-deficient Rab8a. The centrosomal defects induced by pathogenic LRRK2 are associated with a kinase activity-dependent increase in the centrosomal localization of phosphorylated Rab8a, and are prominently reduced upon RNAi of Rab8a. Our findings reveal a new function of LRRK2 mediated by Rab8a phosphorylation and related to various centrosomal defects.

PIP3-independent activation of TorC2 and PKB at the cell’s leading edge mediates chemotaxis

PubMed Central

Kamimura, Yoichiro; Xiong, Yuan; Iglesias, Pablo A.; Hoeller, Oliver; Bolourani, Parvin; Devreotes, Peter N.

2008-01-01

Summary Background Studies show that high phosphotidylinositol 3,4,5 tris phosphate (PIP3) promotes cytoskeletal rearrangements and alters cell motility and chemotaxis, possibly through activation of PKBs. However, chemotaxis can still occur in the absence of PIP3 and the identities of the PIP3 independent pathways remain unknown. Results Here, we outline a PIP3-independent pathway linking temporal and spatial activation of PKBs by Tor complex 2 (TorC2) to the chemotactic response. Within seconds of stimulating Dictyostelium cells with chemoattractant, two PKB homologs, PKBA and PKBR1, mediate transient phosphorylation of at least eight proteins, including Talin, PI4P 5-kinase, two RasGefs, and a RhoGap. Surprisingly, all of the substrates are phosphorylated with normal kinetics in cells lacking PI 3-kinase activity. Cells deficient in TorC2 or PKB activity show reduced phosphorylation of the endogenous substrates and are impaired in chemotaxis. The PKBs are activated through phosphorylation of their hydrophobic motifs via TorC2 and subsequent phosphorylation of their activation loops. These chemoattractant-inducible events restricted to the cell’s leading edge even in the absence of PIP3. Activation of TorC2 depends on heterotrimeric G-protein function and intermediate G-proteins, including Ras GTPases. Conclusions The data lead to a model where cytosolic TorC2, encountering locally activated small G-protein(s) at the leading of the cell, becomes activated and phosphorylates PKBs. These in turn phosphorylate a series of signaling and cytoskeletal proteins, thereby regulating directed migration. PMID:18635356

Phosphorylation of threonine 290 in the activation loop of Tpl2/Cot is necessary but not sufficient for kinase activity.

PubMed

Luciano, Brenda S; Hsu, Sang; Channavajhala, Padma L; Lin, Lih-Ling; Cuozzo, John W

2004-12-10

Cot/Tpl2/MAP3K8 is a serine/threonine kinase known to activate the ERK, p38, and JNK kinase pathways. Studies of Tpl2 knock-out mice reveal a clear defect in tumor necrosis factor-alpha production, although very little detail is known about its regulation and the signaling events involved. In the present study we demonstrated that phosphorylation of Cot was required for its maximal activity as phosphatase treatment of Cot decreased its kinase activity. The Cot sequence contains a conserved threonine at position 290 in the activation loop of the kinase domain. We found that mutation of this residue to alanine eliminated its ability to activate MEK/ERK and NF-kappaB pathways, whereas a phosphomimetic mutation to aspartic acid could rescue the ability to activate MEK. Thr-290 was also required for robust autophosphorylation of Cot. Antibody generated to phospho-Thr-290-Cot recognized both wild-type and kinase-dead Cot, suggesting that phosphorylation of Thr-290 did not occur through autophosphorylation but via another kinase. We showed that Cot was constitutively phosphorylated at Thr-290 in transfected human embryonic kidney 293T cells as well as human monocytes as this residue was phosphorylated in unstimulated and lipopolysaccharide-stimulated cells to the same degree. Treatment with herbimycin A inhibited Cot activity in the MEK/ERK pathway but did not inhibit phosphorylation at Thr-290. Together these results showed that phosphorylation of Cot at Thr-290 is necessary but not sufficient for full kinase activity in the MEK/ERK pathway.

Phosphorylation of the Activation Loop Tyrosine 823 in c-Kit Is Crucial for Cell Survival and Proliferation*

