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Sample records for phosphorylation regulates directional

  1. Bak apoptotic function is not directly regulated by phosphorylation.

    PubMed

    Tran, V H; Bartolo, R; Westphal, D; Alsop, A; Dewson, G; Kluck, R M

    2013-01-01

    During apoptosis, Bak and Bax permeabilize the mitochondrial outer membrane by undergoing major conformational change and oligomerization. This activation process in Bak is reported to require dephosphorylation of tyrosine-108 close to an activation trigger site. To investigate how dephosphorylation of Bak contributes to its activation and conformational change, one-dimensional isoelectric focusing (1D-IEF) and mutagenesis was used to monitor Bak phosphorylation. On 1D-IEF, Bak extracted from a range of cell types migrated as a single band near the predicted isoelectric point of 5.6 both before and after phosphatase treatment, indicating that Bak is not significantly phosphorylated at any residue. In contrast, three engineered 'phosphotagged' Bak variants showed a second band at lower pI, indicating phosphorylation. Apoptosis induced by several stimuli failed to alter Bak pI, indicating little change in phosphorylation status. In addition, alanine substitution of tyrosine-108 and other putative phosphorylation sites failed to enhance Bak activation or pro-apoptotic function. In summary, Bak is not significantly phosphorylated at any residue, and Bak activation during apoptosis does not require dephosphorylation. PMID:23303126

  2. Phosphorylation regulates the Star-PAP-PIPKIα interaction and directs specificity toward mRNA targets

    PubMed Central

    Mohan, Nimmy; AP, Sudheesh; Francis, Nimmy; Anderson, Richard; Laishram, Rakesh S.

    2015-01-01

    Star-PAP is a nuclear non-canonical poly(A) polymerase (PAP) that shows specificity toward mRNA targets. Star-PAP activity is stimulated by lipid messenger phosphatidyl inositol 4,5 bisphoshate (PI4,5P2) and is regulated by the associated Type I phosphatidylinositol-4-phosphate 5-kinase that synthesizes PI4,5P2 as well as protein kinases. These associated kinases act as coactivators of Star-PAP that regulates its activity and specificity toward mRNAs, yet the mechanism of control of these interactions are not defined. We identified a phosphorylated residue (serine 6, S6) on Star-PAP in the zinc finger region, the domain required for PIPKIα interaction. We show that S6 is phosphorylated by CKIα within the nucleus which is required for Star-PAP nuclear retention and interaction with PIPKIα. Unlike the CKIα mediated phosphorylation at the catalytic domain, Star-PAP S6 phosphorylation is insensitive to oxidative stress suggesting a signal mediated regulation of CKIα activity. S6 phosphorylation together with coactivator PIPKIα controlled select subset of Star-PAP target messages by regulating Star-PAP-mRNA association. Our results establish a novel role for phosphorylation in determining Star-PAP target mRNA specificity and regulation of 3′-end processing. PMID:26138484

  3. Directional Phosphorylation and Nuclear Transport of the Splicing Factor SRSF1 Is Regulated by an RNA Recognition Motif.

    PubMed

    Serrano, Pedro; Aubol, Brandon E; Keshwani, Malik M; Forli, Stefano; Ma, Chen-Ting; Dutta, Samit K; Geralt, Michael; Wüthrich, Kurt; Adams, Joseph A

    2016-06-01

    Multisite phosphorylation is required for the biological function of serine-arginine (SR) proteins, a family of essential regulators of mRNA splicing. These modifications are catalyzed by serine-arginine protein kinases (SRPKs) that phosphorylate numerous serines in arginine-serine-rich (RS) domains of SR proteins using a directional, C-to-N-terminal mechanism. The present studies explore how SRPKs govern this highly biased phosphorylation reaction and investigate biological roles of the observed directional phosphorylation mechanism. Using NMR spectroscopy with two separately expressed domains of SRSF1, we showed that several residues in the RNA-binding motif 2 interact with the N-terminal region of the RS domain (RS1). These contacts provide a structural framework that balances the activities of SRPK1 and the protein phosphatase PP1, thereby regulating the phosphoryl content of the RS domain. Disruption of the implicated intramolecular RNA-binding motif 2-RS domain interaction impairs both the directional phosphorylation mechanism and the nuclear translocation of SRSF1 demonstrating that the intrinsic phosphorylation bias is obligatory for SR protein biological function. PMID:27091468

  4. The cAMP Signaling Pathway and Direct Protein Kinase A Phosphorylation Regulate Polycystin-2 (TRPP2) Channel Function.

    PubMed

    Cantero, María del Rocío; Velázquez, Irina F; Streets, Andrew J; Ong, Albert C M; Cantiello, Horacio F

    2015-09-25

    Polycystin-2 (PC2) is a TRP-type, Ca(2+)-permeable non-selective cation channel that plays an important role in Ca(2+) signaling in renal and non-renal cells. The effect(s) of the cAMP pathway and kinase mediated phosphorylation of PC2 seem to be relevant to PC2 trafficking and its interaction with polycystin-1. However, the role of PC2 phosphorylation in channel function is still poorly defined. Here we reconstituted apical membranes of term human syncytiotrophoblast (hST), containing endogenous PC2 (PC2hst), and in vitro translated channel protein (PC2iv). Addition of the catalytic subunit of PKA increased by 566% the spontaneous PC2hst channel activity in the presence of ATP. Interestingly, 8-Br-cAMP also stimulated spontaneous PC2hst channel activity in the absence of the exogenous kinase. Either stimulation was inhibited by addition of alkaline phosphatase, which in turn, was reversed by the phosphatase inhibitor vanadate. Neither maneuver modified the single channel conductance but instead increased channel mean open time. PKA directly phosphorylated PC2, which increased the mean open time but not the single channel conductance of the channel. PKA phosphorylation did not modify either R742X truncated or S829A-mutant PC2iv channel function. The data indicate that the cAMP pathway regulates PC2-mediated cation transport in the hST. The relevant PKA site for PC2 channel regulation centers on a single residue serine 829, in the carboxyl terminus. PMID:26269590

  5. G-protein-coupled receptor regulation of P2X1 receptors does not involve direct channel phosphorylation

    PubMed Central

    2004-01-01

    P2X1 receptors for ATP are ligand-gated cation channels, which mediate smooth muscle contraction, contribute to blood clotting and are co-expressed with a range of GPCRs (G-protein-coupled receptors). Stimulation of Gαq-coupled mGluR1α (metabotropic glutamate receptor 1α), P2Y1 or P2Y2 receptors co-expressed with P2X1 receptors in Xenopus oocytes evoked calcium-activated chloride currents (IClCa) and potentiated subsequent P2X1-receptor-mediated currents by up to 250%. The mGluR1α-receptor-mediated effects were blocked by the phospholipase C inhibitor U-73122. Potentiation was mimicked by treatment with the phor-bol ester PMA. P2X receptors have a conserved intracellular PKC (protein kinase C) site; however, GPCR- and PMA-mediated potentiation was still observed with point mutants in which this site was disrupted. Similarly, the potentiation by GPCRs or PMA was unaffected by chelating the intracellular calcium rise with BAPTA/AM [bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis-(acetoxymethyl ester)] or the PKC inhibitors Ro-32-0432 and bisindolylmaleimide I, suggesting that the regulation does not involve a calcium-sensitive form of PKC. However, both GPCR and PMA potentiation were blocked by the kinase inhibitor staurosporine. Potentiation by phorbol esters was recorded in HEK-293 cells expressing P2X1 receptors, and radiolabelling of phosphorylated proteins in these cells demonstrated that P2X1 receptors are basally phosphorylated and that this level of phosphorylation is unaffected by phorbol ester treatment. This demonstrates that P2X1 regulation does not result directly from phosphorylation of the channel, but more likely by a staurosporine-sensitive phosphorylation of an accessory protein in the P2X1 receptor complex and suggests that in vivo fine-tuning of P2X1 receptors by GPCRs may contribute to cardiovascular control and haemostasis. PMID:15144237

  6. Phosphorylation of the Cav3.2 T-type calcium channel directly regulates its gating properties.

    PubMed

    Blesneac, Iulia; Chemin, Jean; Bidaud, Isabelle; Huc-Brandt, Sylvaine; Vandermoere, Franck; Lory, Philippe

    2015-11-01

    Phosphorylation is a major mechanism regulating the activity of ion channels that remains poorly understood with respect to T-type calcium channels (Cav3). These channels are low voltage-activated calcium channels that play a key role in cellular excitability and various physiological functions. Their dysfunction has been linked to several neurological disorders, including absence epilepsy and neuropathic pain. Recent studies have revealed that T-type channels are modulated by a variety of serine/threonine protein kinase pathways, which indicates the need for a systematic analysis of T-type channel phosphorylation. Here, we immunopurified Cav3.2 channels from rat brain, and we used high-resolution MS to construct the first, to our knowledge, in vivo phosphorylation map of a voltage-gated calcium channel in a mammalian brain. We identified as many as 34 phosphorylation sites, and we show that the vast majority of these sites are also phosphorylated on the human Cav3.2 expressed in HEK293T cells. In patch-clamp studies, treatment of the channel with alkaline phosphatase as well as analysis of dephosphomimetic mutants revealed that phosphorylation regulates important functional properties of Cav3.2 channels, including voltage-dependent activation and inactivation and kinetics. We also identified that the phosphorylation of a locus situated in the loop I-II S442/S445/T446 is crucial for this regulation. Our data show that Cav3.2 channels are highly phosphorylated in the mammalian brain and establish phosphorylation as an important mechanism involved in the dynamic regulation of Cav3.2 channel gating properties. PMID:26483470

  7. Autophagy proteins regulate ERK phosphorylation

    PubMed Central

    Martinez-Lopez, Nuria; Athonvarangkul, Diana; Mishall, Priti; Sahu, Srabani; Singh, Rajat

    2013-01-01

    Autophagy is a conserved pathway that maintains cellular quality control. Extracellular signal-regulated kinase (ERK) controls various aspects of cell physiology including proliferation. Multiple signalling cascades, including ERK, have been shown to regulate autophagy, however whether autophagy proteins (ATG) regulate cell signalling is unknown. Here we show that growth factor exposure increases the interaction of ERK cascade components with ATG proteins in the cytosol and nucleus. ERK and its upstream kinase MEK localize to the extra-luminal face of autophagosomes. ERK2 interacts with ATG proteins via its substrate-binding domains. Deleting Atg7 or Atg5 or blocking LC3 lipidation or ATG5–ATG12 conjugation decreases ERK phosphorylation. Conversely, increasing LC3-II availability by silencing the cysteine protease ATG4B or acute trehalose exposure increases ERK phosphorylation. Decreased ERK phosphorylation in Atg5−/− cells does not occur from overactive phosphatases. Our findings thus reveal an unconventional function of ATG proteins as cellular scaffolds in the regulation of ERK phosphorylation. PMID:24240988

  8. Cellular regulation by protein phosphorylation.

    PubMed

    Fischer, Edmond H

    2013-01-11

    A historical account of the discovery of reversible protein phosphorylation is presented. This process was uncovered in the mid 1950s in a study undertaken with Edwin G. Krebs to elucidate the complex hormonal regulation of skeletal muscle glycogen phosphorylase. Contrary to the known activation of this enzyme by AMP which serves as an allosteric effector, its hormonal regulation results from a phosphorylation of the protein by phosphorylase kinase following the activation of the latter by Ca(2+) and ATP. The study led to the establishment of the first hormonal cascade of successive enzymatic reactions, kinases acting on kinases, initiated by cAMP discovered by Earl Sutherland. It also showed how two different physiological processes, carbohydrate metabolism and muscle contraction, could be regulated in concert. PMID:23058924

  9. AMP-activated protein kinase regulates the vacuolar H+-ATPase via direct phosphorylation of the A subunit (ATP6V1A) in the kidney

    PubMed Central

    Alzamora, Rodrigo; Al-Bataineh, Mohammad M.; Liu, Wen; Gong, Fan; Li, Hui; Thali, Ramon F.; Joho-Auchli, Yolanda; Brunisholz, René A.; Satlin, Lisa M.; Neumann, Dietbert; Pastor-Soler, Núria M.

    2013-01-01

    The vacuolar H+-ATPase (V-ATPase) in intercalated cells contributes to luminal acidification in the kidney collecting duct and nonvolatile acid excretion. We previously showed that the A subunit in the cytoplasmic V1 sector of the V-ATPase (ATP6V1A) is phosphorylated by the metabolic sensor AMP-activated protein kinase (AMPK) in vitro and in kidney cells. Here, we demonstrate that treatment of rabbit isolated, perfused collecting ducts with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) inhibited V-ATPase-dependent H+ secretion from intercalated cells after an acid load. We have identified by mass spectrometry that Ser-384 is a major AMPK phosphorylation site in the V-ATPase A subunit, a result confirmed by comparing AMPK-dependent phosphate labeling of wild-type A-subunit (WT-A) with that of a Ser-384-to-Ala A subunit mutant (S384A-A) in vitro and in intact HEK-293 cells. Compared with WT-A-expressing HEK-293 cells, S384A-A-expressing cells exhibited greater steady-state acidification of HCO3−-containing media. Moreover, AICAR treatment of clone C rabbit intercalated cells expressing the WT-A subunit reduced V-ATPase-dependent extracellular acidification, an effect that was blocked in cells expressing the phosphorylation-deficient S384A-A mutant. Finally, expression of the S384A-A mutant prevented cytoplasmic redistribution of the V-ATPase by AICAR in clone C cells. In summary, direct phosphorylation of the A subunit at Ser-384 by AMPK represents a novel regulatory mechanism of the V-ATPase in kidney intercalated cells. Regulation of the V-ATPase by AMPK may couple V-ATPase activity to cellular metabolic status with potential relevance to ischemic injury in the kidney and other tissues. PMID:23863464

  10. JNK1/2 regulate Bid by direct phosphorylation at Thr59 in response to ALDH1L1

    PubMed Central

    Prakasam, A; Ghose, S; Oleinik, N V; Bethard, J R; Peterson, Y K; Krupenko, N I; Krupenko, S A

    2014-01-01

    BH3 interacting-domain death agonist (Bid) is a BH3-only pro-apoptotic member of the Bcl-2 family of proteins. Its function in apoptosis is associated with the proteolytic cleavage to the truncated form tBid, mainly by caspase-8. tBid translocates to mitochondria and assists Bax and Bak in induction of apoptosis. c-Jun N-terminal kinase (JNK)-dependent alternative processing of Bid to jBid was also reported. We have previously shown that the folate stress enzyme 10-formyltetrahydrofolate dehydrogenase (ALDH1L1) activates JNK1 and JNK2 in cancer cells as a pro-apoptotic response. Here we report that in PC-3 prostate cancer cells, JNK1/2 phosphorylate Bid at Thr59 within the caspase cleavage site in response to ALDH1L1. In vitro, all three JNK isoforms, JNK 1–3, phosphorylated Thr59 of Bid with JNK1 being the least active. Thr59 phosphorylation protected Bid from cleavage by caspase-8, resulting in strong accumulation of the full-length protein and its translocation to mitochondria. Interestingly, although we did not observe jBid in response to ALDH1L1 in PC-3 cells, transient expression of Bid mutants lacking the caspase-8 cleavage site resulted in strong accumulation of jBid. Of note, a T59D mutant mimicking constitutive phosphorylation revealed more profound cleavage of Bid to jBid. JNK-driven Bid accumulation had a pro-apoptotic effect in our study: small interfering RNA silencing of either JNK1/2 or Bid prevented Bid phosphorylation and accumulation, and rescued ALDH1L1-expressing cells. As full-length Bid is a weaker apoptogen than tBid, we propose that the phosphorylation of Bid by JNKs, followed by the accumulation of the full-length protein, delays attainment of apoptosis, and allows the cell to evaluate the stress and make a decision regarding the response strategy. This mechanism perhaps can be modified by the alternative cleavage of phospho-T59 Bid to jBid at some conditions. PMID:25077544

  11. PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

    SciTech Connect

    Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon

    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 Ala (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.

  12. 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. PMID:24485922

  13. Protein Synthesis Initiation Factors: Phosphorylation and Regulation

    SciTech Connect

    Karen S. Browning

    2009-06-15

    The initiation of the synthesis of proteins is a fundamental process shared by all living organisms. Each organism has both shared and unique mechanisms for regulation of this vital process. Higher plants provide for a major amount of fixation of carbon from the environment and turn this carbon into food and fuel sources for our use. However, we have very little understanding of how plants regulate the synthesis of the proteins necessary for these metabolic processes. The research carried out during the grant period sought to address some of these unknowns in the regulation of protein synthesis initiation. Our first goal was to determine if phosphorylation plays a significant role in plant initiation of protein synthesis. The role of phosphorylation, although well documented in mammalian protein synthesis regulation, is not well studied in plants. We showed that several of the factors necessary for the initiation of protein synthesis were targets of plant casein kinase and showed differential phosphorylation by the plant specific isoforms of this kinase. In addition, we identified and confirmed the phosphorylation sites in five of the plant initiation factors. Further, we showed that phosphorylation of one of these factors, eIF5, affected the ability of the factor to participate in the initiation process. Our second goal was to develop a method to make initiation factor 3 (eIF3) using recombinant methods. To date, we successfully cloned and expressed 13/13 subunits of wheat eIF3 in E. coli using de novo gene construction methods. The final step in this process is to place the subunits into three different plasmid operons for co-expression. Successful completion of expression of eIF3 will be an invaluable tool to the plant translation community.

  14. ZDHHC3 Tyrosine Phosphorylation Regulates Neural Cell Adhesion Molecule Palmitoylation.

    PubMed

    Lievens, Patricia Marie-Jeanne; Kuznetsova, Tatiana; Kochlamazashvili, Gaga; Cesca, Fabrizia; Gorinski, Natalya; Galil, Dalia Abdel; Cherkas, Volodimir; Ronkina, Natalia; Lafera, Juri; Gaestel, Matthias; Ponimaskin, Evgeni; Dityatev, Alexander

    2016-09-01

    The neural cell adhesion molecule (NCAM) mediates cell-cell and cell-matrix adhesion. It is broadly expressed in the nervous system and regulates neurite outgrowth, synaptogenesis, and synaptic plasticity. Previous in vitro studies revealed that palmitoylation of NCAM is required for fibroblast growth factor 2 (FGF2)-stimulated neurite outgrowth and identified the zinc finger DHHC (Asp-His-His-Cys)-containing proteins ZDHHC3 and ZDHHC7 as specific NCAM-palmitoylating enzymes. Here, we verified that FGF2 controlled NCAM palmitoylation in vivo and investigated molecular mechanisms regulating NCAM palmitoylation by ZDHHC3. Experiments with overexpression and pharmacological inhibition of FGF receptor (FGFR) and Src revealed that these kinases control tyrosine phosphorylation of ZDHHC3 and that ZDHHC3 is phosphorylated by endogenously expressed FGFR and Src proteins. By site-directed mutagenesis, we found that Tyr18 is an FGFR1-specific ZDHHC3 phosphorylation site, while Tyr295 and Tyr297 are specifically phosphorylated by Src kinase in cell-based and cell-free assays. Abrogation of tyrosine phosphorylation increased ZDHHC3 autopalmitoylation, enhanced interaction with NCAM, and upregulated NCAM palmitoylation. Expression of ZDHHC3 with tyrosine mutated in cultured hippocampal neurons promoted neurite outgrowth. Our findings for the first time highlight that FGFR- and Src-mediated tyrosine phosphorylation of ZDHHC3 modulates ZDHHC3 enzymatic activity and plays a role in neuronal morphogenesis. PMID:27247265

  15. Src kinase regulation by phosphorylation and dephosphorylation

    SciTech Connect

    Roskoski, Robert . E-mail: biocrr@lsuhsc.edu

    2005-05-27

    Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTP{alpha}, PTP{epsilon}, and PTP{lambda}. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined.

  16. Regulation of protein phosphorylation in oat mitochondria

    SciTech Connect

    Pike, C.; Kopeck, K.; Sceppa, E. )

    1989-04-01

    We sought to identify phosphorylated proteins in isolated oat mitocchondria and to characterize the enzymatic and regulatory properties of the protein kinase(s). Mitochondria from oats (Avena sativa L. cv. Garry) were purified on Percoll gradients. Mitochondria were incubated with {sup 32}P-{gamma}-ATP; proteins were separated by SDS-PAGE. A small number of bands was detected on autoradiograms, most prominently at 70 kD and 42 kD; the latter band has been tentatively identified as a subunit of the pyruvate dehydrogenase complex, a well-known phosphoprotein. The protein kinase(s) could also phosphorylate casein, but not histone. Spermine enhanced the phosphorylation of casein and inhibited the phosphorylation of the 42 kD band. These studies were carried out on both intact and burst mitochondria. Control by calcium and other ions was investigated. The question of the action of regulators on protein kinase or protein phosphatase was studied by the use of {sup 35}S-adenosine thiotriphosphate.

  17. Regulation of peroxisome dynamics by phosphorylation.

    PubMed

    Oeljeklaus, Silke; Schummer, Andreas; Mastalski, Thomas; Platta, Harald W; Warscheid, Bettina

    2016-05-01

    Peroxisomes are highly dynamic organelles that can rapidly change in size, abundance, and protein content in response to alterations in nutritional and other environmental conditions. These dynamic changes in peroxisome features, referred to as peroxisome dynamics, rely on the coordinated action of several processes of peroxisome biogenesis. Revealing the regulatory mechanisms of peroxisome dynamics is an emerging theme in cell biology. These mechanisms are inevitably linked to and synchronized with the biogenesis and degradation of peroxisomes. To date, the key players and basic principles of virtually all steps in the peroxisomal life cycle are known, but regulatory mechanisms remained largely elusive. A number of recent studies put the spotlight on reversible protein phosphorylation for the control of peroxisome dynamics and highlighted peroxisomes as hubs for cellular signal integration and regulation. Here, we will present and discuss the results of several studies performed using yeast and mammalian cells that convey a sense of the impact protein phosphorylation may have on the modulation of peroxisome dynamics by regulating peroxisomal matrix and membrane protein import, proliferation, inheritance, and degradation. We further put forward the idea to make use of current data on phosphorylation sites of peroxisomal and peroxisome-associated proteins reported in advanced large-scale phosphoproteomic studies. PMID:26775584

  18. The bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system: regulation by protein phosphorylation and phosphorylation-dependent protein-protein interactions.

    PubMed

    Deutscher, Josef; Aké, Francine Moussan Désirée; Derkaoui, Meriem; Zébré, Arthur Constant; Cao, Thanh Nguyen; Bouraoui, Houda; Kentache, Takfarinas; Mokhtari, Abdelhamid; Milohanic, Eliane; Joyet, Philippe

    2014-06-01

    The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components. PMID:24847021

  19. The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

    PubMed Central

    Aké, Francine Moussan Désirée; Derkaoui, Meriem; Zébré, Arthur Constant; Cao, Thanh Nguyen; Bouraoui, Houda; Kentache, Takfarinas; Mokhtari, Abdelhamid; Milohanic, Eliane; Joyet, Philippe

    2014-01-01

    SUMMARY The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components. PMID:24847021

  20. Regulation of cardiac C-protein phosphorylation

    SciTech Connect

    Titus, F.L.

    1985-01-01

    Molecular mechanisms of cardiac sympathetic and parasympathetic responses were addressed by studying subcellular changes in protein phosphorylation, cAMP-dependent protein kinase activity and protein phosphatase activity in frog hearts. B-adrenergic agonists increased and muscarinic cholinergic agonists decreased (/sup 32/P)phosphate incorporation into C-protein, a thick filament component. Regulation of protein phosphatase activity by Iso and methacholine (MCh) was assayed using extracts of drug treated frog hearts and (/sup 32/P)phospho-C-protein as substrate. Total phosphatase activity decreased 21% in extracts from hearts perfused with 0.1 ..mu..M Iso and 17% in hearts exposed to Iso plus 1 ..mu..M methacholine. This decrease reflected decreased phosphatase-2A activity. No changes in total phosphatase activity were measurable in broken cells treated with Iso or MCh. The results suggest adrenergic stimulation changes contractile activity in frog hearts by activating cAMP-dependent protein kinase associated with particulate cellular elements and inactivating soluble protein phosphatase-2A. This is the first demonstration of coordinated regulation of these enzymes by B-adrenergic agonists favoring phosphorylation of effector proteins. Coordinated regulation by methacholine in the presence of Iso was not observed.

  1. Tyrosine 370 phosphorylation of ATM positively regulates DNA damage response

    PubMed Central

    Lee, Hong-Jen; Lan, Li; Peng, Guang; Chang, Wei-Chao; Hsu, Ming-Chuan; Wang, Ying-Nai; Cheng, Chien-Chia; Wei, Leizhen; Nakajima, Satoshi; Chang, Shih-Shin; Liao, Hsin-Wei; Chen, Chung-Hsuan; Lavin, Martin; Ang, K Kian; Lin, Shiaw-Yih; Hung, Mien-Chie

    2015-01-01

    Ataxia telangiectasia mutated (ATM) mediates DNA damage response by controling irradiation-induced foci formation, cell cycle checkpoint, and apoptosis. However, how upstream signaling regulates ATM is not completely understood. Here, we show that upon irradiation stimulation, ATM associates with and is phosphorylated by epidermal growth factor receptor (EGFR) at Tyr370 (Y370) at the site of DNA double-strand breaks. Depletion of endogenous EGFR impairs ATM-mediated foci formation, homologous recombination, and DNA repair. Moreover, pretreatment with an EGFR kinase inhibitor, gefitinib, blocks EGFR and ATM association, hinders CHK2 activation and subsequent foci formation, and increases radiosensitivity. Thus, we reveal a critical mechanism by which EGFR directly regulates ATM activation in DNA damage response, and our results suggest that the status of ATM Y370 phosphorylation has the potential to serve as a biomarker to stratify patients for either radiotherapy alone or in combination with EGFR inhibition. PMID:25601159

  2. Regulation of the autophagy protein LC3 by phosphorylation

    PubMed Central

    Cherra, Salvatore J.; Kulich, Scott M.; Uechi, Guy; Balasubramani, Manimalha; Mountzouris, John; Day, Billy W.

    2010-01-01

    Macroautophagy is a major catabolic pathway that impacts cell survival, differentiation, tumorigenesis, and neurodegeneration. Although bulk degradation sustains carbon sources during starvation, autophagy contributes to shrinkage of differentiated neuronal processes. Identification of autophagy-related genes has spurred rapid advances in understanding the recruitment of microtubule-associated protein 1 light chain 3 (LC3) in autophagy induction, although braking mechanisms remain less understood. Using mass spectrometry, we identified a direct protein kinase A (PKA) phosphorylation site on LC3 that regulates its participation in autophagy. Both metabolic (rapamycin) and pathological (MPP+) inducers of autophagy caused dephosphorylation of endogenous LC3. The pseudophosphorylated LC3 mutant showed reduced recruitment to autophagosomes, whereas the nonphosphorylatable mutant exhibited enhanced puncta formation. Finally, autophagy-dependent neurite shortening induced by expression of a Parkinson disease–associated G2019S mutation in leucine-rich repeat kinase 2 was inhibited by dibutyryl–cyclic adenosine monophosphate, cytoplasmic expression of the PKA catalytic subunit, or the LC3 phosphorylation mimic. These data demonstrate a role for phosphorylation in regulating LC3 activity. PMID:20713600

  3. Roles of subunit phosphorylation in regulating glutamate receptor function

    PubMed Central

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

    2014-01-01

    Protein phosphorylation is an important mechanism for regulating ionotropic glutamate receptors (iGluRs). Early studies have established that major iGluR subtypes, including α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and N-methyl-D-aspartate (NMDA) receptors, are subject to phosphorylation. Multiple serine, threonine, and tyrosine residues predominantly within the C-terminal regions of AMPA receptor and NMDA receptor subunits have been identified as sensitive phosphorylation sites. These distinct sites undergo either constitutive phosphorylation or activity-dependent phosphorylation induced by changing cellular and synaptic inputs as reversible events. An increasing number of synapse-enriched protein kinases have been found to phosphorylate iGluR. The common kinases include protein kinase A, protein kinase C, Ca2+/calmodulin-dependent protein kinase II, Src/Fyn non-receptor tyrosine kinases, and cyclin dependent kinase-5. Regulated phosphorylation plays a well-documented role in modulating the biochemical, biophysical, and functional properties of the receptor. In the future, identifying the precise mechanisms how phosphorylation regulates iGluR activities and finding the link between iGluR phosphorylation and the pathogenesis of various brain diseases, including psychiatric and neurodegenerative diseases, chronic pain, stroke, Alzheimer’s disease and substance addiction, will be hot topics and could contribute to the development of novel pharmacotherapies, by targeting the defined phosphorylation process, for suppressing iGluR-related disorders. PMID:24291102

  4. Regulation of Endothelial Adherens Junctions by Tyrosine Phosphorylation

    PubMed Central

    Adam, Alejandro Pablo

    2015-01-01

    Endothelial cells form a semipermeable, regulated barrier that limits the passage of fluid, small molecules, and leukocytes between the bloodstream and the surrounding tissues. The adherens junction, a major mechanism of intercellular adhesion, is comprised of transmembrane cadherins forming homotypic interactions between adjacent cells and associated cytoplasmic catenins linking the cadherins to the cytoskeleton. Inflammatory conditions promote the disassembly of the adherens junction and a loss of intercellular adhesion, creating openings or gaps in the endothelium through which small molecules diffuse and leukocytes transmigrate. Tyrosine kinase signaling has emerged as a central regulator of the inflammatory response, partly through direct phosphorylation and dephosphorylation of the adherens junction components. This review discusses the findings that support and those that argue against a direct effect of cadherin and catenin phosphorylation in the disassembly of the adherens junction. Recent findings indicate a complex interaction between kinases, phosphatases, and the adherens junction components that allow a fine regulation of the endothelial permeability to small molecules, leukocyte migration, and barrier resealing. PMID:26556953

  5. Cyclosporine A protects podocytes by regulating WAVE1 phosphorylation

    PubMed Central

    Li, Xuejuan; Ding, Fangrui; Wang, Suxia; Li, Baihong; Ding, Jie

    2015-01-01

    Accumulating evidence suggests that podocytes are direct targets of many classic antiproteinuric drugs. The immunosuppressive drug cyclosporine A (CsA), which is a calcineurin inhibitor, is used to treat proteinuric kidney diseases. One novel mechanism by which CsA reduces proteinuria is by directly stabilizing the podocyte cytoskeleton. Previous studies showed that calcineurin can directly regulate WAVE1 within mouse striatal slices. In this study, WAVE1 was expressed in podocytes and was localized in the podocyte cell bodies and foot processes (FPs). WAVE1 expression increased in both in vivo and in vitro models of puromycin aminonucleoside (PAN)-induced podocyte injury. CsA restored WAVE1 expression and also partially rescued the disordered F-actin arrangement after PAN injury. Co-immunoprecipitation assays showed that calcineurin directly interacted with WAVE1 and regulated WAVE1 phosphorylation in podocytes. Synaptopodin is a well-characterized target of CsA. WAVE1 overexpression and synaptopodin knockdown experiments directly demonstrated that WAVE1 expression is not dependent on synaptopodin expression, and vice versa. Overexpression of WAVE1 using a WAVE1 plasmid disrupted F-actin structure and promoted podocyte migration compared with the empty vector group. Therefore, WAVE1 may be a novel molecular target for the maintenance of podocyte FPs and for antiproteinuric treatment in the future. PMID:26634693

  6. Regulation of CDK9 activity by phosphorylation and dephosphorylation.

    PubMed

    Nekhai, Sergei; Petukhov, Michael; Breuer, Denitra

    2014-01-01

    HIV-1 transcription is regulated by CDK9/cyclin T1, which, unlike a typical cell cycle-dependent kinase, is regulated by associating with 7SK small nuclear ribonuclear protein complex (snRNP). While the protein components of this complex are well studied, the mechanism of the complex formation is still not fully understood. The association of CDK9/cyclin T1 with 7SK snRNP is, in part, regulated by a reversible CDK9 phosphorylation. Here, we present a comprehensive review of the kinases and phosphatases involved in CDK9 phosphorylation and discuss their role in regulation of HIV-1 replication and potential for being targeted for drug development. We propose a novel pathway of HIV-1 transcription regulation via CDK9 Ser-90 phosphorylation by CDK2 and CDK9 Ser-175 dephosphorylation by protein phosphatase-1. PMID:24524087

  7. Phosphorylation Regulates Functions of ZEB1 Transcription Factor.

    PubMed

    Llorens, M Candelaria; Lorenzatti, Guadalupe; Cavallo, Natalia L; Vaglienti, Maria V; Perrone, Ana P; Carenbauer, Anne L; Darling, Douglas S; Cabanillas, Ana M

    2016-10-01

    ZEB1 transcription factor is important in both development and disease, including many TGFβ-induced responses, and the epithelial-to-mesenchymal transition (EMT) by which many tumors undergo metastasis. ZEB1 is differentially phosphorylated in different cell types; however the role of phosphorylation in ZEB1 activity is unknown. Luciferase reporter studies and electrophoresis mobility shift assays (EMSA) show that a decrease in phosphorylation of ZEB1 increases both DNA-binding and transcriptional repression of ZEB1 target genes. Functional analysis of ZEB1 phosphorylation site mutants near the second zinc finger domain (termed ZD2) show that increased phosphorylation (due to either PMA plus ionomycin, or IGF-1) can inhibit transcriptional repression by either a ZEB1-ZD2 domain clone, or full-length ZEB1. This approach identifies phosphosites that have a substantial effect regulating the transcriptional and DNA-binding activity of ZEB1. Immunoprecipitation with anti-ZEB1 antibodies followed by western analysis with a phospho-Threonine-Proline-specific antibody indicates that the ERK consensus site at Thr-867 is phosphorylated in ZEB1. In addition to disrupting in vitro DNA-binding measured by EMSA, IGF-1-induced MEK/ERK phosphorylation is sufficient to disrupt nuclear localization of GFP-ZEB1 fusion clones. These data suggest that phosphorylation of ZEB1 integrates TGFβ signaling with other signaling pathways such as IGF-1. J. Cell. Physiol. 231: 2205-2217, 2016. © 2016 Wiley Periodicals, Inc. PMID:26868487

  8. A secretory kinase complex regulates extracellular protein phosphorylation.

    PubMed

    Cui, Jixin; Xiao, Junyu; Tagliabracci, Vincent S; Wen, Jianzhong; Rahdar, Meghdad; Dixon, Jack E

    2015-01-01

    Although numerous extracellular phosphoproteins have been identified, the protein kinases within the secretory pathway have only recently been discovered, and their regulation is virtually unexplored. Fam20C is the physiological Golgi casein kinase, which phosphorylates many secreted proteins and is critical for proper biomineralization. Fam20A, a Fam20C paralog, is essential for enamel formation, but the biochemical function of Fam20A is unknown. Here we show that Fam20A potentiates Fam20C kinase activity and promotes the phosphorylation of enamel matrix proteins in vitro and in cells. Mechanistically, Fam20A is a pseudokinase that forms a functional complex with Fam20C, and this complex enhances extracellular protein phosphorylation within the secretory pathway. Our findings shed light on the molecular mechanism by which Fam20C and Fam20A collaborate to control enamel formation, and provide the first insight into the regulation of secretory pathway phosphorylation. PMID:25789606

  9. Identification of Phosphorylation Sites Regulating sst3 Somatostatin Receptor Trafficking.

    PubMed

    Lehmann, Andreas; Kliewer, Andrea; Günther, Thomas; Nagel, Falko; Schulz, Stefan

    2016-06-01

    The human somatostatin receptor 3 (sst3) is expressed in about 50% of all neuroendocrine tumors and hence a promising target for multireceptor somatostatin analogs. The sst3 receptor is unique among ssts in that it exhibits a very long intracellular C-terminal tail containing a huge number of potential phosphate acceptor sites. Consequently, our knowledge about the functional role of the C-terminal tail in sst3 receptor regulation is very limited. Here, we have generated a series of phosphorylation-deficient mutants that enabled us to determine crucial sites for its agonist-induced β-arrestin mobilization, internalization, and down-regulation. Based on this information, we generated phosphosite-specific antibodies for C-terminal Ser(337)/Thr(341), Thr(348), and Ser(361) that enabled us to investigate the temporal patterns of sst3 phosphorylation and dephosphorylation. We found that the endogenous ligand somatostatin induced a rapid and robust phosphorylation that was completely blocked by the sst3 antagonist NVP-ACQ090. The stable somatostatin analogs pasireotide and octreotide promoted clearly less phosphorylation compared with somatostatin. We also show that sst3 phosphorylation occurred within seconds to minutes, whereas dephosphorylation of the sst3 receptor occurred at a considerable slower rate. In addition, we also identified G protein-coupled receptor kinases 2 and 3 and protein phosphatase 1α and 1β as key regulators of sst3 phosphorylation and dephosphorylation, respectively. Thus, we here define the C-terminal phosphorylation motif of the human sst3 receptor that regulates its agonist-promoted phosphorylation, β-arrestin recruitment, and internalization of this clinically relevant receptor. PMID:27101376

  10. GIT1 Phosphorylation on Serine 46 by PKD3 Regulates Paxillin Trafficking and Cellular Protrusive Activity*

    PubMed Central

    Huck, Bettina; Kemkemer, Ralf; Franz-Wachtel, Mirita; Macek, Boris; Hausser, Angelika; Olayioye, Monilola A.

    2012-01-01

    The continuous assembly and disassembly of focal adhesions is required for efficient cell spreading and migration. The G-protein-coupled receptor kinase-interacting protein 1 (GIT1) is a multidomain protein whose dynamic localization to sites of cytoskeletal remodeling is critically involved in the regulation of these processes. Here we provide evidence that the subcellular localization of GIT1 is regulated by protein kinase D3 (PKD3) through direct phosphorylation on serine 46. GIT1 phosphorylation on serine 46 was abrograted by PKD3 depletion, thereby identifying GIT1 as the first specific substrate for this kinase. A GIT1 S46D phosphomimetic mutant localized to motile, paxillin-positive cytoplasmic complexes, whereas the phosphorylation-deficient GIT1 S46A was enriched in focal adhesions. We propose that phosphorylation of GIT1 on serine 46 by PKD3 represents a molecular switch by which GIT1 localization, paxillin trafficking, and cellular protrusive activity are regulated. PMID:22893698

  11. JNK phosphorylates β-catenin and regulates adherens junctions

    PubMed Central

    Lee, Meng-Horng; Koria, Piyush; Qu, Jun; Andreadis, Stelios T.