PubMed Central

Agarwal, Shruti; Kazi, Julhash U.; Rönnstrand, Lars

2013-01-01

The receptor tyrosine kinase c-Kit, also known as the stem cell factor receptor, plays a key role in several developmental processes. Activating mutations in c-Kit lead to alteration of these cellular processes and have been implicated in many human cancers such as gastrointestinal stromal tumors, acute myeloid leukemia, testicular seminomas and mastocytosis. Regulation of the catalytic activity of several kinases is known to be governed by phosphorylation of tyrosine residues in the activation loop of the kinase domain. However, in the case of c-Kit phosphorylation of Tyr-823 has been demonstrated to be a late event that is not required for kinase activation. However, because phosphorylation of Tyr-823 is a ligand-activated event, we sought to investigate the functional consequences of Tyr-823 phosphorylation. By using a tyrosine-to-phenylalanine mutant of tyrosine 823, we investigated the impact of Tyr-823 on c-Kit signaling. We demonstrate here that Tyr-823 is crucial for cell survival and proliferation and that mutation of Tyr-823 to phenylalanine leads to decreased sustained phosphorylation and ubiquitination of c-Kit as compared with the wild-type receptor. Furthermore, the mutated receptor was, upon ligand-stimulation, quickly internalized and degraded. Phosphorylation of the E3 ubiquitin ligase Cbl was transient, followed by a substantial reduction in phosphorylation of downstream signaling molecules such as Akt, Erk, p38, Shc, and Gab2. Thus, we propose that activation loop tyrosine 823 is crucial for activation of both the MAPK and PI3K pathways and that its disruption leads to a destabilization of the c-Kit receptor and decreased survival of cells. PMID:23803604

delta opioid receptors stimulate Akt-dependent phosphorylation of c-jun in T cells.

PubMed

Shahabi, Nahid A; McAllen, Kathy; Sharp, Burt M

2006-02-01

Activation of naive T cells markedly up-regulates the expression of delta opioid receptors (DORs). These receptors are bound by DOR peptides released by T cells, modulating T cell functions such as interleukin-2 production, cellular proliferation, and chemotaxis. Previous studies have shown that DOR agonists [e.g., [D-Ala(2)-D-Leu(5)]-enkephalin (DADLE)] modulate T cell antigen receptor signaling through mitogen-activated protein kinases (MAPKs; i.e., extracellular signal-regulated kinases 1 and 2) and that DORs directly induce phosphorylation of activating transcription factor-2 (implicated in cytokine gene transcription) and its association with the MAPK c-jun1 NH(2)-terminal kinase (JNK). Such observations suggest that DORs may induce the phosphorylation of c-jun. These experiments were performed to test this hypothesis and determine the potential roles of phosphoinositide 3-kinase (PI3K) and Akt (protein kinase B). DADLE (10(-10) to 10(-6) M) dose-dependently induced c-jun phosphorylation. This was blocked by pertussis toxin and the DOR-specific antagonist naltindole. Fluorescence flow cytometry showed that DADLE significantly stimulated c-jun phosphorylation by T cells. DADLE stimulated phosphorylation of membrane-associated Akt; wortmannin and LY294002 ([2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]), specific inhibitors of PI3K, abolished the DADLE-induced phosphorylation of c-jun. Finally, inhibitors of Akt and JNK blocked DADLE-induced phosphorylation of c-jun. Thus, activated DORs directly stimulate c-jun phosphorylation through a PI3K-dependent pathway in T cells, apparently involving Akt. This implies that DORs activate JNK through a novel pathway dependent on PI3K and Akt, thereby regulating the function of activator protein-1 transcription complexes containing c-jun and other transcription partners.

Akt-mediated regulation of NFkappaB and the essentialness of NFkappaB for the oncogenicity of PI3K and Akt.

PubMed

Bai, Dong; Ueno, Lynn; Vogt, Peter K

2009-12-15

The serine/threonine kinase Akt (cellular homolog of murine thymoma virus akt8 oncogene), also known as PKB (protein kinase B), is activated by lipid products of phosphatidylinositol 3-kinase (PI3K). Akt phosphorylates numerous protein targets that control cell survival, proliferation and motility. Previous studies suggest that Akt regulates transcriptional activity of the nuclear factor-kappaB (NFkappaB) by inducing phosphorylation and subsequent degradation of inhibitor of kappaB (IkappaB). We show here that NFkappaB-driven transcription increases in chicken embryonic fibroblasts (CEF) transformed by myristylated Akt (myrAkt). Accordingly, both a dominant negative mutant of Akt and Akt inhibitors repress NFkappaB-dependent transcription. The degradation of the IkappaB protein is strongly enhanced in Akt-transformed cells, and the loss of NFkappaB activity by introduction of a super-repressor of NFkappaB, IkappaBSR, interferes with PI3K- and Akt-induced oncogenic transformation of CEF. The phosphorylation of the p65 subunit of NFkappaB at serine 534 is also upregulated in Akt-transformed cells. Our data suggest that the stimulation of NFkappaB by Akt is dependent on the phosphorylation of p65 at S534, mediated by IKK (IkappaB kinase) alpha and beta. Akt phosphorylates IKKalpha on T23, and this phosphorylation event is a prerequisite for the phosphorylation of p65 at S534 by IKKalpha and beta. Our results demonstrate two separate functions of the IKK complex in NFkappaB activation in cells with constitutive Akt activity: the phosphorylation and consequent degradation of IkappaB and the phosphorylation of p65. The data further support the conclusion that NFkappaB activity is essential for PI3K- and Akt-induced oncogenic transformation. Copyright (c) 2009 UICC.

Biological role of site-specific O-glycosylation in cell adhesion activity and phosphorylation of osteopontin.

PubMed

Oyama, Midori; Kariya, Yoshinobu; Kariya, Yukiko; Matsumoto, Kana; Kanno, Mayumi; Yamaguchi, Yoshiki; Hashimoto, Yasuhiro

2018-05-09

Osteopontin (OPN) is an extracellular glycosylated phosphoprotein that promotes cell adhesion by interacting with several integrin receptors. We previously reported that an OPN mutant lacking five O-glycosylation sites (Thr 134 /Thr 138 /Thr 143 /Thr 147 /Thr 152 ) in the threonine/proline-rich region increased cell adhesion activity and phosphorylation compared with the wild type. However, the role of O-glycosylation in cell adhesion activity and phosphorylation of OPN remains to be clarified. Here, we show that site-specific O-glycosylation in the threonine/proline-rich region of OPN affects its cell adhesion activity and phosphorylation independently and/or synergistically. Using site-directed mutagenesis, we found that OPN mutants with substitution sets of Thr 134 /Thr 138 or Thr 143 /Thr 147 /Thr 152 had decreased and increased cell adhesion activity, respectively. In contrast, the introduction of a single mutation into the O-glycosylation sites had no effect on OPN cell adhesion activity. An adhesion assay using function-blocking antibodies against αvβ3 and β1 integrins, as well as αvβ3 integrin-overexpressing A549 cells, revealed that site-specific O-glycosylation affected the association of OPN with the two integrins. Phosphorylation analyses using phos-tag and LC-MS/MS indicated that phosphorylation levels and sites were influenced by the O-glycosylation status, although the number of O-glycosylation sites was not correlated with the phosphorylation level in OPN. Furthermore, a correlation analysis between phosphorylation level and cell adhesion activity in OPN mutants with the site-specific O-glycosylation showed that they were not always correlated. These results provide conclusive evidence of a novel regulatory mechanism of cell adhesion activity and phosphorylation of OPN by site-specific O-glycosylation. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

The Impact of Phosphorylation on Electron Capture Dissociation of Proteins: A Top-Down Perspective

NASA Astrophysics Data System (ADS)

Chen, Bifan; Guo, Xiao; Tucholski, Trisha; Lin, Ziqing; McIlwain, Sean; Ge, Ying

2017-09-01

Electron capture dissociation (ECD) is well suited for the characterization of phosphoproteins, with which labile phosphate groups are generally preserved during the fragmentation process. However, the impact of phosphorylation on ECD fragmentation of intact proteins remains unclear. Here, we have performed a systematic investigation of the phosphorylation effect on ECD of intact proteins by comparing the ECD cleavages of mono-phosphorylated α-casein, multi-phosphorylated β-casein, and immunoaffinity-purified phosphorylated cardiac troponin I with those of their unphosphorylated counterparts, respectively. In contrast to phosphopeptides, phosphorylation has significantly reduced deleterious effects on the fragmentation of intact proteins during ECD. On a global scale, the fragmentation patterns are highly comparable between unphosphorylated and phosphorylated precursors under the same ECD conditions, despite a slight decrease in the number of fragment ions observed for the phosphorylated forms. On a local scale, single phosphorylation of intact proteins imposes minimal effects on fragmentation near the phosphorylation sites, but multiple phosphorylations in close proximity result in a significant reduction of ECD bond cleavages. [Figure not available: see fulltext.

Extracellular Protein Kinase A Modulates Intracellular Calcium/Calmodulin-Dependent Protein Kinase II, Nitric Oxide Synthase, and the Glutamate-Nitric Oxide-cGMP Pathway in Cerebellum. Differential Effects in Hyperammonemia.