    2009-01-01

    The c-Jun amino-terminal kinase (JNK) is an important player in inflammation, proliferation, and apoptosis. More recently, JNK was found to regulate cell migration by phosphorylating paxillin. Here, we report a novel role of JNK in cell adhesion. Specifically, we provide evidence that JNK binds to E-cadherin/β-catenin complex and phosphorylates β-catenin at serine 37 and threonine 41, the sites also phosphorylated by GSK-3β. Inhibition of JNK kinase activity using dominant-negative constructs reduces phosphorylation of β-catenin and promotes localization of E-cadherin/β-catenin complex to cell-cell contact sites. Conversely, activation of JNK induces β-catenin phosphorylation and disruption of cell contacts, which are prevented by JNK siRNA. We propose that JNK binds to β-catenin and regulates formation of adherens junctions, ultimately controlling cell-to-cell adhesion.—Lee, M.-H., Koria, P., Qu, J., Andreadis, S. T. JNK phosphorylates β-catenin and regulates adherens junctions. PMID:19667122

  12. Phosphorylation-Dependent Regulation of G-Protein Cycle during Nodule Formation in Soybean[OPEN

    PubMed Central

    2015-01-01

    Signaling pathways mediated by heterotrimeric G-protein complexes comprising Gα, Gβ, and Gγ subunits and their regulatory RGS (Regulator of G-protein Signaling) protein are conserved in all eukaryotes. We have shown that the specific Gβ and Gγ proteins of a soybean (Glycine max) heterotrimeric G-protein complex are involved in regulation of nodulation. We now demonstrate the role of Nod factor receptor 1 (NFR1)-mediated phosphorylation in regulation of the G-protein cycle during nodulation in soybean. We also show that during nodulation, the G-protein cycle is regulated by the activity of RGS proteins. Lower or higher expression of RGS proteins results in fewer or more nodules, respectively. NFR1 interacts with RGS proteins and phosphorylates them. Analysis of phosphorylated RGS protein identifies specific amino acids that, when phosphorylated, result in significantly higher GTPase accelerating activity. These data point to phosphorylation-based regulation of G-protein signaling during nodule development. We propose that active NFR1 receptors phosphorylate and activate RGS proteins, which help maintain the Gα proteins in their inactive, trimeric conformation, resulting in successful nodule development. Alternatively, RGS proteins might also have a direct role in regulating nodulation because overexpression of their phospho-mimic version leads to partial restoration of nodule formation in nod49 mutants. PMID:26498905

  13. Regulation of Microtubule Dynamics through Phosphorylation on Stathmin by Epstein-Barr Virus Kinase BGLF4*

    PubMed Central

    Chen, Po-Wen; Lin, Sue-Jane; Tsai, Shu-Chun; Lin, Jiun-Han; Chen, Mei-Ru; Wang, Jiin-Tarng; Lee, Chung-Pei; Tsai, Ching-Hwa

    2010-01-01

    Stathmin is an important microtubule (MT)-destabilizing protein, and its activity is differently attenuated by phosphorylation at one or more of its four phosphorylatable serine residues (Ser-16, Ser-25, Ser-38, and Ser-63). This phosphorylation of stathmin plays important roles in mitotic spindle formation. We observed increasing levels of phosphorylated stathmin in Epstein-Barr virus (EBV)-harboring lymphoblastoid cell lines (LCLs) and nasopharyngeal carcinoma (NPC) cell lines during the EBV lytic cycle. These suggest that EBV lytic products may be involved in the regulation of stathmin phosphorylation. BGLF4 is an EBV-encoded kinase and has similar kinase activity to cdc2, an important kinase that phosphorylates serine residues 25 and 38 of stathmin during mitosis. Using an siRNA approach, we demonstrated that BGLF4 contributes to the phosphorylation of stathmin in EBV-harboring NPC. Moreover, we confirmed that BGLF4 interacts with and phosphorylates stathmin using an in vitro kinase assay and an in vivo two-dimensional electrophoresis assay. Interestingly, unlike cdc2, BGLF4 was shown to phosphorylate non-proline directed serine residues of stathmin (Ser-16) and it mediated phosphorylation of stathmin predominantly at serines 16, 25, and 38, indicating that BGLF4 can down-regulate the activity of stathmin. Finally, we demonstrated that the pattern of MT organization was changed in BGLF4-expressing cells, possibly through phosphorylation of stathmin. In conclusion, we have shown that a viral Ser/Thr kinase can directly modulate the activity of stathmin and this contributes to alteration of cellular MT dynamics and then may modulate the associated cellular processes. PMID:20110360

  14. Threonine phosphorylation prevents promoter DNA binding of the Group B Streptococcus response regulator CovR.

    PubMed

    Lin, Wan-Jung; Walthers, Don; Connelly, James E; Burnside, Kellie; Jewell, Kelsea A; Kenney, Linda J; Rajagopal, Lakshmi

    2009-03-01

    All living organisms communicate with the external environment for their survival and existence. In prokaryotes, communication is achieved by two-component systems (TCS) comprising histidine kinases and response regulators. In eukaryotes, signalling is accomplished by serine/threonine and tyrosine kinases. Although TCS and serine/threonine kinases coexist in prokaryotes, direct cross-talk between these families was first described in Group B Streptococcus (GBS). A serine/threonine kinase (Stk1) and a TCS (CovR/CovS) co-regulate toxin expression in GBS. Typically, promoter binding of regulators like CovR is controlled by phosphorylation of the conserved active site aspartate (D53). In this study, we show that Stk1 phosphorylates CovR at threonine 65. The functional consequence of threonine phosphorylation of CovR in GBS was evaluated using phosphomimetic and silencing substitutions. GBS encoding the phosphomimetic T65E allele are deficient for CovR regulation unlike strains encoding the non-phosphorylated T65A allele. Further, compared with wild-type or T65A CovR, the T65E CovR is unable to bind promoter DNA and is decreased for phosphorylation at D53, similar to Stk1-phosphorylated CovR. Collectively, we provide evidence for a novel mechanism of response regulator control that enables GBS (and possibly other prokaryotes) to fine-tune gene expression for environmental adaptation. PMID:19170889

  15. NHE3 function and phosphorylation are regulated by a calyculin A-sensitive phosphatase

    PubMed Central

    Dynia, Diane W.; Steinmetz, Amy G.

    2010-01-01

    Na+/H+ exchanger 3 (NHE3) is phosphorylated and regulated by multiple kinases, including PKA, SGK1, and CK2; however, the role of phosphatases in the dephosphorylation and regulation of NHE3 remains unknown. The purpose of this study was to determine whether serine/threonine phosphatases alter NHE3 activity and phosphorylation and, if so, at which sites. To this end, we first examined the effects of calyculin A [a combined protein phosphatase 1 (PP1) and PP2A inhibitor] and okadaic acid (a PP2A inhibitor) on general and site-specific NHE3 phosphorylation. Calyculin A induced a phosphorylation-dependent NHE3 gel mobility shift and increased NHE3 phosphorylation at serines 552 and 605. No change in NHE3 phosphorylation was detected after okadaic acid treatment. An NHE3 gel mobility shift was also evident in calyculin A-treated COS-7 cells transfected with either wild-type or mutant (S552A, S605G, S661A, S716A) rat NHE3. Since the NHE3 gel mobility shift occurred despite mutation of known phosphorylation sites, novel sites of phosphorylation must also exist. Next, we assayed NHE3 activity in response to calyculin A and okadaic acid and found that calyculin A induced a 24% inhibition of NHE3 activity, whereas okadaic acid had no effect. When all known NHE3 phosphorylation sites were mutated, calyculin A induced a stimulation of NHE3 activity, demonstrating a functional significance for the novel phosphorylation sites. Finally, we established that the PP1 catalytic subunit can directly dephosphorylate immunopurified NHE3 in vitro. In conclusion, our data demonstrate that a calyculin A-sensitive phosphatase, most likely PP1, is involved in the regulation and dephosphorylation of NHE3 at known and novel sites. PMID:20015946

  16. Regulation of ABC Transporter Function Via Phosphorylation by Protein Kinases

    PubMed Central

    Stolarczyk, Elzbieta I.; Reiling, Cassandra J.; Paumi, Christian M.

    2011-01-01

    ATP-binding cassette (ABC) transporters are multispanning membrane proteins that utilize ATP to move a broad range of substrates across cellular membranes. ABC transporters are involved in a number of human disorders and diseases [1]. Overexpression of a subset of the transporters has been closely linked to multidrug resistance in both bacteria and viruses and in cancer. A poorly understood and important aspect of ABC transporter biology is the role of phosphorylation as a mechanism to regulate transporter function. In this review, we summarize the current literature addressing the role of phosphorylation in regulating ABC transporter function. A comprehensive list of all the phosphorylation sites that have been identified for the human ABC transporters is presented, and we discuss the role of individual kinases in regulating transporter function. We address the potential pitfalls and difficulties associated with identifying phosphorylation sites and the corresponding kinase(s), and we discuss novel techniques that may circumvent these problems. We conclude by providing a brief perspective on studying ABC transporter phosphorylation. PMID:21118091

  17. Dynamic phosphorylation of Histone Deacetylase 1 by Aurora kinases during mitosis regulates zebrafish embryos development

    PubMed Central

    Loponte, Sara; Segré, Chiara V.; Senese, Silvia; Miccolo, Claudia; Santaguida, Stefano; Deflorian, Gianluca; Citro, Simona; Mattoscio, Domenico; Pisati, Federica; Moser, Mirjam A.; Visintin, Rosella; Seiser, Christian; Chiocca, Susanna

    2016-01-01

    Histone deacetylases (HDACs) catalyze the removal of acetyl molecules from histone and non-histone substrates playing important roles in chromatin remodeling and control of gene expression. Class I HDAC1 is a critical regulator of cell cycle progression, cellular proliferation and differentiation during development; it is also regulated by many post-translational modifications (PTMs). Herein we characterize a new mitosis-specific phosphorylation of HDAC1 driven by Aurora kinases A and B. We show that this phosphorylation affects HDAC1 enzymatic activity and it is critical for the maintenance of a proper proliferative and developmental plan in a complex organism. Notably, we find that Aurora-dependent phosphorylation of HDAC1 regulates histone acetylation by modulating the expression of genes directly involved in the developing zebrafish central nervous system. Our data represent a step towards the comprehension of HDAC1 regulation by its PTM code, with important implications in unravelling its roles both in physiology and pathology. PMID:27458029

  18. Dynamic phosphorylation of Histone Deacetylase 1 by Aurora kinases during mitosis regulates zebrafish embryos development.

    PubMed

    Loponte, Sara; Segré, Chiara V; Senese, Silvia; Miccolo, Claudia; Santaguida, Stefano; Deflorian, Gianluca; Citro, Simona; Mattoscio, Domenico; Pisati, Federica; Moser, Mirjam A; Visintin, Rosella; Seiser, Christian; Chiocca, Susanna

    2016-01-01

    Histone deacetylases (HDACs) catalyze the removal of acetyl molecules from histone and non-histone substrates playing important roles in chromatin remodeling and control of gene expression. Class I HDAC1 is a critical regulator of cell cycle progression, cellular proliferation and differentiation during development; it is also regulated by many post-translational modifications (PTMs). Herein we characterize a new mitosis-specific phosphorylation of HDAC1 driven by Aurora kinases A and B. We show that this phosphorylation affects HDAC1 enzymatic activity and it is critical for the maintenance of a proper proliferative and developmental plan in a complex organism. Notably, we find that Aurora-dependent phosphorylation of HDAC1 regulates histone acetylation by modulating the expression of genes directly involved in the developing zebrafish central nervous system. Our data represent a step towards the comprehension of HDAC1 regulation by its PTM code, with important implications in unravelling its roles both in physiology and pathology. PMID:27458029

  19. Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis.

    PubMed

    Chen, J-Y; Lin, Y-Y; Jou, T-S

    2012-06-01

    We demonstrated a protein kinase C (PKC)-dependent phosphorylation of canine ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50) at serine 347/348 by site-directed mutagenesis and a phospho-specific antibody. Cell fractionation and confocal imaging revealed the relocation of EBP50 from the plasma membrane to cytosol that accompanied this phosphorylation event. Increased phosphorylation at these serine residues led to the dissociation of EBP50 from ezrin and β-PIX, which are two upstream regulators of Rac1 activation. Cells overexpressing an EBP50 mutant, mimicking serine 347/348 phosphorylation, became refractory to hepatocyte growth factor-induced cell spreading and scattering, which is normally mediated by Rac1 activation. Detachment of cells from the substratum also elicited an increase in EBP50 phosphorylation, apparently due to counteracting activities of PKC and protein phosphastase 2A, which resulted in decreased Rac1 activation and induction of anoikis. Cells overexpressing an EBP50 mutant defective in serine 347/348 phosphorylation did not undergo apoptosis in suspension culture. These studies reveal a signaling cascade in which different phosphorylation states and subcellular localization of EBP50 regulate Rac1 function. PMID:22301917

  20. Serine/Threonine/Tyrosine Protein Kinase Phosphorylates Oleosin, a Regulator of Lipid Metabolic Functions1[OA

    PubMed Central

    Parthibane, Velayoudame; Iyappan, Ramachandiran; Vijayakumar, Anitha; Venkateshwari, Varadarajan; Rajasekharan, Ram

    2012-01-01

    Plant oils are stored in oleosomes or oil bodies, which are surrounded by a monolayer of phospholipids embedded with oleosin proteins that stabilize the structure. Recently, a structural protein, Oleosin3 (OLE3), was shown to exhibit both monoacylglycerol acyltransferase and phospholipase A2 activities. The regulation of these distinct dual activities in a single protein is unclear. Here, we report that a serine/threonine/tyrosine protein kinase phosphorylates oleosin. Using bimolecular fluorescence complementation analysis, we demonstrate that this kinase interacts with OLE3 and that the fluorescence was associated with chloroplasts. Oleosin-green fluorescent protein fusion protein was exclusively associated with the chloroplasts. Phosphorylated OLE3 exhibited reduced monoacylglycerol acyltransferase and increased phospholipase A2 activities. Moreover, phosphatidylcholine and diacylglycerol activated oleosin phosphorylation, whereas lysophosphatidylcholine, oleic acid, and Ca2+ inhibited phosphorylation. In addition, recombinant peanut (Arachis hypogaea) kinase was determined to predominantly phosphorylate serine residues, specifically serine-18 in OLE3. Phosphorylation levels of OLE3 during seed germination were determined to be higher than in developing peanut seeds. These findings provide direct evidence for the in vivo substrate selectivity of the dual-specificity kinase and demonstrate that the bifunctional activities of oleosin are regulated by phosphorylation. PMID:22434039

  1. Negative regulation of Vps34 by Cdk mediated phosphorylation

    PubMed Central

    Furuya, Tsuyoshi; Kim, Minsu; Lipinski, Marta; Li, Juying; Kim, Dohoon; Lu, Tao; Shen, Yong; Rameh, Lucia; Yankner, Bruce; Tsai, Li-Huei; Yuan, Junying

    2010-01-01

    Summary Vps34 (vacuolar protein sorting 34) complexes, the class III PtdIns3 kinase, specifically phosphorylate the D3-position of PtdIns to produce PtdIns3P. Vps34 is involved in the control of multiple key intracellular membrane trafficking pathways including endocytic sorting and autophagy. In mammalian cells, Vps34 interacts with Beclin 1, an orthologue of Atg6 in yeast, to regulate the production of PtdIns3P and autophagy. We show that Vps34 is phosphorylated on Thr159 by Cdk1, which negatively regulates its interaction with Beclin1 during mitosis. Cdk5/p25, a neuronal cdk shown to play a role in Alzheimer’s disease, can also phosphorylate Thr159 of Vps34. Phosphorylation of Vps34 on Thr159 inhibits its interaction with Beclin 1. We propose that phosphorylation of Thr159 in Vps34 is a key regulatory mechanism that controls the class III PtdIns3 kinase activity in cell cycle progression, development and human diseases including neurodegeneration and cancers. PMID:20513426

  2. 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

  3. Regulation of cilia assembly, disassembly, and length by protein phosphorylation.

    PubMed

    Cao, Muqing; Li, Guihua; Pan, Junmin

    2009-01-01

    The exact mechanism by which cells are able to assemble, regulate, and disassemble cilia or flagella is not yet completely understood. Recent studies in several model systems, including Chlamydomonas, Tetrahymena, Leishmania, Caenorhabditis elegans, and mammals, provide increasing biochemical and genetic evidence that phosphorylation of multiple protein kinases plays a key role in cilia assembly, disassembly, and length regulation. Members of several protein kinase families--including aurora kinases, never in mitosis A (NIMA)-related protein kinases, mitogen-activated protein (MAP) kinases, and a novel cyclin-dependent protein kinase--are involved in the ciliary regulation process. Among the newly identified protein kinase substrates are Chlamydomonas kinesin-13 (CrKinesin13), a microtubule depolymerizer, and histone deacetylase 6 (HDAC6), a microtubule deacetylase. Chlamydomonas aurora/Ipl1p-like protein kinase (CALK) and CrKinesin13 are two proteins that undergo phosphorylation changes correlated with flagellar assembly or disassembly. CALK becomes phosphorylated when flagella are lost, whereas CrKinesin13 is phosphorylated when new flagella are assembled. Conversely, suppressing CrKinesin13 expression results in cells with shorter flagella. PMID:20362099

  4. Regulation of Monoamine Transporters: Role of Transporter Phosphorylation

    PubMed Central

    Ramamoorthy, Sammanda; Shippenberg, Toni S.; Jayanthi, Lankupalle D.

    2010-01-01

    Presynaptic biogenic amine transporters mediate reuptake of released amines from the synapse, thus regulating serotonin, dopamine and norepinephrine neurotransmission. Medications utilized in the treatment of depression, attention deficit-hyperactivity disorder and other psychiatric disorders possess high affinity for amine transporters. In addition, amine transporters are targets for psychostimulants. Altered expression of biogenic amine transporters has long been implicated in several psychiatric and degenerative disorders. Therefore, appropriate regulation and maintenance of biogenic amine transporter activity is critical for the maintenance of normal amine homoeostasis. Accumulating evidence suggests that cellular protein kinases and phosphatases regulate amine transporter expression, activity, trafficking and degradation. Amine transporters are phosphoproteins that undergo dynamic control under the influence of various kinase and phosphatase activities. This review presents a brief overview of the role of amine transporter phosphorylation in the regulation of amine transport in the normal and diseased brain. Understanding the molecular mechanisms by which phosphorylation events affect amine transporter activity is essential for understanding the contribution of transporter phosphorylation to the regulation of monoamine neurotransmission and for identifying potential new targets for the treatment of various brain diseases. PMID:20951731

  5. Protein phosphorylation and regulation of adaptive responses in bacteria.

    PubMed Central

    Stock, J B; Ninfa, A J; Stock, A M

    1989-01-01

    Bacteria continuously adapt to changes in their environment. Responses are largely controlled by signal transduction systems that contain two central enzymatic components, a protein kinase that uses adenosine triphosphate to phosphorylate itself at a histidine residue and a response regulator that accepts phosphoryl groups from the kinase. This conserved phosphotransfer chemistry is found in a wide range of bacterial species and operates in diverse systems to provide different regulatory outputs. The histidine kinases are frequently membrane receptor proteins that respond to environmental signals and phosphorylate response regulators that control transcription. Four specific regulatory systems are discussed in detail: chemotaxis in response to attractant and repellent stimuli (Che), regulation of gene expression in response to nitrogen deprivation (Ntr), control of the expression of enzymes and transport systems that assimilate phosphorus (Pho), and regulation of outer membrane porin expression in response to osmolarity and other culture conditions (Omp). Several additional systems are also examined, including systems that control complex developmental processes such as sporulation and fruiting-body formation, systems required for virulent infections of plant or animal host tissues, and systems that regulate transport and metabolism. Finally, an attempt is made to understand how cross-talk between parallel phosphotransfer pathways can provide a global regulatory curcuitry. PMID:2556636

  6. Phosphorylation of K+ channels at single residues regulates memory formation

    PubMed Central

    Vernon, Jeffrey; Irvine, Elaine E.; Peters, Marco; Jeyabalan, Jeshmi

    2016-01-01

    Phosphorylation is a ubiquitous post-translational modification of proteins, and a known physiological regulator of K+ channel function. Phosphorylation of K+ channels by kinases has long been presumed to regulate neuronal processing and behavior. Although circumstantial evidence has accumulated from behavioral studies of vertebrates and invertebrates, the contribution to memory of single phosphorylation sites on K+ channels has never been reported. We have used gene targeting in mice to inactivate protein kinase A substrate residues in the fast-inactivating subunit Kv4.2 (T38A mutants), and in the small-conductance Ca2+-activated subunit SK1 (S105A mutants). Both manipulations perturbed a specific form of memory, leaving others intact. T38A mutants had enhanced spatial memory for at least 4 wk after training, whereas performance in three tests of fear memory was unaffected. S105A mutants were impaired in passive avoidance memory, sparing fear, and spatial memory. Together with recent findings that excitability governs the participation of neurons in a memory circuit, this result suggests that the memory type supported by neurons may depend critically on the phosphorylation of specific K+ channels at single residues. PMID:26980786

  7. A secretory kinase complex regulates extracellular protein phosphorylation

    PubMed Central

    Cui, Jixin; Xiao, Junyu; Tagliabracci, Vincent S; Wen, Jianzhong; Rahdar, Meghdad; Dixon, Jack E

    2015-01-01

    Although numerous extracellular phosphoproteins have been identified, the protein kinases within the secretory pathway have only recently been discovered, and their regulation is virtually unexplored. Fam20C is the physiological Golgi casein kinase, which phosphorylates many secreted proteins and is critical for proper biomineralization. Fam20A, a Fam20C paralog, is essential for enamel formation, but the biochemical function of Fam20A is unknown. Here we show that Fam20A potentiates Fam20C kinase activity and promotes the phosphorylation of enamel matrix proteins in vitro and in cells. Mechanistically, Fam20A is a pseudokinase that forms a functional complex with Fam20C, and this complex enhances extracellular protein phosphorylation within the secretory pathway. Our findings shed light on the molecular mechanism by which Fam20C and Fam20A collaborate to control enamel formation, and provide the first insight into the regulation of secretory pathway phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.06120.001 PMID:25789606

  8. TARP phosphorylation regulates synaptic AMPA receptors through lipid bilayers

    PubMed Central

    Sumioka, Akio; Yan, Dan; Tomita, Susumu

    2010-01-01

    Summary Neurons use neurotransmitters to communicate across synapses, constructing neural circuits in the brain. AMPA-type glutamate receptors are the predominant excitatory neurotransmitter receptors mediating fast synaptic transmission. AMPA receptors localize at synapses by forming protein complexes with transmembrane AMPA receptor regulatory proteins (TARPs) and PSD-95-like MAGUKs. Among the three classes of ionotropic glutamate receptors (AMPA-, NMDA, kainate-type), AMPA receptor activity is most regulatable by neuronal activity to adjust synaptic strength. Here, we mutated the prototypical TARP, stargazin, and found that TARP phosphorylation regulates synaptic AMPA receptor activity in vivo. We also found that stargazin interacts with negatively-charged lipid bilayers in its phosphorylation dependent manner, and that the lipid interaction inhibited stargazin binding to PSD-95. Cationic lipids dissociated stargazin from lipid bilayers and enhanced synaptic AMPA receptor activity in a stargazin phosphorylation-dependent manner. Thus, TARP phosphorylation plays a critical role in regulating AMPA receptor-mediated synaptic transmission via a lipid bilayer interaction. PMID:20547132

  9. Retinoids regulate TGFβ signaling at the level of Smad2 phosphorylation and nuclear accumulation

    PubMed Central

    Hoover, Loretta L.; Burton, Elizabeth G.; O'Neill, Megan L.; Brooks, Bonnie A.; Sreedharan, Shilpa; Dawson, Nineveh A.; Kubalak, Steven W.

    2008-01-01

    Indirect regulation of transforming growth factor (TGF)-β signaling by retinoids occurs on a long-term timescale, secondary to transcriptional events. Studies by our group show loss of retinoid X receptor (RXR) alpha results in increased TGFβ2 in the midgestational heart, which may play a role in the cardiac defects seen in this model[1]. Acute and direct interactions between retinoid and TGFβ signaling, however, are not clearly understood. Treatment of dispersed hearts and NIH3T3 cells for one-hour with TGFβ and retinoids (dual treatment) resulted in increased phosphorylated Smad2 and Smad3 when compared to treatment with TGFβ alone. Of all dual treatments, those with the RXR agonist Bexarotene, resulted in the highest level of phosphorylated Smad2, a 7-fold increase over TGFβ2 alone. Additionally, during dual treatment phosphorylation of Smad2 occurs via the TGFβ type I receptor but not by increased activation of the receptor. As loss of RXRα results in increased levels of Smad2 phosphorylation in response to TGFβ treatment and since nuclear accumulation of phosphorylated Smad2 is decreased during dual treatment, we propose that RXRα directly regulates the activities of Smad2. These data show retinoid signaling influences the TGFβ pathway in an acute and direct manner that has been unappreciated until now. PMID:18773928

  10. Phosphorylation and SCF-mediated degradation regulate CREB-H transcription of metabolic targets

    PubMed Central

    Barbosa, Sónia; Carreira, Suzanne; Bailey, Daniel; Abaitua, Fernando; O'Hare, Peter

    2015-01-01

    CREB‑H, an endoplasmic reticulum–anchored transcription factor, plays a key role in regulating secretion and in metabolic and inflammatory pathways, but how its activity is modulated remains unclear. We examined processing of the nuclear active form and identified a motif around S87–S90 with homology to DSG-type phosphodegrons. We show that this region is subject to multiple phosphorylations, which regulate CREB-H stability by targeting it to the SCFFbw1a E3 ubiquitin ligase. Data from phosphatase treatment, use of phosophospecific antibody, and substitution of serine residues demonstrate phosphorylation of candidate serines in the region, with the core S87/S90 motif representing a critical determinant promoting proteasome-mediated degradation. Candidate kinases CKII and GSK-3b phosphorylate CREB-H in vitro with specificities for different serines. Prior phosphorylation with GSK-3 at one or more of the adjacent serines substantially increases S87/S90-dependent phosphorylation by CKII. In vivo expression of a dominant-negative Cul1 enhances steady-state levels of CREB‑H, an effect augmented by Fbw1a. CREB-H directly interacts with Fbw1a in a phosphorylation-dependent manner. Finally, mutations within the phosphodegron, when incorporated into the full-length protein, result in increased levels of constitutively cleaved nuclear protein and increased transcription and secretion of a key endogenous target gene, apolipoprotein A IV. PMID:26108621

  11. Phosphorylation by Nek1 regulates opening and closing of voltage dependent anion channel 1

    SciTech Connect

    Chen, Yumay; Gaczynska, Maria; Osmulski, Pawel; Polci, Rosaria; Riley, Daniel J.

    2010-04-09

    VDAC1 is a key component of the mitochondrial permeability transition pore. To initiate apoptosis and certain other forms of cell death, mitochondria become permeable such that cytochrome c and other pre-apoptotic molecules resident inside the mitochondria enter the cytosol and activate apoptotic cascades. We have shown recently that VDAC1 interacts directly with never-in-mitosis A related kinase 1 (Nek1), and that Nek1 phosphorylates VDAC1 on Ser193 to prevent excessive cell death after injury. How this phosphorylation regulates the activity of VDAC1, however, has not yet been reported. Here, we use atomic force microscopy (AFM) and cytochrome c conductance studies to examine the configuration of VDAC1 before and after phosphorylation by Nek1. Wild-type VDAC1 assumes an open configuration, but closes and prevents cytochrome c efflux when phosphorylated by Nek1. A VDAC1-Ser193Ala mutant, which cannot be phosphorylated by Nek1 under identical conditions, remains open and constitutively allows cytochrome c efflux. Conversely, a VDAC1-Ser193Glu mutant, which mimics constitutive phosphorylation by Nek1, remains closed by AFM and prevents cytochrome c leakage in the same liposome assays. Our data provide a mechanism to explain how Nek1 regulates cell death by affecting the opening and closing of VDAC1.

  12. Serine/threonine/tyrosine phosphorylation regulates DNA binding of bacterial transcriptional regulators.

    PubMed

    Kalantari, Aida; Derouiche, Abderahmane; Shi, Lei; Mijakovic, Ivan

    2015-09-01

    Reversible phosphorylation of bacterial transcriptional regulators (TRs) belonging to the family of two-component systems (TCSs) is a well-established mechanism for regulating gene expression. Recent evidence points to the fact that reversible phosphorylation of bacterial TRs on other types of residue, i.e. serine, threonine, tyrosine and cysteine, is also quite common. The phosphorylation of the ester type (phospho-serine/threonine/tyrosine) is more stable than the aspartate phosphorylation of TCSs. The kinases which catalyse these phosphorylation events (Hanks-type serine/threonine protein kinases and bacterial protein tyrosine kinases) are also much more promiscuous than the TCS kinases, i.e. each of them can phosphorylate several substrate proteins. As a consequence, the dynamics and topology of the signal transduction networks depending on these kinases differ significantly from the TCSs. Here, we present an overview of different classes of bacterial TR phosphorylated and regulated by serine/threonine and tyrosine kinases. Particular attention is given to examples when serine/threonine and tyrosine kinases interact with TCSs, phosphorylating either the histidine kinases or the response regulators. We argue that these promiscuous kinases connect several signal transduction pathways and serve the role of signal integration. PMID:26220449

  13. Protein Phosphorylation: A Major Switch Mechanism for Metabolic Regulation.

    PubMed

    Humphrey, Sean J; James, David E; Mann, Matthias

    2015-12-01

    Metabolism research is undergoing a renaissance because many diseases are increasingly recognized as being characterized by perturbations in intracellular metabolic regulation. Metabolic changes can be conferred through changes to the expression of metabolic enzymes, the concentrations of substrates or products that govern reaction kinetics, or post-translational modification (PTM) of the proteins that facilitate these reactions. On the 60th anniversary since its discovery, reversible protein phosphorylation is widely appreciated as an essential PTM regulating metabolism. With the ability to quantitatively measure dynamic changes in protein phosphorylation on a global scale - hereafter referred to as phosphoproteomics - we are now entering a new era in metabolism research, with mass spectrometry (MS)-based proteomics at the helm. PMID:26498855

  14. PR65A Phosphorylation Regulates PP2A Complex Signaling

    PubMed Central

    Kotlo, Kumar; Xing, Yongna; Lather, Sonia; Grillon, Jean Michel; Johnson, Keven; Skidgel, Randal A.; Solaro, R. John; Danziger, Robert S.

    2014-01-01

    Serine-threonine Protein phosphatase 2 A (PP2A), a member of the PPP family of phosphatases, regulates a variety of essential cellular processes, including cell-cycling, DNA replication, transcription, translation, and secondary signaling pathways. In the heart, increased PP2A activity/signaling has been linked to cardiac remodeling, contractile dysfunction and, in failure, arrythmogenicity. The core PP2A complex is a hetero-trimeric holoenzyme consisting of a 36 kDa catalytic subunit (PP2Ac); a regulatory scaffold subunit of 65 kDa (PR65A or PP2Aa); and one of at least 18 associated variable regulatory proteins (B subunits) classified into 3 families. In the present study, three in vivo sites of phosphorylation in cardiac PR65A are identified (S303, T268, S314). Using HEK cells transfected with recombinant forms of PR65A with phosphomimetic (P-PR65A) and non-phosphorylated (N-PR65A) amino acid substitutions at these sites, these phosphorylations were shown to inhibit the interaction of PR65A with PP2Ac and PP2A holoenzyme signaling. Forty-seven phospho-proteins were increased in abundance in HEK cells transfected with P-PR65A versus N-PR65A by phospho-protein profiling using 2D-DIGE analysis on phospho-enriched whole cell protein extracts. Among these proteins were elongation factor 1α (EF1A), elongation factor 2, heat shock protein 60 (HSP60), NADPH-dehydrogenase 1 alpha sub complex, annexin A, and PR65A. Compared to controls, failing hearts from the Dahl rat had less phosphorylated PR65A protein abundance and increased PP2A activity. Thus, PR65A phosphorylation is an in vivo mechanism for regulation of the PP2A signaling complex and increased PP2A activity in heart failure. PMID:24465463

  15. Hedgehog-regulated atypical PKC promotes phosphorylation and activation of Smoothened and Cubitus interruptus in Drosophila.

    PubMed

    Jiang, Kai; Liu, Yajuan; Fan, Junkai; Epperly, Garretson; Gao, Tianyan; Jiang, Jin; Jia, Jianhang

    2014-11-11

    Smoothened (Smo) is essential for transduction of the Hedgehog (Hh) signal in both insects and vertebrates. Cell surface/cilium accumulation of Smo is thought to play an important role in Hh signaling, but how the localization of Smo is controlled remains poorly understood. In this study, we demonstrate that atypical PKC (aPKC) regulates Smo phosphorylation and basolateral accumulation in Drosophila wings. Inactivation of aPKC by either RNAi or a mutation inhibits Smo basolateral accumulation and attenuates Hh target gene expression. In contrast, expression of constitutively active aPKC elevates basolateral accumulation of Smo and promotes Hh signaling. The aPKC-mediated phosphorylation of Smo at Ser680 promotes Ser683 phosphorylation by casein kinase 1 (CK1), and these phosphorylation events elevate Smo activity in vivo. Moreover, aPKC has an additional positive role in Hh signaling by regulating the activity of Cubitus interruptus (Ci) through phosphorylation of the Zn finger DNA-binding domain. Finally, the expression of aPKC is up-regulated by Hh signaling in a Ci-dependent manner. Our findings indicate a direct involvement of aPKC in Hh signaling beyond its role in cell polarity. PMID:25349414

  16. Hedgehog-regulated atypical PKC promotes phosphorylation and activation of Smoothened and Cubitus interruptus in Drosophila

    PubMed Central

    Jiang, Kai; Liu, Yajuan; Fan, Junkai; Epperly, Garretson; Gao, Tianyan; Jiang, Jin; Jia, Jianhang

    2014-01-01

    Smoothened (Smo) is essential for transduction of the Hedgehog (Hh) signal in both insects and vertebrates. Cell surface/cilium accumulation of Smo is thought to play an important role in Hh signaling, but how the localization of Smo is controlled remains poorly understood. In this study, we demonstrate that atypical PKC (aPKC) regulates Smo phosphorylation and basolateral accumulation in Drosophila wings. Inactivation of aPKC by either RNAi or a mutation inhibits Smo basolateral accumulation and attenuates Hh target gene expression. In contrast, expression of constitutively active aPKC elevates basolateral accumulation of Smo and promotes Hh signaling. The aPKC-mediated phosphorylation of Smo at Ser680 promotes Ser683 phosphorylation by casein kinase 1 (CK1), and these phosphorylation events elevate Smo activity in vivo. Moreover, aPKC has an additional positive role in Hh signaling by regulating the activity of Cubitus interruptus (Ci) through phosphorylation of the Zn finger DNA-binding domain. Finally, the expression of aPKC is up-regulated by Hh signaling in a Ci-dependent manner. Our findings indicate a direct involvement of aPKC in Hh signaling beyond its role in cell polarity. PMID:25349414

  17. Regulation and Function of Phosphorylation on VP8, the Major Tegument Protein of Bovine Herpesvirus 1

    PubMed Central

    Zhang, Kuan; Afroz, Sharmin; Brownlie, Robert; Snider, Marlene

    2015-01-01

    ABSTRACT The major tegument protein of bovine herpesvirus 1 (BoHV-1), VP8, is essential for virus replication in cattle. VP8 is phosphorylated in vitro by casein kinase 2 (CK2) and BoHV-1 unique short protein 3 (US3). In this study, VP8 was found to be phosphorylated in both transfected and infected cells but was detected as a nonphosphorylated form in mature virions. This suggests that phosphorylation of VP8 is strictly controlled during different stages of the viral life cycle. The regulation and function of VP8 phosphorylation by US3 and CK2 were further analyzed. An in vitro kinase assay, site-directed mutagenesis, and liquid chromatography-mass spectrometry were used to identify the active sites for US3 and CK2. The two kinases phosphorylate VP8 at different sites, resulting in distinct phosphopeptide patterns. S16 is a primary phosphoreceptor for US3, and it subsequently triggers phosphorylation at S32. CK2 has multiple active sites, among which T107 appears to be the preferred residue. Additionally, CK2 consensus motifs in the N terminus of VP8 are essential for phosphorylation. Based on these results, a nonphosphorylated VP8 mutant was constructed and used for further studies. In transfected cells phosphorylation was not required for nuclear localization of VP8. Phosphorylated VP8 appeared to recruit promyelocytic leukemia (PML) protein and to remodel the distribution of PML in the nucleus; however, PML protein did not show an association with nonphosphorylated VP8. This suggests that VP8 plays a role in resisting PML-related host antiviral defenses by redistributing PML protein and that this function depends on the phosphorylation of VP8. IMPORTANCE The progression of VP8 phosphorylation over time and its function in BoHV-1 replication have not been characterized. This study demonstrates that activation of S16 initiates further phosphorylation at S32 by US3. Additionally, VP8 is phosphorylated by CK2 at several residues, with T107 having the highest level

  18. SIRT1 phosphorylation by AMP-activated protein kinase regulates p53 acetylation

    PubMed Central

    Lau, Alan W; Liu, Pengda; Inuzuka, Hiroyuki; Gao, Daming

    2014-01-01

    The deacetylase SIRT1 regulates multiple biological processes including cellular metabolism and aging. Importantly, SIRT1 can also inactivate the p53 tumor suppressor via deacetylation, suggesting a role in oncogenesis. Recently, SIRT1 was shown to be released from its endogenous inhibitor DBC1 by a process requiring AMPK and the phosphorylation of SIRT1 by yet undefined kinase(s). Here we provide further evidence that AMPK directly phosphorylates SIRT1 on T344, releasing it from DBC1. Furthermore, a phospho-mimetic SIRT1 (T334E) showed decreased binding to DBC1, supporting the importance of this phosphorylation in AMPK-mediated regulation of SIRT1 activity. In addition, inhibition of AMPK by Compound C led to increased p53 acetylation, suggesting a role for the AMPK/SIRT1 pathway in regulating p53 signaling. Together, our results support a hypothesis that AMPK negatively regulates p53 acetylation via phosphorylation of SIRT1 on T344. Furthermore, our findings also define the AMPK/SIRT1 axis as a possible targetable pathway to regulate p53 function. PMID:24959379

  19. Thr-161 phosphorylation of monomeric Cdc2. Regulation by protein phosphatase 2C in Xenopus oocytes.

    PubMed

    De Smedt, Veronique; Poulhe, Robert; Cayla, Xavier; Dessauge, Frederic; Karaiskou, Anthi; Jessus, Catherine; Ozon, Rene

    2002-08-01

    Fully grown Xenopus oocyte is arrested at prophase I of meiosis. Re-entry into meiosis depends on the activation of MPF (M-phase promoting factor or cyclin B.Cdc2 complex), triggered by progesterone. The prophase-arrested oocyte contains a store of Cdc2. Most of the protein is present as a monomer whereas a minor fraction, called pre-MPF, is found to be associated with cyclin B. Activation of Cdc2 depends on two key events: cyclin binding and an activating phosphorylation on Thr-161 residue located in the T-loop. To get new insights into the regulation of Thr-161 phosphorylation of Cdc2, monomeric Cdc2 was isolated from prophase oocytes. Based on its activation upon cyclin addition and detection by an antibody directed specifically against Cdc2 phosphorylated on Thr-161, we show for the first time that the prophase oocyte contains a significant amount of monomeric Cdc2 phosphorylated on Thr-161. PP2C, a Mg2+-dependent phosphatase, negatively controls Thr-161 phosphorylation of Cdc2. The unexpected presence of a population of free Cdc2 already phosphorylated on Thr-161 could contribute to the generation of the Cdc2 kinase activity threshold required to initiate MPF amplification. PMID:12036957

  20. Importance of Phosphorylation for Osteopontin Regulation of Biomineralization

    PubMed Central

    Gericke, A.; Qin, C.; Spevak, L.; Fujimoto, Y.; Butler, W. T.; Sørensen, E. S.; Boskey, A. L.