PubMed

Cabrera-Pastor, Andrea; Llansola, Marta; Felipo, Vicente

2016-12-21

Extracellular protein kinases, including cAMP-dependent protein kinase (PKA), modulate neuronal functions including N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation. NMDA receptor activation increases calcium, which binds to calmodulin and activates nitric oxide synthase (NOS), increasing nitric oxide (NO), which activates guanylate cyclase, increasing cGMP, which is released to the extracellular fluid, allowing analysis of this glutamate-NO-cGMP pathway in vivo by microdialysis. The function of this pathway is impaired in hyperammonemic rats. The aims of this work were to assess (1) whether the glutamate-NO-cGMP pathway is modulated in cerebellum in vivo by an extracellular PKA, (2) the role of phosphorylation and activity of calcium/calmodulin-dependent protein kinase II (CaMKII) and NOS in the pathway modulation by extracellular PKA, and (3) whether the effects are different in hyperammonemic and control rats. The pathway was analyzed by in vivo microdialysis. The role of extracellular PKA was analyzed by inhibiting it with a membrane-impermeable inhibitor. The mechanisms involved were analyzed in freshly isolated cerebellar slices from control and hyperammonemic rats. In control rats, inhibiting extracellular PKA reduces the glutamate-NO-cGMP pathway function in vivo. This is due to reduction of CaMKII phosphorylation and activity, which reduces NOS phosphorylation at Ser1417 and NOS activity, resulting in reduced guanylate cyclase activation and cGMP formation. In hyperammonemic rats, under basal conditions, CaMKII phosphorylation and activity are increased, increasing NOS phosphorylation at Ser847, which reduces NOS activity, guanylate cyclase activation, and cGMP. Inhibiting extracellular PKA in hyperammonemic rats normalizes CaMKII phosphorylation and activity, NOS phosphorylation, NOS activity, and cGMP, restoring normal function of the pathway.

Regulation of flagellar assembly by glycogen synthase kinase 3 in Chlamydomonas reinhardtii.

PubMed

Wilson, Nedra F; Lefebvre, Paul A

2004-10-01

Chlamydomonas reinhardtii controls flagellar assembly such that flagella are of an equal and predetermined length. Previous studies demonstrated that lithium, an inhibitor of glycogen synthase kinase 3 (GSK3), induced flagellar elongation, suggesting that a lithium-sensitive signal transduction pathway regulated flagellar length (S. Nakamura, H. Takino, and M. K. Kojima, Cell Struct. Funct. 12:369-374, 1987). Here, we demonstrate that lithium treatment depletes the pool of flagellar proteins from the cell body and that the heterotrimeric kinesin Fla10p accumulates in flagella. We identify GSK3 in Chlamydomonas and demonstrate that its kinase activity is inhibited by lithium in vitro. The tyrosine-phosphorylated, active form of GSK3 was enriched in flagella and GSK3 associated with the axoneme in a phosphorylation-dependent manner. The level of active GSK3 correlated with flagellar length; early during flagellar regeneration, active GSK3 increased over basal levels. This increase in active GSK3 was rapidly lost within 30 min of regeneration as the level of active GSK3 decreased relative to the predeflagellation level. Taken together, these results suggest a possible role for GSK3 in regulating the assembly and length of flagella.

Carbachol-induced rabbit bladder smooth muscle contraction: roles of protein kinase C and Rho kinase.

PubMed

Wang, Tanchun; Kendig, Derek M; Smolock, Elaine M; Moreland, Robert S

2009-12-01

Smooth muscle contraction is regulated by phosphorylation of the myosin light chain (MLC) catalyzed by MLC kinase and dephosphorylation catalyzed by MLC phosphatase. Agonist stimulation of smooth muscle results in the inhibition of MLC phosphatase activity and a net increase in MLC phosphorylation and therefore force. The two pathways believed to be primarily important for inhibition of MLC phosphatase activity are protein kinase C (PKC)-catalyzed CPI-17 phosphorylation and Rho kinase (ROCK)-catalyzed myosin phosphatase-targeting subunit (MYPT1) phosphorylation. The goal of this study was to determine the roles of PKC and ROCK and their downstream effectors in regulating MLC phosphorylation levels and force during the phasic and sustained phases of carbachol-stimulated contraction in intact bladder smooth muscle. These studies were performed in the presence and absence of the PKC inhibitor bisindolylmaleimide-1 (Bis) or the ROCK inhibitor H-1152. Phosphorylation levels of Thr(38)-CPI-17 and Thr(696)/Thr(850)-MYPT1 were measured at different times during carbachol stimulation using site-specific antibodies. Thr(38)-CPI-17 phosphorylation increased concurrently with carbachol-stimulated force generation. This increase was reduced by inhibition of PKC during the entire contraction but was only reduced by ROCK inhibition during the sustained phase of contraction. MYPT1 showed high basal phosphorylation levels at both sites; however, only Thr(850) phosphorylation increased with carbachol stimulation; the increase was abolished by the inhibition of either ROCK or PKC. Our results suggest that during agonist stimulation, PKC regulates MLC phosphatase activity through phosphorylation of CPI-17. In contrast, ROCK phosphorylates both Thr(850)-MYPT1 and CPI-17, possibly through cross talk with a PKC pathway, but is only significant during the sustained phase of contraction. Last, our results demonstrate that there is a constitutively activate pool of ROCK that phosphorylates MYPT1 in the basal state, which may account for the high resting levels of MLC phosphorylation measured in rabbit bladder smooth muscle.

Combining Microinjection and Immunoblotting to Analyze MAP Kinase Phosphorylation in Single Starfish Oocytes and Eggs

NASA Astrophysics Data System (ADS)

Carroll, David J.; Hua, Wei

The starfish oocyte has proven useful for studies involving microinjection because it is relatively large (190 μm) and optically clear. These oocytes are easily obtained from the ovary arrested at prophase of meiosis I, making them useful as a model system for the study of cell cycle-related events. In this chapter, a method for combining microinjection with immunoblotting of single cells is described. Individual starfish oocytes are injected, removed from the microinjection chamber, and analyzed by immunoblotting for the dual-phosphorylated form of mitogen-activated protein kinase (MAPK). This method will allow for experiments testing the regulation of MAPK in single cells and for the manipulation of these cells by a quantitative microinjection technique.

CYCLIC AMP-DEPENDENT PROTEIN KINASE INDUCTION BY POLYCHLORINATED BIPHENYLS (PCBS) STIMULATES CREB PHOSPHORYLATION VIA A CALCIUM-DEPENDENT, PKC-INDEPENDENT PATHWAY IN CORTICAL NEURONS.

EPA Science Inventory

We have previously demonstrated that the PCB mixture, Aroclor 1254 (A1254), increases the phosphorylated form of CREB (pCREB), the cAMP-responsive element binding protein. This transcription factor is important in nervous system development and plasticity. Phosphorylation
of C...

Phosphorylation of paxillin via the ERK mitogen-activated protein kinase cascade in EL4 thymoma cells.

PubMed

Ku, H; Meier, K E

2000-04-14

Intracellular signals can regulate cell adhesion via several mechanisms in a process referred to as "inside-out" signaling. In phorbol ester-sensitive EL4 thymoma cells, phorbol-12-myristate 13-acetate (PMA) induces activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases and promotes cell adhesion. In this study, clonal EL4 cell lines with varying abilities to activate ERKs in response to PMA were used to examine signaling events occurring downstream of ERK activation. Paxillin, a multifunctional docking protein involved in cell adhesion, was phosphorylated on serine/threonine residues in response to PMA treatment. This response was correlated with the extent and time course of ERK activation. PMA-induced phosphorylation of paxillin was inhibited by compounds that block the ERK activation pathway in EL4 cells, primary murine thymocytes, and primary murine splenocytes. Paxillin was phosphorylated in vitro by purified active ERK2. Two-dimensional electrophoresis revealed that PMA treatment generated a complex pattern of phosphorylated paxillin species in intact cells, some of which were generated by ERK-mediated phosphorylation in vitro. An ERK pathway inhibitor interfered with PMA-induced adhesion of sensitive EL4 cells to substrate. These findings describe a novel inside-out signaling pathway by which the ERK cascade may regulate events involved in adhesion.

Abscisic acid-activated SNRK2 protein kinases function in the gene-regulation pathway of ABA signal transduction by phosphorylating ABA response element-binding factors.

PubMed

Kobayashi, Yuhko; Murata, Michiharu; Minami, Hideyuki; Yamamoto, Shuhei; Kagaya, Yasuaki; Hobo, Tokunori; Yamamoto, Akiko; Hattori, Tsukaho

2005-12-01

The plant hormone abscisic acid (ABA) induces gene expression via the ABA-response element (ABRE) present in the promoters of ABA-regulated genes. A group of bZIP proteins have been identified as ABRE-binding factors (ABFs) that activate transcription through this cis element. A rice ABF, TRAB1, has been shown to be activated via ABA-dependent phosphorylation. While a large number of signalling factors have been identified that are involved in stomatal regulation by ABA, relatively less is known about the ABA-signalling pathway that leads to gene expression. We have shown recently that three members of the rice SnRK2 protein kinase family, SAPK8, SAPK9 and SAPK10, are activated by ABA signal as well as by hyperosmotic stress. Here we show that transient overexpression in cultured cell protoplasts of these ABA-activated SnRK2 protein kinases leads to the activation of an ABRE-regulated promoter, suggesting that these kinases are involved in the gene-regulation pathway of ABA signalling. We further show several lines of evidence that these ABA-activated SnRK2 protein kinases directly phosphorylate TRAB1 in response to ABA. Kinetic analysis of SAPK10 activation and TRAB1 phosphorylation indicated that the latter immediately followed the former. TRAB1 was found to be phosphorylated not only in response to ABA, but also in response to hyperosmotic stress, which was interpreted as the consequence of phosphorylation of TRAB1 by hyperosmotically activated SAPKs. Physical interaction between TRAB1 and SAPK10 in vivo was demonstrated by a co-immunoprecipitation experiment. Finally, TRAB1 was phosphorylated in vitro by the ABA-activated SnRK2 protein kinases at Ser102, which is phosphorylated in vivo in response to ABA and is critical for the activation function.