    2006-01-01

    Previous in vitro and in vivo studies demonstrated that osteopontin (OPN) is an inhibitor of the formation and growth of hydroxyapatite (HA) and other biominerals. The present study tests the hypotheses that the interaction of OPN with HA is determined by the extent of protein phosphorylation and that this interaction regulates the mineralization process. Bone OPN as previously reported inhibited HA formation and HA-seeded growth in a gelatin-gel system. A transglutaminase-linked OPN polymer had similar effects. Recombinant, nonphosphorylated OPN and chemically dephosphorylated OPN, had no effect on HA formation or growth in this system. In contrast, highly phosphorylated milk OPN (mOPN) promoted HA formation. The mOPN stabilized the conversion of amorphous calcium phosphate (a noncrystalline constituent of milk) to HA, whereas bone OPN had a lesser effect on this conversion. Mixtures of OPN and osteocalcin known to form a complex in vitro, unexpectedly promoted HA formation. To test the hypothesis that small alterations in protein conformation caused by phosphorylation account for the differences in the observed ability of OPN to interact with HA, the conformation of bone OPN and mOPN in the presence and absence of crystalline HA was determined by attenuated total reflection (ATR) infrared (IR) spectroscopy. Both proteins exhibited a predominantly random coil structure, which was unaffected by the addition of Ca2+. Binding to HA did not alter the secondary structure of bone OPN, but induced a small increase of β-sheet (few percent) in mOPN. These data taken together suggest that the phosphorylation of OPN is an important factor in regulating the OPN-mediated mineralization process. PMID:16007483

  1. Hydrogen Peroxide-Induced Akt Phosphorylation Regulates Bax Activation

    PubMed Central

    Sadidi, Mahdieh; Lentz, Stephen I.; Feldman, Eva L.

    2009-01-01

    Reactive oxygen species such as hydrogen peroxide (H2O2) are involved in many cellular processes that positively and negatively regulate cell fate. H2O2, acting as an intracellular messenger, activates phosphatidylinositol-3 kinase (PI3K) and its downstream target Akt, and promotes cell survival. The aim of the current study was to understand the mechanism by which PI3K/Akt signaling promotes survival in SH-SY5Y neuroblastoma cells. We demonstrate that PI3K/Akt mediates phosphorylation of the pro-apoptotic Bcl-2 family member Bax. This phosphorylation suppresses apoptosis and promotes cell survival. Increased survival in the presence of H2O2 was blocked by LY294002, an inhibitor of PI3K activation. LY294002 prevented Bax phosphorylation and resulted in Bax translocation to the mitochondria, cytochrome c release, caspase-3 activation, and cell death. Collectively, these findings reveal a mechanism by which H2O2-induced activation of PI3K/Akt influences posttranslational modification of Bax and inactivate a key component of the cell death machinery. PMID:19278624

  2. PKC phosphorylates HEXIM1 and regulates P-TEFb activity

    PubMed Central

    Fujinaga, Koh; Barboric, Matjaz; Li, Qintong; Luo, Zeping; Price, David H.; Peterlin, B. Matija

    2012-01-01

    The positive transcription elongation factor b (P-TEFb) regulates RNA polymerase II elongation. In cells, P-TEFb partitions between small active and larger inactive states. In the latter, HEXIM1 binds to 7SK snRNA and recruits as well as inactivates P-TEFb in the 7SK snRNP. Several stimuli can affect this P-TEFb equilibrium. In this study, we demonstrate that protein kinase C (PKC) phosphorylates the serine at position158 (S158) in HEXIM1. This phosphorylated HEXIM1 protein neither binds to 7SK snRNA nor inhibits P-TEFb. Phorbol esters or the engagement of the T cell antigen receptor, which activate PKC and the expression of the constitutively active (CA) PKCθ protein, which is found in T cells, inhibit the formation of the 7SK snRNP. All these stimuli increase P-TEFb-dependent transcription. In contrast, the kinase-negative PKCθ and the mutant HEXIM1 (S158A) proteins block effects of these PKC-activating stimuli. These results indicate that the phosphorylation of HEXIM1 by PKC represents a major regulatory step of P-TEFb activity in cells. PMID:22821562

  3. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization

    PubMed Central

    Lee, Wei Lin; Grimes, Jonathan M; Robinson, Robert C

    2016-01-01

    Pathogenic Yersinia species evade host immune systems through the injection of Yersinia outer proteins (Yops) into phagocytic cells. One Yop, YopO, also known as YpkA, induces actin-filament disruption, impairing phagocytosis. Here we describe the X-ray structure of Yersinia enterocolitica YopO in complex with actin, which reveals that YopO binds to an actin monomer in a manner that blocks polymerization yet allows the bound actin to interact with host actin-regulating proteins. SILAC-MS and biochemical analyses confirm that actin-polymerization regulators such as VASP, EVL, WASP, gelsolin and the formin diaphanous 1 are directly sequestered and phosphorylated by YopO through formation of ternary complexes with actin. This leads to a model in which YopO at the membrane sequesters actin from polymerization while using the bound actin as bait to recruit, phosphorylate and misregulate host actin-regulating proteins to disrupt phagocytosis. PMID:25664724

  4. Phosphorylation Regulates the Bound Structure of an Intrinsically Disordered Protein: The p53-TAZ2 Case

    PubMed Central

    Ithuralde, Raúl Esteban; Turjanski, Adrián Gustavo

    2016-01-01

    Disordered regions and Intrinsically Disordered Proteins (IDPs) are involved in critical cellular processes and may acquire a stable three-dimensional structure only upon binding to their partners. IDPs may follow a folding-after-binding process, known as induced folding, or a folding-before-binding process, known as conformational selection. The transcription factor p53 is involved in the regulation of cellular events that arise upon stress or DNA damage. The p53 domain structure is composed of an N-terminal transactivation domain (p53TAD), a DNA Binding Domain and a tetramerization domain. The activity of TAD is tightly regulated by interactions with cofactors, inhibitors and phosphorylation. To initiate transcription, p53TAD binds to the TAZ2 domain of CBP, a co-transcription factor, and undergoes a folding and binding process, as revealed by the recent NMR structure of the complex. The activity of p53 is regulated by phosphorylation at multiple sites on the TAD domain and recent studies have shown that modifications at three residues affect the binding towards TAZ2. However, we still do not know how these phosphorylations affect the structure of the bound state and, therefore, how they regulate the p53 function. In this work, we have used computational simulations to understand how phosphorylation affects the structure of the p53TAD:TAZ2 complex and regulates the recognition mechanism. Phosphorylation has been proposed to enhance binding by direct interaction with the folded protein or by changing the unbound conformation of IDPs, for example by pre-folding the protein favoring the recognition mechanism. Here, we show an interesting turn in the p53 case: phosphorylation mainly affects the bound structure of p53TAD, highlighting the complexity of IDP protein-protein interactions. Our results are in agreement with previous experimental studies, allowing a clear picture of how p53 is regulated by phosphorylation and giving new insights into how post

  5. PAS kinase is activated by direct SNF1-dependent phosphorylation and mediates inhibition of TORC1 through the phosphorylation and activation of Pbp1

    PubMed Central

    DeMille, Desiree; Badal, Bryan D.; Evans, J. Brady; Mathis, Andrew D.; Anderson, Joseph F.; Grose, Julianne H.

    2015-01-01

    We describe the interplay between three sensory protein kinases in yeast: AMP-regulated kinase (AMPK, or SNF1 in yeast), PAS kinase 1 (Psk1 in yeast), and the target of rapamycin complex 1 (TORC1). This signaling cascade occurs through the SNF1-dependent phosphorylation and activation of Psk1, which phosphorylates and activates poly(A)- binding protein binding protein 1 (Pbp1), which then inhibits TORC1 through sequestration at stress granules. The SNF1-dependent phosphorylation of Psk1 appears to be direct, in that Snf1 is necessary and sufficient for Psk1 activation by alternate carbon sources, is required for altered Psk1 protein mobility, is able to phosphorylate Psk1 in vitro, and binds Psk1 via its substrate-targeting subunit Gal83. Evidence for the direct phosphorylation and activation of Pbp1 by Psk1 is also provided by in vitro and in vivo kinase assays, including the reduction of Pbp1 localization at distinct cytoplasmic foci and subsequent rescue of TORC1 inhibition in PAS kinase–deficient yeast. In support of this signaling cascade, Snf1-deficient cells display increased TORC1 activity, whereas cells containing hyperactive Snf1 display a PAS kinase–dependent decrease in TORC1 activity. This interplay between yeast SNF1, Psk1, and TORC1 allows for proper glucose allocation during nutrient depletion, reducing cell growth and proliferation when energy is low. PMID:25428989

  6. Protein kinase C regulates the phosphorylation and oligomerization of ERM binding phosphoprotein 50

    SciTech Connect

    Fouassier, Laura; Nichols, Matthew T.; Gidey, Elizabeth; McWilliams, Ryan R.; Robin, Helene; Finnigan, Claire; Howell, Kathryn E.; Housset, Chantal; Doctor, R. Brian . E-mail: brian.doctor@uchsc.edu

    2005-05-15

    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-293 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.

  7. Molecular Features of Phosphatase and Tensin Homolog (PTEN) Regulation by C-terminal Phosphorylation.

    PubMed

    Chen, Zan; Dempsey, Daniel R; Thomas, Stefani N; Hayward, Dawn; Bolduc, David M; Cole, Philip A

    2016-07-01

    PTEN is a tumor suppressor that functions to negatively regulate the PI3K/AKT pathway as the lipid phosphatase for phosphatidylinositol 3,4,5-triphosphate. Phosphorylation of a cluster of Ser/Thr residues (amino acids 380-385) on the C-terminal tail serves to alter the conformational state of PTEN from an open active state to a closed inhibited state, resulting in a reduction of plasma membrane localization and inhibition of enzyme activity. The relative contribution of each phosphorylation site to PTEN autoinhibition and the structural basis for the conformational closure is still unclear. To further the structural understanding of PTEN regulation by C-terminal tail phosphorylation, we used protein semisynthesis to insert stoichiometric and site-specific phospho-Ser/Thr(s) in the C-terminal tail of PTEN. Additionally, we employed photo-cross-linking to map the intramolecular PTEN interactions of the phospho-tail. Systematic evaluation of the PTEN C-tail phospho-cluster showed autoinhibition, and conformational closure was influenced by the aggregate effect of multiple phospho-sites rather than dominated by a single phosphorylation site. Moreover, photo-cross-linking suggested a direct interaction between the PTEN C-tail and a segment in the N-terminal region of the catalytic domain. Mutagenesis experiments provided additional insights into how the PTEN phospho-tail interacts with both the C2 and catalytic domains. PMID:27226612

  8. Regulation of DNA methylation patterns by CK2-mediated phosphorylation of Dnmt3a.

    PubMed

    Deplus, Rachel; Blanchon, Loïc; Rajavelu, Arumugam; Boukaba, Abdelhalim; Defrance, Matthieu; Luciani, Judith; Rothé, Françoise; Dedeurwaerder, Sarah; Denis, Hélène; Brinkman, Arie B; Simmer, Femke; Müller, Fabian; Bertin, Benjamin; Berdasco, Maria; Putmans, Pascale; Calonne, Emilie; Litchfield, David W; de Launoit, Yvan; Jurkowski, Tomasz P; Stunnenberg, Hendrik G; Bock, Christoph; Sotiriou, Christos; Fraga, Mario F; Esteller, Manel; Jeltsch, Albert; Fuks, François

    2014-08-01

    DNA methylation is a central epigenetic modification that is established by de novo DNA methyltransferases. The mechanisms underlying the generation of genomic methylation patterns are still poorly understood. Using mass spectrometry and a phosphospecific Dnmt3a antibody, we demonstrate that CK2 phosphorylates endogenous Dnmt3a at two key residues located near its PWWP domain, thereby downregulating the ability of Dnmt3a to methylate DNA. Genome-wide DNA methylation analysis shows that CK2 primarily modulates CpG methylation of several repeats, most notably of Alu SINEs. This modulation can be directly attributed to CK2-mediated phosphorylation of Dnmt3a. We also find that CK2-mediated phosphorylation is required for localization of Dnmt3a to heterochromatin. By revealing phosphorylation as a mode of regulation of de novo DNA methyltransferase function and by uncovering a mechanism for the regulation of methylation at repetitive elements, our results shed light on the origin of DNA methylation patterns. PMID:25066127

  9. Phosphorylation of Plk1 at Ser326 regulates its functions during mitotic progression

    PubMed Central

    Tang, J; Yang, X; Liu, X

    2009-01-01

    Polo-like kinase 1 (Plk1), the best characterized member of the mammalian polo-like kinase family, is well regulated throughout the cell cycle at the protein expression level. Moreover, it is known that Plk1 kinase activity is also regulated at the post-translational level through phosphorylation. However, the upstream kinases of Plk1 have not been identified. Although the involvement of the p38 MAP kinase pathway in cellular responses to stress has been well documented, the role of this pathway in normal cell cycle progression is unclear. Here, we show that phosphorylated p38 and MAP kinase-activated protein kinase 2 (MK2) are colocalized with Plk1 to the spindle poles during prophase and metaphase. Specific depletion of various members of the p38 MAP kinase pathway by the use of RNA interference revealed that the pathway is required for mitotic progression under normal growth conditions. Furthermore, MK2 directly phosphorylates Ser326 of Plk1. Ectopic expression of Plk1-S326A completely blocked cells at mitosis, likely due to the defect of bipolar spindle formation and subsequent activation of the spindle checkpoint. Only Plk1-S326E, but not the Plk1-S326A, efficiently rescued the p38 or MK2-depletion-induced mitotic defects, further solidifying the requirement of S326 phosphorylation during mitotic progression. PMID:18695677

  10. Src-Dependent Phosphorylation of ASAP1 Regulates Podosomes▿

    PubMed Central

    Bharti, Sanita; Inoue, Hiroki; Bharti, Kapil; Hirsch, Dianne S.; Nie, Zhongzhen; Yoon, Hye-Young; Artym, Vira; Yamada, Kenneth M.; Mueller, Susette C.; Barr, Valarie A.; Randazzo, Paul A.

    2007-01-01

    Invadopodia are Src-induced cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing Src homology 3 (SH3) and Bin, amphiphysin, and RVS161/167 (BAR) domains, is a substrate of Src that controls invadopodia. We have examined the structural requirements for ASAP1-dependent formation of invadopodia and related structures in NIH 3T3 fibroblasts called podosomes. We found that both predominant splice variants of ASAP1 (ASAP1a and ASAP1b) associated with invadopodia and podosomes. Podosomes were highly dynamic, with rapid turnover of both ASAP1 and actin. Reduction of ASAP1 levels by small interfering RNA blocked formation of invadopodia and podosomes. Podosomes were formed in NIH 3T3 fibroblasts in which endogenous ASAP1 was replaced with either recombinant ASAP1a or ASAP1b. ASAP1 mutants that lacked the Src binding site or GAP activity functioned as well as wild-type ASAP1 in the formation of podosomes. Recombinant ASAP1 lacking the BAR domain, the SH3 domain, or the Src phosphorylation site did not support podosome formation. Based on these results, we conclude that ASAP1 is a critical target of tyrosine kinase signaling involved in the regulation of podosomes and invadopodia and speculate that ASAP1 may function as a coincidence detector of simultaneous protein association through the ASAP1 SH3 domain and phosphorylation by Src. PMID:17893324

  11. Quantitative phosphoproteomics of protein kinase SnRK1 regulated protein phosphorylation in Arabidopsis under submergence.

    PubMed

    Cho, Hsing-Yi; Wen, Tuan-Nan; Wang, Ying-Tsui; Shih, Ming-Che

    2016-04-01

    SNF1 RELATED PROTEIN KINASE 1 (SnRK1) is proposed to be a central integrator of the plant stress and energy starvation signalling pathways. We observed that the Arabidopsis SnRK1.1 dominant negative mutant (SnRK1.1 (K48M) ) had lower tolerance to submergence than the wild type, suggesting that SnRK1.1-dependent phosphorylation of target proteins is important in signalling pathways triggered by submergence. We conducted quantitative phosphoproteomics and found that the phosphorylation levels of 57 proteins increased and the levels of 27 proteins decreased in Col-0 within 0.5-3h of submergence. Among the 57 proteins with increased phosphorylation in Col-0, 38 did not show increased phosphorylation levels in SnRK1.1 (K48M) under submergence. These proteins are involved mainly in sugar and protein synthesis. In particular, the phosphorylation of MPK6, which is involved in regulating ROS responses under abiotic stresses, was disrupted in the SnRK1.1 (K48M) mutant. In addition, PTP1, a negative regulator of MPK6 activity that directly dephosphorylates MPK6, was also regulated by SnRK1.1. We also showed that energy conservation was disrupted in SnRK1.1 (K48M) , mpk6, and PTP1 (S7AS8A) under submergence. These results reveal insights into the function of SnRK1 and the downstream signalling factors related to submergence. PMID:27029354

  12. Quantitative phosphoproteomics of protein kinase SnRK1 regulated protein phosphorylation in Arabidopsis under submergence

    PubMed Central

    Cho, Hsing-Yi; Wen, Tuan-Nan; Wang, Ying-Tsui; Shih, Ming-Che

    2016-01-01

    SNF1 RELATED PROTEIN KINASE 1 (SnRK1) is proposed to be a central integrator of the plant stress and energy starvation signalling pathways. We observed that the Arabidopsis SnRK1.1 dominant negative mutant (SnRK1.1 K48M) had lower tolerance to submergence than the wild type, suggesting that SnRK1.1-dependent phosphorylation of target proteins is important in signalling pathways triggered by submergence. We conducted quantitative phosphoproteomics and found that the phosphorylation levels of 57 proteins increased and the levels of 27 proteins decreased in Col-0 within 0.5–3h of submergence. Among the 57 proteins with increased phosphorylation in Col-0, 38 did not show increased phosphorylation levels in SnRK1.1 K48M under submergence. These proteins are involved mainly in sugar and protein synthesis. In particular, the phosphorylation of MPK6, which is involved in regulating ROS responses under abiotic stresses, was disrupted in the SnRK1.1 K48M mutant. In addition, PTP1, a negative regulator of MPK6 activity that directly dephosphorylates MPK6, was also regulated by SnRK1.1. We also showed that energy conservation was disrupted in SnRK1.1 K48M, mpk6, and PTP1 S7AS8A under submergence. These results reveal insights into the function of SnRK1 and the downstream signalling factors related to submergence. PMID:27029354

  13. Phosphorylation and linear ubiquitin direct A20 inhibition of inflammation.

    PubMed

    Wertz, Ingrid E; Newton, Kim; Seshasayee, Dhaya; Kusam, Saritha; Lam, Cynthia; Zhang, Juan; Popovych, Nataliya; Helgason, Elizabeth; Schoeffler, Allyn; Jeet, Surinder; Ramamoorthi, Nandhini; Kategaya, Lorna; Newman, Robert J; Horikawa, Keisuke; Dugger, Debra; Sandoval, Wendy; Mukund, Susmith; Zindal, Anuradha; Martin, Flavius; Quan, Clifford; Tom, Jeffrey; Fairbrother, Wayne J; Townsend, Michael; Warming, Søren; DeVoss, Jason; Liu, Jinfeng; Dueber, Erin; Caplazi, Patrick; Lee, Wyne P; Goodnow, Christopher C; Balazs, Mercedesz; Yu, Kebing; Kolumam, Ganesh; Dixit, Vishva M

    2015-12-17

    Inactivation of the TNFAIP3 gene, encoding the A20 protein, is associated with critical inflammatory diseases including multiple sclerosis, rheumatoid arthritis and Crohn's disease. However, the role of A20 in attenuating inflammatory signalling is unclear owing to paradoxical in vitro and in vivo findings. Here we utilize genetically engineered mice bearing mutations in the A20 ovarian tumour (OTU)-type deubiquitinase domain or in the zinc finger-4 (ZnF4) ubiquitin-binding motif to investigate these discrepancies. We find that phosphorylation of A20 promotes cleavage of Lys63-linked polyubiquitin chains by the OTU domain and enhances ZnF4-mediated substrate ubiquitination. Additionally, levels of linear ubiquitination dictate whether A20-deficient cells die in response to tumour necrosis factor. Mechanistically, linear ubiquitin chains preserve the architecture of the TNFR1 signalling complex by blocking A20-mediated disassembly of Lys63-linked polyubiquitin scaffolds. Collectively, our studies reveal molecular mechanisms whereby A20 deubiquitinase activity and ubiquitin binding, linear ubiquitination, and cellular kinases cooperate to regulate inflammation and cell death. PMID:26649818

  14. Brain phosphorylation of MeCP2 at serine 164 is developmentally regulated and globally alters its chromatin association

    PubMed Central

    Stefanelli, Gilda; Gandaglia, Anna; Costa, Mario; Cheema, Manjinder S.; Di Marino, Daniele; Barbiero, Isabella; Kilstrup-Nielsen, Charlotte; Ausió, Juan; Landsberger, Nicoletta

    2016-01-01

    MeCP2 is a transcriptional regulator whose functional alterations are responsible for several autism spectrum and mental disorders. Post-translational modifications (PTMs), and particularly differential phosphorylation, modulate MeCP2 function in response to diverse stimuli. Understanding the detailed role of MeCP2 phosphorylation is thus instrumental to ascertain how MeCP2 integrates the environmental signals and directs its adaptive transcriptional responses. The evolutionarily conserved serine 164 (S164) was found phosphorylated in rodent brain but its functional role has remained uncharacterized. We show here that phosphorylation of S164 in brain is dynamically regulated during neuronal maturation. S164 phosphorylation highly impairs MeCP2 binding to DNA in vitro and largely affects its nucleosome binding and chromatin affinity in vivo. Strikingly, the chromatin-binding properties of the global MeCP2 appear also extensively altered during the course of brain maturation. Functional assays reveal that proper temporal regulation of S164 phosphorylation controls the ability of MeCP2 to regulate neuronal morphology. Altogether, our results support the hypothesis of a complex PTM-mediated functional regulation of MeCP2 potentially involving a still poorly characterized epigenetic code. Furthermore, they demonstrate the relevance of the Intervening Domain of MeCP2 for binding to DNA. PMID:27323888

  15. Brain phosphorylation of MeCP2 at serine 164 is developmentally regulated and globally alters its chromatin association.

    PubMed

    Stefanelli, Gilda; Gandaglia, Anna; Costa, Mario; Cheema, Manjinder S; Di Marino, Daniele; Barbiero, Isabella; Kilstrup-Nielsen, Charlotte; Ausió, Juan; Landsberger, Nicoletta

    2016-01-01

    MeCP2 is a transcriptional regulator whose functional alterations are responsible for several autism spectrum and mental disorders. Post-translational modifications (PTMs), and particularly differential phosphorylation, modulate MeCP2 function in response to diverse stimuli. Understanding the detailed role of MeCP2 phosphorylation is thus instrumental to ascertain how MeCP2 integrates the environmental signals and directs its adaptive transcriptional responses. The evolutionarily conserved serine 164 (S164) was found phosphorylated in rodent brain but its functional role has remained uncharacterized. We show here that phosphorylation of S164 in brain is dynamically regulated during neuronal maturation. S164 phosphorylation highly impairs MeCP2 binding to DNA in vitro and largely affects its nucleosome binding and chromatin affinity in vivo. Strikingly, the chromatin-binding properties of the global MeCP2 appear also extensively altered during the course of brain maturation. Functional assays reveal that proper temporal regulation of S164 phosphorylation controls the ability of MeCP2 to regulate neuronal morphology. Altogether, our results support the hypothesis of a complex PTM-mediated functional regulation of MeCP2 potentially involving a still poorly characterized epigenetic code. Furthermore, they demonstrate the relevance of the Intervening Domain of MeCP2 for binding to DNA. PMID:27323888

  16. FSCB phosphorylation regulates mouse spermatozoa capacitation through suppressing SUMOylation of ROPN1/ROPN1L

    PubMed Central

    Zhang, Xinqi; Chen, Mingrui; Yu, Renyi; Liu, Benli; Tian, Zhiqiang; Liu, Shunli

    2016-01-01

    Fibrous sheath CABYR binding protein (FSCB) is regulated by protein kinase A (PKA)-mediated tyrosine phosphorylation in the spermatozoa capacitation. Recently, we showed that FSCB phosphorylation activated spermatozoa motility. Nevertheless, the underlying mechanisms have not been completely elucidated. Here, we showed that FSCB phosphorylation inhibited SUMOylation of two crucial proteins ROPN1/ROPN1L that are associated with PKA/A kinase activity and spermatozoa motility. Suppression of SUMOylation of ROPN1/ROPN1L mimicked the effects of FSCB phosphorylation on spermatozoa motility. Immunoprecipitation assay showed that phosphorylated FSCB had a significantly higher affinity to ROPN1/ROPN1L than non-phosphorylated FSCB. Together, our data suggest that FSCB phosphorylation may regulate mouse spermatozoa capacitation through suppressing SUMOylation of ROPN1/ROPN1L, which sheds new light on creating a therapeutic strategy targeting FSCB phosphorylation in the study of infertility.

  17. Mouse Sphingosine Kinase 1a Is Negatively Regulated through Conventional PKC-Dependent Phosphorylation at S373 Residue

    PubMed Central

    Oh, Yong-Seok; Bae, Sun Sik; Park, Jong Bae; Ha, Sang Hoon; Ryu, Sung Ho; Suh, Pann-Ghill

    2015-01-01

    Sphingosine kinase is a lipid kinase that converts sphingosine into sphingosine-1-phosphate, an important signaling molecule with intracellular and extracellular functions. Although diverse extracellular stimuli influence cellular sphingosine kinase activity, the molecular mechanisms underlying its regulation remain to be clarified. In this study, we investigated the phosphorylation-dependent regulation of mouse sphingosine kinase (mSK) isoforms 1 and 2. mSK1a was robustly phosphorylated in response to extracellular stimuli such as phorbol ester, whereas mSK2 exhibited a high basal level of phosphorylation in quiescent cells regardless of agonist stimulation. Interestingly, phorbol ester-induced phosphorylation of mSK1a correlated with suppression of its activity. Chemical inhibition of conventional PKCs (cPKCs) abolished mSK1a phosphorylation, while overexpression of PKCα, a cPKC isoform, potentiated the phosphorylation, in response to phorbol ester. Furthermore, an in vitro kinase assay showed that PKCα directly phosphorylated mSK1a. In addition, phosphopeptide mapping analysis determined that the S373 residue of mSK1a was the only site phosphorylated by cPKC. Interestingly, alanine substitution of S373 made mSK1a refractory to the inhibitory effect of phorbol esters, whereas glutamate substitution of the same residue resulted in a significant reduction in mSK1a activity, suggesting the significant role of this phosphorylation event. Taken together, we propose that mSK1a is negatively regulated through cPKC-dependent phosphorylation at S373 residue. PMID:26642194

  18. Signaling-dependent Phosphorylation of Mitotic Centromere-associated Kinesin Regulates Microtubule Depolymerization and Its Centrosomal Localization*

    PubMed Central

    Pakala, Suresh B.; Nair, Vasudha S.; Reddy, Sirigiri DivijendraNatha; Kumar, Rakesh

    2012-01-01

    Although p21-activated kinase 1 (PAK1) and microtubule (MT) dynamics regulate numerous fundamental processes including cytoskeleton remodeling, directional motility, and mitotic functions, the significance of PAK1 signaling in regulating the functions of MT-destabilizing protein mitotic centromere-associated kinesin (MCAK) remains unknown. Here we found that MCAK is a cognate substrate of PAK1 wherein PAK1 phosphorylates MCAK on serines 192 and 111 both in vivo and in vitro. Furthermore, we found that PAK1 phosphorylation of MCAK on serines 192 and 111 preferentially regulates its microtubule depolymerization activity and localization to centrosomes, respectively, in the mammalian cells. PMID:23055517

  19. How Phosphorylation and ATPase Activity Regulate Anion Flux though the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

    PubMed

    Zwick, Matthias; Esposito, Cinzia; Hellstern, Manuel; Seelig, Anna

    2016-07-01

    The cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), mutations of which cause cystic fibrosis, belongs to the ATP-binding cassette (ABC) transporter family and works as a channel for small anions, such as chloride and bicarbonate. Anion channel activity is known to depend on phosphorylation by cAMP-dependent protein kinase A (PKA) and CFTR-ATPase activity. Whereas anion channel activity has been extensively investigated, phosphorylation and CFTR-ATPase activity are still poorly understood. Here, we show that the two processes can be measured in a label-free and non-invasive manner in real time in live cells, stably transfected with CFTR. This study reveals three key findings. (i) The major contribution (≥90%) to the total CFTR-related ATP hydrolysis rate is due to phosphorylation by PKA and the minor contribution (≤10%) to CFTR-ATPase activity. (ii) The mutant CFTR-E1371S that is still conductive, but defective in ATP hydrolysis, is not phosphorylated, suggesting that phosphorylation requires a functional nucleotide binding domain and occurs in the post-hydrolysis transition state. (iii) CFTR-ATPase activity is inversely related to CFTR anion flux. The present data are consistent with a model in which CFTR is in a closed conformation with two ATPs bound. The open conformation is induced by ATP hydrolysis and corresponds to the post-hydrolysis transition state that is stabilized by phosphorylation and binding of chloride channel potentiators. PMID:27226582

  20. G Protein-Coupled Receptors Directly Bind Filamin A with High Affinity and Promote Filamin Phosphorylation

    PubMed Central

    2015-01-01

    Although interaction of a few G protein-coupled receptors (GPCRs) with Filamin A, a key actin cross-linking and biomechanical signal transducer protein, has been observed, a comprehensive structure–function analysis of this interaction is lacking. Through a systematic sequence-based analysis, we found that a conserved filamin binding motif is present in the cytoplasmic domains of >20% of the 824 GPCRs encoded in the human genome. Direct high-affinity interaction of filamin binding motif peptides of select GPCRs with the Ig domain of Filamin A was confirmed by nuclear magnetic resonance spectroscopy and isothermal titration calorimetric experiments. Engagement of the filamin binding motif with the Filamin A Ig domain induced the phosphorylation of filamin by protein kinase A in vitro. In transfected cells, agonist activation as well as constitutive activation of representative GPCRs dramatically elicited recruitment and phosphorylation of cellular Filamin A, a phenomenon long known to be crucial for regulating the structure and dynamics of the cytoskeleton. Our data suggest a molecular mechanism for direct GPCR–cytoskeleton coupling via filamin. Until now, GPCR signaling to the cytoskeleton was predominantly thought to be indirect, through canonical G protein-mediated signaling cascades involving GTPases, adenylyl cyclases, phospholipases, ion channels, and protein kinases. We propose that the GPCR-induced filamin phosphorylation pathway is a conserved, novel biochemical signaling paradigm. PMID:26460884

  1. Auxin-regulated changes in protein phosphorylation in pea epicotyl segments

    SciTech Connect

    Reddy, A.S.N.; Chengappa, S.; Raghothama, K.G.; Poovaiah, B.W.

    1987-04-01

    Auxin-regulated changes in protein phosphorylation were studied by labeling pea epicotyl segments with (/sup 32/P) PO/sub 4//sup 3 -/ and analyzing the phosphoproteins by two dimensional (2-D) gel electrophoresis. Analysis of phosphoproteins revealed auxin-regulated changes in the phosphorylation of specific polypeptides. In the presence of auxin, phosphorylation of 23,000, 82,000, 105,000 and 110,000 molecular weight polypeptides was markedly decreased whereas phosphorylation of 19,000, 24,000, 28,000 molecular weight polypeptides was increased. Some of these changes are very rapid and could be observed within minutes. Furthermore, their studies with calmodulin antagonists indicate the possible involvement of calmodulin-dependent protein kinases and/or phosphatases in auxin-regulated changes in protein phosphorylation. In view of these results, they suggest that auxin-regulated protein phosphorylation could be the one of the earliest events in regulating diverse physiological processes by this hormone.

  2. Regulation of the cystic fibrosis transmembrane conductance regulator anion channel by tyrosine phosphorylation.

    PubMed

    Billet, Arnaud; Jia, Yanlin; Jensen, Tim; Riordan, John R; Hanrahan, John W

    2015-09-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) channel is activated by PKA phosphorylation of a regulatory domain that interacts dynamically with multiple CFTR domains and with other proteins. The large number of consensus sequences for phosphorylation by PKA has naturally focused most attention on regulation by this kinase. We report here that human CFTR is also phosphorylated by the tyrosine kinases p60c-Src (proto-oncogene tyrosine-protein kinase) and the proline-rich tyrosine kinase 2 (Pyk2), and they can also cause robust activation of quiescent CFTR channels. In excised patch-clamp experiments, CFTR activity during exposure to Src or Pyk2 reached ∼80% of that stimulated by PKA. Exposure to PKA after Src or Pyk2 caused a further increase to the level induced by PKA alone, implying a common limiting step. Channels became spontaneously active when v-Src or the catalytic domain of Pyk2 was coexpressed with CFTR and were further stimulated by the tyrosine phosphatase inhibitor dephostatin. Exogenous Src also activated 15SA-CFTR, a variant that lacks 15 potential PKA sites and has little response to PKA. PKA-independent activation by tyrosine phosphorylation has implications for the mechanism of regulation by the R domain and for the physiologic functions of CFTR. PMID:26062600

  3. Pknox1/Prep1 regulates mitochondrial oxidative phosphorylation components in skeletal muscle.

    PubMed

    Kanzleiter, Timo; Rath, Michaela; Penkov, Dmitry; Puchkov, Dmytro; Schulz, Nadja; Blasi, Francesco; Schürmann, Annette

    2014-01-01

    The homeodomain transcription factor Prep1 was previously shown to regulate insulin sensitivity. Our aim was to study the specific role of Prep1 for the regulation of energy metabolism in skeletal muscle. Muscle-specific ablation of Prep1 resulted in increased expression of respiratory chain subunits. This finding was consistent with an increase in mitochondrial enzyme activity without affecting mitochondrial volume fraction as assessed by electron microscopy. Metabolic phenotyping revealed no differences in daily energy expenditure or body composition. However, during treadmill exercise challenge, Prep1 ablation resulted in a higher maximal oxidative capacity and better endurance. Elevated PGC-1α expression was identified as a cause for increased mitochondrial capacity in Prep1 ablated mice. Prep1 stabilizes p160 Mybbp1a, a known inhibitor of PGC-1α activity. Thereby, p160 protein levels were significantly lower in the muscle of Prep1 ablated mice. By a chromatin immunoprecipitation-sequencing (ChIP-seq) approach, PREP1 binding sites in genes encoding mitochondrial components (e.g., Ndufs2) were identified that might be responsible for elevated proteins involved in oxidative phosphorylation (OXPHOS) in the muscle of Prep1 null mutants. These results suggest that Prep1 exhibits additional direct effects on regulation of mitochondrial proteins. We therefore conclude that Prep1 is a regulator of oxidative phosphorylation components via direct and indirect mechanisms. PMID:24216763

  4. Pknox1/Prep1 Regulates Mitochondrial Oxidative Phosphorylation Components in Skeletal Muscle

    PubMed Central

    Rath, Michaela; Penkov, Dmitry; Puchkov, Dmytro; Schulz, Nadja; Blasi, Francesco; Schürmann, Annette

    2014-01-01

    The homeodomain transcription factor Prep1 was previously shown to regulate insulin sensitivity. Our aim was to study the specific role of Prep1 for the regulation of energy metabolism in skeletal muscle. Muscle-specific ablation of Prep1 resulted in increased expression of respiratory chain subunits. This finding was consistent with an increase in mitochondrial enzyme activity without affecting mitochondrial volume fraction as assessed by electron microscopy. Metabolic phenotyping revealed no differences in daily energy expenditure or body composition. However, during treadmill exercise challenge, Prep1 ablation resulted in a higher maximal oxidative capacity and better endurance. Elevated PGC-1α expression was identified as a cause for increased mitochondrial capacity in Prep1 ablated mice. Prep1 stabilizes p160 Mybbp1a, a known inhibitor of PGC-1α activity. Thereby, p160 protein levels were significantly lower in the muscle of Prep1 ablated mice. By a chromatin immunoprecipitation-sequencing (ChIP-seq) approach, PREP1 binding sites in genes encoding mitochondrial components (e.g., Ndufs2) were identified that might be responsible for elevated proteins involved in oxidative phosphorylation (OXPHOS) in the muscle of Prep1 null mutants. These results suggest that Prep1 exhibits additional direct effects on regulation of mitochondrial proteins. We therefore conclude that Prep1 is a regulator of oxidative phosphorylation components via direct and indirect mechanisms. PMID:24216763

  5. Phosphorylation of K[superscript +] Channels at Single Residues Regulates Memory Formation

    ERIC Educational Resources Information Center

    Vernon, Jeffrey; Irvine, Elaine E.; Peters, Marco; Jeyabalan, Jeshmi; Giese, K. Peter

    2016-01-01

    Phosphorylation is a ubiquitous post-translational modification of proteins, and a known physiological regulator of K[superscript +] channel function. Phosphorylation of K[superscript +] channels by kinases has long been presumed to regulate neuronal processing and behavior. Although circumstantial evidence has accumulated from behavioral studies…

  6. Dynamic regulation of FGF23 by Fam20C phosphorylation, GalNAc-T3 glycosylation, and furin proteolysis

    PubMed Central

    Tagliabracci, Vincent S.; Engel, James L.; Wiley, Sandra E.; Xiao, Junyu; Gonzalez, David J.; Nidumanda Appaiah, Hitesh; Koller, Antonius; Nizet, Victor; White, Kenneth E.; Dixon, Jack E.