soc-2 encodes a leucine-rich repeat protein implicated in fibroblast growth factor receptor signaling

PubMed Central

Selfors, Laura M.; Schutzman, Jennifer L.; Borland, Christina Z.; Stern, Michael J.

1998-01-01

Activation of fibroblast growth factor (FGF) receptors elicits diverse cellular responses including growth, mitogenesis, migration, and differentiation. The intracellular signaling pathways that mediate these important processes are not well understood. In Caenorhabditis elegans, suppressors of clr-1 identify genes, termed soc genes, that potentially mediate or activate signaling through the EGL-15 FGF receptor. We demonstrate that three soc genes, soc-1, soc-2, and sem-5, suppress the activity of an activated form of the EGL-15 FGF receptor, consistent with the soc genes functioning downstream of EGL-15. We show that soc-2 encodes a protein composed almost entirely of leucine-rich repeats, a domain implicated in protein–protein interactions. We identified a putative human homolog, SHOC-2, which is 54% identical to SOC-2. We find that shoc-2 maps to 10q25, shoc-2 mRNA is expressed in all tissues assayed, and SHOC-2 protein is cytoplasmically localized. Within the leucine-rich repeats of both SOC-2 and SHOC-2 are two YXNX motifs that are potential tyrosine-phosphorylated docking sites for the SEM-5/GRB2 Src homology 2 domain. However, phosphorylation of these residues is not required for SOC-2 function in vivo, and SHOC-2 is not observed to be tyrosine phosphorylated in response to FGF stimulation. We conclude that this genetic system has allowed for the identification of a conserved gene implicated in mediating FGF receptor signaling in C. elegans. PMID:9618511

E-box-independent regulation of transcription and differentiation by MYC.

PubMed

Uribesalgo, Iris; Buschbeck, Marcus; Gutiérrez, Arantxa; Teichmann, Sophia; Demajo, Santiago; Kuebler, Bernd; Nomdedéu, Josep F; Martín-Caballero, Juan; Roma, Guglielmo; Benitah, Salvador Aznar; Di Croce, Luciano

2011-10-23

MYC proto-oncogene is a key player in cell homeostasis that is commonly deregulated in human carcinogenesis(1). MYC can either activate or repress target genes by forming a complex with MAX (ref. 2). MYC also exerts MAX-independent functions that are not yet fully characterized(3). Cells possess an intrinsic pathway that can abrogate MYC-MAX dimerization and E-box interaction, by inducing phosphorylation of MYC in a PAK2-dependent manner at three residues located in its helix-loop-helix domain(4). Here we show that these carboxy-terminal phosphorylation events switch MYC from an oncogenic to a tumour-suppressive function. In undifferentiated cells, MYC-MAX is targeted to the promoters of retinoic-acid-responsive genes by its direct interaction with the retinoic acid receptor-α (RARα). MYC-MAX cooperates with RARα to repress genes required for differentiation, in an E-box-independent manner. Conversely, on C-terminal phosphorylation of MYC during differentiation, the complex switches from a repressive to an activating function, by releasing MAX and recruiting transcriptional co-activators. Phospho-MYC synergizes with retinoic acid to eliminate circulating leukaemic cells and to decrease the level of tumour invasion. Our results identify an E-box-independent mechanism for transcriptional regulation by MYC that unveils previously unknown functions for MYC in differentiation. These may be exploited to develop alternative targeted therapies.

Phosphorylation of glutaminase by PKCε is essential for its enzymatic activity and critically contributes to tumorigenesis.

PubMed

Han, Tianyu; Zhan, Weihua; Gan, Mingxi; Liu, Fanrong; Yu, Bentong; Chin, Y Eugene; Wang, Jian-Bin

2018-06-01

Glutamine metabolism plays an important role in cancer development and progression. Glutaminase C (GAC), the first enzyme in glutaminolysis, has emerged as an important target for cancer therapy and many studies have focused on the mechanism of enhanced GAC expression in cancer cells. However, little is known about the post-translational modification of GAC. Here, we report that phosphorylation is a crucial post-translational modification of GAC, which is responsible for the higher glutaminase activity in lung tumor tissues and cancer cells. We identify the key Ser314 phosphorylation site on GAC that is regulated by the NF-κB-PKCε axis. Blocking Ser314 phosphorylation by the S314A mutation in lung cancer cells inhibits the glutaminase activity, triggers genetic reprogramming, and alleviates tumor malignancy. Furthermore, we find that a high level of GAC phosphorylation correlates with poor survival rate of lung cancer patients. These findings highlight a previously unappreciated mechanism for activation of GAC by phosphorylation and demonstrate that targeting glutaminase activity can inhibit oncogenic transformation.

A phosphorylation-and-ubiquitylation circuitry driving ATR activation and homologous recombination

PubMed Central

Dubois, Jean-Christophe; Yates, Maïlyn; Gaudreau-Lapierre, Antoine; Clément, Geneviève; Cappadocia, Laurent; Gaudreau, Luc

2017-01-01

Abstract RPA-coated single-stranded DNA (RPA–ssDNA), a nucleoprotein structure induced by DNA damage, promotes ATR activation and homologous recombination (HR). RPA is hyper-phosphorylated and ubiquitylated after DNA damage. The ubiquitylation of RPA by PRP19 and RFWD3 facilitates ATR activation and HR, but how it is stimulated by DNA damage is still unclear. Here, we show that RFWD3 binds RPA constitutively, whereas PRP19 recognizes RPA after DNA damage. The recruitment of PRP19 by RPA depends on PIKK-mediated RPA phosphorylation and a positively charged pocket in PRP19. An RPA32 mutant lacking phosphorylation sites fails to recruit PRP19 and support RPA ubiquitylation. PRP19 mutants unable to bind RPA or lacking ubiquitin ligase activity also fail to support RPA ubiquitylation and HR. These results suggest that RPA phosphorylation enhances the recruitment of PRP19 to RPA–ssDNA and stimulates RPA ubiquitylation through a process requiring both PRP19 and RFWD3, thereby triggering a phosphorylation-ubiquitylation circuitry that promotes ATR activation and HR. PMID:28666352

Calcium/calmodulin-dependent kinase II phosphorylation of the GABAA receptor alpha1 subunit modulates benzodiazepine binding.

PubMed

Churn, Severn B; Rana, Aniruddha; Lee, Kangmin; Parsons, J Travis; De Blas, Angel; Delorenzo, Robert J

2002-09-01

gamma-Aminobutyric acid (GABA) is the primary neurotransmitter that is responsible for the fast inhibitory synaptic transmission in the central nervous system. A major post-translational mechanism that can rapidly regulate GABAAR function is receptor phosphorylation. This study was designed to test the effect of endogenous calcium and calmodulin-dependent kinase II (CaM kinase II) activation on both allosteric modulator binding and GABAA receptor subunit phosphorylation. Endogenous CaM kinase II activity was stimulated, and GABAA receptors were subsequently analyzed for bothallosteric modulator binding properties and immunoprecipitated and analyzed for subunit phosphorylation levels. A significant increase in allosteric-modulator binding of the GABAAR was observed under conditions maximal for CaM kinase II activation. In addition, CaM kinase II activation resulted in a direct increase in phosphorylation of the GABAA receptor alpha1 subunit. The data suggest that the CaM kinase II-dependent phosphorylation of the GABAA receptor alpha1 subunit modulated allosteric modulator binding to the GABAA receptor.

Neutral Sphingomyelinase 2 Activity and Protein Stability Are Modulated by Phosphorylation of Five Conserved Serines*

PubMed Central

Filosto, Simone; Ashfaq, Majid; Chung, Samuel; Fry, William; Goldkorn, Tzipora

2012-01-01

We previously presented that the neutral sphingomyelinase 2 (nSMase2) is the only SMase activated in human airway epithelial (HAE) cells following exposure to oxidative stress (ox-stress), yielding ceramide accumulation and thereby inducing apoptosis. Furthermore, we reported that nSMase2 is a phospho-protein in which the level of phosphorylation controls nSMase2 activation induced by ox-stress. Here we identify five specific serines that are phosphorylated in nSMase2 and demonstrate that their phosphorylation controls the nSMase2 activity upon ox-stress exposure in an interdependent manner. Furthermore, we show that the nSMase2 protein stability and thus its level of expression is also post-translationally regulated by these five serine phosphorylation sites. This study provides initial structure/function insights regarding nSMase2 phosphorylation sites and offers some new links for future studies aiming to fully elucidate nSMase2 regulatory machinery. PMID:22074919

Dynamic phosphorylation of Ebola virus VP30 in NP-induced inclusion bodies.