    2014-01-01

    The family with sequence similarity 20, member C (Fam20C) has recently been identified as the Golgi casein kinase. Fam20C phosphorylates secreted proteins on Ser-x-Glu/pSer motifs and loss-of-function mutations in the kinase cause Raine syndrome, an often-fatal osteosclerotic bone dysplasia. Fam20C is potentially an upstream regulator of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23), because humans with FAM20C mutations and Fam20C KO mice develop hypophosphatemia due to an increase in full-length, biologically active FGF23. However, the mechanism by which Fam20C regulates FGF23 is unknown. Here we show that Fam20C directly phosphorylates FGF23 on Ser180, within the FGF23 R176XXR179/S180AE subtilisin-like proprotein convertase motif. This phosphorylation event inhibits O-glycosylation of FGF23 by polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-T3), and promotes FGF23 cleavage and inactivation by the subtilisin-like proprotein convertase furin. Collectively, our results provide a molecular mechanism by which FGF23 is dynamically regulated by phosphorylation, glycosylation, and proteolysis. Furthermore, our findings suggest that cross-talk between phosphorylation and O-glycosylation of proteins in the secretory pathway may be an important mechanism by which secreted proteins are regulated. PMID:24706917

  7. Phosphorylation-Dependent Regulation of the DNA Damage Response of Adaptor Protein KIBRA in Cancer Cells.

    PubMed

    Mavuluri, Jayadev; Beesetti, Swarnalatha; Surabhi, Rohan; Kremerskothen, Joachim; Venkatraman, Ganesh; Rayala, Suresh K

    2016-05-01

    Multifunctional adaptor proteins encompassing various protein-protein interaction domains play a central role in the DNA damage response pathway. In this report, we show that KIBRA is a physiologically interacting reversible substrate of ataxia telangiectasia mutated (ATM) kinase. We identified the site of phosphorylation in KIBRA as threonine 1006, which is embedded within the serine/threonine (S/T) Q consensus motif, by site-directed mutagenesis, and we further confirmed the same with a phospho-(S/T) Q motif-specific antibody. Results from DNA repair functional assays such as the γ-H2AX assay, pulsed-field gel electrophoresis (PFGE), Comet assay, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and clonogenic cell survival assay using stable overexpression clones of wild-type (wt.) KIBRA and active (T1006E) and inactive (T1006A) KIBRA phosphorylation mutants showed that T1006 phosphorylation on KIBRA is essential for optimal DNA double-strand break repair in cancer cells. Further, results from stable retroviral short hairpin RNA-mediated knockdown (KD) clones of KIBRA and KIBRA knockout (KO) model cells generated by a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that depleting KIBRA levels compromised the DNA repair functions in cancer cells upon inducing DNA damage. All these phenotypic events were reversed upon reconstitution of KIBRA into cells lacking KIBRA knock-in (KI) model cells. All these results point to the fact that phosphorylated KIBRA might be functioning as a scaffolding protein/adaptor protein facilitating the platform for further recruitment of other DNA damage response factors. In summary, these data demonstrate the imperative functional role of KIBRAper se(KIBRA phosphorylation at T1006 site as a molecular switch that regulates the DNA damage response, possibly via the nonhomologous end joining [NHEJ] pathway), suggesting that KIBRA could be a potential

  8. Nucleolin regulates phosphorylation and nuclear export of fibroblast growth factor 1 (FGF1).

    PubMed

    Sletten, Torunn; Kostas, Michal; Bober, Joanna; Sorensen, Vigdis; Yadollahi, Mandana; Olsnes, Sjur; Tomala, Justyna; Otlewski, Jacek; Zakrzewska, Malgorzata; Wiedlocha, Antoni

    2014-01-01

    Extracellular fibroblast growth factor 1 (FGF1) acts through cell surface tyrosine kinase receptors, but FGF1 can also act directly in the cell nucleus, as a result of nuclear import of endogenously produced, non-secreted FGF1 or by transport of extracellular FGF1 via endosomes and cytosol into the nucleus. In the nucleus, FGF1 can be phosphorylated by protein kinase C δ (PKCδ), and this event induces nuclear export of FGF1. To identify intracellular targets of FGF1 we performed affinity pull-down assays and identified nucleolin, a nuclear multifunctional protein, as an interaction partner of FGF1. We confirmed a direct nucleolin-FGF1 interaction by surface plasmon resonance and identified residues of FGF1 involved in the binding to be located within the heparin binding site. To assess the biological role of the nucleolin-FGF1 interaction, we studied the intracellular trafficking of FGF1. In nucleolin depleted cells, exogenous FGF1 was endocytosed and translocated to the cytosol and nucleus, but FGF1 was not phosphorylated by PKCδ or exported from the nucleus. Using FGF1 mutants with reduced binding to nucleolin and a FGF1-phosphomimetic mutant, we showed that the nucleolin-FGF1 interaction is critical for the intranuclear phosphorylation of FGF1 by PKCδ and thereby the regulation of nuclear export of FGF1. PMID:24595027

  9. Motile properties of the bi-directional kinesin-5 Cin8 are affected by phosphorylation in its motor domain

    PubMed Central

    Shapira, Ofer; Gheber, Larisa

    2016-01-01

    The Saccharomyces cerevisiae kinesin-5 Cin8 performs essential mitotic functions in spindle assembly and anaphase B spindle elongation. Recent work has shown that Cin8 is a bi-directional motor which moves towards the minus-end of microtubules (MTs) under high ionic strength (IS) conditions and changes directionality in low IS conditions and when bound between anti-parallel microtubules. Previous work from our laboratory has also indicated that Cin8 is differentially phosphorylated during late anaphase at cyclin-dependent kinase 1 (Cdk1)-specific sites located in its motor domain. In vivo, such phosphorylation causes Cin8 detachment from spindles and reduces the spindle elongation rate, while maintaining proper spindle morphology. To study the effect of phosphorylation on Cin8 motor function, we examined in vitro motile properties of wild type Cin8, as well as its phosphorylation using phospho-deficient and phospho-mimic variants, in a single molecule fluorescence motility assay. Analysis was performed on whole cell extracts and on purified Cin8 samples. We found that addition of negative charges in the phospho-mimic mutant weakened the MT-motor interaction, increased motor velocity and promoted minus-end-directed motility. These results indicate that phosphorylation in the catalytic domain of Cin8 regulates its motor function. PMID:27216310

  10. Phosphorylation of KasB Regulates Virulence and Acid-Fastness in Mycobacterium tuberculosis

    PubMed Central

    Vilchèze, Catherine; Molle, Virginie; Carrère-Kremer, Séverine; Leiba, Jade; Mourey, Lionel; Shenai, Shubhada; Baronian, Grégory; Tufariello, Joann; Hartman, Travis; Veyron-Churlet, Romain; Trivelli, Xavier; Tiwari, Sangeeta; Weinrick, Brian; Alland, David; Guérardel, Yann; Jacobs, William R.; Kremer, Laurent

    2014-01-01

    Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4–6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of Ser/Thr kinase

  11. Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1.

    PubMed

    Callender, Tracy L; Laureau, Raphaelle; Wan, Lihong; Chen, Xiangyu; Sandhu, Rima; Laljee, Saif; Zhou, Sai; Suhandynata, Ray T; Prugar, Evelyn; Gaines, William A; Kwon, YoungHo; Börner, G Valentin; Nicolas, Alain; Neiman, Aaron M; Hollingsworth, Nancy M

    2016-08-01

    During meiosis, programmed double strand breaks (DSBs) are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH) bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i) phosphorylation of Rad54 by Mek1 and (ii) binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1. PMID:27483004

  12. Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1

    PubMed Central

    Callender, Tracy L.; Laljee, Saif; Zhou, Sai; Suhandynata, Ray T.; Gaines, William A.; Kwon, YoungHo; Börner, G. Valentin; Nicolas, Alain; Neiman, Aaron M.

    2016-01-01

    During meiosis, programmed double strand breaks (DSBs) are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH) bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i) phosphorylation of Rad54 by Mek1 and (ii) binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1. PMID:27483004

  13. Cholinergic regulation of protein phosphorylation in bovine adrenal chromaffin cells

    SciTech Connect

    Haycock, J.W.; Browning, M.D.; Greengard, P.

    1988-03-01

    Chromaffin cells were isolated from bovine adrenal medullae and maintained in primary culture. After prelabeling with /sup 32/PO/sub 4/, exposure of the chromaffin cells to acetylcholine increased the phosphorylation of a M/sub r/ approx. = 100,000 protein and a M/sub r/ approx. = 60,000 protein (tyrosine hydroxylase), visualized after separation of total cellular proteins in NaDodSO/sub 4//polyacrylamide gels. Immunoprecipitation with antibodies to three known phosphoproteins (100-kDa, 87-kDa, and protein III) revealed an acetylcholine-dependent phosphorylation of these proteins. These three proteins were also shown to be present in bovine adrenal chromaffin cells by immunolabeling techniques. 100-kDa is a M/sub r/ approx. = 100,000 protein selectively phosphorylated by calcium/calmodulin-dependent protein kinase III, 87-kDa is a M/sub r/ approx. = 87,000 protein selectively phosphorylated by protein kinase C, and protein III is a phosphoprotein doublet of M/sub r/ approx. = 74,000 (IIIa) and M/sub r/ approx. = 55,000 (IIIb) phosphorylated by cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase I. The data demonstrate that cholinergic activation of chromaffin cells increases the phosphorylation of several proteins and that several protein kinase systems may be involved in these effects.

  14. Regulation of AMPA receptor extrasynaptic insertion by 4.1N, phosphorylation and palmitoylation.

    PubMed

    Lin, Da-Ting; Makino, Yuichi; Sharma, Kamal; Hayashi, Takashi; Neve, Rachael; Takamiya, Kogo; Huganir, Richard L

    2009-07-01

    The insertion of AMPA receptors (AMPARs) into the plasma membrane is an important step in the synaptic delivery of AMPARs during the expression of synaptic plasticity. However, the molecular mechanisms regulating AMPAR insertion remain elusive. By directly visualizing individual insertion events of the AMPAR subunit GluR1 in rodents, we found that the protein 4.1N was required for activity-dependent GluR1 insertion. Protein kinase C (PKC) phosphorylation of the serine 816 (S816) and S818 residues of GluR1 enhanced 4.1N binding to GluR1 and facilitated GluR1 insertion. In addition, palmitoylation of GluR1 C811 residue modulated PKC phosphorylation and GluR1 insertion. Finally, disrupting 4.1N-dependent GluR1 insertion decreased surface expression of GluR1 and the expression of long-term potentiation. Our study uncovers a previously unknown mechanism that governs activity-dependent GluR1 trafficking, reveals an interaction between AMPAR palmitoylation and phosphorylation, and underscores the functional importance of 4.1N in AMPAR trafficking and synaptic plasticity. PMID:19503082

  15. Regulation of AMPA receptor extrasynaptic insertion by 4.1N, phosphorylation and palmitoylation

    PubMed Central

    Lin, Da–Ting; Makino, Yuichi; Sharma, Kamal; Hayashi, Takashi; Neve, Rachael; Takamiya, Kogo; Huganir, Richard L.

    2009-01-01

    The insertion of alpha–amino–3–hydroxy–5–methyl–4–isoxazolepropionic acid receptors (AMPARs) into the plasma membrane is a key step in synaptic delivery of AMPARs during the expression of synaptic plasticity. However, the molecular mechanisms regulating AMPAR insertion remain elusive. By directly visualizing individual insertion events of the AMPAR subunit GluR1, we demonstrate that Protein 4.1N is required for activity dependent GluR1 insertion. PKC phosphorylation of GluR1 S816 and S818 residues enhances 4.1N binding to GluR1, and facilitates GluR1 insertion. In addition, palmitoylation of GluR1 C811 residue modulates PKC phosphorylation and GluR1 insertion. Finally, disrupting 4.1N dependent GluR1 insertion decreases surface expression of GluR1 and the expression of long–term potentiation (LTP). Our study uncovers a novel mechanism that governs activity dependent GluR1 trafficking, reveals an interesting interplay between AMPAR palmitoylation and phosphorylation, and underscores the functional significance of the 4.1N protein in AMPAR trafficking and synaptic plasticity. PMID:19503082

  16. Regulation of photoreceptor gap junction phosphorylation by adenosine in zebrafish retina

    PubMed Central

    Li, Hongyan; Chuang, Alice Z.; O’Brien, John

    2014-01-01

    Electrical coupling of photoreceptors through gap junctions suppresses voltage noise, routes rod signals into cone pathways, expands the dynamic range of rod photoreceptors in high scotopic and mesopic illumination, and improves detection of contrast and small stimuli. In essentially all vertebrates, connexin 35/36 (gene homologues Cx36 in mammals, Cx35 in other vertebrates) is the major gap junction protein observed in photoreceptors, mediating rod-cone, cone-cone, and possibly rod-rod communication. Photoreceptor coupling is dynamically controlled by the day/night cycle and light/dark adaptation, and is directly correlated with phosphorylation of Cx35/36 at two sites, serine110 and serine 276/293 (homologous sites in teleost fish and mammals respectively). Activity of protein kinase A (PKA) plays a key role during this process. Previous studies have shown that activation of dopamine D4 receptors on photoreceptors inhibits adenylyl cyclase, down-regulates cAMP and PKA activity, and leads to photoreceptor uncoupling, imposing the daytime/light condition. In this study we explored the role of adenosine, a nighttime signal with a high extracellular concentration at night and a low concentration in the day, in regulating photoreceptor coupling by examining photoreceptor Cx35 phosphorylation in zebrafish retina. Adenosine enhanced photoreceptor Cx35 phosphorylation in daytime, but with a complex dose-response curve. Selective pharmacological manipulations revealed that adenosine A2a receptors provide a potent positive drive to phosphorylate photoreceptor Cx35 under the influence of endogenous adenosine at night. A2a receptors can be activated in the daytime as well by micromolar exogenous adenosine. However, the higher affinity adenosine A1 receptors are also present and have an antagonistic though less potent effect. Thus the nighttime/darkness signal adenosine provides a net positive drive on Cx35 phosphorylation at night, working in opposition to dopamine to

  17. Regulation of photoreceptor gap junction phosphorylation by adenosine in zebrafish retina.

    PubMed

    Li, Hongyan; Chuang, Alice Z; O'Brien, John

    2014-05-01

    Electrical coupling of photoreceptors through gap junctions suppresses voltage noise, routes rod signals into cone pathways, expands the dynamic range of rod photoreceptors in high scotopic and mesopic illumination, and improves detection of contrast and small stimuli. In essentially all vertebrates, connexin 35/36 (gene homologs Cx36 in mammals, Cx35 in other vertebrates) is the major gap junction protein observed in photoreceptors, mediating rod-cone, cone-cone, and possibly rod-rod communication. Photoreceptor coupling is dynamically controlled by the day/night cycle and light/dark adaptation, and is directly correlated with phosphorylation of Cx35/36 at two sites, serine110 and serine 276/293 (homologous sites in teleost fish and mammals, respectively). Activity of protein kinase A (PKA) plays a key role during this process. Previous studies have shown that activation of dopamine D4 receptors on photoreceptors inhibits adenylyl cyclase, down-regulates cAMP and PKA activity, and leads to photoreceptor uncoupling, imposing the daytime/light condition. In this study, we explored the role of adenosine, a nighttime signal with a high extracellular concentration at night and a low concentration in the day, in regulating photoreceptor coupling by examining photoreceptor Cx35 phosphorylation in zebrafish retina. Adenosine enhanced photoreceptor Cx35 phosphorylation in daytime, but with a complex dose-response curve. Selective pharmacological manipulations revealed that adenosine A2a receptors provide a potent positive drive to phosphorylate photoreceptor Cx35 under the influence of endogenous adenosine at night. A2a receptors can be activated in the daytime as well by micromolar exogenous adenosine. However, the higher affinity adenosine A1 receptors are also present and have an antagonistic though less potent effect. Thus, the nighttime/darkness signal adenosine provides a net positive drive on Cx35 phosphorylation at night, working in opposition to dopamine to

  18. Abundant protein phosphorylation potentially regulates Arabidopsis anther development

    PubMed Central

    Ye, Juanying; Zhang, Zaibao; You, Chenjiang; Zhang, Xumin; Lu, Jianan; Ma, Hong

    2016-01-01

    As the male reproductive organ of flowering plants, the stamen consists of the anther and filament. Previous studies on stamen development mainly focused on single gene functions by genetic methods or gene expression changes using comparative transcriptomic approaches, especially in model plants such as Arabidopsis thaliana. However, studies on Arabidopsis anther protein expression and post-translational modifications are still lacking. Here we report proteomic and phosphoproteomic studies on developing Arabidopsis anthers at stages 4–7 and 8–12. We identified 3908 high-confidence phosphorylation sites corresponding to 1637 phosphoproteins. Among the 1637 phosphoproteins, 493 were newly identified, with 952 phosphorylation sites. Phosphopeptide enrichment prior to LC-MS analysis facilitated the identification of low-abundance proteins and regulatory proteins, thereby increasing the coverage of proteomic analysis, and facilitated the analysis of more regulatory proteins. Thirty-nine serine and six threonine phosphorylation motifs were uncovered from the anther phosphoproteome and further analysis supports that phosphorylation of casein kinase II, mitogen-activated protein kinases, and 14-3-3 proteins is a key regulatory mechanism in anther development. Phosphorylated residues were preferentially located in variable protein regions among family members, but they were they were conserved across angiosperms in general. Moreover, phosphorylation might reduce activity of reactive oxygen species scavenging enzymes and hamper brassinosteroid signaling in early anther development. Most of the novel phosphoproteins showed tissue-specific expression in the anther according to previous microarray data. This study provides a community resource with information on the abundance and phosphorylation status of thousands of proteins in developing anthers, contributing to understanding post-translational regulatory mechanisms during anther development. PMID:27531888

  19. Abundant protein phosphorylation potentially regulates Arabidopsis anther development.

    PubMed

    Ye, Juanying; Zhang, Zaibao; You, Chenjiang; Zhang, Xumin; Lu, Jianan; Ma, Hong

    2016-09-01

    As the male reproductive organ of flowering plants, the stamen consists of the anther and filament. Previous studies on stamen development mainly focused on single gene functions by genetic methods or gene expression changes using comparative transcriptomic approaches, especially in model plants such as Arabidopsis thaliana However, studies on Arabidopsis anther protein expression and post-translational modifications are still lacking. Here we report proteomic and phosphoproteomic studies on developing Arabidopsis anthers at stages 4-7 and 8-12. We identified 3908 high-confidence phosphorylation sites corresponding to 1637 phosphoproteins. Among the 1637 phosphoproteins, 493 were newly identified, with 952 phosphorylation sites. Phosphopeptide enrichment prior to LC-MS analysis facilitated the identification of low-abundance proteins and regulatory proteins, thereby increasing the coverage of proteomic analysis, and facilitated the analysis of more regulatory proteins. Thirty-nine serine and six threonine phosphorylation motifs were uncovered from the anther phosphoproteome and further analysis supports that phosphorylation of casein kinase II, mitogen-activated protein kinases, and 14-3-3 proteins is a key regulatory mechanism in anther development. Phosphorylated residues were preferentially located in variable protein regions among family members, but they were they were conserved across angiosperms in general. Moreover, phosphorylation might reduce activity of reactive oxygen species scavenging enzymes and hamper brassinosteroid signaling in early anther development. Most of the novel phosphoproteins showed tissue-specific expression in the anther according to previous microarray data. This study provides a community resource with information on the abundance and phosphorylation status of thousands of proteins in developing anthers, contributing to understanding post-translational regulatory mechanisms during anther development. PMID:27531888

  20. 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

  1. Kin of IRRE-like Protein 2 Is a Phosphorylated Glycoprotein That Regulates Basal Insulin Secretion.

    PubMed

    Yesildag, Burcak; Bock, Thomas; Herrmanns, Karolin; Wollscheid, Bernd; Stoffel, Markus

    2015-10-23

    Direct interactions among pancreatic β-cells via cell surface proteins inhibit basal and enhance stimulated insulin secretion. Here, we functionally and biochemically characterized Kirrel2, an immunoglobulin superfamily protein with β-cell-specific expression in the pancreas. Our results show that Kirrel2 is a phosphorylated glycoprotein that co-localizes and interacts with the adherens junction proteins E-cadherin and β-catenin in MIN6 cells. We further demonstrate that the phosphosites Tyr(595-596) are functionally relevant for the regulation of Kirrel2 stability and localization. Analysis of the extracellular and intracellular domains of Kirrel2 revealed that it is cleaved and shed from MIN6 cells and that the remaining membrane spanning cytoplasmic domain is processed by γ-secretase complex. Kirrel2 knockdown with RNA interference in MIN6 cells and ablation of Kirrel2 from mice with genetic deletion resulted in increased basal insulin secretion from β-cells, with no immediate influence on stimulated insulin secretion, total insulin content, or whole body glucose metabolism. Our results show that in pancreatic β-cells Kirrel2 localizes to adherens junctions, is regulated by multiple post-translational events, including glycosylation, extracellular cleavage, and phosphorylation, and engages in the regulation of basal insulin secretion. PMID:26324709

  2. C-terminal phosphorylation is essential for regulation of ethylene synthesizing ACC synthase enzyme.

    PubMed

    Choudhury, Swarup Roy; Roy, Sujit; Sengupta, Dibyendu N

    2013-02-01

    The genetic and molecular biological studies mainly in Arabidopsis and in some other plants have begun to uncover the various components of ripening signaling pathway in plants. Although transcriptional regulation of major ripening genes have been studied in detail, information on role of phosphorylation in regulating the activity and stability of core ripening pathway associated proteins in relation to ethylene biosynthesis during fruit ripening is still limited. Recently we have demonstrated the evidence for post-translational regulation of MA-ACS1 (Musa acuminata ACC synthase 1), the rate limiting step enzyme regulating ripening ethylene production in banana, through phosphorylation at the C-terminal Ser 476 and 479 residues by a 41-kDa Ser/Thr protein kinase. (1) Here we have further discussed role of protein phosphorylation in regulation of stability and activity of ACS enzymes and the mechanistic and evolutionary perspective of phosphorylation pattern of Type I ACC synthase enzymes. PMID:23221778

  3. Dual function of partitioning-defective 3 in the regulation of YAP phosphorylation and activation

    PubMed Central

    Zhang, Peng; Wang, Shuting; Wang, Sai; Qiao, Jing; Zhang, Lei; Zhang, Zhe; Chen, Zhengjun

    2016-01-01

    Partitioning-defective 3 (Par3), a key component of the evolutionarily conserved polarity PAR complex (Par3/Par6/aPKC), controls cell polarity and contributes to cell migration, proliferation and tumor development. Emerging evidence indicates that cell polarity proteins function as upstream modulators that regulate the Hippo pathway. However, little is known about Par3’s involvement in the Hippo pathway. Here, we find Par3 and YAP dynamically co-localize in different subcellular compartments; that is, the membrane, cytoplasm and nucleus, in a cell-density-dependent manner. Interestingly, Par3 knockdown promotes YAP phosphorylation, leading to a significant impairment of YAP nuclear translocation at low cell density, but not at high density, in MDCK cells. Furthermore, via its third PDZ domain, Par3 directly binds to the PDZ-binding motif of YAP. The interaction is required for regulating YAP phosphorylation and nuclear localization. Mechanistically, Par3, as a scaffold protein, associates with LATS1 and protein phosphatase 1, α subunit (PP1A) in the cytoplasm and nucleus. Par3 promotes the dephosphorylation of LATS1 and YAP, thus enhancing YAP activation and cell proliferation. Strikingly, we also find that under the condition of PP1A knockdown, Par3 expression promotes YAP hyperphosphorylation, leading to the suppression of YAP activity and its downstream targets. Par3 expression results in differential effects on YAP phosphorylation and activation in different tumor cell lines. These findings indicate that Par3 may have a dual role in regulating the activation of the Hippo pathway, in a manner possibly dependent on cellular context or cell type in response to cell–cell contact and cell polarity signals. PMID:27462467

  4. NHE3 phosphorylation via PKCη marks the polarity and orientation of directionally migrating cells

    PubMed Central

    Bola, Sharanya; Zhang, Lei; Reid, Brian; Fu, Guo; Funk, Richard H. W.; Zhao, Min

    2015-01-01

    Endogenous electric fields (EF) may provide an overriding cue for directional cell migration during wound closure. Perceiving a constant direction requires active sodium-hydrogen exchanger (pNHE3) at the leading edge of HEK 293 cells but its activation mechanism is not yet fully understood. Because protein kinase C (PKC) is required in electrotaxis, we asked whether NHE3 is activated by PKC during wound healing. Using pharmacological (pseudosubstrate and edelfosine) inhibition, we showed that inhibition of PKCη isoform impairs directional cell migration in HEK 293 cells in the presence of a persistent directional cue (0.25–0.3 V/mm of EF for 2 h). Further, we found that pNHE3 forms complexes with both PKCη and ɣ-tubulin, suggesting that these molecules may regulate the microtubule-organizing center. In addition, cellular pNHE3 content was reduced significantly when PKCη was inhibited during directional cell migration. Taken together, these data suggest that PKCη-dependent phosphorylation of NHE3 and the formation of pNHE3/PKCη/ɣ-tubulin complexes at the leading edge of the cell are required for directional cell migration in an EF. PMID:24788043

  5. HEY1 functions are regulated by its phosphorylation at Ser-68.

    PubMed

    López-Mateo, Irene; Arruabarrena-Aristorena, Amaia; Artaza-Irigaray, Cristina; López, Juan A; Calvo, Enrique; Belandia, Borja

    2016-07-01

    HEY1 (hairy/enhancer-of-split related with YRPW motif 1) is a member of the basic helix-loop-helix-orange (bHLH-O) family of transcription repressors that mediate Notch signalling. HEY1 acts as a positive regulator of the tumour suppressor p53 via still unknown mechanisms. A MALDI-TOF/TOF MS analysis has uncovered a novel HEY1 regulatory phosphorylation event at Ser-68. Strikingly, this single phosphorylation event controls HEY1 stability and function: simulation of HEY1 Ser-68 phosphorylation increases HEY1 protein stability but inhibits its ability to enhance p53 transcriptional activity. Unlike wild-type HEY1, expression of the phosphomimetic mutant HEY1-S68D failed to induce p53-dependent cell cycle arrest and it did not sensitize U2OS cells to p53-activating chemotherapeutic drugs. We have identified two related kinases, STK38 (serine/threonine kinase 38) and STK38L (serine/threonine kinase 38 like), which interact with and phosphorylate HEY1 at Ser-68. HEY1 is phosphorylated at Ser-68 during mitosis and it accumulates in the centrosomes of mitotic cells, suggesting a possible integration of HEY1-dependent signalling in centrosome function. Moreover, HEY1 interacts with a subset of p53-activating ribosomal proteins. Ribosomal stress causes HEY1 relocalization from the nucleoplasm to perinucleolar structures termed nucleolar caps. HEY1 interacts physically with at least one of the ribosomal proteins, RPL11, and both proteins cooperate in the inhibition of MDM2-mediated p53 degradation resulting in a synergistic positive effect on p53 transcriptional activity. HEY1 itself also interacts directly with MDM2 and it is subjected to MDM2-mediated degradation. Simulation of HEY1 Ser-68 phosphorylation prevents its interaction with p53, RPL11 and MDM2 and abolishes HEY1 migration to nucleolar caps upon ribosomal stress. Our findings uncover a novel mechanism for cross-talk between Notch signalling and nucleolar stress. PMID:27129302

  6. TCR-induced Akt serine 473 phosphorylation is regulated by protein kinase C-alpha

    SciTech Connect

    Yang, Lifen; Qiao, Guilin; Ying, Haiyan; Zhang, Jian; Yin, Fei

    2010-09-10

    Research highlights: {yields} Conventional PKC positively regulates TCR-induced phosphorylation of Akt. {yields} PKC-alpha is the PDK-2 responsible for phosphorylating Akt at Ser{sup 473} upon TCR stimulation. {yields} Knockdown of PKC-alpha decreases TCR-induced Akt phosphorylation. -- Abstract: Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser{sup 473} in the hydrophobic motif, along with Thr{sup 308} in its activation loop, is considered necessary for Akt function. It is widely accepted that phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr{sup 308}, but the kinase(s) responsible for phosphorylating Akt at Ser{sup 473} (PDK-2) remains elusive. The existence of PDK-2 is considered to be specific to cell type and stimulus. PDK-2 in T cells in response to TCR stimulation has not been clearly defined. In this study, we found that conventional PKC positively regulated TCR-induced Akt Ser{sup 473} phosphorylation. PKC-alpha purified from T cells can phosphorylate Akt at Ser{sup 473} in vitro upon TCR stimulation. Knockdown of PKC-alpha in T-cell-line Jurkat cells reduced TCR-induced phosphorylation of Akt as well as its downstream targets. Thus our results suggest that PKC-alpha is a candidate for PDK-2 in T cells upon TCR stimulation.

  7. Tyrosine Phosphorylation of SGEF Regulates RhoG Activity and Cell Migration

    PubMed Central

    Okuyama, Yusuke; Umeda, Kentaro; Negishi, Manabu; Katoh, Hironori

    2016-01-01

    SGEF and Ephexin4 are members of the Ephexin subfamily of RhoGEFs that specifically activate the small GTPase RhoG. It is reported that Ephexin1 and Ephexin5, two well-characterized Ephexin subfamily RhoGEFs, are tyrosine-phosphorylated by Src, and that their phosphorylation affect their activities and functions. In this study, we show that SGEF, but not Ephexin4, is tyrosine-phosphorylated by Src. Tyrosine phosphorylation of SGEF suppresses its interaction with RhoG, the elevation of RhoG activity, and SGEF-mediated promotion of cell migration. We identified tyrosine 530 (Y530), which is located within the Dbl homology domain, as a major phosphorylation site of SGEF by Src, and Y530F mutation blocked the inhibitory effect of Src on SGEF. Taken together, these results suggest that the activity of SGEF is negatively regulated by tyrosine phosphorylation of the DH domain. PMID:27437949

  8. Phosphorylation events in the multiple gene regulator of group A Streptococcus significantly influence global gene expression and virulence.

    PubMed

    Sanson, Misu; Makthal, Nishanth; Gavagan, Maire; Cantu, Concepcion; Olsen, Randall J; Musser, James M; Kumaraswami, Muthiah

    2015-06-01

    Whole-genome sequencing analysis of ∼800 strains of group A Streptococcus (GAS) found that the gene encoding the multiple virulence gene regulator of GAS (mga) is highly polymorphic in serotype M59 strains but not in strains of other serotypes. To help understand the molecular mechanism of gene regulation by Mga and its contribution to GAS pathogenesis in serotype M59 GAS, we constructed an isogenic mga mutant strain. Transcriptome studies indicated a significant regulatory influence of Mga and altered metabolic capabilities conferred by Mga-regulated genes. We assessed the phosphorylation status of Mga in GAS cell lysates with Phos-tag gels. The results revealed that Mga is phosphorylated at histidines in vivo. Using phosphomimetic and nonphosphomimetic substitutions at conserved phosphoenolpyruvate:carbohydrate phosphotransferase regulation domain (PRD) histidines of Mga, we demonstrated that phosphorylation-mimicking aspartate replacements at H207 and H273 of PRD-1 and at H327 of PRD-2 are inhibitory to Mga-dependent gene expression. Conversely, non-phosphorylation-mimicking alanine substitutions at H273 and H327 relieved inhibition, and the mutant strains exhibited a wild-type phenotype. The opposing regulatory profiles observed for phosphorylation- and non-phosphorylation-mimicking substitutions at H273 extended to global gene regulation by Mga. Consistent with these observations, the H273D mutant strain attenuated GAS virulence, whereas the H273A strain exhibited a wild-type virulence phenotype in a mouse model of necrotizing fasciitis. Together, our results demonstrate phosphoregulation of Mga and its direct link to virulence in M59 GAS strains. These data also lay a foundation toward understanding how naturally occurring gain-of-function variations in mga, such as H201R, may confer an advantage to the pathogen and contribute to M59 GAS pathogenesis. PMID:25824840

  9. Phosphorylation Events in the Multiple Gene Regulator of Group A Streptococcus Significantly Influence Global Gene Expression and Virulence

    PubMed Central

    Sanson, Misu; Makthal, Nishanth; Gavagan, Maire; Cantu, Concepcion; Olsen, Randall J.; Musser, James M.

    2015-01-01

    Whole-genome sequencing analysis of ∼800 strains of group A Streptococcus (GAS) found that the gene encoding the multiple virulence gene regulator of GAS (mga) is highly polymorphic in serotype M59 strains but not in strains of other serotypes. To help understand the molecular mechanism of gene regulation by Mga and its contribution to GAS pathogenesis in serotype M59 GAS, we constructed an isogenic mga mutant strain. Transcriptome studies indicated a significant regulatory influence of Mga and altered metabolic capabilities conferred by Mga-regulated genes. We assessed the phosphorylation status of Mga in GAS cell lysates with Phos-tag gels. The results revealed that Mga is phosphorylated at histidines in vivo. Using phosphomimetic and nonphosphomimetic substitutions at conserved phosphoenolpyruvate:carbohydrate phosphotransferase regulation domain (PRD) histidines of Mga, we demonstrated that phosphorylation-mimicking aspartate replacements at H207 and H273 of PRD-1 and at H327 of PRD-2 are inhibitory to Mga-dependent gene expression. Conversely, non-phosphorylation-mimicking alanine substitutions at H273 and H327 relieved inhibition, and the mutant strains exhibited a wild-type phenotype. The opposing regulatory profiles observed for phosphorylation- and non-phosphorylation-mimicking substitutions at H273 extended to global gene regulation by Mga. Consistent with these observations, the H273D mutant strain attenuated GAS virulence, whereas the H273A strain exhibited a wild-type virulence phenotype in a mouse model of necrotizing fasciitis. Together, our results demonstrate phosphoregulation of Mga and its direct link to virulence in M59 GAS strains. These data also lay a foundation toward understanding how naturally occurring gain-of-function variations in mga, such as H201R, may confer an advantage to the pathogen and contribute to M59 GAS pathogenesis. PMID:25824840

  10. BRAF, KIT and NRAS mutations and expression of c-KIT, phosphorylated extracellular signal-regulated kinase and phosphorylated AKT in Japanese melanoma patients.

    PubMed

    Oyama, Satomi; Funasaka, Yoko; Watanabe, Atsushi; Takizawa, Toshihiro; Kawana, Seiji; Saeki, Hidehisa

    2015-05-01

    To clarify the status of gene mutation and activation of growth signal in melanoma of Japanese patients in vivo, we analyzed the mutation of BRAF exon 15, NRAS exon 2, and KIT exons 9, 11, 13, 17 and 18 in melanoma cells obtained by laser capture microdissection, and performed direct sequencing in 20 cases of acral lentiginous melanoma (ALM) and 17 cases of superficial spreading melanoma (SSM). In the study of the mutation of BRAF, pyrosequencing was also done. To examine the cell proliferation signaling, immunohistochemistry for phosphorylated extracellular signal-regulated kinase (pERK), phosphorylated AKT (phosphorylated AKT) and c-KIT was done. The mutation of BRAF p.V600E was detected in 13 cases of ALM (65.0%) and 12 cases of SSM (70.6%). No NRAS mutation was found in all cases. The mutation in exons 9, 11, and 18 of KIT was detected in nine cases. The mutation of BRAF and KIT showed no correlation with clinical stage, lymph node metastasis, tumor thickness, ulceration and histology. pERK and pAKT was observed in small population of melanoma cells and there was no correlation with gene mutation. Our results indicate that the mutations of BRAF and KIT exist in Japanese melanoma patients, however, the cell growth signaling may be regulated by not only these mutated genes, but by other unknown regulatory factors, which may affect the prognosis of melanoma. PMID:25766129

  11. Phosphorylation acts positively and negatively to regulate MRTF-A subcellular localisation and activity.

    PubMed

    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. PMID:27304076

  12. 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

  13. Phosphorylation and inactivation of yeast 6-phosphofructo-2-kinase contribute to the regulation of glycolysis under hypotonic stress.

    PubMed

    Dihazi, H; Kessler, R; Eschrich, K

    2001-12-01

    Phosphorylation of yeast 6-phosphofructo-2-kinase and its role for the regulation of glycolysis under hypoosmotic conditions were investigated. 6-Phosphofructo-2-kinase was found to be phosphorylated in vitro by protein kinase C at serine 652 and thereby inactivated. Protein phosphatase 2A reversed the phosphorylative inhibition of the enzyme. When yeast cells were shifted to hypotonic media, 6-phosphofructo-2-kinase was found to be phosphorylated and inactivated. Under in vivo conditions, two phosphate residues were incorporated into the enzyme. One of them is bound to serine 652, indicating that this modification was probably caused by yeast protein kinase C1. The second phosphate is bound to Ser8 within the N-terminal peptide T(1-41) which contains several serine residues but no protein kinase C recognition sequence. Site-directed mutagenesis confirmed that the phosphorylation of serine 652 but not the N-terminal modification is responsible for the in vivo inactivation of 6-phosphofructo-2-kinase. The obtained results suggest that the phosphorylation of 6-phosphofructo-2-kinase mediates a response of the cells to an activation of the hypoosmolarity MAP kinase pathway. Via a suppression of glycolysis, the inactivation of 6-phosphofructo-2-kinase is expected to be responsible for the observed accumulation of glucose 6-phosphate, an essential precursor of the cell wall glucans, and the decrease of glycerol, an important osmolyte. PMID:11724581

  14. Phosphorylation Affects DNA-Binding of the Senescence-Regulating bZIP Transcription Factor GBF1

    PubMed Central

    Smykowski, Anja; Fischer, Stefan M.; Zentgraf, Ulrike

    2015-01-01

    Massive changes in the transcriptome of Arabidopsis thaliana during onset and progression of leaf senescence imply a central role for transcription factors. While many transcription factors are themselves up- or down-regulated during senescence, the bZIP transcription factor G-box-binding factor 1 (GBF1/bZIP41) is constitutively expressed in Arabidopsis leaf tissue but at the same time triggers the onset of leaf senescence, suggesting posttranscriptional mechanisms for senescence-specific GBF1 activation. Here we show that GBF1 is phosphorylated by the threonine/serine CASEIN KINASE II (CKII) in vitro and that CKII phosphorylation had a negative effect on GBF1 DNA-binding to G-boxes of two direct target genes, CATALASE2 and RBSCS1a. Phosphorylation mimicry at three serine positions in the basic region of GBF1 also had a negative effect on DNA-binding. Kinase assays revealed that CKII phosphorylates at least one serine in the basic domain but has additional phosphorylation sites outside this domain. Two different ckII α subunit1 and one α subunit2 T-DNA insertion lines showed no visible senescence phenotype, but in all lines the expression of the senescence marker gene SAG12 was remarkably diminished. A model is presented suggesting that senescence-specific GBF1 activation might be achieved by lowering the phosphorylation of GBF1 by CKII. PMID:27135347

  15. Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation

    PubMed Central

    Gone, Swapna; Alfonso-Prieto, Mercedes; Paudyal, Samridhdi; Nicholson, Allen W.

    2016-01-01

    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. PMID:27150669

  16. 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.

  17. JAK2 Tyrosine Kinase Phosphorylates and Is Negatively Regulated by Centrosomal Protein Ninein

    PubMed Central

    Jay, Jennifer; Hammer, Alan; Nestor-Kalinoski, Andrea

    2014-01-01

    JAK2 is a cytoplasmic tyrosine kinase critical for cytokine signaling. In this study, we have identified a novel centrosome-associated complex containing ninein and JAK2. We have found that active JAK2 localizes around the mother centrioles, where it partly colocalizes with ninein, a protein involved in microtubule (MT) nucleation and anchoring. We demonstrated that JAK2 is an important regulator of centrosome function. Depletion of JAK2 or use of JAK2-null cells causes defects in MT anchoring and increased numbers of cells with mitotic defects; however, MT nucleation is unaffected. We showed that JAK2 directly phosphorylates the N terminus of ninein while the C terminus of ninein inhibits JAK2 kinase activity in vitro. Overexpressed wild-type (WT) or C-terminal (amino acids 1179 to 1931) ninein inhibits JAK2. This ninein-dependent inhibition of JAK2 significantly decreases prolactin- and interferon gamma (IFN-γ)-induced tyrosyl phosphorylation of STAT1 and STAT5. Downregulation of ninein enhances JAK2 activation. These results indicate that JAK2 is a novel member of centrosome-associated complex and that this localization regulates both centrosomal function and JAK2 kinase activity, thus controlling cytokine-activated molecular pathways. PMID:25332239

  18. A tyrosine-phosphorylated 55-kilodalton motility-associated bovine sperm protein is regulated by cyclic adenosine 3',5'-monophosphates and calcium.