PubMed

Lier, Clemens; Becker, Stephan; Biedenkopf, Nadine

2017-12-01

Zaire Ebolavirus (EBOV) causes a severe feverish disease with high case fatality rates. Transcription of EBOV is dependent on the activity of the nucleocapsid protein VP30 which represents an essential viral transcription factor. Activity of VP30 is regulated via phosphorylation at six N-terminal serine residues. Recent data demonstrated that dynamic phosphorylation and dephosphorylation of serine residue 29 is essential for transcriptional support activity of VP30. To analyze the spatio/temporal dynamics of VP30 phosphorylation, we generated a peptide antibody recognizing specifically VP30 phosphorylated at serine 29. Using this antibody we could demonstrate that (i) the majority of VP30 molecules in EBOV-infected cells is dephosphorylated at the crucial position serine 29, (ii) both, VP30 phosphorylation and dephosphorylation take place in viral inclusion bodies that are induced by the nucleoprotein NP and (iii) NP influences the phosphorylation state of VP30. Copyright © 2017 Elsevier Inc. All rights reserved.

Rho-GTPase effector ROCK phosphorylates cofilin in actin-meditated cytokinesis during mouse oocyte meiosis.

PubMed

Duan, Xing; Liu, Jun; Dai, Xiao-Xin; Liu, Hong-Lin; Cui, Xiang-Shun; Kim, Nam-Hyung; Wang, Zhen-Bo; Wang, Qiang; Sun, Shao-Chen

2014-02-01

During oocyte meiosis, a spindle forms in the central cytoplasm and migrates to the cortex. Subsequently, the oocyte extrudes a small body and forms a highly polarized egg; this process is regulated primarily by actin. ROCK is a Rho-GTPase effector that is involved in various cellular functions, such as stress fiber formation, cell migration, tumor cell invasion, and cell motility. In this study, we investigated possible roles for ROCK in mouse oocyte meiosis. ROCK was localized around spindles after germinal vesicle breakdown and was colocalized with cytoplasmic actin and mitochondria. Disrupting ROCK activity by RNAi or an inhibitor resulted in cell cycle progression and polar body extrusion failure. Time-lapse microscopy showed that this may have been due to spindle migration and cytokinesis defects, as chromosomes segregated but failed to extrude a polar body and then realigned. Actin expression at oocyte membranes and in cytoplasm was significantly decreased after these treatments. Actin caps were also disrupted, which was confirmed by a failure to form cortical granule-free domains. The mitochondrial distribution was also disrupted, which indicated that mitochondria were involved in the ROCK-mediated actin assembly. In addition, the phosphorylation levels of Cofilin, a downstream molecule of ROCK, decreased after disrupting ROCK activity. Thus, our results indicated that a ROCK-Cofilin-actin pathway regulated meiotic spindle migration and cytokinesis during mouse oocyte maturation.

Fanconi anemia proteins FANCD2 and FANCI exhibit different DNA damage responses during S-phase

PubMed Central

Sareen, Archana; Chaudhury, Indrajit; Adams, Nicole; Sobeck, Alexandra

2012-01-01

Fanconi anemia (FA) pathway members, FANCD2 and FANCI, contribute to the repair of replication-stalling DNA lesions. FA pathway activation relies on phosphorylation of FANCI by the ataxia telangiectasia and Rad3-related (ATR) kinase, followed by monoubiquitination of FANCD2 and FANCI by the FA core complex. FANCD2 and FANCI are thought to form a functional heterodimer during DNA repair, but it is unclear how dimer formation is regulated or what the functions of the FANCD2–FANCI complex versus the monomeric proteins are. We show that the FANCD2–FANCI complex forms independently of ATR and FA core complex, and represents the inactive form of both proteins. DNA damage-induced FA pathway activation triggers dissociation of FANCD2 from FANCI. Dissociation coincides with FANCD2 monoubiquitination, which significantly precedes monoubiquitination of FANCI; moreover, monoubiquitination responses of FANCD2 and FANCI exhibit distinct DNA substrate specificities. A phosphodead FANCI mutant fails to dissociate from FANCD2, whereas phosphomimetic FANCI cannot interact with FANCD2, indicating that FANCI phosphorylation is the molecular trigger for FANCD2–FANCI dissociation. Following dissociation, FANCD2 binds replicating chromatin prior to—and independently of—FANCI. Moreover, the concentration of chromatin-bound FANCD2 exceeds that of FANCI throughout replication. Our results suggest that FANCD2 and FANCI function separately at consecutive steps during DNA repair in S-phase. PMID:22753026