    PubMed

    Vijayaraghavan, S; Trautman, K D; Goueli, S A; Carr, D W

    1997-06-01

    Sperm motility is regulated by protein phosphorylation. We have recently shown that a serine/threonine phosphatase system is involved in motility regulation. Two of the components of the phosphatase system, GSK-3 and PP1gamma2, are regulated by tyrosine phosphorylation. During our investigation of sperm tyrosine-phosphorylated proteins we discovered a 55-kDa protein whose tyrosine phosphorylation correlates closely to the motility state of sperm. This protein is tyrosine phosphorylated to a much higher degree in motile caudal than in immotile caput epididymal sperm. Motility inhibition of caudal epididymal sperm by protein kinase A (PKA) anchoring inhibition or by ionomycin-induced calcium overload led to the virtual disappearance of tyrosine phosphorylation of the 55-kDa protein. Conversely, treatment of sperm with motility activators, isobutylmethylxanthine or 8-bromo-cAMP, resulted in increased tyrosine phosphorylation of the protein. The protein was present in the soluble 100 000 x g supernatants of sperm extracts and was heat labile. Chromatography through diethylaminoethyl-cellulose and Western blot analysis showed that this 55-kDa protein is not a regulatory subunit of PKA or alpha-tubulin. Our results represent the identification of a soluble protein whose tyrosine phosphorylation varies directly with motility and suggest that motility regulation may involve cross talk between PKA, calcium, and tyrosine kinase pathways. PMID:9166697

  19. Direct evidence for a role of intramitochondrial Ca2+ in the regulation of oxidative phosphorylation in the stimulated rat heart. Studies using 31P n.m.r. and ruthenium red.

    PubMed Central

    Unitt, J F; McCormack, J G; Reid, D; MacLachlan, L K; England, P J

    1989-01-01

    1. The concentrations of free ATP, phosphocreatine (PCr), Pi, H+ and ADP (calculated) were monitored in perfused rat hearts by 31P n.m.r. before and during positive inotropic stimulation. Data were accumulated in 20 s blocks. 2. Administration of 0.1 microM-(-)-isoprenaline resulted in no significant changes in ATP, transient decreases in PCr, and transient increases in ADP and Pi. However, the concentrations of all of these metabolites returned to pre-stimulated values within 1 min, whereas cardiac work and O2 uptake remained elevated. 3. In contrast, in hearts perfused continuously with Ruthenium Red (2.5 micrograms/ml), a potent inhibitor of mitochondrial Ca2+ uptake, administration of isoprenaline caused significant decreases in ATP, and also much larger and more prolonged changes in the concentrations of ADP, PCr and Pi. In this instance values did not fully return to pre-stimulated concentrations. Administration of Ruthenium Red alone to unstimulated hearts had minor effects. 4. It is proposed that, in the absence of Ruthenium Red, the transmission of changes in cytoplasmic Ca2+ across the mitochondrial inner membrane is able to maintain the phosphorylation potential of the heart during positive inotropic stimulation, through activation of the Ca2+-sensitive intramitochondrial dehydrogenases (pyruvate, NAD+-isocitrate and 2-oxoglutarate dehydrogenases) leading to enhanced NADH production. 5. This mechanism is unavailable in the presence of Ruthenium Red, and oxidative phosphorylation must be stimulated primarily by a fall in phosphorylation potential, in accordance with the classical concept of respiratory control. However, the full oxidative response of the heart to stimulation may not be achievable under such circumstances. PMID:2479373

  20. Heat Shock Proteins Regulate Activation-induced Proteasomal Degradation of the Mature Phosphorylated Form of Protein Kinase C*

    PubMed Central

    Lum, Michelle A.; Balaburski, Gregor M.; Murphy, Maureen E.; Black, Adrian R.; Black, Jennifer D.

    2013-01-01

    Although alterations in stimulus-induced degradation of PKC have been implicated in disease, mechanistic understanding of this process remains limited. Evidence supports the existence of both proteasomal and lysosomal mechanisms of PKC processing. An established pathway involves rate-limiting priming site dephosphorylation of the activated enzyme and proteasomal clearance of the dephosphorylated protein. However, here we show that agonists promote down-regulation of endogenous PKCα with minimal accumulation of a nonphosphorylated species in multiple cell types. Furthermore, proteasome and lysosome inhibitors predominantly protect fully phosphorylated PKCα, pointing to this form as a substrate for degradation. Failure to detect substantive dephosphorylation of activated PKCα was not due to rephosphorylation because inhibition of Hsp70/Hsc70, which is required for re-priming, had only a minor effect on agonist-induced accumulation of nonphosphorylated protein. Thus, PKC degradation can occur in the absence of dephosphorylation. Further analysis revealed novel functions for Hsp70/Hsc70 and Hsp90 in the control of agonist-induced PKCα processing. These chaperones help to maintain phosphorylation of activated PKCα but have opposing effects on degradation of the phosphorylated protein; Hsp90 is protective, whereas Hsp70/Hsc70 activity is required for proteasomal processing of this species. Notably, down-regulation of nonphosphorylated PKCα shows little Hsp70/Hsc70 dependence, arguing that phosphorylated and nonphosphorylated species are differentially targeted for proteasomal degradation. Finally, lysosomal processing of activated PKCα is not regulated by phosphorylation or Hsps. Collectively, these data demonstrate that phosphorylated PKCα is a direct target for agonist-induced proteasomal degradation via an Hsp-regulated mechanism, and highlight the existence of a novel pathway of PKC desensitization in cells. PMID:23900841

  1. Phosphorylation-dependent localization of the response regulator FrzZ signals cell reversals in Myxococcus xanthus

    PubMed Central

    Kaimer, Christine; Zusman, David R.

    2013-01-01

    Summary The life cycle of Myxococcus xanthus includes coordinated group movement and fruiting body formation, and requires directed motility and controlled cell reversals. Reversals are achieved by inverting cell polarity and re-organizing many motility proteins. The Frz chemosensory pathway regulates the frequency of cell reversals. While it has been established that phosphotransfer from the kinase FrzE to the response regulator FrzZ is required, it is unknown how phosphorylated FrzZ, the putative output of the pathway, targets the cell polarity axis. In this study, we used Phos-tag SDS-PAGE to determine the cellular level of phospho-FrzZ under different growth conditions and in Frz signaling mutants. We detected consistent FrzZ phosphorylation, albeit with a short half-life, in cells grown on plates, but not from liquid culture. The available pool of phospho-FrzZ correlated with reversal frequencies, with higher levels found in hyper-reversing mutants. Phosphorylation was not detected in hypo-reversing mutants. Fluorescence microscopy revealed that FrzZ is recruited to the leading cell pole upon phosphorylation and switches to the opposite pole during reversals. These results are consistent with the hypothesis that the Frz pathway modulates reversal frequency through a localized response regulator that targets cell polarity regulators at the leading cell pole. PMID:23551551

  2. Phosphorylation of Yeast Pah1 Phosphatidate Phosphatase by Casein Kinase II Regulates Its Function in Lipid Metabolism.

    PubMed

    Hsieh, Lu-Sheng; Su, Wen-Min; Han, Gil-Soo; Carman, George M

    2016-05-01

    Pah1 phosphatidate phosphatase in Saccharomyces cerevisiae catalyzes the penultimate step in the synthesis of triacylglycerol (i.e. the production of diacylglycerol by dephosphorylation of phosphatidate). The enzyme playing a major role in lipid metabolism is subject to phosphorylation (e.g. by Pho85-Pho80, Cdc28-cyclin B, and protein kinases A and C) and dephosphorylation (e.g. by Nem1-Spo7) that regulate its cellular location, catalytic activity, and stability/degradation. In this work, we show that Pah1 is a substrate for casein kinase II (CKII); its phosphorylation was time- and dose-dependent and was dependent on the concentrations of Pah1 (Km = 0.23 μm) and ATP (Km = 5.5 μm). By mass spectrometry, truncation analysis, site-directed mutagenesis, phosphopeptide mapping, and phosphoamino acid analysis, we identified that >90% of its phosphorylation occurs on Thr-170, Ser-250, Ser-313, Ser-705, Ser-814, and Ser-818. The CKII-phosphorylated Pah1 was a substrate for the Nem1-Spo7 protein phosphatase and was degraded by the 20S proteasome. The prephosphorylation of Pah1 by protein kinase A or protein kinase C reduced its subsequent phosphorylation by CKII. The prephosphorylation of Pah1 by CKII reduced its subsequent phosphorylation by protein kinase A but not by protein kinase C. The expression of Pah1 with combined mutations of S705D and 7A, which mimic its phosphorylation by CKII and lack of phosphorylation by Pho85-Pho80, caused an increase in triacylglycerol content and lipid droplet number in cells expressing the Nem1-Spo7 phosphatase complex. PMID:27044741

  3. Nanog Increases Focal Adhesion Kinase (FAK) Promoter Activity and Expression and Directly Binds to FAK Protein to Be Phosphorylated*

    PubMed Central

    Ho, Baotran; Olson, Gretchen; Figel, Sheila; Gelman, Irwin; Cance, William G.; Golubovskaya, Vita M.

    2012-01-01

    Nanog and FAK were shown to be overexpressed in cancer cells. In this report, the Nanog overexpression increased FAK expression in 293, SW480, and SW620 cancer cells. Nanog binds the FAK promoter and up-regulates its activity, whereas Nanog siRNA decreases FAK promoter activity and FAK mRNA. The FAK promoter contains four Nanog-binding sites. The site-directed mutagenesis of these sites significantly decreased up-regulation of FAK promoter activity by Nanog. EMSA showed the specific binding of Nanog to each of the four sites, and binding was confirmed by ChIP assay. Nanog directly binds the FAK protein by pulldown and immunoprecipitation assays, and proteins co-localize by confocal microscopy. Nanog binds the N-terminal domain of FAK. In addition, FAK directly phosphorylates Nanog in a dose-dependent manner by in vitro kinase assay and in cancer cells in vivo. The site-directed mutagenesis of Nanog tyrosines, Y35F and Y174F, blocked phosphorylation and binding by FAK. Moreover, overexpression of wild type Nanog increased filopodia/lamellipodia formation, whereas mutant Y35F and Y174F Nanog did not. The wild type Nanog increased cell invasion that was inhibited by the FAK inhibitor and increased by FAK more significantly than with the mutants Y35F and Y174F Nanog. Down-regulation of Nanog with siRNA decreased cell growth reversed by FAK overexpression. Thus, these data demonstrate the regulation of the FAK promoter by Nanog, the direct binding of the proteins, the phosphorylation of Nanog by FAK, and the effect of FAK and Nanog cross-regulation on cancer cell morphology, invasion, and growth that plays a significant role in carcinogenesis. PMID:22493428

  4. Nanog increases focal adhesion kinase (FAK) promoter activity and expression and directly binds to FAK protein to be phosphorylated.

    PubMed

    Ho, Baotran; Olson, Gretchen; Figel, Sheila; Gelman, Irwin; Cance, William G; Golubovskaya, Vita M

    2012-05-25

    Nanog and FAK were shown to be overexpressed in cancer cells. In this report, the Nanog overexpression increased FAK expression in 293, SW480, and SW620 cancer cells. Nanog binds the FAK promoter and up-regulates its activity, whereas Nanog siRNA decreases FAK promoter activity and FAK mRNA. The FAK promoter contains four Nanog-binding sites. The site-directed mutagenesis of these sites significantly decreased up-regulation of FAK promoter activity by Nanog. EMSA showed the specific binding of Nanog to each of the four sites, and binding was confirmed by ChIP assay. Nanog directly binds the FAK protein by pulldown and immunoprecipitation assays, and proteins co-localize by confocal microscopy. Nanog binds the N-terminal domain of FAK. In addition, FAK directly phosphorylates Nanog in a dose-dependent manner by in vitro kinase assay and in cancer cells in vivo. The site-directed mutagenesis of Nanog tyrosines, Y35F and Y174F, blocked phosphorylation and binding by FAK. Moreover, overexpression of wild type Nanog increased filopodia/lamellipodia formation, whereas mutant Y35F and Y174F Nanog did not. The wild type Nanog increased cell invasion that was inhibited by the FAK inhibitor and increased by FAK more significantly than with the mutants Y35F and Y174F Nanog. Down-regulation of Nanog with siRNA decreased cell growth reversed by FAK overexpression. Thus, these data demonstrate the regulation of the FAK promoter by Nanog, the direct binding of the proteins, the phosphorylation of Nanog by FAK, and the effect of FAK and Nanog cross-regulation on cancer cell morphology, invasion, and growth that plays a significant role in carcinogenesis. PMID:22493428

  5. Protein Kinase Cβ Phosphorylates Occludin Regulating Tight Junction Trafficking in Vascular Endothelial Growth Factor–Induced Permeability In Vivo

    PubMed Central

    Murakami, Tomoaki; Frey, Tiffany; Lin, Chengmao; Antonetti, David A.

    2012-01-01

    Vascular endothelial growth factor (VEGF)–induced breakdown of the blood-retinal barrier requires protein kinase C (PKC)β activation. However, the molecular mechanisms related to this process remain poorly understood. In this study, the role of occludin phosphorylation and ubiquitination downstream of PKCβ activation in tight junction (TJ) trafficking and endothelial permeability was investigated. Treatment of bovine retinal endothelial cells and intravitreal injection of PKCβ inhibitors as well as expression of dominant-negative kinase was used to determine the contribution of PKCβ to endothelial permeability and occludin phosphorylation at Ser490 detected with a site-specific antibody. In vitro kinase assay was used to demonstrate direct occludin phosphorylation by PKCβ. Ubiquitination was measured by immunoblotting after occludin immunoprecipitation. Confocal microscopy revealed organization of TJ proteins. The results reveal that inhibition of VEGF-induced PKCβ activation blocks occludin Ser490 phosphorylation, ubiquitination, and TJ trafficking in retinal vascular endothelial cells both in vitro and in vivo and prevents VEGF-stimulated vascular permeability. Occludin Ser490 is a direct target of PKCβ, and mutating Ser490 to Ala (S490A) blocks permeability downstream of PKCβ. Therefore, PKCβ activation phosphorylates occludin on Ser490, leading to ubiquitination required for VEGF-induced permeability. These data demonstrate a novel mechanism for PKCβ targeted inhibitors in regulating vascular permeability. PMID:22438576

  6. ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis

    SciTech Connect

    Inesta-Vaquera, Francisco A.; Campbell, David G.; Arthur, J. Simon C.; Cuenda, Ana

    2010-08-13

    Research highlights: {yields} hDlg is phosphorylated during mitosis in multiple residues. {yields} Prospho-hDlg is excluded from the midbody during mitosis. {yields} hDlg is not phosphorylated by p38{gamma} or JNK1/2 during mitosis. {yields} ERK5 pathway mediates hDlg phosphorylation in mitosis. -- Abstract: Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.

  7. Thylakoid protein phosphorylation: Regulation of light energy distribution in photosynthesis

    SciTech Connect

    Coughlan, S.J.

    1990-01-01

    It has become apparent that green plants possess the ability to adapt to changes in the spectral quality of ambient light. This phenomenon, state transitions, involves a reversible distribution of light energy between the two photosystems in response to changes in the excitation state of photosystems 1 and 2. Thus, the quantum efficiency of photosynthetic electron transport is maintained under different illumination conditions, and damage caused by excessive energetic input of light (photoinhibition) is prevented. This model comprises a phosphorylation/dephosphorylation cycle of three major components: substrates, the protein kinase(s) and protein phosphatase(s) responsible for the specific phosphorylation and dephosphorylation of these of substrates, and the control mechanisms whereby the protein kinase(s) is activated/deactivated in response to redox and /or conformational changes in the thylakoid. This report considers the three components in some detail.

  8. Structure of a bacterial enzyme regulated by phosphorylation, isocitrate dehydrogenase.

    PubMed

    Hurley, J H; Thorsness, P E; Ramalingam, V; Helmers, N H; Koshland, D E; Stroud, R M

    1989-11-01

    The structure of isocitrate dehydrogenase [threo-DS-isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42] from Escherichia coli has been solved and refined at 2.5 A resolution and is topologically different from that of any other dehydrogenase. This enzyme, a dimer of identical 416-residue subunits, is inactivated by phosphorylation at Ser-113, which lies at the edge of an interdomain pocket that also contains many residues conserved between isocitrate dehydrogenase and isopropylmalate dehydrogenase. Isocitrate dehydrogenase contains an unusual clasp-like domain in which both polypeptide chains in the dimer interlock. Based on the structure of isocitrate dehydrogenase and conservation with isopropylmalate dehydrogenase, we suggest that the active site lies in an interdomain pocket close to the phosphorylation site. PMID:2682654

  9. Nitrogen regulates CRY1 phosphorylation and circadian clock input pathways.

    PubMed

    Zhou, Yang-Hong; Zhang, Zhong-Wei; Zheng, Chong; Yuan, Shu; He, Yikun

    2016-09-01

    The delayed flowering phenotype caused by nitrogen (N) fertilizer application has been known for a long time, but we know little about the specific molecular mechanism for this phenomenon before. Our study indicated that low nitrogen increases the NADPH/NADP(+) and ATP/AMP ratios which affect adenosine monophosphate-activated protein kinase (AMPK) activity and phosphorylation and abundance of nuclear CRY1 protein. Then CRY1 acts in the N signal input pathway to the circadian clock. Here we further discuss: (1) the role of C/N ratio in flowering, (2) circadian oscillation of plant AMPK transcripts and proteins, (3) conservation of nutrition-mediated CRY1 phosphorylation and degradation, and (4) crosstalks between nitrogen signals and nitric oxide (NO) signals in flowering. PMID:27617369

  10. AMP-activated Protein Kinase Directly Phosphorylates and Destabilizes Hedgehog Pathway Transcription Factor GLI1 in Medulloblastoma

    PubMed Central

    Li, Yen-Hsing; Luo, Jia; Mosley, Yung-Yi C.; Hedrick, Victoria E.; Paul, Lake N.; Chang, Julia; Zhang, GuangJun; Wang, Yu-Kuo; Banko, Max R.; Brunet, Anne; Kuang, Shihuan; Wu, Jen-Leih; Chang, Chun-Ju; Scott, Matthew P.; Yang, Jer-Yen

    2015-01-01

    Summary The Hedgehog (Hh) pathway regulates cell differentiation and proliferation during development by controlling the Gli transcription factors. Cell fate decisions and progression toward organ and tissue maturity must be coordinated and how energy sensor regulates Hh pathway is not clear. AMP-activated Protein Kinase (AMPK) is an important sensor of energy stores that controls protein synthesis and other energy-intensive processes. AMPK is directly responsive to intracellular AMP levels, inhibiting a wide range of cell activities if ATP is low and AMP is high. Thus, AMPK can affect development by influencing protein synthesis and other processes needed for growth and differentiation. Activation of AMPK reduces GLI1 protein levels and stability, thus blocking Sonic hedgehog-induced transcriptional activity. AMPK phosphorylates GLI1 at serines 102 and 408 and threonine 1074. Mutation of these three sites into alanine prevents phosphorylation by AMPK. This in turn leads to increased GLI1 protein stability, transcriptional activity, and oncogenic potency. PMID:26190112

  11. Phosphorylation of Serine 402 Regulates RacGAP Protein Activity of FilGAP Protein.

    PubMed

    Morishita, Yuji; Tsutsumi, Koji; Ohta, Yasutaka

    2015-10-23

    FilGAP is a Rho GTPase-activating protein (GAP) that specifically regulates Rac. FilGAP is phosphorylated by ROCK, and this phosphorylation stimulates its RacGAP activity. However, it is unclear how phosphorylation regulates cellular functions and localization of FilGAP. We found that non-phosphorylatable FilGAP (ST/A) mutant is predominantly localized to the cytoskeleton along actin filaments and partially co-localized with vinculin around cell periphery, whereas phosphomimetic FilGAP (ST/D) mutant is diffusely cytoplasmic. Moreover, phosphorylated FilGAP detected by Phos-tag is also mainly localized in the cytoplasm. Of the six potential phosphorylation sites in FilGAP tested, only mutation of serine 402 to alanine (S402A) resulted in decreased cell spreading on fibronectin. FilGAP phosphorylated at Ser-402 is localized to the cytoplasm but not at the cytoskeleton. Although Ser-402 is highly phosphorylated in serum-starved quiescent cells, dephosphorylation of Ser-402 is accompanied with the cell spreading on fibronectin. Treatment of the cells expressing wild-type FilGAP with calyculin A, a Ser/Thr phosphatase inhibitor, suppressed cell spreading on fibronectin, whereas cells transfected with FilGAP S402A mutant were not affected by calyculin A. Expression of constitutively activate Arf6 Q67L mutant stimulated membrane blebbing activity of both non-phosphorylatable (ST/A) and phosphomimetic (ST/D) FilGAP mutants. Conversely, depletion of endogenous Arf6 suppressed membrane blebbing induced by FilGAP (ST/A) and (ST/D) mutants. Our study suggests that Arf6 and phosphorylation of FilGAP may regulate FilGAP, and phosphorylation of Ser-402 may play a role in the regulation of cell spreading on fibronectin. PMID:26359494

  12. Phosphorylation in intrinsically disordered regions regulates the activity of Neurogenin2

    PubMed Central

    2014-01-01

    Background Neuronal differentiation is largely under the control of basic Helix-Loop-Helix (bHLH) proneural transcription factors that play key roles during development of the embryonic nervous system. In addition to well-characterised regulation of their expression, increasing evidence is emerging for additional post-translational regulation of proneural protein activity. Of particular interest is the bHLH proneural factor Neurogenin2 (Ngn2), which orchestrates progression from neural progenitor to differentiated neuron in several regions of the central nervous system. Previous studies have demonstrated a key role for cell cycle-dependent multi-site phosphorylation of Ngn2 protein at Serine-Proline (SP) sites for regulation of its neuronal differentiation activity, although the potential structural and functional consequences of phosphorylation at different regions of the protein are unclear. Results Here we characterise the role of phosphorylation of specific regions of Ngn2 on the stability of Ngn2 protein and on its neuronal differentiation activity in vivo in the developing embryo, demonstrating clearly that the location of SP sites is less important than the number of SP sites available for control of Ngn2 activity in vivo. We also provide structural evidence that Ngn2 contains large, intrinsically disordered regions that undergo phosphorylation by cyclin-dependent kinases (cdks). Conclusions Phosphorylation of Ngn2 occurs in both the N- and C-terminal regions, either side of the conserved basic Helix-Loop-Helix domain. While these phosphorylation events do not change the intrinsic stability of Ngn2, phosphorylation on multiple sites acts to limit its ability to drive neuronal differentiation in vivo. Phosphorylated regions of Ngn2 are predicted to be intrinsically disordered and cdk-dependent phosphorylation of these intrinsically disordered regions contributes to Ngn2 regulation. PMID:25374254

  13. Constitutive Phosphorylation by Protein Kinase C Regulates D1 Dopamine Receptor Signaling

    PubMed Central

    Rankin, Michele L.; Sibley, David R.

    2010-01-01

    The D1 dopamine receptor (D1DAR) is robustly phosphorylated by multiple protein kinases, yet the phosphorylation sites and functional consequences of these modifications are not fully understood. Here, we report that the D1DAR is phosphorylated by protein kinase C (PKC) in the absence of agonist stimulation. Phosphorylation of the D1DAR by PKC is constitutive in nature, can be induced by phorbol ester treatment or through activation of Gq-mediated signal transduction pathways, and is abolished by PKC inhibitors. We demonstrate that most, but not all, isoforms of PKC are capable of phosphorylating the receptor. To directly assess the functional role of PKC phosphorylation of the D1DAR, a site-directed mutagenesis approach was used to identify the PKC sites within the receptor. Five serine residues were found to mediate the PKC phosphorylation. Replacement of these residues had no effect on D1DAR expression or agonist-induced desensitization; however, G protein coupling and cAMP accumulation were significantly enhanced in PKC-null D1DAR. Thus, constitutive or heterologous PKC phosphorylation of the D1DAR dampens dopamine activation of the receptor, most likely occurring in a context-specific manner, mediated by the repertoire of PKC isozymes within the cell. PMID:20969574

  14. The tumor suppressor Smad4/DPC4 is regulated by phosphorylations that integrate FGF, Wnt, and TGF-β signaling.

    PubMed

    Demagny, Hadrien; Araki, Tatsuya; De Robertis, Edward M

    2014-10-23

    Smad4 is a major tumor suppressor currently thought to function constitutively in the transforming growth factor β (TGF-β)-signaling pathway. Here, we report that Smad4 activity is directly regulated by the Wnt and fibroblast growth factor (FGF) pathways through GSK3 and mitogen-activated protein kinase (MAPK) phosphorylation sites. FGF activates MAPK, which primes three sequential GSK3 phosphorylations that generate a Wnt-regulated phosphodegron bound by the ubiquitin E3 ligase β-TrCP. In the presence of FGF, Wnt potentiates TGF-β signaling by preventing Smad4 GSK3 phosphorylations that inhibit a transcriptional activation domain located in the linker region. When MAPK is not activated, the Wnt and TGF-β signaling pathways remain insulated from each other. In Xenopus embryos, these Smad4 phosphorylations regulate germ-layer specification and Spemann organizer formation. The results show that three major signaling pathways critical in development and cancer are integrated at the level of Smad4. PMID:25373906

  15. Cortactin phosphorylation regulates cell invasion through a pH-dependent pathway

    PubMed Central

    Magalhaes, Marco A. O.; Larson, Daniel R.; Mader, Christopher C.; Bravo-Cordero, Jose Javier; Gil-Henn, Hava; Oser, Matthew; Chen, Xiaoming; Koleske, Anthony J.

    2011-01-01

    Invadopodia are invasive protrusions with proteolytic activity uniquely found in tumor cells. Cortactin phosphorylation is a key step during invadopodia maturation, regulating Nck1 binding and cofilin activity. The precise mechanism of cortactin-dependent cofilin regulation and the roles of this pathway in invadopodia maturation and cell invasion are not fully understood. We provide evidence that cortactin–cofilin binding is regulated by local pH changes at invadopodia that are mediated by the sodium–hydrogen exchanger NHE1. Furthermore, cortactin tyrosine phosphorylation mediates the recruitment of NHE1 to the invadopodium compartment, where it locally increases the pH to cause the release of cofilin from cortactin. We show that this mechanism involving cortactin phosphorylation, local pH increase, and cofilin activation regulates the dynamic cycles of invadopodium protrusion and retraction and is essential for cell invasion in 3D. Together, these findings identify a novel pH-dependent regulation of cell invasion. PMID:22105349

  16. Phosphorylation sites in BubR1 that regulate kinetochore attachment, tension, and mitotic exit

    PubMed Central

    Huang, Haomin; Hittle, James; Zappacosta, Francesca; Annan, Roland S.; Hershko, Avram; Yen, Timothy J.

    2008-01-01

    BubR1 kinase is essential for the mitotic checkpoint and also for kinetochores to establish microtubule attachments. In this study, we report that BubR1 is phosphorylated in mitosis on four residues that differ from sites recently reported to be phosphorylated by Plk1 (Elowe, S., S. Hummer, A. Uldschmid, X. Li, and E.A. Nigg. 2007. Genes Dev. 21:2205–2219; Matsumura, S., F. Toyoshima, and E. Nishida. 2007. J. Biol. Chem. 282:15217–15227). S670, the most conserved residue, is phosphorylated at kinetochores at the onset of mitosis and dephosphorylated before anaphase onset. Unlike the Plk1-dependent S676 phosphorylation, S670 phosphorylation is sensitive to microtubule attachments but not to kinetochore tension. Functionally, phosphorylation of S670 is essential for error correction and for kinetochores with end-on attachments to establish tension. Furthermore, in vitro data suggest that the phosphorylation status of BubR1 is important for checkpoint inhibition of the anaphase-promoting complex/cyclosome. Finally, RNA interference experiments show that Mps1 is a major but not the exclusive kinase that specifies BubR1 phosphorylation in vivo. The combined data suggest that BubR1 may be an effector of multiple kinases that are involved in discrete aspects of kinetochore attachments and checkpoint regulation. PMID:19015317

  17. 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

  18. Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation.

    PubMed Central

    Johansson, I; Karlsson, M; Shukla, V K; Chrispeels, M J; Larsson, C; Kjellbom, P

    1998-01-01

    PM28A is a major intrinsic protein of the spinach leaf plasma membrane and the major phosphoprotein. Phosphorylation of PM28A is dependent in vivo on the apoplastic water potential and in vitro on submicromolar concentrations of Ca2+. Here, we demonstrate that PM28A is an aquaporin and that its water channel activity is regulated by phosphorylation. Wild-type and mutant forms of PM28A, in which putative phosphorylation sites had been knocked out, were expressed in Xenopus oocytes, and the resulting increase in osmotic water permeability was measured in the presence or absence of an inhibitor of protein kinases (K252a) or of an inhibitor of protein phosphatases (okadaic acid). The results indicate that the water channel activity of PM28A is regulated by phosphorylation of two serine residues, Ser-115 in the first cytoplasmic loop and Ser-274 in the C-terminal region. Labeling of spinach leaves with 32P-orthophosphate and subsequent sequencing of PM28A-derived peptides demonstrated that Ser-274 is phosphorylated in vivo, whereas phosphorylation of Ser-115, a residue conserved among all plant plasma membrane aquaporins, could not be demonstrated. This identifies Ser-274 of PM28A as the amino acid residue being phosphorylated in vivo in response to increasing apoplastic water potential and dephosphorylated in response to decreasing water potential. Taken together, our results suggest an active role for PM28A in maintaining cellular water balance. PMID:9501117

  19. Whi5 Regulation by Site Specific CDK-Phosphorylation in Saccharomyces cerevisiae

    PubMed Central

    Wagner, Michelle V.; Smolka, Marcus B.; de Bruin, Rob A. M.; Zhou, Huilin; Wittenberg, Curt; Dowdy, Steven F.

    2009-01-01

    The Whi5 transcriptional repressor is a negative regulator of G1 cell cycle progression in Saccharomyces cerevisiae and is functionally equivalent to the Retinoblastoma (Rb) tumor suppressor protein in mammals. In early G1, Whi5 binds to and inhibits SBF (Swi4/Swi6) transcriptional complexes. At Start, Cln:Cdc28 kinases phosphorylate and inactivate Whi5, causing its dissociation from SBF promoters and nuclear export, allowing activation of SBF transcription and entry into late G1. In an analysis of Whi5 phosphorylation, we found that 10 of the 12 putative CDK phosphorylation sites on Whi5 were occupied in vivo in asynchronously growing cells. In addition, we identified 6 non-CDK Whi5 phosphorylation sites. Whi5 CDK and non-CDK phosphorylation mutants were functional and able to rescue the small cell size of whi5Δ cells. However, the Whi5 CDK mutant with all 12 putative CDK sites changed to alanine causes a dramatic cell cycle phenotype when expressed with a Swi6 CDK phosphorylation mutant. Mutational analysis of Whi5 determined that only four C-terminal CDK sites were necessary and sufficient for Whi5 inactivation when Swi6 CDK sites were also mutated. Although these four Whi5 CDK sites do not wholly determine Whi5 nuclear export, they do impact regulation of cell size. Taken together, these observations begin to dissect the regulatory role of specific phosphorylation sites on Whi5. PMID:19172996

  20. Allosteric Activation of Bacterial Response Regulators: the Role of the Cognate Histidine Kinase Beyond Phosphorylation

    PubMed Central

    Trajtenberg, Felipe; Albanesi, Daniela; Ruétalo, Natalia; Botti, Horacio; Mechaly, Ariel E.; Nieves, Marcos; Aguilar, Pablo S.; Cybulski, Larisa; Larrieux, Nicole; de Mendoza, Diego

    2014-01-01

    ABSTRACT Response regulators are proteins that undergo transient phosphorylation, connecting specific signals to adaptive responses. Remarkably, the molecular mechanism of response regulator activation remains elusive, largely because of the scarcity of structural data on multidomain response regulators and histidine kinase/response regulator complexes. We now address this question by using a combination of crystallographic data and functional analyses in vitro and in vivo, studying DesR and its cognate sensor kinase DesK, a two-component system that controls membrane fluidity in Bacillus subtilis. We establish that phosphorylation of the receiver domain of DesR is allosterically coupled to two distinct exposed surfaces of the protein, controlling noncanonical dimerization/tetramerization, cooperative activation, and DesK binding. One of these surfaces is critical for both homodimerization- and kinase-triggered allosteric activations. Moreover, DesK induces a phosphorylation-independent activation of DesR in vivo, uncovering a novel and stringent level of specificity among kinases and regulators. Our results support a model that helps to explain how response regulators restrict phosphorylation by small-molecule phosphoryl donors, as well as cross talk with noncognate sensors. PMID:25406381

  1. Ribosomal Protein S6 Phosphorylation in the Nervous System: From Regulation to Function

    PubMed Central

    Biever, Anne; Valjent, Emmanuel; Puighermanal, Emma

    2015-01-01

    Since the discovery of the phosphorylation of the 40S ribosomal protein S6 (rpS6) about four decades ago, much effort has been made to uncover the molecular mechanisms underlying the regulation of this post-translational modification. In the field of neuroscience, rpS6 phosphorylation is commonly used as a readout of the mammalian target of rapamycin complex 1 signaling activation or as a marker for neuronal activity. Nevertheless, its biological role in neurons still remains puzzling. Here we review the pharmacological and physiological stimuli regulating this modification in the nervous system as well as the pathways that transduce these signals into rpS6 phosphorylation. Altered rpS6 phosphorylation observed in various genetic and pathophysiological mouse models is also discussed. Finally, we examine the current state of knowledge on the physiological role of this post-translational modification and highlight the questions that remain to be addressed. PMID:26733799

  2. Phosphorylation of Src by phosphoinositide 3-kinase regulates beta-adrenergic receptor-mediated EGFR transactivation.

    PubMed

    Watson, Lewis J; Alexander, Kevin M; Mohan, Maradumane L; Bowman, Amber L; Mangmool, Supachoke; Xiao, Kunhong; Naga Prasad, Sathyamangla V; Rockman, Howard A

    2016-10-01

    β2-Adrenergic receptors (β2AR) transactivate epidermal growth factor receptors (EGFR) through formation of a β2AR-EGFR complex that requires activation of Src to mediate signaling. Here, we show that both lipid and protein kinase activities of the bifunctional phosphoinositide 3-kinase (PI3K) enzyme are required for β2AR-stimulated EGFR transactivation. Mechanistically, the generation of phosphatidylinositol (3,4,5)-tris-phosphate (PIP3) by the lipid kinase function stabilizes β2AR-EGFR complexes while the protein kinase activity of PI3K regulates Src activation by direct phosphorylation. The protein kinase activity of PI3K phosphorylates serine residue 70 on Src to enhance its activity and induce EGFR transactivation following βAR stimulation. This newly identified function for PI3K, whereby Src is a substrate for the protein kinase activity of PI3K, is of importance since Src plays a key role in pathological and physiological signaling. PMID:27169346

  3. Protein kinase C-mediated phosphorylation and functional regulation of dopamine transporters in striatal synaptosomes.

    PubMed

    Vaughan, R A; Huff, R A; Uhl, G R; Kuhar, M J

    1997-06-13

    Dopamine transporters (DATs) are members of a family of Na+- and Cl--dependent neurotransmitter transporters responsible for the rapid clearance of dopamine from synaptic clefts. The predicted primary sequence of DAT contains numerous consensus phosphorylation sites. In this report we demonstrate that DATs undergo endogenous phosphorylation in striatal synaptosomes that is regulated by activators of protein kinase C. Rat striatal synaptosomes were metabolically labeled with [32P]orthophosphate, and solubilized homogenates were subjected to immunoprecipitation with an antiserum specific for DAT. Basal phosphorylation occurred in the absence of exogenous treatments, and the phosphorylation level was rapidly increased when synaptosomes were treated with the phosphatase inhibitors okadaic acid or calyculin. Treatment of synaptosomes with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) also increased the level of phosphate incorporation. This occurred within 10 min and was dosedependent between 0.1 and 1 microM PMA. DAT phosphorylation was also significantly increased by two other protein kinase C activators, (-)-indolactam V and 1-oleoyl-2-acetyl-sn-glycerol. The inactive phorbol ester 4alpha-phorbol 12,13-didecanoate at 10 microM was without effect, and PMA-induced phosphorylation was blocked by treatment of synaptosomes with the protein kinase C inhibitors staurosporine and bisindoylmaleimide. These results indicate that DATs undergo rapid in vivo phosphorylation in response to protein kinase C activation and that a robust mechanism exists in synaptosomes for DAT dephosphorylation. Dopamine transport activity in synaptosomes was reduced by all treatments that promoted DAT phosphorylation, with comparable dose, time, and inhibitor characteristics. The change in transport activity was produced by a reduction in Vmax with no significant effect on the Km for dopamine. These results suggest that synaptosomal dopamine transport activity is regulated by

  4. Tandem phosphorylation within an intrinsically disordered region regulates ACTN4 function

    PubMed Central

    Travers, Timothy; Shao, Hanshuang; Joughin, Brian A.; Lauffenburger, Douglas A.; Wells, Alan; Camacho, Carlos J.

    2015-01-01

    Phosphorylated residues occur preferentially in the intrinsically disordered regions of eukaryotic proteins. In the disordered N-terminal region of human α-actinin-4 (ACTN4), Tyr4 and Tyr31 are phosphorylated in cells stimulated with epidermal growth factor (EGF), and a mutant with phosphorylation-mimicking mutations of both tyrosines exhibits reduced interaction with actin in vitro. Cleavage of ACTN4 by m-calpain, a protease that in motile cells is predominantly activated at the rear, removes the Tyr4 site. Here, we found that introducing a phosphomimetic mutation at only Tyr31 was sufficient to inhibit the interaction with actin in vitro. However, molecular dynamics simulations predicted that Tyr31 is mostly buried and that phosphorylation of Tyr4 would increase the solvent exposure and thus kinase accessibility of Tyr31. In fibroblast cells, EGF stimulation increased tyrosine phosphorylation of a mutant form of ACTN4 with a phosphorylation-mimicking residue at Tyr4, whereas a truncation mutant representing the product of m-calpain cleavage exhibited EGF-stimulated tyrosine phosphorylation at the background amount similar to that observed for a double phosphomimetic mutant of Tyr4 and Tyr31. We also found that inhibition of the receptor tyrosine kinases of the TAM family, such as AXL, blocked EGF-stimulated tyrosine phosphorylation of ACTN4. Mathematical modeling predicted that the kinetics of phosphorylation at Tyr31 can be dictated by the kinase affinity for Tyr4. This study suggests that tandem-site phosphorylation within intrinsically disordered regions provides a mechanism for a site to function as a switch to reveal a nearby function-regulating site. PMID:26012634

  5. Regulation of Auxin Transport by Phosphorylation and Flavonoids during Gravitropism in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Muday, Gloria K.

    2005-01-01

    The focus of this research includes: 1) Regulation of Axin transport by flavonoids during gravitropism; 2) Phosphorylation control of auxin transport during gravity response; 3) Ethylene regulation of gravitropic curvature; 4) IBA transport and gravitropic response; and 5) Other collaborative projects.

  6. ERK/MAPK Regulates Hippocampal Histone Phosphorylation Following Contextual Fear Conditioning

    ERIC Educational Resources Information Center

    Levenson, Jonathan M.; Sweatt, J. David; Chwang, Wilson B.; O'Riordan, Kenneth J.

    2006-01-01

    Long-term memory formation is regulated by many distinct molecular mechanisms that control gene expression. An emerging model for effecting a stable, coordinated pattern of gene transcription involves epigenetic tagging through modifications of histones or DNA. In this study, we investigated the regulation of histone phosphorylation in the…

  7. Cofilin Oligomer Formation Occurs In Vivo and Is Regulated by Cofilin Phosphorylation

    PubMed Central

    Goyal, Pankaj; Pandey, Dharmendra; Brünnert, Daniela; Hammer, Elke; Zygmunt, Marek; Siess, Wolfgang

    2013-01-01

    Background ADF/cofilin proteins are key regulators of actin dynamics. Their function is inhibited by LIMK-mediated phosphorylation at Ser-3. Previous in vitro studies have shown that dependent on its concentration, cofilin either depolymerizes F-actin (at low cofilin concentrations) or promotes actin polymerization (at high cofilin concentrations). Methodology/Principal Findings We found that after in vivo cross-linking with different probes, a cofilin oligomer (65 kDa) could be detected in platelets and endothelial cells. The cofilin oligomer did not contain actin. Notably, ADF that only depolymerizes F-actin was present mainly in monomeric form. Furthermore, we found that formation of the cofilin oligomer is regulated by Ser-3 cofilin phosphorylation. Cofilin but not phosphorylated cofilin was present in the endogenous cofilin oligomer. In vitro, formation of cofilin oligomers was drastically reduced after phosphorylation by LIMK2. In endothelial cells, LIMK-mediated cofilin phosphorylation after thrombin-stimulation of EGFP- or DsRed2-tagged cofilin transfected cells reduced cofilin aggregate formation, whereas inhibition of cofilin phosphorylation after Rho-kinase inhibitor (Y27632) treatment of endothelial cells promoted formation of cofilin aggregates. In platelets, cofilin dephosphorylation after thrombin-stimulation and Y27632 treatment led to an increased formation of the cofilin oligomer. Conclusion/Significance Based on our results, we propose that an equilibrium exists between the monomeric and oligomeric forms of cofilin in intact cells that is regulated by cofilin phosphorylation. Cofilin phosphorylation at Ser-3 may induce conformational changes on the protein-protein interacting surface of the cofilin oligomer, thereby preventing and/or disrupting cofilin oligomer formation. Cofilin oligomerization might explain the dual action of cofilin on actin dynamics in vivo. PMID:23951242

  8. Regulation of myosin light chain phosphorylation in the trabecular meshwork: role in aqueous humour outflow facility.

    PubMed

    Rao, P Vasantha; Deng, Peifeng; Sasaki, Yasuharu; Epstein, David L

    2005-02-01

    Cellular contraction and relaxation and integrity of the actin cytoskeleton in trabecular meshwork (TM) tissue have been thought to influence aqueous humour outflow. However, the cellular pathways that regulate these events in TM cells are not well understood. In this study, we investigated physiological agonist-mediated regulation of myosin light chain (MLC) phosphorylation in the TM, and correlated such effects with alterations in aqueous outflow facility, since MLC phosphorylation is a critical biochemical determinant of cellular contraction in TM cells. Treatment of serum starved human TM cells with endothelin-1 (0.1 microM), thromboxane A2 mimetic U-46619 (1.0 microM), or angiotensin II (1 microM), all of which are agonists of G-protein coupled receptors, triggered activation of MLC phosphorylation, as determined by urea/glycerol-based Western blot analysis. Agonist-stimulated increase in MLC phosphorylation was associated with activation of Rho GTPase in TM cells, as determined in pull-down assays. In contrast, treatment of human TM cells with a novel Rho-kinase inhibitor H-1152 (0.1-2 microM), in the presence of serum reduced basal MLC phosphorylation. H-1152 also increased aqueous outflow facility significantly in a dose-dependent fashion, in perfusion studies with cadaver porcine eyes. This effect of H-1152 on outflow facility was associated with decreased MLC phosphorylation in TM tissue of drug-perfused eyes. Collectively, this study identifies potential physiological regulators of MLC phosphorylation in human TM cells and demonstrates the significance of Rho/Rho-kinase pathway-mediated MLC phosphorylation in modulation of aqueous outflow facility through TM. PMID:15670798

  9. Direct Phosphorylation and Activation of a Mitogen-Activated Protein Kinase by a Calcium-Dependent Protein Kinase in Rice[C][W

    PubMed Central

    Xie, Kabin; Chen, Jianping; Wang, Qin; Yang, Yinong

    2014-01-01

    The mitogen-activated protein kinase (MAPK) is a pivotal point of convergence for many signaling pathways in eukaryotes. In the classical MAPK cascade, a signal is transmitted via sequential phosphorylation and activation of MAPK kinase kinase, MAPK kinase (MKK), and MAPK. The activation of MAPK is dependent on dual phosphorylation of a TXY motif by an MKK, which is considered the sole kinase to phosphorylate and activate MAPK. Here, we report a novel regulatory mechanism of MAPK phosphorylation and activation besides the canonical MAPK cascade. A rice (Oryza sativa) calcium-dependent protein kinase (CDPK), CPK18, was identified as an upstream kinase of MAPK (MPK5) in vitro and in vivo. Curiously, CPK18 was shown to phosphorylate and activate MPK5 without affecting the phosphorylation of its TXY motif. Instead, CPK18 was found to predominantly phosphorylate two Thr residues (Thr-14 and Thr-32) that are widely conserved in MAPKs from land plants. Further analyses reveal that the newly identified CPK18-MPK5 pathway represses defense gene expression and negatively regulates rice blast resistance. Our results suggest that land plants have evolved an MKK-independent phosphorylation pathway that directly connects calcium signaling to the MAPK machinery. PMID:25035404

  10. Regulation of the gating of cystic fibrosis transmembrane conductance regulator C1 channels by phosphorylation and ATP hydrolysis.

    PubMed Central

    Hwang, T C; Nagel, G; Nairn, A C; Gadsby, D C

    1994-01-01

    Opening of cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels requires their phosphorylation by protein kinase A followed by exposure to ATP. We examined the interaction between nucleotides and phosphorylated CFTR channels by recording currents in intact cardiac myocytes and in excised patches. We found that, although the hydrolysis-resistant ATP analogue 5'-adenosine(beta,gamma- imino)triphosphate (AMP-PNP) cannot open phosphorylated CFTR channels, it can cause channels opened by ATP to remain open for many minutes. This suggests that ATP action at one site on CFTR is a prerequisite for AMP-PNP action at a second site. However, this action of AMP-PNP is restricted to highly phosphorylated CFTR channels, which, in the presence of ATP, display a relatively high open probability, but is not seen in partially phosphorylated CFTR channels, which have a low open probability in the presence of ATP. Our findings argue that incremental phosphorylation differentially regulates the interactions between nucleotides and the two nucleotide binding domains of CFTR. The nature of those interactions suggests that ATP hydrolysis at one nucleotide binding domain controls channel opening and ATP hydrolysis at the other regulates channel closing. Images PMID:7515176

  11. Regulation of the gating of cystic fibrosis transmembrane conductance regulator C1 channels by phosphorylation and ATP hydrolysis.

    PubMed

    Hwang, T C; Nagel, G; Nairn, A C; Gadsby, D C

    1994-05-24

    Opening of cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels requires their phosphorylation by protein kinase A followed by exposure to ATP. We examined the interaction between nucleotides and phosphorylated CFTR channels by recording currents in intact cardiac myocytes and in excised patches. We found that, although the hydrolysis-resistant ATP analogue 5'-adenosine(beta,gamma- imino)triphosphate (AMP-PNP) cannot open phosphorylated CFTR channels, it can cause channels opened by ATP to remain open for many minutes. This suggests that ATP action at one site on CFTR is a prerequisite for AMP-PNP action at a second site. However, this action of AMP-PNP is restricted to highly phosphorylated CFTR channels, which, in the presence of ATP, display a relatively high open probability, but is not seen in partially phosphorylated CFTR channels, which have a low open probability in the presence of ATP. Our findings argue that incremental phosphorylation differentially regulates the interactions between nucleotides and the two nucleotide binding domains of CFTR. The nature of those interactions suggests that ATP hydrolysis at one nucleotide binding domain controls channel opening and ATP hydrolysis at the other regulates channel closing. PMID:7515176

  12. Nephrin phosphorylation regulates podocyte adhesion through the PINCH-1-ILK-α-parvin complex

    PubMed Central

    Zha, Dongqing; Chen, Cheng; Liang, Wei; Chen, Xinghua; Ma, Tean; Yang, Hongxia; van Goor, Harry; Ding, Guohua

    2013-01-01

    Nephrin, a structural molecule, is also a signaling molecule after phosphorylation. Inhibition of nephrin phosphorylation is correlated with podocyte injury. The PINCH-1-ILK-α-parvin (PIP) complex plays a crucial role in cell adhesion and cytoskeleton formation. We hypothesized that nephrin phosphorylation influenced cytoskeleton and cell adhesion in podocytes by regulating the PIP complex. The nephrin phosphorylation, PIP complex formation, and F-actin in Wistar rats intraperitoneally injected with puromycin aminonucleoside were gradually decreased but increased with time, coinciding with the recovery from glomerular/podocyte injury and proteinuria. In cultured podocytes, PIP complex knockdown resulted in cytoskeleton reorganization and decreased cell adhesion and spreading. Nephrin and its phosphorylation were unaffected after PIP complex knockdown. Furthermore, inhibition of nephrin phosphorylation suppressed PIP complex expression, disorganized podocyte cytoskeleton, and decreased cell adhesion and spreading. These findings indicate that alterations in nephrin phosphorylation disorganize podocyte cytoskeleton and decrease cell adhesion through a PIP complex-dependent mechanism. [BMB Reports 2013; 46(4): 230-235] PMID:23615266

  13. Regulation of AMPA receptor subunit GluA1 surface expression by PAK3 phosphorylation

    PubMed Central

    Hussain, Natasha K.; Thomas, Gareth M.; Luo, Junjie; Huganir, Richard L.

    2015-01-01

    AMPA receptors (AMPARs) are the major excitatory receptors of the brain and are fundamental to synaptic plasticity, memory, and cognition. Dynamic recycling of AMPARs in neurons is regulated through several types of posttranslational modification, including phosphorylation. Here, we identify a previously unidentified signal transduction cascade that modulates phosphorylation of serine residue 863 (S863) in the GluA1 AMPAR subunit and controls surface trafficking of GluA1 in neurons. Activation of the EphR–Ephrin signal transduction pathway enhances S863 phosphorylation. Further, EphB2 can interact with Zizimin1, a guanine–nucleotide exchange factor that activates Cdc42 and stimulates S863 phosphorylation in neurons. Among the numerous targets downstream of Cdc42, we determined that the p21-activated kinase-3 (PAK3) phosphorylates S863 in vitro. Moreover, specific loss of PAK3 expression and pharmacological inhibition of PAK both disrupt activity-dependent phosphorylation of S863 in cortical neurons. EphB2, Cdc42, and PAKs are broadly capable of controlling dendritic spine formation and synaptic plasticity and are implicated in multiple cognitive disorders. Collectively, these data delineate a novel signal cascade regulating AMPAR trafficking that may contribute to the molecular mechanisms that govern learning and cognition. PMID:26460013

  14. Differential regulation of the histone chaperone HIRA during muscle cell differentiation by a phosphorylation switch

    PubMed Central

    Yang, Jae-Hyun; Song, Tae-Yang; Jo, Chanhee; Park, Jinyoung; Lee, Han-Young; Song, Ilang; Hong, Suji; Jung, Kwan Young; Kim, Jaehoon; Han, Jeung-Whan; Youn, Hong-Duk; Cho, Eun-Jung

    2016-01-01

    Replication-independent incorporation of variant histone H3.3 has a profound impact on chromatin function and numerous cellular processes, including the differentiation of muscle cells. The histone chaperone HIRA and H3.3 have essential roles in MyoD regulation during myoblast differentiation. However, the precise mechanism that determines the onset of H3.3 deposition in response to differentiation signals is unclear. Here we show that HIRA is phosphorylated by Akt kinase, an important signaling modulator in muscle cells. By generating a phosphospecific antibody, we found that a significant amount of HIRA was phosphorylated in myoblasts. The phosphorylation level of HIRA and the occupancy of phosphorylated protein on muscle genes gradually decreased during cellular differentiation. Remarkably, the forced expression of the phosphomimic form of HIRA resulted in reduced H3.3 deposition and suppressed the activation of muscle genes in myotubes. Our data show that HIRA phosphorylation limits the expression of myogenic genes, while the dephosphorylation of HIRA is required for proficient H3.3 deposition and gene activation, demonstrating that the phosphorylation switch is exploited to modulate HIRA/H3.3-mediated muscle gene regulation during myogenesis. PMID:27515126

  15. Filamin a regulates neural progenitor proliferation and cortical size through Wee1-dependent Cdk1 phosphorylation.

    PubMed

    Lian, Gewei; Lu, Jie; Hu, Jianjun; Zhang, Jingping; Cross, Sally H; Ferland, Russell J; Sheen, Volney L

    2012-05-30

    Cytoskeleton-associated proteins play key roles not only in regulating cell morphology and migration but also in proliferation. Mutations in the cytoskeleton-associated gene filamin A (FlnA) cause the human disorder periventricular heterotopia (PH). PH is a disorder of neural stem cell development that is characterized by disruption of progenitors along the ventricular epithelium and subsequent formation of ectopic neuronal nodules. FlnA-dependent regulation of cytoskeletal dynamics is thought to direct neural progenitor migration and proliferation. Here we show that embryonic FlnA-null mice exhibited a reduction in brain size and decline in neural progenitor numbers over time. The drop in the progenitor population was not attributable to cell death or changes in premature differentiation, but to prolonged cell cycle duration. Suppression of FlnA led to prolongation of the entire cell cycle length, principally in M phase. FlnA loss impaired degradation of cyclin B1-related proteins, thereby delaying the onset and progression through mitosis. We found that the cdk1 kinase Wee1 bound FlnA, demonstrated increased expression levels after loss of FlnA function, and was associated with increased phosphorylation of cdk1. Phosphorylation of cdk1 inhibited activation of the anaphase promoting complex degradation system, which was responsible for cyclin B1 degradation and progression through mitosis. Collectively, our results demonstrate a molecular mechanism whereby FlnA loss impaired G2 to M phase entry, leading to cell cycle prolongation, compromised neural progenitor proliferation, and reduced brain size. PMID:22649246

  16. Polo inhibits progenitor self-renewal and regulates Numb asymmetry by phosphorylating Pon

    PubMed Central

    Wang, Hongyan; Ouyang, Yingshi; Somers, W. Gregory; Chia, William; Lu, Bingwei

    2011-01-01

    Self-renewal and differentiation are cardinal features of stem cells. Asymmetric cell division provides one fundamental mechanism by which stem cell self-renewal and differentiation are balanced1,2. A failure of this balance could lead to diseases such as cancer3–6. During asymmetric division of stem cells, factors controlling their self-renewal and differentiation are unequally segregated between daughter cells. Numb is one such factor that is segregated to the differentiating daughter cell during the stem-cell-like neuroblast divisions in Drosophila melanogaster7, where it inhibits self-renewal8,9. The localization and function of Numb is cell-cycle-dependent7,10–12. Here we show that Polo (ref. 13), a key cell cycle regulator, the mammalian counterparts of which have been implicated as oncogenes as well as tumour suppressors14,15, acts as a tumour suppressor in the larval brain. Supernumerary neuroblasts are produced at the expense of neurons in polo mutants. Polo directly phosphorylates Partner of Numb (Pon, ref. 16), an adaptor protein for Numb, and this phosphorylation event is important for Pon to localize Numb. In polo mutants, the asymmetric localization of Pon, Numb and atypical protein kinase C are disrupted, whereas other polarity markers are largely unaffected. Overexpression of Numb suppresses neuroblast over-proliferation caused by polo mutations, suggesting that Numb has a major role in mediating this effect of Polo. Our results reveal a biochemical link between the cell cycle and the asymmetric protein localization machinery, and indicate that Polo can inhibit progenitor self-renewal by regulating the localization and function of Numb. PMID:17805297

  17. PML tumor suppressor is regulated by HIPK2-mediated phosphorylation in response to DNA damage.

    PubMed

    Gresko, E; Ritterhoff, S; Sevilla-Perez, J; Roscic, A; Fröbius, K; Kotevic, I; Vichalkovski, A; Hess, D; Hemmings, B A; Schmitz, M L

    2009-02-01

    The promyelocytic leukemia (PML) tumor suppressor protein, a central regulator of cell proliferation and apoptosis, is frequently fused to the retinoic acid receptor-alpha (RARalpha) in acute PML. Here we show the interaction of PML with another tumor suppressor protein, the serine/threonine kinase homeodomain-interacting protein kinase (HIPK2). In response to DNA damage, HIPK2 phosphorylates PML at serines 8 and 38. Although HIPK2-mediated phosphorylation of PML occurs early during the DNA damage response, the oncogenic PML-RARalpha fusion protein is phosphorylated with significantly delayed kinetics. DNA damage or HIPK2 expression leads to the stabilization of PML and PML-RARalpha proteins. The N-terminal phosphorylation sites contribute to the DNA damage-induced PML SUMOylation and are required for the ability of PML to cooperate with HIPK2 for the induction of cell death. PMID:19015637

  18. Intramolecular Regulation of Phosphorylation Status of the Circadian Clock Protein KaiC

    PubMed Central

    Xu, Yao; Mori, Tetsuya; Qin, Ximing; Yan, Heping; Egli, Martin; Johnson, Carl Hirschie

    2009-01-01

    Background KaiC, a central clock protein in cyanobacteria, undergoes circadian oscillations between hypophosphorylated and hyperphosphorylated forms in vivo and in vitro. Structural analyses of KaiC crystals have identified threonine and serine residues in KaiC at three residues (T426, S431, and T432) as potential sites at which KaiC is phosphorylated; mutation of any of these three sites to alanine abolishes rhythmicity, revealing an essential clock role for each residue separately and for KaiC phosphorylation in general. Mass spectrometry studies confirmed that the S431 and T432 residues are key phosphorylation sites, however, the role of the threonine residue at position 426 was not clear from the mass spectrometry measurements. Methodology and Principal Findings Mutational approaches and biochemical analyses of KaiC support a key role for T426 in control of the KaiC phosphorylation status in vivo and in vitro and demonstrates that alternative amino acids at residue 426 dramatically affect KaiC's properties in vivo and in vitro, especially genetic dominance/recessive relationships, KaiC dephosphorylation, and the formation of complexes of KaiC with KaiA and KaiB. These mutations alter key circadian properties, including period, amplitude, robustness, and temperature compensation. Crystallographic analyses indicate that the T426 site is phosphorylatible under some conditions, and in vitro phosphorylation assays of KaiC demonstrate labile phosphorylation of KaiC when the primary S431 and T432 sites are blocked. Conclusions and Significance T426 is a crucial site that regulates KaiC phosphorylation status in vivo and in vitro and these studies underscore the importance of KaiC phosphorylation status in the essential cyanobacterial circadian functions. The regulatory roles of these phosphorylation sites–including T426–within KaiC enhance our understanding of the molecular mechanism underlying circadian rhythm generation in cyanobacteria. PMID:19946629

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

    PubMed

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

    1995-07-01

    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) PMID:7542585

  20. Regulation of NHE3 by lysophosphatidic acid is mediated by phosphorylation of NHE3 by RSK2.

    PubMed

    No, Yi Ran; He, Peijian; Yoo, Byong Kwon; Yun, C Chris

    2015-07-01

    Na(+)/H(+) exchange by Na(+)/H(+) exchanger 3 (NHE3) is a major route of sodium absorption in the intestine and kidney. We have shown previously that lysophosphatidic acid (LPA), a small phospholipid produced ubiquitously by all types of cells, stimulates NHE3 via LPA5 receptor. Stimulation of NHE3 activity by LPA involves LPA5 transactivating EGF receptor (EGFR) in the apical membrane. EGFR activates proline-rich tyrosine kinase 2 (Pyk2) and ERK, both of which are necessary for NHE3 regulation. However, Pyk2 and ERK are regulated by EGFR via independent pathways and appear to converge on an unidentified intermediate that ultimately targets NHE3. The p90 ribosomal S6 kinase (RSK) family of Ser/Thr protein kinases is a known effector of EGFR and ERK. Hence, we hypothesized that RSK may be the convergent effector of Pyk2 and ERK although it is not known whether Pyk2 regulates RSK. In this study, we show that Pyk2 is necessary for the maintenance of phosphoinositide-dependent kinase 1 (PDK1) autophosphorylation, and knockdown of Pyk2 or PDK1 mitigated LPA-induced phosphorylation of RSK and stimulation of NHE3 activity. Additionally, we show that RSK2, but not RSK1, is responsible for NHE3 regulation. RSK2 interacts with NHE3 at the apical membrane domain, where it phosphorylates NHE3. Alteration of S663 of NHE3 ablated LPA-induced phosphorylation of NHE3 and stimulation of the transport activity. Our study identifies RSK2 as a new kinase that regulates NHE3 activity by direct phosphorylation. PMID:25855080

  1. Regulation of NHE3 by lysophosphatidic acid is mediated by phosphorylation of NHE3 by RSK2

    PubMed Central

    No, Yi Ran; He, Peijian; Yoo, Byong Kwon

    2015-01-01

    Na+/H+ exchange by Na+/H+ exchanger 3 (NHE3) is a major route of sodium absorption in the intestine and kidney. We have shown previously that lysophosphatidic acid (LPA), a small phospholipid produced ubiquitously by all types of cells, stimulates NHE3 via LPA5 receptor. Stimulation of NHE3 activity by LPA involves LPA5 transactivating EGF receptor (EGFR) in the apical membrane. EGFR activates proline-rich tyrosine kinase 2 (Pyk2) and ERK, both of which are necessary for NHE3 regulation. However, Pyk2 and ERK are regulated by EGFR via independent pathways and appear to converge on an unidentified intermediate that ultimately targets NHE3. The p90 ribosomal S6 kinase (RSK) family of Ser/Thr protein kinases is a known effector of EGFR and ERK. Hence, we hypothesized that RSK may be the convergent effector of Pyk2 and ERK although it is not known whether Pyk2 regulates RSK. In this study, we show that Pyk2 is necessary for the maintenance of phosphoinositide-dependent kinase 1 (PDK1) autophosphorylation, and knockdown of Pyk2 or PDK1 mitigated LPA-induced phosphorylation of RSK and stimulation of NHE3 activity. Additionally, we show that RSK2, but not RSK1, is responsible for NHE3 regulation. RSK2 interacts with NHE3 at the apical membrane domain, where it phosphorylates NHE3. Alteration of S663 of NHE3 ablated LPA-induced phosphorylation of NHE3 and stimulation of the transport activity. Our study identifies RSK2 as a new kinase that regulates NHE3 activity by direct phosphorylation. PMID:25855080

  2. 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

  3. PPM1D controls nucleolar formation by up-regulating phosphorylation of nucleophosmin.

    PubMed

    Kozakai, Yuuki; Kamada, Rui; Furuta, Junya; Kiyota, Yuhei; Chuman, Yoshiro; Sakaguchi, Kazuyasu

    2016-01-01

    An increase of nucleolar number and size has made nucleoli essential markers for cytology and tumour development. However, the underlying basis for their structural integrity and abundance remains unclear. Protein phosphatase PPM1D was found to be up-regulated in different carcinomas including breast cancers. Here, we demonstrate for the first time that PPM1D regulates nucleolar formation via inducing an increased phosphorylation of the nucleolar protein NPM. We show that PPM1D overexpression induces an increase in the nucleolar number regardless of p53 status. We also demonstrated that specific sequential phosphorylation of NPM is important for nucleolar formation and that PPM1D is a novel upstream regulator of this phosphorylation pathway. These results enhance our understanding of the molecular mechanisms that govern nucleoli formation by demonstrating that PPM1D regulates nucleolar formation by regulating NPM phosphorylation status through a novel signalling pathway, PPM1D-CDC25C-CDK1-PLK1. PMID:27619510

  4. Early Steps in Autophagy Depend on Direct Phosphorylation of Atg9 by the Atg1 Kinase

    PubMed Central

    Papinski, Daniel; Schuschnig, Martina; Reiter, Wolfgang; Wilhelm, Larissa; Barnes, Christopher A.; Maiolica, Alessio; Hansmann, Isabella; Pfaffenwimmer, Thaddaeus; Kijanska, Monika; Stoffel, Ingrid; Lee, Sung Sik; Brezovich, Andrea; Lou, Jane Hua; Turk, Benjamin E.; Aebersold, Ruedi; Ammerer, Gustav; Peter, Matthias; Kraft, Claudine

    2014-01-01

    Summary Bulk degradation of cytoplasmic material is mediated by a highly conserved intracellular trafficking pathway termed autophagy. This pathway is characterized by the formation of double-membrane vesicles termed autophagosomes engulfing the substrate and transporting it to the vacuole/lysosome for breakdown and recycling. The Atg1/ULK1 kinase is essential for this process; however, little is known about its targets and the means by which it controls autophagy. Here we have screened for Atg1 kinase substrates using consensus peptide arrays and identified three components of the autophagy machinery. The multimembrane-spanning protein Atg9 is a direct target of this kinase essential for autophagy. Phosphorylated Atg9 is then required for the efficient recruitment of Atg8 and Atg18 to the site of autophagosome formation and subsequent expansion of the isolation membrane, a prerequisite for a functioning autophagy pathway. These findings show that the Atg1 kinase acts early in autophagy by regulating the outgrowth of autophagosomal membranes. PMID:24440502

  5. Regulation of lamin properties and functions: does phosphorylation do it all?

    PubMed Central

    Machowska, Magdalena; Piekarowicz, Katarzyna; Rzepecki, Ryszard

    2015-01-01

    The main functions of lamins are their mechanical and structural roles as major building blocks of the karyoskeleton. They are also involved in chromatin structure regulation, gene expression, intracellular signalling pathway modulation and development. All essential lamin functions seem to depend on their capacity for assembly or disassembly after the receipt of specific signals, and after specific, selective and precisely regulated interactions through their various domains. Reversible phosphorylation of lamins is crucial for their functions, so it is important to understand how lamin polymerization and interactions are modulated, and which sequences may undergo such modifications. This review combines experimental data with results of our in silico analyses focused on lamin phosphorylation in model organisms to show the presence of evolutionarily conserved sequences and to indicate specific in vivo phosphorylations that affect particular functions. PMID:26581574

  6. Regulation of GABA-modulin phosphorylation and GABA receptor binding by excitatory amino acids

    SciTech Connect

    Vaccarino, F.; Guidotti, A.

    1987-05-01

    Primary cultures of cerebellar granule cells phosphorylate numerous proteins including GABA-modulin (GM), which is a putative allosteric modulator of GABA receptors. Cell depolarization and treatment with dicarboxylic excitatory amino acids, which activate PI turnover, Ca/sup 2 +/ influx and guanylate cyclase in granule cells increase the phosphorylation of specific proteins. To determine GM phosphorylation by endogenous protein kinases in living granule cell cultures, GM was isolated by immunoprecipitation and reverse-phase HPLC. High K/sup +/, veratridine, glutamate and NMDA treatment stimulated GM phosphorylation over 2-fold. This increase was abolished by the absence of extracellular Ca/sup 2 +/ and was antagonized by Mg/sup 2 +/ ions and by AVP. The excitatory amino acid action was mimicked by phorbol esters but not by forskolin or by cGMP, and thus may be mediated by an activation of protein kinase C (PKC). Moreover, excitatory amino acids increase /sup 3/H-labelled phorbol ester binding sites in granule cell membrane. The same cultures, treated with glutamate or kainate, showed a 50-fold greater efficacy of muscimol for the stimulation of benzodiazepine (BZ) binding. These data-suggest that excitatory amino acid stimulation of neurons triggers PKC translocation and the activated enzyme phosphorylates GM. The extent of GM phosphorylation may regulate the coupling between GABA and BZ binding sites.

  7. Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation

    PubMed Central

    Montagnoli, Alessia; Fiore, Francesca; Eytan, Esther; Carrano, Andrea C.; Draetta, Giulio F.; Hershko, Avram; Pagano, Michele

    1999-01-01

    The cellular abundance of the cyclin-dependent kinase (Cdk) inhibitor p27 is regulated by the ubiquitin–proteasome system. Activation of p27 degradation is seen in proliferating cells and in many types of aggressive human carcinomas. p27 can be phosphorylated on threonine 187 by Cdks, and cyclin E/Cdk2 overexpression can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, whether threonine 187 phosphorylation stimulates p27 degradation through the ubiquitin–proteasome system or an alternative pathway is still not known. Here, we demonstrate that p27 ubiquitination (as assayed in vivo and in an in vitro reconstituted system) is cell-cycle regulated and that Cdk activity is required for the in vitro ubiquitination of p27. Furthermore, ubiquitination of wild-type p27, but not of p27(T187A), can occur in G1-enriched extracts only upon addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreonine 187 site-specific antibody for p27, we show that threonine 187 phosphorylation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G1 cells. Finally, we show that in addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires formation of a trimeric complex with the cyclin and Cdk subunits. In fact, cyclin B/Cdk1 which can phosphorylate p27 efficiently, but cannot form a stable complex with it, is unable to stimulate p27 ubiquitination by G1 extracts. Furthermore, another p27 mutant [p27(CK−)] that can be phosphorylated by cyclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquitination. Thus throughout the cell cycle, both phosphorylation and trimeric complex formation act as signals for the ubiquitination of a Cdk inhibitor. PMID:10323868

  8. Extensive Crosstalk Between O-GlcNAcylation and Phosphorylation Regulates Cytokinesis

    PubMed Central

    Wang, Zihao; Udeshi, Namrata D.; Slawson, Chad; Compton, Philip D.; Sakabe, Kaoru; Cheung, Win D.; Shabanowitz, Jeffrey; Hunt, Donald F.; Hart, Gerald W.

    2010-01-01

    Like phosphorylation, the addition of O-linked β-N-acetylglucosamine (O-GlcNAcylation) is a ubiquitous, reversible process that modifies serine and threonine residues on nuclear and cytoplasmic proteins. Overexpression of the enzyme that adds O-GlcNAc to target proteins, O-GlcNAc transferase (OGT), perturbs cytokinesis and promotes polyploidy, but the molecular targets of OGT that are important for its cell cycle functions are unknown. Here, we identify 141 previously unknown O-GlcNAc sites on proteins that function in spindle assembly and cytokinesis. Many of these O-GlcNAcylation sites are either identical to known phosphorylation sites or in close proximity to them. Furthermore, we found that O-GlcNAcylation altered the phosphorylation of key proteins associated with the mitotic spindle and midbody. Forced overexpression of OGT increased the inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1) and reduced the phosphorylation of CDK1 target proteins. The increased phosphorylation of CDK1 is explained by increased activation of its upstream kinase, MYT1, and by a concomitant reduction in the transcript for the CDK1 phosphatase, CDC25C. OGT overexpression also caused a reduction in both messenger RNA expression and protein abundance of Polo-like kinase 1, which is upstream of both MYT1 and CDC25C. The data not only illustrate the crosstalk between O-GlcNAcylation and phosphorylation of proteins that are regulators of crucial signaling pathways, but also uncover a mechanism for the role of O-GlcNAcylation in regulation of cell division. PMID:20068230

  9. The Intracellular Concentration of Acetyl Phosphate in Escherichia coli Is Sufficient for Direct Phosphorylation of Two-Component Response Regulators▿

    PubMed Central

    Klein, Adam H.; Shulla, Ana; Reimann, Sylvia A.; Keating, David H.; Wolfe, Alan J.

    2007-01-01

    Acetyl phosphate, the intermediate of the AckA-Pta pathway, acts as a global signal in Escherichia coli. Although acetyl phosphate clearly signals through two-component response regulators, it remains unclear whether acetyl phosphate acts as a direct phospho donor or functions through an indirect mechanism. We used two-dimensional thin-layer chromatography to measure the relative concentrations of acetyl phosphate, acetyl coenzyme A, ATP, and GTP over the course of the entire growth curve. We estimated that the intracellular concentration of acetyl phosphate in wild-type cells reaches at least 3 mM, a concentration sufficient to activate two-component response regulators via direct phosphoryl transfer. PMID:17545286

  10. Phosphorylation of filamin A by Cdk1 regulates filamin A localization and daughter cell separation.

    PubMed

    Szeto, Sandy G Y; Williams, Elizabeth C; Rudner, Adam D; Lee, Jonathan M

    2015-01-15

    In cell culture, many adherent mammalian cells undergo substantial actin cytoskeleton rearrangement prior to mitosis as they detach from the extracellular matrix and become spherical. At the end of mitosis, the actin cytoskeleton is required for cytokinesis and the reassembly of interphase structures as cells spread and reattach to substrate. To understand the processes regulating mitotic cytoskeletal remodeling, we studied how mitotic phosphorylation regulates filamin A (FLNa). FLNa is an actin-crosslinking protein that was previously identified as a cyclin-dependent kinase 1 (Cdk1) binding partner and substrate in vitro. Using quantitative label-based mass spectrometry, we find that FLNa serines 1084, 1459 and 1533 are phosphorylated in mitotic HeLa cells and all three sites match the phosphorylation consensus sequence of Cdk1. To investigate the functional role of mitotic FLNa phosphorylation, we mutated serines 1084, 1459 and 1533 to nonphosphorylatable alanine residues and expressed GFP-tagged FLNa(S1084A,S1459A,S1533A) (FLNa-AAA GFP) in a FLNa-deficient human melanoma cell line called M2. M2 cells expressing FLNa-AAA GFP have enhanced FLNa-AAA GFP and actin localization at sites of contact between daughter cells, impaired post-mitotic daughter cell separation and defects in cell migration. Therefore, mitotic phosphorylation of FLNa is important for successful cell division and interphase cell behavior. PMID:25445790

  11. Cdk9 T-loop Phosphorylation is Regulated by the Calcium Signaling Pathway

    PubMed Central

    Ramakrishnan, Rajesh; Rice, Andrew P.

    2011-01-01

    Eukaryotic RNA polymerase II transcriptional elongation is a tightly regulated process and is dependent upon positive transcription elongation factor-b (P-TEFb). The core P-TEFb complex is composed of Cdk9 and Cyclin T and is essential for the expression of most protein coding genes. Cdk9 kinase function is dependent upon phosphorylation of Thr186 in its T-loop. In this study, we examined kinases and signaling pathways that influence Cdk9 T-loop phosphorylation. Using an RNAi screen in HeLa cells, we found that Cdk9 T-loop phosphorylation is regulated by Calcium/Calmodulin- dependent kinase 1D (CaMK1D). Using small molecules inhibitors in HeLa cells and primary CD4+ T lymphocytes, we found that the Ca2+ signaling pathway is required for Cdk9 T-loop phosphorylation. Inhibition of Ca2+ signaling led to dephosphorylation of Thr186 on Cdk9. In reporter plasmid assays, inhibition of the Ca2+ signaling pathway repressed the PCNA promoter and HIV-1 Tat transactivation of the HIV-1 LTR, but not HTLV-1 Tax transactivation of the HTLV-1 LTR, suggesting that perturbation of the Ca2+ pathway and reduction of Cdk9 T-loop phosphorylation inhibits transcription units that have a rigorous requirement for P-TEFb function. PMID:21448926

  12. Role of individual R domain phosphorylation sites in CFTR regulation by protein kinase A.

    PubMed

    Hegedus, Tamás; Aleksandrov, Andrei; Mengos, April; Cui, Liying; Jensen, Timothy J; Riordan, John R

    2009-06-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in transcellular ion transport and when defective, results in the genetic disease cystic fibrosis. CFTR is novel in the ATP-binding cassette superfamily as an ion channel that is enabled by a unique unstructured regulatory domain. This R domain contains multiple protein kinase A sites, which when phosphorylated allow channel gating. Most of the sites have been indicated to stimulate channel activity, while two of them have been suggested to be inhibitory. It is unknown whether individual sites act coordinately or distinctly. To address this issue, we raised monoclonal antibodies recognizing the unphosphorylated, but not the phosphorylated states of four functionally relevant sites (700, 737, 768, and 813). This enabled simultaneous monitoring of their phosphorylation and dephosphorylation and revealed that both processes occurred rapidly at the first three sites, but more slowly at the fourth. The parallel phosphorylation rates of the stimulatory 700 and the putative inhibitory 737 and 768 sites prompted us to reexamine the role of the latter two. With serines 737 and 768 reintroduced individually into a PKA insensitive variant, in which serines at 15 sites had been replaced by alanines, a level of channel activation by PKA was restored, showing that these sites can mediate stimulation. Thus, we have provided new tools to study the CFTR regulation by phosphorylation and found that sites proposed to inhibit channel activity can also participate in stimulation. PMID:19328185

  13. VANADL SULFATE INHIBITS NO PRODUCTION BY DIFFERENTIALLY REGULATING SERINE/THREONINE PHOSPHORYLATION OF ENOS

    EPA Science Inventory

    VANADYL SULFATE INHIBITS NO PRODUCTION BY DIFFERENTIALLY REGULATING SERINE/THREONINE PHOSPHORYLATION OF eNOS. Zhuowei Li, Jacqueline D. Carter, Lisa A. Dailey, Joleen Soukup, Yuh-Chin T. Huang. CEMALB, University of North Carolina and ORD, US EPA, Chapel Hill, North Carolina
    V...

  14. VANADYL SULFATE INHIBITS NO PRODUCTION BY DIFFERENTIALLY REGULATING SERINE/THREONINE PHOSPHORYLATION OF ENOS

    EPA Science Inventory

    VANADYL SULFATE INHIBITS NO PRODUCTION BY DIFFERENTIALLY REGULATING SERINE/THREONINE PHOSPHORYLATION OF eNOS.

    Zhuowei Li, Jacqueline D. Carter, Lisa A. Dailey, Joleen Soukup, Yuh-Chin T. Huang. CEMALB, University of North Carolina and NHEERL, US EPA, Chapel Hill, North Ca...

  15. NEK6-mediated phosphorylation of human TPP1 regulates telomere length through telomerase recruitment.

    PubMed

    Hirai, Yugo; Tamura, Miki; Otani, Junji; Ishikawa, Fuyuki

    2016-08-01

    Shelterin component TPP1 plays critical roles in chromosome end protection and telomere length regulation. Specifically, TPP1 contains an OB-fold domain that provides an interface to recruit telomerase. However, it remains largely unknown how telomerase recruitment is regulated by cell cycle regulators. We show that TPP1 interacts with the cell cycle regulator kinase NEK6 in human cells. We found that NEK6-mediated phosphorylation of TPP1 Ser255 in G2/M phase regulates the association between telomerase activity and TPP1. Furthermore, we found evidence that POT1 negatively regulates TPP1 phosphorylation because the level of Ser255 phosphorylation was elevated when telomeres were elongated by a POT1 mutant lacking its OB-fold domains. Ser255 is located in the intervening region between the telomerase-recruiting OB-fold and the POT1 recruitment domains. Ser255 and the surrounding amino acids are conserved among vertebrates. These observations suggest that a region adjacent to the OB-fold domain of TPP1 is involved in telomere length regulation via telomerase recruitment. PMID:27396482

  16. Regulation of AU-Rich Element RNA Binding Proteins by Phosphorylation and the Prolyl Isomerase Pin1

    PubMed Central

    Shen, Zhong-Jian; Malter, James S.

    2015-01-01

    The accumulation of 3' untranslated region (3'-UTR), AU-rich element (ARE) containing mRNAs, are predominantly controlled at the post-transcriptional level. Regulation appears to rely on a variable and dynamic interaction between mRNA target and ARE-specific binding proteins (AUBPs). The AUBP-ARE mRNA recognition is directed by multiple intracellular signals that are predominantly targeted at the AUBPs. These include (but are unlikely limited to) methylation, acetylation, phosphorylation, ubiquitination and isomerization. These regulatory events ultimately affect ARE mRNA location, abundance, translation and stability. In this review, we describe recent advances in our understanding of phosphorylation and its impact on conformation of the AUBPs, interaction with ARE mRNAs and highlight the role of Pin1 mediated prolyl cis-trans isomerization in these biological process. PMID:25874604

  17. Regulation of Tyrosine Hydroxylase Expression and Phosphorylation in Dopamine Transporter-Deficient Mice.

    PubMed

    Salvatore, Michael F; Calipari, Erin S; Jones, Sara R

    2016-07-20

    Tyrosine hydroxylase (TH) and dopamine transporters (DATs) regulate dopamine (DA) neurotransmission at the biosynthesis and reuptake steps, respectively. Dysfunction or loss of these proteins occurs in impaired locomotor or addictive behavior, but little is known about the influence of DAT expression on TH function. Differences in TH phosphorylation, DA tissue content, l-DOPA biosynthesis, and DA turnover exist between the somatodendritic and terminal field compartments of nigrostriatal and mesoaccumbens pathways. We examined whether differential DAT expression affects these compartmental differences in DA regulation by comparing TH expression and phosphorylation at ser31 and ser40. In heterozygous DAT knockout (KO) (+/-) mice, DA tissue content and DA turnover were unchanged relative to wild-type mice, despite a 40% reduction in DAT protein expression. In DAT KO (-/-) mice, DA turnover increased in all DA compartments, but DA tissue content decreased (90-96%) only in terminal fields. TH protein expression and phosphorylation were differentially affected within DA pathway compartments by relative expression of DAT. TH protein decreased (∼74%), though to a significantly lesser extent than DA, in striatum and nucleus accumbens (NAc) in DAT -/- mice, with no decrease in substantia nigra or ventral tegmental area. Striatal ser31 TH phosphorylation and recovery of DA relative to TH protein expression in DAT +/- and DAT -/- mice decreased, whereas ser40 TH phosphorylation increased ∼2- to 3-fold in striatum and NAc of DAT -/- mice. These results suggest that DAT expression affects TH expression and phosphorylation largely in DA terminal field compartments, further corroborating evidence for dichotomous regulation of TH between somatodendritic and terminal field compartments of the nigrostriatal and mesoaccumbens pathways. PMID:27124386

  18. Phosphorylation of FEZ1 by Microtubule Affinity Regulating Kinases regulates its function in presynaptic protein trafficking

    PubMed Central

    Butkevich, Eugenia; Härtig, Wolfgang; Nikolov, Miroslav; Erck, Christian; Grosche, Jens; Urlaub, Henning; Schmidt, Christoph F.; Klopfenstein, Dieter R.; Chua, John Jia En

    2016-01-01

    Adapters bind motor proteins to cargoes and therefore play essential roles in Kinesin-1 mediated intracellular transport. The regulatory mechanisms governing adapter functions and the spectrum of cargoes recognized by individual adapters remain poorly defined. Here, we show that cargoes transported by the Kinesin-1 adapter FEZ1 are enriched for presynaptic components and identify that specific phosphorylation of FEZ1 at its serine 58 regulatory site is mediated by microtubule affinity-regulating kinases (MARK/PAR-1). Loss of MARK/PAR-1 impairs axonal transport, with adapter and cargo abnormally co-aggregating in neuronal cell bodies and axons. Presynaptic specializations are markedly reduced and distorted in FEZ1 and MARK/PAR-1 mutants. Strikingly, abnormal co-aggregates of unphosphorylated FEZ1, Kinesin-1 and its putative cargoes are present in brains of transgenic mice modelling aspects of Alzheimer’s disease, a neurodegenerative disorder exhibiting impaired axonal transport and altered MARK activity. Our findings suggest that perturbed FEZ1-mediated synaptic delivery of proteins arising from abnormal signalling potentially contributes to the process of neurodegeneration. PMID:27247180

  19. Phosphorylation of FEZ1 by Microtubule Affinity Regulating Kinases regulates its function in presynaptic protein trafficking.

    PubMed

    Butkevich, Eugenia; Härtig, Wolfgang; Nikolov, Miroslav; Erck, Christian; Grosche, Jens; Urlaub, Henning; Schmidt, Christoph F; Klopfenstein, Dieter R; Chua, John Jia En

    2016-01-01

    Adapters bind motor proteins to cargoes and therefore play essential roles in Kinesin-1 mediated intracellular transport. The regulatory mechanisms governing adapter functions and the spectrum of cargoes recognized by individual adapters remain poorly defined. Here, we show that cargoes transported by the Kinesin-1 adapter FEZ1 are enriched for presynaptic components and identify that specific phosphorylation of FEZ1 at its serine 58 regulatory site is mediated by microtubule affinity-regulating kinases (MARK/PAR-1). Loss of MARK/PAR-1 impairs axonal transport, with adapter and cargo abnormally co-aggregating in neuronal cell bodies and axons. Presynaptic specializations are markedly reduced and distorted in FEZ1 and MARK/PAR-1 mutants. Strikingly, abnormal co-aggregates of unphosphorylated FEZ1, Kinesin-1 and its putative cargoes are present in brains of transgenic mice modelling aspects of Alzheimer's disease, a neurodegenerative disorder exhibiting impaired axonal transport and altered MARK activity. Our findings suggest that perturbed FEZ1-mediated synaptic delivery of proteins arising from abnormal signalling potentially contributes to the process of neurodegeneration. PMID:27247180

  20. Regulation of Beclin 1 Protein Phosphorylation and Autophagy by Protein Phosphatase 2A (PP2A) and Death-associated Protein Kinase 3 (DAPK3).

    PubMed

    Fujiwara, Nobuyuki; Usui, Tatsuya; Ohama, Takashi; Sato, Koichi

    2016-05-13

    Autophagy is an evolutionarily conserved intracellular degradation system that is involved in cell survival and activated in various diseases, including cancer. Beclin 1 is a central scaffold protein that assembles components for promoting or inhibiting autophagy. Association of Beclin 1 with its interacting proteins is regulated by the phosphorylation of Beclin 1 by various Ser/Thr kinases, but the Ser/Thr phosphatases that regulate these phosphorylation events remain unknown. Here we identify Ser-90 in Beclin 1 as a regulatory site whose phosphorylation is markedly enhanced in cells treated with okadaic acid, an inhibitor of protein phosphatase 2A (PP2A). Beclin 1 Ser-90 phosphorylation is induced in skeletal muscle tissues isolated from starved mice. The Beclin 1 S90A mutant blocked starvation-induced autophagy. We found association of PP2A B55α with Beclin 1, which dissociate by starvation. We also found that death-associated protein kinase 3 directly phosphorylates Beclin 1 Ser-90. We propose that physiological regulation of Beclin 1 Ser-90 phosphorylation by PP2A and death-associated protein kinase 3 controls autophagy. PMID:26994142

  1. O-GlcNAcylation stabilizes β-catenin through direct competition with phosphorylation at threonine 41

    PubMed Central

    Olivier-Van Stichelen, Stéphanie; Dehennaut, Vanessa; Buzy, Armelle; Zachayus, Jean-Luc; Guinez, Céline; Mir, Anne-Marie; El Yazidi-Belkoura, Ikram; Copin, Marie-Christine; Boureme, Didier; Loyaux, Denis; Ferrara, Pascual; Lefebvre, Tony

    2014-01-01

    Dysfunctions in Wnt signaling increase β-catenin stability and are associated with cancers, including colorectal cancer. In addition, β-catenin degradation is decreased by nutrient-dependent O-GlcNAcylation. Human colon tumors and colons from mice fed high-carbohydrate diets exhibited higher amounts of β-catenin and O-GlcNAc relative to healthy tissues and mice fed a standard diet, respectively. Administration of the O-GlcNAcase inhibitor thiamet G to mice also increased colonic expression of β-catenin. By ETD-MS/MS, we identified 4 O-GlcNAcylation sites at the N terminus of β-catenin (S23/T40/T41/T112). Furthermore, mutation of serine and threonine residues within the D box of β-catenin reduced O-GlcNAcylation by 75%. Interestingly, elevating O-GlcNAcylation in human colon cell lines drastically reduced phosphorylation at T41, a key residue of the D box responsible for β-catenin stability. Analyses of β-catenin O-GlcNAcylation mutants reinforced T41 as the most crucial residue that controls the β-catenin degradation rate. Finally, inhibiting O-GlcNAcylation decreased the β-catenin/α-catenin interaction necessary for mucosa integrity, whereas O-GlcNAcase silencing improved this interaction. These results suggest that O-GlcNAcylation regulates not only the stability of β-catenin, but also affects its localization at the level of adherens junctions. Accordingly, we propose that O-GlcNAcylation of β-catenin is a missing link between the glucose metabolism deregulation observed in metabolic disorders and the development of cancer.—Olivier-Van Stichelen, S., Dehennaut, V., Buzy, A., Zachayus, J.-L., Guinez, C., Mir, A.-M., El Yazidi-Belkoura, I., Copin, M.-C., Boureme, D., Loyaux, D., Ferrara, P., Lefebvre, T. O-GlcNAcylation stabilizes β-catenin through direct competition with phosphorylation at threonine 41. PMID:24744147

  2. PKC-2 phosphorylation of UNC-18 Ser322 in AFD neurons regulates temperature dependency of locomotion.

    PubMed

    Edwards, Mark R; Johnson, James R; Rankin, Kimberley; Jenkins, Rosalind E; Maguire, Carl; Morgan, Alan; Burgoyne, Robert D; Barclay, Jeff W

    2012-05-16

    Diacylglycerol (DAG)/protein kinase C (PKC) signaling plays an integral role in the regulation of neuronal function. This is certainly true in Caenorhabditis elegans and in particular for thermosensory signaling and behavior. Downstream molecular targets for transduction of this signaling cascade remain, however, virtually uncharacterized. We investigated whether PKC phosphorylation of Munc18-1, an essential protein in vesicle trafficking and exocytosis, was the downstream effector for DAG regulation of thermosensory behavior. We demonstrate here that the C. elegans ortholog of Munc18-1, UNC-18, was phosphorylated in vitro at Ser322. Transgenic rescue of unc-18-null worms with Ser322 phosphomutants displayed altered thermosensitivity. C. elegans expresses three DAG-regulated PKCs, and blocking UNC-18 Ser322 phosphorylation was phenocopied only by deletion of calcium-activated PKC-2. Expression of nonphosphorylatable UNC-18 S322A, either pan-neuronally or specifically in AFD thermosensory neurons, converted wild-type worms to a pkc-2-null phenotype. These data demonstrate that an individual DAG-dependent thermosensory behavior of an organism is effected specifically by the downstream PKC-2 phosphorylation of UNC-18 on Ser322 in AFD neurons. PMID:22593072

  3. Phosphorylation of the RNA-binding protein Dazl by MAPKAP kinase 2 regulates spermatogenesis.

    PubMed

    Williams, Patrick A; Krug, Michael S; McMillan, Emily A; Peake, Jasmine D; Davis, Tara L; Cocklin, Simon; Strochlic, Todd I

    2016-08-01

    Developing male germ cells are exquisitely sensitive to environmental insults such as heat and oxidative stress. An additional characteristic of these cells is their unique dependence on RNA-binding proteins for regulating posttranscriptional gene expression and translational control. Here we provide a mechanistic link unifying these two features. We show that the germ cell-specific RNA-binding protein deleted in azoospermia-like (Dazl) is phosphorylated by MAPKAP kinase 2 (MK2), a stress-induced protein kinase activated downstream of p38 MAPK. We demonstrate that phosphorylation of Dazl by MK2 on an evolutionarily conserved serine residue inhibits its interaction with poly(A)-binding protein, resulting in reduced translation of Dazl-regulated target RNAs. We further show that transgenic expression of wild-type human Dazl but not a phosphomimetic form in the Drosophila male germline can restore fertility to flies deficient in boule, the Drosophila orthologue of human Dazl. These results illuminate a novel role for MK2 in spermatogenesis, expand the repertoire of RNA-binding proteins phosphorylated by this kinase, and suggest that signaling by the p38-MK2 pathway is a negative regulator of spermatogenesis via phosphorylation of Dazl. PMID:27280388

  4. AMP-activated protein kinase phosphorylates CtBP1 and down-regulates its activity

    SciTech Connect

    Kim, Jae-Hwan; Choi, Soo-Youn; Kang, Byung-Hee; Lee, Soon-Min; Cho, Eun-Jung; Youn, Hong-Duk

    2013-02-01

    Highlights: ► AMPK phosphorylates CtBP1 on serine 158. ► AMPK-mediated phosphorylation of CtBP1 causes the ubiquitination and nuclear export of CtBP1. ► AMPK downregulates the CtBP1-mediated repression of Bax transcription. -- Abstract: CtBP is a transcriptional repressor which plays a significant role in the regulation of cell proliferation and tumor progression. It was reported that glucose withdrawal causes induction of Bax due to the dissociation of CtBP from the Bax promoter. However, the precise mechanism involved in the regulation of CtBP still remains unclear. In this study, we found that an activated AMP-activated protein kinase (AMPK) phosphorylates CtBP1 on Ser-158 upon metabolic stresses. Moreover, AMPK-mediated phosphorylation of CtBP1 (S158) attenuates the repressive function of CtBP1. We also confirmed that triggering activation of AMPK by various factors resulted in an increase of Bax gene expression. These findings provide connections of AMPK with CtBP1-mediated regulation of Bax expression for cell death under metabolic stresses.

  5. Occludin S490 Phosphorylation Regulates Vascular Endothelial Growth Factor-Induced Retinal Neovascularization.

    PubMed

    Liu, Xuwen; Dreffs, Alyssa; Díaz-Coránguez, Monica; Runkle, E Aaron; Gardner, Thomas W; Chiodo, Vince A; Hauswirth, William W; Antonetti, David A

    2016-09-01

    Occludin is a transmembrane tight junction protein that contributes to diverse cellular functions, including control of barrier properties, cell migration, and proliferation. Vascular endothelial growth factor (VEGF) induces phosphorylation of occludin at S490, which is required for VEGF-induced endothelial permeability. Herein, we demonstrate that occludin S490 phosphorylation also regulates VEGF-induced retinal endothelial cell proliferation and neovascularization. Using a specific antibody, phospho-occludin was located in centrosomes in endothelial cell cultures, animal models, and human surgical samples of retinal neovessels. Occludin S490 phosphorylation was found to increase with endothelial tube formation in vitro and in vivo during retinal neovascularization after induction of VEGF expression. More important, expression of occludin mutated at S490 to Ala, completely inhibited angiogenesis in cell culture models and in vivo. Collectively, these data suggest a novel role for occludin in regulation of endothelial proliferation and angiogenesis in a phosphorylation-dependent manner. These findings may lead to methods of regulating pathological neovascularization by specifically targeting endothelial cell proliferation. PMID:27423695

  6. Synaptic Clustering of PSD-95 Is Regulated by c-Abl through Tyrosine Phosphorylation

    PubMed Central

    de Arce, Karen Perez; Varela-Nallar, Lorena; Farias, Olivia; Cifuentes, Alejandra; Bull, Paulina; Couch, Brian A.; Koleske, Anthony J.; Inestrosa, Nibaldo C.; Alvarez, Alejandra R.

    2010-01-01

    The c-Abl tyrosine kinase is present in mouse brain synapses, but its precise synaptic function is unknown. We found that c-Abl levels in the rat hippocampus increase postnatally, with expression peaking at the first postnatal week. In 14 d in vitro hippocampal neuron cultures, c-Abl localizes primarily to the postsynaptic compartment, in which it colocalizes with the postsynaptic scaffold protein postsynaptic density protein-95 (PSD-95) in apposition to presynaptic markers. c-Abl associates with PSD-95, and chemical or genetic inhibition of c-Abl kinase activity reduces PSD-95 tyrosine phosphorylation, leading to reduced PSD-95 clustering and reduced synapses in treated neurons. c-Abl can phosphorylate PSD-95 on tyrosine 533, and mutation of this residue reduces the ability of PSD-95 to cluster at postsynaptic sites. Our results indicate that c-Abl regulates synapse formation by mediating tyrosine phosphorylation and clustering of PSD-95. PMID:20220006

  7. From inventory to functional mechanisms: regulation of the mitochondrial protein import machinery by phosphorylation.

    PubMed

    Gerbeth, Carolin; Mikropoulou, Despina; Meisinger, Chris

    2013-10-01

    For decades, the pyruvate dehydrogenase complex in the mitochondrial matrix was considered as a rare example of how protein kinases and phosphatases can regulate important functions within this organelle. During the last decade, several proteomic studies revealed that a large fraction of mitochondrial proteins are indeed phosphorylated. A surprisingly high number of phosphorylation sites was found at the preprotein import machinery, TOM, in the outer membrane that provides the central protein import gate for most mitochondrial precursors synthesized in the cytosol. This review describes current knowledge of the mitochondrial phosphoproteome and introduces the first regulatory mechanisms of protein import dynamics by reversible phosphorylation, which have been uncovered mainly in the model organism Saccharomyces cerevisiae. PMID:23895388

  8. Phosphorylation Regulates Id2 Degradation and Mediates the Proliferation of Neural Precursor Cells

    PubMed Central

    Sullivan, Jaclyn M.; Havrda, Matthew C.; Kettenbach, Arminja N.; Paolella, Brenton R.; Zhang, Zhonghua; Gerber, Scott A.; Israel, Mark A.

    2016-01-01

    Inhibitor of DNA binding proteins (Id1-Id4) function to inhibit differentiation and promote proliferation of many different cell types. Among the Id family members, Id2 has been most extensively studied in the central nervous system (CNS). Id2 contributes to cultured neural precursor cell (NPC) proliferation as well as to the proliferation of CNS tumors such as glioblastoma that are likely to arise from NPC-like cells. We identified three phosphorylation sites near the N-terminus of Id2 in NPCs. To interrogate the importance of Id2 phosphorylation, Id2−/− NPCs were modified to express wild type (WT) Id2 or an Id2 mutant protein that could not be phosphorylated at the identified sites. We observed that NPCs expressing this mutant lacking phosphorylation near the N-terminus had higher steady-state levels of Id2 when compared to NPCs expressing WT Id2. This elevated level was the result of a longer half-life and reduced proteasome-mediated degradation. Moreover, NPCs expressing constitutively de-phosphorylated Id2 proliferated more rapidly than NPCs expressing WT Id2, a finding consistent with the well-characterized function of Id2 in driving proliferation. Observing that phosphorylation of Id2 modulates the degradation of this important cell-cycle regulator, we sought to identify a phosphatase that would stabilize Id2 enhancing its activity in NPCs and extended our analysis to include human glioblastoma-derived stem cells (GSCs). We found that expression of the phosphatase PP2A altered Id2 levels. Our findings suggest that inhibition of PP2A may be a novel strategy to regulate the proliferation of normal NPCs and malignant GSCs by decreasing Id2 levels. PMID:26756672

  9. LATS1 phosphorylates forkhead L2 and regulates its transcriptional activity.

    PubMed

    Pisarska, Margareta D; Kuo, Fang-Ting; Bentsi-Barnes, Ikuko K; Khan, Salma; Barlow, Gillian M

    2010-07-01

    Forkhead L2 (FOXL2) is expressed in the ovary and acts as a transcriptional repressor of the steroidogenic acute regulatory (StAR) gene, a marker of granulosa cell differentiation. Human FOXL2 mutations that produce truncated proteins lacking the COOH terminus result in blepharophimosis/ptosis/epicanthus inversus (BPES) syndrome type I, which is associated with premature ovarian failure (POF). In this study, we investigated whether FOXL2's activity as a transcriptional repressor is regulated by phosphorylation. We found that FOXL2 is phosphorylated at a serine residue and, using yeast two-hybrid screening, identified LATS1 as a potential FOXL2-interacting protein. LATS1 is a serine/threonine kinase whose deletion in mice results in an ovarian phenotype similar to POF. Using coimmunoprecipitation and kinase assays, we confirmed that LATS1 binds to FOXL2 and demonstrated that LATS1 phosphorylates FOXL2 at a serine residue. Moreover, we found that FOXL2 and LATS1 are coexpressed in developing mouse gonads and in granulosa cells of small and medium follicles in the mouse ovary. Last, we demonstrated that coexpression with LATS1 enhances FOXL2's activity as a repressor of the StAR promoter, and this results from the kinase activity of LATS1. These results provide novel evidence that FOXL2 is phosphorylated by LATS1 and that this phosphorylation enhances the transcriptional repression of the StAR gene, a marker of granulosa cell differentiation. These data support our hypothesis that phosphorylation of FOXL2 may be a control mechanism regulating the rate of granulosa cell differentiation and hence, follicle maturation, and its dysregulation may contribute to accelerated follicular development and POF in BPES type I. PMID:20407010

  10. Protein kinase Cζ exhibits constitutive phosphorylation and phosphatidylinositol-3,4,5-triphosphate-independent regulation

    PubMed Central

    Tobias, Irene S.; Kaulich, Manuel; Kim, Peter K.; Simon, Nitya; Jacinto, Estela; Dowdy, Steven F.; King, Charles C.; Newton, Alexandra C.

    2016-01-01

    Atypical protein kinase C (aPKC) isoenzymes are key modulators of insulin signalling, and their dysfunction correlates with insulin-resistant states in both mice and humans. Despite the engaged interest in the importance of aPKCs to type 2 diabetes, much less is known about the molecular mechanisms that govern their cellular functions than for the conventional and novel PKC isoenzymes and the functionally-related protein kinase B (Akt) family of kinases. Here we show that aPKC is constitutively phosphorylated and, using a genetically-encoded reporter for PKC activity, basally active in cells. Specifically, we show that phosphorylation at two key regulatory sites, the activation loop and turn motif, of the aPKC PKCζ in multiple cultured cell types is constitutive and independently regulated by separate kinases: ribosome-associated mammalian target of rapamycin complex 2 (mTORC2) mediates co-translational phosphorylation of the turn motif, followed by phosphorylation at the activation loop by phosphoinositide-dependent kinase-1 (PDK1). Live cell imaging reveals that global aPKC activity is constitutive and insulin unresponsive, in marked contrast to the insulin-dependent activation of Akt monitored by an Akt-specific reporter. Nor does forced recruitment to phosphoinositides by fusing the pleckstrin homology (PH) domain of Akt to the kinase domain of PKCζ alter either the phosphorylation or activity of PKCζ. Thus, insulin stimulation does not activate PKCζ through the canonical phosphatidylinositol-3,4,5-triphosphate-mediated pathway that activates Akt, contrasting with previous literature on PKCζ activation. These studies support a model wherein an alternative mechanism regulates PKCζ-mediated insulin signalling that does not utilize conventional activation via agonist-evoked phosphorylation at the activation loop. Rather, we propose that scaffolding near substrates drives the function of PKCζ. PMID:26635352

  11. Extracellular signal-regulated kinase phosphorylation in forebrain neurones contributes to osmoregulatory mechanisms

    PubMed Central

    Dine, Julien; Ducourneau, Vincent R R; Fénelon, Valérie S; Fossat, Pascal; Amadio, Aurélie; Eder, Matthias; Israel, Jean-Marc; Oliet, Stéphane H R; Voisin, Daniel L

    2014-01-01

    Vasopressin secretion from the magnocellular neurosecretory cells (MNCs) is crucial for body fluid homeostasis. Osmotic regulation of MNC activity involves the concerted modulation of intrinsic mechanosensitive ion channels, taurine release from local astrocytes as well as excitatory inputs derived from osmosensitive forebrain regions. Extracellular signal-regulated protein kinases (ERK) are mitogen-activated protein kinases that transduce extracellular stimuli into intracellular post-translational and transcriptional responses, leading to changes in intrinsic neuronal properties and synaptic function. Here, we investigated whether ERK activation (i.e. phosphorylation) plays a role in the functioning of forebrain osmoregulatory networks. We found that within 10 min after intraperitoneal injections of hypertonic saline (3 m, 6 m) in rats, many phosphoERK-immunopositive neurones were observed in osmosensitive forebrain regions, including the MNC containing supraoptic nuclei. The intensity of ERK labelling was dose-dependent. Reciprocally, slow intragastric infusions of water that lower osmolality reduced basal ERK phosphorylation. In the supraoptic nucleus, ERK phosphorylation predominated in vasopressin neurones vs. oxytocin neurones and was absent from astrocytes. Western blot experiments confirmed that phosphoERK expression in the supraoptic nucleus was dose dependent. Intracerebroventricular administration of the ERK phosphorylation inhibitor U 0126 before a hyperosmotic challenge reduced the number of both phosphoERK-immunopositive neurones and Fos expressing neurones in osmosensitive forebrain regions. Blockade of ERK phosphorylation also reduced hypertonically induced depolarization and an increase in firing of the supraoptic MNCs recorded in vitro. It finally reduced hypertonically induced vasopressin release in the bloodstream. Altogether, these findings identify ERK phosphorylation as a new element contributing to the osmoregulatory mechanisms of

  12. Phosphorylation Regulates Id2 Degradation and Mediates the Proliferation of Neural Precursor Cells.

    PubMed

    Sullivan, Jaclyn M; Havrda, Matthew C; Kettenbach, Arminja N; Paolella, Brenton R; Zhang, Zhonghua; Gerber, Scott A; Israel, Mark A

    2016-05-01

    Inhibitor of DNA binding proteins (Id1-Id4) function to inhibit differentiation and promote proliferation of many different cell types. Among the Id family members, Id2 has been most extensively studied in the central nervous system (CNS). Id2 contributes to cultured neural precursor cell (NPC) proliferation as well as to the proliferation of CNS tumors such as glioblastoma that are likely to arise from NPC-like cells. We identified three phosphorylation sites near the N-terminus of Id2 in NPCs. To interrogate the importance of Id2 phosphorylation, Id2(-/-) NPCs were modified to express wild type (WT) Id2 or an Id2 mutant protein that could not be phosphorylated at the identified sites. We observed that NPCs expressing this mutant lacking phosphorylation near the N-terminus had higher steady-state levels of Id2 when compared to NPCs expressing WT Id2. This elevated level was the result of a longer half-life and reduced proteasome-mediated degradation. Moreover, NPCs expressing constitutively de-phosphorylated Id2 proliferated more rapidly than NPCs expressing WT Id2, a finding consistent with the well-characterized function of Id2 in driving proliferation. Observing that phosphorylation of Id2 modulates the degradation of this important cell-cycle regulator, we sought to identify a phosphatase that would stabilize Id2 enhancing its activity in NPCs and extended our analysis to include human glioblastoma-derived stem cells (GSCs). We found that expression of the phosphatase PP2A altered Id2 levels. Our findings suggest that inhibition of PP2A may be a novel strategy to regulate the proliferation of normal NPCs and malignant GSCs by decreasing Id2 levels. Stem Cells 2016;34:1321-1331. PMID:26756672

  13. Myosin Vb and Rab11a regulate phosphorylation of ezrin in enterocytes.

    PubMed

    Dhekne, Herschel S; Hsiao, Nai-Hua; Roelofs, Pieter; Kumari, Meena; Slim, Christiaan L; Rings, Edmond H H M; van Ijzendoorn, Sven C D

    2014-03-01

    Microvilli at the apical surface of enterocytes allow the efficient absorption of nutrients in the intestine. Ezrin activation by its phosphorylation at T567 is important for microvilli development, but how such ezrin phosphorylation is controlled is not well understood. We demonstrate that a subset of kinases that phosphorylate ezrin closely co-distributes with apical recycling endosome marker Rab11a in the subapical domain. Expression of dominant-negative Rab11a mutant or depletion of the Rab11a-binding motor protein myosin Vb prevents the subapical enrichment of Rab11a and these kinases and inhibits ezrin phosphorylation and microvilli development, without affecting the polarized distribution of ezrin itself. We observe a similar loss of the subapical enrichment of Rab11a and the kinases and reduced phosphorylation of ezrin in microvillus inclusion disease, which is associated with MYO5B mutations, intestinal microvilli atrophy and malabsorption. Thus, part of the machinery for ezrin activation depends on recycling endosomes controlled by myosin Vb and Rab11a which, we propose, might act as subapical signaling platforms that enterocytes use to regulate development of microvilli and maintain human intestinal function. PMID:24413175

  14. FLCN, a novel autophagy component, interacts with GABARAP and is regulated by ULK1 phosphorylation.

    PubMed

    Dunlop, Elaine A; Seifan, Sara; Claessens, Tijs; Behrends, Christian; Kamps, Miriam Af; Rozycka, Ewelina; Kemp, Alain J; Nookala, Ravi K; Blenis, John; Coull, Barry J; Murray, James T; van Steensel, Maurice Am; Wilkinson, Simon; Tee, Andrew R

    2014-10-01

    Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, currently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1. PMID:25126726

  15. Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast.

    PubMed

    Miao, Yansong; Han, Xuemei; Zheng, Liangzhen; Xie, Ying; Mu, Yuguang; Yates, John R; Drubin, David G

    2016-01-01

    Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin-Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism. PMID:27068241

  16. Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast

    PubMed Central

    Miao, Yansong; Han, Xuemei; Zheng, Liangzhen; Xie, Ying; Mu, Yuguang; Yates, John R.; Drubin, David G.

    2016-01-01

    Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin–Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism. PMID:27068241

  17. PLK1-dependent activation of LRRK1 regulates spindle orientation by phosphorylating CDK5RAP2.

    PubMed

    Hanafusa, Hiroshi; Kedashiro, Shin; Tezuka, Motohiro; Funatsu, Motoki; Usami, Satoshi; Toyoshima, Fumiko; Matsumoto, Kunihiro

    2015-08-01

    Correct formation of the cell division axis requires the initial precise orientation of the mitotic spindle. Proper spindle orientation depends on centrosome maturation, and Polo-like kinase 1 (PLK1) is known to play a crucial role in this process. However, the molecular mechanisms that function downstream of PLK1 are not well understood. Here we show that LRRK1 is a PLK1 substrate that is phosphorylated on Ser 1790. PLK1 phosphorylation is required for CDK1-mediated activation of LRRK1 at the centrosomes, and this in turn regulates mitotic spindle orientation by nucleating the growth of astral microtubules from the centrosomes. Interestingly, LRRK1 in turn phosphorylates CDK5RAP2(Cep215), a human homologue of Drosophila Centrosomin (Cnn), in its γ-tubulin-binding motif, thus promoting the interaction of CDK5RAP2 with γ-tubulin. LRRK1 phosphorylation of CDK5RAP2 Ser 140 is necessary for CDK5RAP2-dependent microtubule nucleation. Thus, our findings provide evidence that LRRK1 regulates mitotic spindle orientation downstream of PLK1 through CDK5RAP2-dependent centrosome maturation. PMID:26192437

  18. Keratin 8 phosphorylation regulates keratin reorganization and migration of epithelial tumor cells

    PubMed Central

    Busch, Tobias; Armacki, Milena; Eiseler, Tim; Joodi, Golsa; Temme, Claudia; Jansen, Julia; von Wichert, Götz; Omary, M. Bishr; Spatz, Joachim; Seufferlein, Thomas

    2012-01-01

    Cell migration and invasion are largely dependent on the complex organization of the various cytoskeletal components. Whereas the role of actin filaments and microtubules in cell motility is well established, the role of intermediate filaments in this process is incompletely understood. Organization and structure of the keratin cytoskeleton, which consists of heteropolymers of at least one type 1 and one type 2 intermediate filament, are in part regulated by post-translational modifications. In particular, phosphorylation events influence the properties of the keratin network. Sphingosylphosphorylcholine (SPC) is a bioactive lipid with the exceptional ability to change the organization of the keratin cytoskeleton, leading to reorganization of keratin filaments, increased elasticity, and subsequently increased migration of epithelial tumor cells. Here we investigate the signaling pathways that mediate SPC-induced keratin reorganization and the role of keratin phosphorylation in this process. We establish that the MEK–ERK signaling cascade regulates both SPC-induced keratin phosphorylation and reorganization in human pancreatic and gastric cancer cells and identify Ser431 in keratin 8 as the crucial residue whose phosphorylation is required and sufficient to induce keratin reorganization and consequently enhanced migration of human epithelial tumor cells. PMID:22344252

  19. Bub1 autophosphorylation feeds back to regulate kinetochore docking and promote localized substrate phosphorylation

    PubMed Central

    Asghar, Adeel; Lajeunesse, Audrey; Dulla, Kalyan; Combes, Guillaume; Thebault, Philippe; Nigg, Erich A.; Elowe, Sabine

    2015-01-01

    During mitosis, Bub1 kinase phosphorylates histone H2A-T120 to promote centromere sister chromatid cohesion through recruitment of shugoshin (Sgo) proteins. The regulation and dynamics of H2A-T120 phosphorylation are poorly understood. Using quantitative phosphoproteomics we show that Bub1 is autophosphorylated at numerous sites. We confirm mitosis-specific autophosphorylation of a several residues and show that Bub1 activation is primed in interphase but fully achieved only in mitosis. Mutation of a single autophosphorylation site T589 alters kinetochore turnover of Bub1 and results in uniform H2A-T120 phosphorylation and Sgo recruitment along chromosome arms. Consequently, improper sister chromatid resolution and chromosome segregation errors are observed. Kinetochore tethering of Bub1-T589A refocuses H2A-T120 phosphorylation and Sgo1 to centromeres. Recruitment of the Bub1-Bub3-BubR1 axis to kinetochores has recently been extensively studied. Our data provide novel insight into the regulation and kinetochore residency of Bub1 and indicate that its localization is dynamic and tightly controlled through feedback autophosphorylation. PMID:26399325

  20. SUMOylation and phosphorylation of GluK2 regulate kainate receptor trafficking and synaptic plasticity

    PubMed Central

    Chamberlain, Sophie E L; González-González, Inmaculada M; Wilkinson, Kevin A; Konopacki, Filip A; Kantamneni, Sriharsha; Henley, Jeremy M; Mellor, Jack R

    2012-01-01

    Summary Phosphorylation or SUMOylation of the kainate receptor (KAR) subunit GluK2 have both individually been shown to regulate KAR surface expression. However, it is unknown if phosphorylation and SUMOylation of GluK2 are important for activity-dependent KAR synaptic plasticity. Here, we show that PKC-mediated phosphorylation of GluK2 at serine 868 promotes GluK2 SUMOylation at lysine 886 and that both these events are necessary for the internalization of GluK2 containing KARs that occurs during long-term depression of KAR-mediated synaptic transmission at rat hippocampal mossy fiber synapses. Conversely, phosphorylation of GluK2 at serine 868 in the absence of SUMOylation leads to an increase in KAR surface expression by facilitating receptor recycling between endosomal compartments and the plasma membrane. Thus, we describe a role for the dynamic control of synaptic SUMOylation in the regulation of KAR synaptic transmission and plasticity. PMID:22522402

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

    SciTech Connect

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

    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, thus 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.

  2. Regulation of Myosin II Dynamics by Phosphorylation and Dephosphorylation of Its Light Chain in Epithelial Cells

    PubMed Central

    Watanabe, Toshiyuki; Hosoya, Hiroshi

    2007-01-01

    Nonmuscle myosin II, an actin-based motor protein, plays an essential role in actin cytoskeleton organization and cellular motility. Although phosphorylation of its regulatory light chain (MRLC) is known to be involved in myosin II filament assembly and motor activity in vitro, it remains unclear exactly how MRLC phosphorylation regulates myosin II dynamics in vivo. We established clones of Madin Darby canine kidney II epithelial cells expressing MRLC-enhanced green fluorescent protein or its mutants. Time-lapse imaging revealed that both phosphorylation and dephosphorylation are required for proper dynamics of myosin II. Inhibitors affecting myosin phosphorylation and MRLC mutants indicated that monophosphorylation of MRLC is required and sufficient for maintenance of stress fibers. Diphosphorylated MRLC stabilized myosin II filaments and was distributed locally in regions of stress fibers where contraction occurs, suggesting that diphosphorylation is involved in the spatial regulation of myosin II assembly and contraction. We further found that myosin phosphatase or Zipper-interacting protein kinase localizes to stress fibers depending on the activity of myosin II ATPase. PMID:17151359

  3. Sites of Regulated Phosphorylation that Control K-Cl Cotransporter Activity

    PubMed Central

    Rinehart, Jesse; Maksimova, Yelena D.; Tanis, Jessica E.; Stone, Kathryn L.; Hodson, Caleb A.; Zhang, Junhui; Risinger, Mary; Pan, Weijun; Wu, Dianqing; Colangelo, Christopher M.; Forbush, Biff; Joiner, Clinton H.; Gulcicek, Erol E.; Gallagher, Patrick G.; Lifton, Richard P.

    2010-01-01

    Summary Modulation of intracellular chloride concentration ([Cl−]i) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl− exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl−]I; however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3's intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl−]i and have implications for control of cell volume and neuronal function. PMID:19665974

  4. Direct inhibition of Retinoblastoma phosphorylation by Nimbolide causes cell cycle arrest and suppresses glioblastoma growth

    PubMed Central

    Anderson, Jane; Liu, Xiaona; Henry, Heather; Gasilina, Anjelika; Nassar, Nicholas; Ghosh, Jayeeta; Clark, Jason P; Kumar, Ashish; Pauletti, Giovanni M.; Ghosh, Pradip K; Dasgupta, Biplab

    2013-01-01

    Purpose Classical pharmacology allows the use and development of conventional phytomedicine faster and more economically than conventional drugs. This approach should be tested for their efficacy in terms of complementarity and disease control. The purpose of this study was to determine the molecular mechanisms by which nimbolide, a triterpenoid found in the well-known medicinal plant Azadirachta indica controls glioblastoma (GBM) growth. Experimental Design Using in vitro signaling, anchorage-independent growth, kinase assays, and xenograft models, we investigated the mechanisms of its growth inhibition in glioblastoma. Results We show that nimbolide or an ethanol soluble fraction of A. indica leaves (Azt) that contains nimbolide as the principal cytotoxic agent is highly cytotoxic against GBM in vitro and in vivo. Azt caused cell cycle arrest, most prominently at the G1-S stage in GBM cells expressing EGFRvIII, an oncogene present in about 20-25% of GBMs. Azt/nimbolide directly inhibited CDK4/CDK6 kinase activity leading to hypophosphorylation of the retinoblastoma (RB) protein, cell cycle arrest at G1-S and cell death. Independent of RB hypophosphorylation, Azt also significantly reduced proliferative and survival advantage of GBM cells in vitro and in tumor xenografts by downregulating Bcl2 and blocking growth factor induced phosphorylation of Akt, Erk1/2 and STAT3. These effects were specific since Azt did not affect mTOR or other cell cycle regulators. In vivo, Azt completely prevented initiation and inhibited progression of GBM growth. Conclusions Our preclinical findings demonstrate Nimbolide as a potent anti-glioma agent that blocks cell cycle and inhibits glioma growth in vitro and in vivo. PMID:24170547

  5. TPX2 phosphorylation maintains metaphase spindle length by regulating microtubule flux

    PubMed Central

    Fu, Jingyan; Bian, Minglei; Xin, Guangwei; Deng, Zhaoxuan; Luo, Jia; Guo, Xiao; Chen, Hao; Wang, Yao; Jiang, Qing

    2015-01-01

    A steady-state metaphase spindle maintains constant length, although the microtubules undergo intensive dynamics. Tubulin dimers are incorporated at plus ends of spindle microtubules while they are removed from the minus ends, resulting in poleward movement. Such microtubule flux is regulated by the microtubule rescue factors CLASPs at kinetochores and depolymerizing protein Kif2a at the poles, along with other regulators of microtubule dynamics. How microtubule polymerization and depolymerization are coordinated remains unclear. Here we show that TPX2, a microtubule-bundling protein and activator of Aurora A, plays an important role. TPX2 was phosphorylated by Aurora A during mitosis. Its phospho-null mutant caused short metaphase spindles coupled with low microtubule flux rate. Interestingly, phosphorylation of TPX2 regulated its interaction with CLASP1 but not Kif2a. The effect of its mutant in shortening the spindle could be rescued by codepletion of CLASP1 and Kif2a that abolished microtubule flux. Together we propose that Aurora A–dependent TPX2 phosphorylation controls mitotic spindle length through regulating microtubule flux. PMID:26240182

  6. EGFR Signaling Regulates Maspin/SerpinB5 Phosphorylation and Nuclear Localization in Mammary Epithelial Cells

    PubMed Central

    Reina, Jeffrey; Morais Freitas, Vanessa

    2016-01-01

    Maspin (SerpinB5) is a non-inhibitory serpin (serine protease inhibitor) with very diverse biological activities including regulation of cell adhesion, migration, death, control of gene expression and oxidative stress response. Initially described as a tumor and metastasis suppressor, clinical data brought controversies to the field, as some studies reported no correlation between SerpinB5 expression and prognosis value. These data underscore the importance of understanding SerpinB5 function in a normal physiological context and the molecular mechanism involved. Several SerpinB5 phosphoforms have been detected in different cell lines, but the signaling pathways involved and the biological significance of this post-translational modification in vivo remains to be explored. In this study we investigated SerpinB5 expression, subcellular localization and phosphorylation in different stages of the mouse mammary gland development and the signaling pathway involved. Here we show that SerpinB5 is first detected in late pregnancy, reaches its highest levels in lactation and remains at constant levels during post-lactational regression (involution). Using high resolution isoelectric focusing followed but immunoblot, we found at least 8 different phosphoforms of SerpinB5 during lactation, which decreases steadily at the onset of involution. In order to investigate the signaling pathway involved in SerpinB5 phosphorylation, we took advantage of the non-transformed MCF-10A model system, as we have previously observed SerpinB5 phosphorylation in these cells. We detected basal levels of SerpinB5 phosphorylation in serum- and growth factor-starved cells, which is due to amphiregulin autocrine activity on MCF-10A cells. EGF and TGF alpha, two other EGFR ligands, promote important SerpinB5 phosphorylation. Interestingly, EGF treatment is followed by SerpinB5 nuclear accumulation. Altogether, these data indicate that SerpinB5 expression and phosphorylation are developmentally

  7. Phosphorylation of the group A Streptococcal CovR response regulator causes dimerization and promoter-specific recruitment by RNA polymerase.

    PubMed

    Gusa, Asiya A; Gao, Jinxin; Stringer, Virginia; Churchward, Gordon; Scott, June R

    2006-07-01

    The group A streptococcus (GAS), Streptococcus pyogenes, is an important human pathogen that causes infections ranging in severity from self-limiting pharyngitis to severe invasive diseases that are associated with significant morbidity and mortality. The pathogenic effects of GAS are mediated by the expression of virulence factors, one of which is the hyaluronic acid capsule (encoded by genes in the has operon). The expression of these virulence factors is controlled by the CovR/S (CsrR/S) two-component regulatory system of GAS which regulates, directly or indirectly, the expression of about 15% of the genome. CovR is a member of the OmpR/PhoB family of transcriptional regulators. Here we show that phosphorylation by acetyl phosphate results in dimerization of CovR. Dimerization was not observed using a D53A mutant of CovR, indicating that D53 is the site of phosphorylation in CovR. Phosphorylation stimulated binding of CovR to a DNA fragment containing the promoter of the has operon (Phas) approximately twofold. Binding of CovR D53A mutant protein to Phas was indistinguishable from the binding of wild-type unphosphorylated CovR. In vitro transcription, using purified GAS RNA polymerase, showed that wild-type CovR repressed transcription, and repression was stimulated more than sixfold by phosphorylation. In the presence of RNA polymerase, binding at Phas of phosphorylated, but not unphosphorylated, CovR was stimulated about fourfold, which accounts for the difference in the effect of phosphorylation on repression versus DNA binding. Thus, regulation of Phas by CovR is direct, and the degree of repression of Phas is controlled by the phosphorylation of CovR. PMID:16788170

  8. A Genome-Wide siRNA Screen in Mammalian Cells for Regulators of S6 Phosphorylation

    PubMed Central

    Papageorgiou, Angela; Rapley, Joseph; Mesirov, Jill P.; Tamayo, Pablo; Avruch, Joseph

    2015-01-01

    mTOR complex1, the major regulator of mRNA translation in all eukaryotic cells, is strongly activated in most cancers. We performed a genome-wide RNAi screen in a human cancer cell line, seeking genes that regulate S6 phosphorylation, readout of mTORC1 activity. Applying a stringent selection, we retrieved nearly 600 genes wherein at least two RNAis gave significant reduction in S6-P. This cohort contains known regulators of mTOR complex 1 and is significantly enriched in genes whose depletion affects the proliferation/viability of the large set of cancer cell lines in the Achilles database in a manner paralleling that caused by mTOR depletion. We next examined the effect of RNAi pools directed at 534 of these gene products on S6-P in TSC1 null mouse embryo fibroblasts. 76 RNAis reduced S6 phosphorylation significantly in 2 or 3 replicates. Surprisingly, among this cohort of genes the only elements previously associated with the maintenance of mTORC1 activity are two subunits of the vacuolar ATPase and the CUL4 subunit DDB1. RNAi against a second set of 84 targets reduced S6-P in only one of three replicates. However, an indication that this group also bears attention is the presence of rpS6KB1 itself, Rac1 and MAP4K3, a protein kinase that supports amino acid signaling to rpS6KB1. The finding that S6 phosphorylation requires a previously unidentified, functionally diverse cohort of genes that participate in fundamental cellular processes such as mRNA translation, RNA processing, DNA repair and metabolism suggests the operation of feedback pathways in the regulation of mTORC1 operating through novel mechanisms. PMID:25790369

  9. Tyrosine Phosphorylation Based Homo-dimerization of Arabidopsis RACK1A Proteins Regulates Oxidative Stress Signaling Pathways in Yeast

    PubMed Central

    Sabila, Mercy; Kundu, Nabanita; Smalls, Deana; Ullah, Hemayet

    2016-01-01

    Scaffold proteins are known as important cellular regulators that can interact with multiple proteins to modulate diverse signal transduction pathways. RACK1 (Receptor for Activated C Kinase 1) is a WD-40 type scaffold protein, conserved in eukaryotes, from Chlamydymonas to plants and humans, plays regulatory roles in diverse signal transduction and stress response pathways. RACK1 in humans has been implicated in myriads of neuropathological diseases including Alzheimer and alcohol addictions. Model plant Arabidopsis thaliana genome maintains three different RACK1 genes termed RACK1A, RACK1B, and RACK1C with a very high (85–93%) sequence identity among them. Loss of function mutation in Arabidopsis indicates that RACK1 proteins regulate diverse environmental stress signaling pathways including drought and salt stress resistance pathway. Recently deduced crystal structure of Arabidopsis RACK1A- very first among all of the RACK1 proteins, indicates that it can potentially be regulated by post-translational modifications, like tyrosine phosphorylations and sumoylation at key residues. Here we show evidence that RACK1A proteins, depending on diverse environmental stresses, are tyrosine phosphorylated. Utilizing site-directed mutagenesis of key tyrosine residues, it is found that tyrosine phosphorylation can potentially dictate the homo-dimerization of RACK1A proteins. The homo-dimerized RACK1A proteins play a role in providing UV-B induced oxidative stress resistance. It is proposed that RACK1A proteins ability to function as scaffold protein may potentially be regulated by the homo-dimerized RACK1A proteins to mediate diverse stress signaling pathways. PMID:26941753

  10. Tyrosine Phosphorylation Based Homo-dimerization of Arabidopsis RACK1A Proteins Regulates Oxidative Stress Signaling Pathways in Yeast.

    PubMed

    Sabila, Mercy; Kundu, Nabanita; Smalls, Deana; Ullah, Hemayet

    2016-01-01

    Scaffold proteins are known as important cellular regulators that can interact with multiple proteins to modulate diverse signal transduction pathways. RACK1 (Receptor for Activated C Kinase 1) is a WD-40 type scaffold protein, conserved in eukaryotes, from Chlamydymonas to plants and humans, plays regulatory roles in diverse signal transduction and stress response pathways. RACK1 in humans has been implicated in myriads of neuropathological diseases including Alzheimer and alcohol addictions. Model plant Arabidopsis thaliana genome maintains three different RACK1 genes termed RACK1A, RACK1B, and RACK1C with a very high (85-93%) sequence identity among them. Loss of function mutation in Arabidopsis indicates that RACK1 proteins regulate diverse environmental stress signaling pathways including drought and salt stress resistance pathway. Recently deduced crystal structure of Arabidopsis RACK1A- very first among all of the RACK1 proteins, indicates that it can potentially be regulated by post-translational modifications, like tyrosine phosphorylations and sumoylation at key residues. Here we show evidence that RACK1A proteins, depending on diverse environmental stresses, are tyrosine phosphorylated. Utilizing site-directed mutagenesis of key tyrosine residues, it is found that tyrosine phosphorylation can potentially dictate the homo-dimerization of RACK1A proteins. The homo-dimerized RACK1A proteins play a role in providing UV-B induced oxidative stress resistance. It is proposed that RACK1A proteins ability to function as scaffold protein may potentially be regulated by the homo-dimerized RACK1A proteins to mediate diverse stress signaling pathways. PMID:26941753

  11. Mitotic protein kinase CDK1 phosphorylation of mRNA translation regulator 4E-BP1 Ser83 may contribute to cell transformation.

    PubMed

    Velásquez, Celestino; Cheng, Erdong; Shuda, Masahiro; Lee-Oesterreich, Paula J; Pogge von Strandmann, Lisa; Gritsenko, Marina A; Jacobs, Jon M; Moore, Patrick S; Chang, Yuan

    2016-07-26

    Mammalian target of rapamycin (mTOR)-directed eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation promotes cap-dependent translation and tumorigenesis. During mitosis, cyclin-dependent kinase 1 (CDK1) substitutes for mTOR and fully phosphorylates 4E-BP1 at canonical sites (T37, T46, S65, and T70) and the noncanonical S83 site, resulting in a mitosis-specific hyperphosphorylated δ isoform. Colocalization studies with a phospho-S83 specific antibody indicate that 4E-BP1 S83 phosphorylation accumulates at centrosomes during prophase, peaks at metaphase, and decreases through telophase. Although S83 phosphorylation of 4E-BP1 does not affect general cap-dependent translation, expression of an alanine substitution mutant 4E-BP1.S83A partially reverses rodent cell transformation induced by Merkel cell polyomavirus small T antigen viral oncoprotein. In contrast to inhibitory mTOR 4E-BP1 phosphorylation, these findings suggest that mitotic CDK1-directed phosphorylation of δ-4E-BP1 may yield a gain of function, distinct from translation regulation, that may be important in tumorigenesis and mitotic centrosome function. PMID:27402756

  12. Mitotic protein kinase CDK1 phosphorylation of mRNA translation regulator 4E-BP1 Ser83 may contribute to cell transformation

    PubMed Central

    Velásquez, Celestino; Cheng, Erdong; Shuda, Masahiro; Lee-Oesterreich, Paula J.; Pogge von Strandmann, Lisa; Gritsenko, Marina A.; Jacobs, Jon M.; Moore, Patrick S.; Chang, Yuan

    2016-01-01

    Mammalian target of rapamycin (mTOR)-directed eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation promotes cap-dependent translation and tumorigenesis. During mitosis, cyclin-dependent kinase 1 (CDK1) substitutes for mTOR and fully phosphorylates 4E-BP1 at canonical sites (T37, T46, S65, and T70) and the noncanonical S83 site, resulting in a mitosis-specific hyperphosphorylated δ isoform. Colocalization studies with a phospho-S83 specific antibody indicate that 4E-BP1 S83 phosphorylation accumulates at centrosomes during prophase, peaks at metaphase, and decreases through telophase. Although S83 phosphorylation of 4E-BP1 does not affect general cap-dependent translation, expression of an alanine substitution mutant 4E-BP1.S83A partially reverses rodent cell transformation induced by Merkel cell polyomavirus small T antigen viral oncoprotein. In contrast to inhibitory mTOR 4E-BP1 phosphorylation, these findings suggest that mitotic CDK1-directed phosphorylation of δ-4E-BP1 may yield a gain of function, distinct from translation regulation, that may be important in tumorigenesis and mitotic centrosome function. PMID:27402756

  13. Regulation and function of stimulus-induced phosphorylation of MeCP2

    PubMed Central

    Li, Hongda; Chang, Qiang

    2014-01-01

    DNA methylation-dependent epigenetic regulation plays important roles in the development and function of the mammalian nervous system. MeCP2 is a key player in recognizing methylated DNA and interpreting the epigenetic information encoded in different DNA methylation patterns. Mutations in the MECP2 gene cause Rett syndrome, a devastating neurological disease that shares many features with autism. One interesting aspect of MeCP2 function is that it can be phosphorylated in response to diverse stimuli. Insights into the regulation and function of MeCP2 phosphorylation will help improve our understanding of how MeCP2 integrates environmental stimuli in neuronal nuclei to generate adaptive responses and may eventually lead to treatments for patients. PMID:25568644

  14. Differential regulation of a CLC anion channel by SPAK kinase ortholog-mediated multisite phosphorylation

    PubMed Central

    Miyazaki, Hiroaki

    2012-01-01

    Shrinkage-induced inhibition of the Caenorhabditis elegans cell volume and cell cycle-dependent CLC anion channel CLH-3b occurs by concomitant phosphorylation of S742 and S747, which are located on a 175 amino acid linker domain between cystathionine-β-synthase 1 (CBS1) and CBS2. Phosphorylation is mediated by the SPAK kinase homolog GCK-3 and is mimicked by substituting serine residues with glutamate. Type 1 serine/threonine protein phosphatases mediate swelling-induced channel dephosphorylation. S742E/S747E double mutant channels are constitutively inactive and cannot be activated by cell swelling. S742E and S747E mutant channels were fully active in the absence of GCK-3 and were inactive when coexpressed with the kinase. Both channels responded to cell volume changes. However, the S747E mutant channel activated and inactivated in response to cell swelling and shrinkage, respectively, much more slowly than either wild-type or S742E mutant channels. Slower activation and inactivation of S747E was not due to altered rates of dephosphorylation or dephosphorylation-dependent conformational changes. GCK-3 binds to the 175 amino acid inter-CBS linker domain. Coexpression of wild-type CLH-3b and GCK-3 with either wild-type or S742E linkers gave rise to similar channel activity and regulation. In contrast, coexpression with the S747E linker greatly enhanced basal channel activity and increased the rate of shrinkage-induced channel inactivation. Our findings suggest the intriguing possibility that the phosphorylation state of S742 in S747E mutant channels modulates GCK-3/channel interaction and hence channel phosphorylation. These results provide a foundation for further detailed studies of the role of multisite phosphorylation in regulating CLH-3b and GCK-3 activity. PMID:22357738

  15. The heterocyst differentiation transcriptional regulator HetR of the filamentous cyanobacterium Anabaena forms tetramers and can be regulated by phosphorylation.

    PubMed

    Valladares, Ana; Flores, Enrique; Herrero, Antonia

    2016-02-01

    Many filamentous cyanobacteria respond to the external cue of nitrogen scarcity by the differentiation of heterocysts, cells specialized in the fixation of atmospheric nitrogen in oxic environments. Heterocysts follow a spatial pattern along the filament of two heterocysts separated by ca. 10-15 vegetative cells performing oxygenic photosynthesis. HetR is a transcriptional regulator that directs heterocyst differentiation. In the model strain Anabaena sp. PCC 7120, the HetR protein was observed in various oligomeric forms in vivo, including a tetramer that peaked with maximal hetR expression during differentiation. Tetramers were not detected in a hetR point mutant incapable of differentiation, but were conspicuous in an over-differentiating strain lacking the PatS inhibitor. In differentiated filaments the HetR tetramer was restricted to heterocysts, being undetectable in vegetative cells. HetR co-purified with RNA polymerase from Anabaena mainly as a tetramer. In vitro, purified recombinant HetR was distributed between monomers, dimers, trimers and tetramers, and it was phosphorylated when incubated with (γ-(32)P)ATP. Phosphorylation and PatS hampered the accumulation of HetR tetramers and impaired HetR binding to DNA. In summary, tetrameric HetR appears to represent a functionally relevant form of HetR, whose abundance in the Anabaena filament could be negatively regulated by phosphorylation and by PatS. PMID:26552991

  16. Regulation of the plasma membrane proton pump (H(+)-ATPase) by phosphorylation.

    PubMed

    Haruta, Miyoshi; Gray, William M; Sussman, Michael R

    2015-12-01

    In plants and fungi, energetics at the plasma membrane is provided by a large protonmotive force (PMF) generated by the family of P-type ATPases specialized for proton transport (commonly called PM H(+)-ATPases or, in Arabidopsis, AHAs for Arabidopsis H(+)-ATPases). Studies have demonstrated that this 100-kDa protein is essential for plant growth and development. Posttranslational modifications of the H(+)-ATPase play crucial roles in its regulation. Phosphorylation of several Thr and Ser residues within the carboxy terminal regulatory domain composed of ∼100 amino acids change in response to environmental stimuli, endogenous hormones, and nutrient conditions. Recently developed mass spectrometric technologies provide a means to carefully quantify these changes in H(+)-ATPase phosphorylation at the different sites. These chemical modifications can then be genetically tested in planta by complementing the loss-of-function aha mutants with phosphomimetic mutations. Interestingly, recent data suggest that phosphatase-mediated changes in PM H(+)-ATPase phosphorylation are important in mediating auxin-regulated growth. Thus, as with another hormone (abscisic acid), dephosphorylation by phosphatases, rather than kinase mediated phosphorylation, may be an important focal point for regulation during plant signal transduction. Although interactions with other proteins have also been implicated in ATPase regulation, the very hydrophobic nature and high concentration of this polytopic protein presents special challenges in evaluating the biological significance of these interactions. Only by combining biochemical and genetic experiments can we attempt to meet these challenges to understand the essential molecular details by which this protein functions in planta. PMID:26476298

  17. Research Resource: Identification of Novel Growth Hormone-Regulated Phosphorylation Sites by Quantitative Phosphoproteomics

    PubMed Central

    Ray, Bridgette N.; Kweon, Hye Kyong; Argetsinger, Lawrence S.; Fingar, Diane C.; Andrews, Philip C.

    2012-01-01

    GH and GH receptors are expressed throughout life, and GH elicits a diverse range of responses, including growth and altered metabolism. It is therefore important to understand the full spectrum of GH signaling pathways and cellular responses. We applied mass spectrometry-based phosphoproteomics combined with stable isotope labeling with amino acids in cell culture to identify proteins rapidly phosphorylated in response to GH in 3T3-F442A preadipocytes. We identified 132 phosphosites in 95 proteins that exhibited rapid (5 or 15 min) GH-dependent statistically significant increases in phosphorylation by more than or equal to 50% and 96 phosphosites in 46 proteins that were down-regulated by GH by more than or equal to 30%. Several of the GH-stimulated phosphorylation sites were known (e.g. regulatory Thr/Tyr in Erks 1 and 2, Tyr in signal transducers and activators of transcription (Stat) 5a and 5b, Ser939 in tuberous sclerosis protein (TSC) 2 or tuberin). The remaining 126 GH-stimulated sites were not previously associated with GH. Kyoto Encyclopedia of Genes and Genomes pathway analysis of GH-stimulated sites indicated enrichment in proteins associated with the insulin and mammalian target of rapamycin (mTOR) pathways, regulation of the actin cytoskeleton, and focal adhesions. Akt/protein kinase A consensus sites (RXRXXS/T) were the most commonly phosphorylated consensus sites. Immunoblotting confirmed GH-stimulated phosphorylation of all seven novel GH-dependent sites tested [regulatory sites in proline-rich Akt substrate, 40 kDA (PRAS40), regulatory associated protein of mTOR, ATP-citrate lyase, Na+/H+ exchanger-1, N-myc downstream regulated gene 1, and Shc]). The immunoblot results suggest that many, if not most, of the GH-stimulated phosphosites identified in this large-scale quantitative phosphoproteomics analysis, including sites in multiple proteins in the Akt/ mTOR complex 1 pathway, are phosphorylated in response to GH. Their identification significantly

  18. Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics.

    PubMed

    Colson, Brett A; Thompson, Andrew R; Espinoza-Fonseca, L Michel; Thomas, David D

    2016-03-22

    We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electron-electron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiac myosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0-C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein's flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein-protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility. PMID:26908877

  19. STIM2 regulates PKA-dependent phosphorylation and trafficking of AMPARs

    PubMed Central

    Garcia-Alvarez, Gisela; Lu, Bo; Yap, Kenrick An Fu; Wong, Loo Chin; Thevathasan, Jervis Vermal; Lim, Lynette; Ji, Fang; Tan, Kia Wee; Mancuso, James J.; Tang, Willcyn; Poon, Shou Yu; Augustine, George J.; Fivaz, Marc

    2015-01-01

    STIMs (STIM1 and STIM2 in mammals) are transmembrane proteins that reside in the endoplasmic reticulum (ER) and regulate store-operated Ca2+ entry (SOCE). The function of STIMs in the brain is only beginning to be explored, and the relevance of SOCE in nerve cells is being debated. Here we identify STIM2 as a central organizer of excitatory synapses. STIM2, but not its paralogue STIM1, influences the formation of dendritic spines and shapes basal synaptic transmission in excitatory neurons. We further demonstrate that STIM2 is essential for cAMP/PKA-dependent phosphorylation of the AMPA receptor (AMPAR) subunit GluA1. cAMP triggers rapid migration of STIM2 to ER–plasma membrane (PM) contact sites, enhances recruitment of GluA1 to these ER-PM junctions, and promotes localization of STIM2 in dendritic spines. Both biochemical and imaging data suggest that STIM2 regulates GluA1 phosphorylation by coupling PKA to the AMPAR in a SOCE-independent manner. Consistent with a central role of STIM2 in regulating AMPAR phosphorylation, STIM2 promotes cAMP-dependent surface delivery of GluA1 through combined effects on exocytosis and endocytosis. Collectively our results point to a unique mechanism of synaptic plasticity driven by dynamic assembly of a STIM2 signaling complex at ER-PM contact sites. PMID:25609091

  20. Correction: Learning from each other: ABC transporter regulation by protein phosphorylation in plant and mammalian systems.

    PubMed

    Aryal, Bibek; Laurent, Christophe; Geisler, Markus

    2016-04-15

    The ABC (ATP-binding cassette) transporter family in higher plants is highly expanded compared with those of mammalians. Moreover, some members of the plant ABCB subfamily display very high substrate specificity compared with their mammalian counterparts that are often associated with multidrug resistance (MDR) phenomena. In this review we highlight prominent functions of plant and mammalian ABC transporters and summarize our knowledge on their post-transcriptional regulation with a focus on protein phosphorylation. A deeper comparison of regulatory events of human cystic fibrosis transmembrane conductance regulator (CFTR) and ABCB1 from the model plantArabidopsisreveals a surprisingly high degree of similarity. Both physically interact with orthologues of the FK506-binding proteins (FKBPs) that chaperon both transporters to the plasma membrane in an action that seems to involve Hsp90. Further both transporters are phosphorylated at regulatory domains that connect both nucleotide-binding folds. Taken together it appears that ABC transporters exhibit an evolutionary conserved but complex regulation by protein phosphorylation, which apparently is, at least in some cases, tightly connected with protein-protein interactions (PPI). PMID:27068986

  1. Serine 1179 Phosphorylation of Endothelial Nitric Oxide Synthase Increases Superoxide Generation and Alters Cofactor Regulation

    PubMed Central

    Harbeck, Mark C.; He, Donghong; Xie, Lishi; Chen, Weiguo

    2015-01-01

    Endothelial nitric oxide synthase (eNOS) is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis. In addition to producing NO, eNOS can also generate superoxide (O2-.) in the absence of the cofactor tetrahydrobiopterin (BH4). Previous studies have shown that bovine eNOS serine 1179 (Serine 1177/human) phosphorylation critically modulates NO synthesis. However, the effect of serine 1179 phosphorylation on eNOS superoxide generation is unknown. Here, we used the phosphomimetic form of eNOS (S1179D) to determine the effect of S1179 phosphorylation on superoxide generating activity, and its sensitivity to regulation by BH4, Ca2+, and calmodulin (CAM). S1179D eNOS exhibited significantly increased superoxide generating activity and NADPH consumption compared to wild-type eNOS (WT eNOS). The superoxide generating activities of S1179D eNOS and WT eNOS did not differ significantly in their sensitivity to regulation by either Ca2+ or CaM. The sensitivity of the superoxide generating activity of S1179D eNOS to inhibition by BH4 was significantly reduced compared to WT eNOS. In eNOS-overexpressing 293 cells, BH4 depletion with 10mM DAHP for 48 hours followed by 50ng/ml VEGF for 30 min to phosphorylate eNOS S1179 increased ROS accumulation compared to DAHP-only treated cells. Meanwhile, MTT assay indicated that overexpression of eNOS in HEK293 cells decreased cellular viability compared to control cells at BH4 depletion condition (P<0.01). VEGF-mediated Serine 1179 phosphorylation further decreased the cellular viability in eNOS-overexpressing 293 cells (P<0.01). Our data demonstrate that eNOS serine 1179 phosphorylation, in addition to enhancing NO production, also profoundly affects superoxide generation: S1179 phosphorylation increases superoxide production while decreasing sensitivity to the inhibitory effect of BH4 on this activity. PMID:26560496

  2. Inhibition of CRMP2 phosphorylation repairs CNS by regulating neurotrophic and inhibitory responses.

    PubMed

    Nagai, Jun; Owada, Kazuki; Kitamura, Yoshiteru; Goshima, Yoshio; Ohshima, Toshio

    2016-03-01

    Central nervous system (CNS) regeneration is restricted by both the lack of neurotrophic responses and the presence of inhibitory factors. As of yet, a common mediator of these two pathways has not been identified. Microtubule dynamics is responsible for several key processes after CNS injuries: intracellular trafficking of receptors for neurotrophic factors, axonal retraction by inhibitory factors, and secondary tissue damages by inflammation and scarring. Kinases regulating microtubule organization, such as Cdk5 or GSK3β, may play pivotal roles during CNS recovery, but the molecular mechanisms remain to be elucidated. Collapsin response mediator protein 2 (CRMP2) stabilizes cytoskeletal polymerization, while CRMP2 phosphorylation by Cdk5 and GSK3β loses its affinity for cytoskeleton proteins, leading to the inhibition of axonal growth. Here, we characterized CRMP2 phosphorylation as the first crucial factor regulating neurotrophic and inhibitory responses after spinal cord injury (SCI). We found that pharmacological inhibition of GSK3β enhanced brain-derived neurotrophic factor (BDNF)-induced axonal growth response in cultured dorsal root ganglion (DRG) neurons. DRG neurons from CRMP2 knock-in (Crmp2KI/KI) mice, where CRMP2 phosphorylation was eliminated, showed elevated sensitivity to BDNF as well. Additionally, cultured Crmp2KI/KI neurons exhibited suppressed axonal growth inhibition by chondroitin sulfate proteoglycan (CSPG). These data suggest a couple of new molecular insights: the BDNF/GSK3β/CRMP2 and CSPG/GSK3β/CRMP2 pathways. Next, we tested the significance of CRMP2 phosphorylation after CNS injury in vivo. The phosphorylation level of CRMP2 was enhanced in the injured spinal cord. Crmp2KI/KI mice exhibited prominent recovery of locomotive and nociceptive functions after SCI, which correlated with the enhanced axonal growth of the motor and sensory neurons. Neuroprotective effects against SCI, such as microtubule stabilization, reduced inflammation

  3. Cyclin-dependent kinase regulates the length of S phase through TICRR/TRESLIN phosphorylation

    PubMed Central

    Goins, Duane; Siefert, Joseph C.; Clowdus, Emily A.

    2015-01-01

    S-phase cyclin-dependent kinases (CDKs) stimulate replication initiation and accelerate progression through the replication timing program, but it is unknown which CDK substrates are responsible for these effects. CDK phosphorylation of the replication factor TICRR (TopBP1-interacting checkpoint and replication regulator)/TRESLIN is required for DNA replication. We show here that phosphorylated TICRR is limiting for S-phase progression. Overexpression of a TICRR mutant with phosphomimetic mutations at two key CDK-phosphorylated residues (TICRRTESE) stimulates DNA synthesis and shortens S phase by increasing replication initiation. This effect requires the TICRR region that is necessary for its interaction with MDM two-binding protein. Expression of TICRRTESE does not grossly alter the spatial organization of replication forks in the nucleus but does increase replication clusters and the number of replication forks within each cluster. In contrast to CDK hyperactivation, the acceleration of S-phase progression by TICRRTESE does not induce DNA damage. These results show that CDK can stimulate initiation and compress the replication timing program by phosphorylating a single protein, suggesting a simple mechanism by which S-phase length is controlled. PMID:25737283

  4. Phosphorylation of the Scc2 cohesin deposition complex subunit regulates chromosome condensation through cohesin integrity

    PubMed Central

    Woodman, Julie; Hoffman, Matthew; Dzieciatkowska, Monika; Hansen, Kirk C.; Megee, Paul C.

    2015-01-01

    The cohesion of replicated sister chromatids promotes chromosome biorientation, gene regulation, DNA repair, and chromosome condensation. Cohesion is mediated by cohesin, which is deposited on chromosomes by a separate conserved loading complex composed of Scc2 and Scc4 in Saccharomyces cerevisiae. Although it is known to be required, the role of Scc2/Scc4 in cohesin deposition remains enigmatic. Scc2 is a phosphoprotein, although the functions of phosphorylation in deposition are unknown. We identified 11 phosphorylated residues in Scc2 by mass spectrometry. Mutants of SCC2 with substitutions that mimic constitutive phosphorylation retain normal Scc2–Scc4 interactions and chromatin association but exhibit decreased viability, sensitivity to genotoxic agents, and decreased stability of the Mcd1 cohesin subunit in mitotic cells. Cohesin association on chromosome arms, but not pericentromeric regions, is reduced in the phosphomimetic mutants but remains above a key threshold, as cohesion is only modestly perturbed. However, these scc2 phosphomimetic mutants exhibit dramatic chromosome condensation defects that are likely responsible for their high inviability. From these data, we conclude that normal Scc2 function requires modulation of its phosphorylation state and suggest that scc2 phosphomimetic mutants cause an increased incidence of abortive cohesin deposition events that result in compromised cohesin complex integrity and Mcd1 turnover. PMID:26354421

  5. PKA Regulates PINK1 Stability and Parkin Recruitment to Damaged Mitochondria through Phosphorylation of MIC60.

    PubMed

    Akabane, Shiori; Uno, Midori; Tani, Naoki; Shimazaki, Shunta; Ebara, Natsumi; Kato, Hiroki; Kosako, Hidetaka; Oka, Toshihiko

    2016-05-01

    A mitochondrial kinase, PTEN-induced putative kinase 1 (PINK1), selectively recruits the ubiquitin ligase Parkin to damaged mitochondria, which modifies mitochondria by polyubiquitination, leading to mitochondrial autophagy. Here, we report that treatment with an adenylate cyclase agonist or expression of protein kinase A (PKA) impairs Parkin recruitment to damaged mitochondria and decreases PINK1 protein levels. We identified a mitochondrial membrane protein, MIC60 (also known as mitofilin), as a PKA substrate. Mutational and mass spectrometric analyses revealed that the Ser528 residue of MIC60 undergoes PKA-dependent phosphorylation. MIC60 transiently interacts with PINK1, and MIC60 downregulation leads to a reduction in PINK1 and mislocalization of Parkin. Phosphorylation-mimic mutants of MIC60 fail to restore the defect in Parkin recruitment in MIC60-knocked down cells, whereas a phosphorylation-deficient MIC60 mutant facilitates the mitochondrial localization of Parkin. Our findings indicate that PKA-mediated phosphorylation of MIC60 negatively regulates mitochondrial clearance that is initiated by PINK1 and Parkin. PMID:27153535

  6. Structural Basis of Human p70 Ribosomal S6 Kinase-1 Regulation by Activation Loop Phosphorylation

    SciTech Connect

    Sunami, Tomoko; Byrne, Noel; Diehl, Ronald E.; Funabashi, Kaoru; Hall, Dawn L.; Ikuta, Mari; Patel, Sangita B.; Shipman, Jennifer M.; Smith, Robert F.; Takahashi, Ikuko; Zugay-Murphy, Joan; Iwasawa, Yoshikazu; Lumb, Kevin J.; Munshi, Sanjeev K.; Sharma, Sujata

    2010-03-04

    p70 ribosomal S6 kinase (p70S6K) is a downstream effector of the mTOR signaling pathway involved in cell proliferation, cell growth, cell-cycle progression, and glucose homeostasis. Multiple phosphorylation events within the catalytic, autoinhibitory, and hydrophobic motif domains contribute to the regulation of p70S6K. We report the crystal structures of the kinase domain of p70S6K1 bound to staurosporine in both the unphosphorylated state and in the 3{prime}-phosphoinositide-dependent kinase-1-phosphorylated state in which Thr-252 of the activation loop is phosphorylated. Unphosphorylated p70S6K1 exists in two crystal forms, one in which the p70S6K1 kinase domain exists as a monomer and the other as a domain-swapped dimer. The crystal structure of the partially activated kinase domain that is phosphorylated within the activation loop reveals conformational ordering of the activation loop that is consistent with a role in activation. The structures offer insights into the structural basis of the 3{prime}-phosphoinositide-dependent kinase-1-induced activation of p70S6K and provide a platform for the rational structure-guided design of specific p70S6K inhibitors.

  7. Allosteric Regulation of GRASP Protein-dependent Golgi Membrane Tethering by Mitotic Phosphorylation*

    PubMed Central

    Truschel, Steven T.; Zhang, Ming; Bachert, Collin; Macbeth, Mark R.; Linstedt, Adam D.

    2012-01-01

    Mitotic phosphorylation of the conserved GRASP domain of GRASP65 disrupts its self-association, leading to a loss of Golgi membrane tethering, cisternal unlinking, and Golgi breakdown. Recently, the structural basis of the GRASP self-interaction was determined, yet the mechanism by which phosphorylation disrupts this activity is unknown. Here, we present the crystal structure of a GRASP phosphomimic containing an aspartic acid substitution for a serine residue (Ser-189) that in GRASP65 is phosphorylated by PLK1, causing a block in membrane tethering and Golgi ribbon formation. The structure revealed a conformational change in the GRASP internal ligand that prevented its insertion into the PDZ binding pocket, and gel filtration assays showed that this phosphomimic mutant exhibited a significant reduction in dimer formation. Interestingly, the structure also revealed an apparent propagation of conformational change from the site of phosphorylation to the shifted ligand, and alanine substitution of two residues (Glu-145 and Ser-146) at penultimate positions in this chain rescued dimer formation by the phosphomimic. These data reveal the structural basis of the phosphoinhibition of GRASP-mediated membrane tethering and provide a mechanism for its allosteric regulation. PMID:22523075

  8. Allosteric regulation of GRASP protein-dependent Golgi membrane tethering by mitotic phosphorylation.

    PubMed

    Truschel, Steven T; Zhang, Ming; Bachert, Collin; Macbeth, Mark R; Linstedt, Adam D

    2012-06-01

    Mitotic phosphorylation of the conserved GRASP domain of GRASP65 disrupts its self-association, leading to a loss of Golgi membrane tethering, cisternal unlinking, and Golgi breakdown. Recently, the structural basis of the GRASP self-interaction was determined, yet the mechanism by which phosphorylation disrupts this activity is unknown. Here, we present the crystal structure of a GRASP phosphomimic containing an aspartic acid substitution for a serine residue (Ser-189) that in GRASP65 is phosphorylated by PLK1, causing a block in membrane tethering and Golgi ribbon formation. The structure revealed a conformational change in the GRASP internal ligand that prevented its insertion into the PDZ binding pocket, and gel filtration assays showed that this phosphomimic mutant exhibited a significant reduction in dimer formation. Interestingly, the structure also revealed an apparent propagation of conformational change from the site of phosphorylation to the shifted ligand, and alanine substitution of two residues (Glu-145 and Ser-146) at penultimate positions in this chain rescued dimer formation by the phosphomimic. These data reveal the structural basis of the phosphoinhibition of GRASP-mediated membrane tethering and provide a mechanism for its allosteric regulation. PMID:22523075

  9. Altered regulation of tau phosphorylation in a mouse model of down syndrome aging

    PubMed Central

    Sheppard, Olivia; Plattner, Florian; Rubin, Anna; Slender, Amy; Linehan, Jacqueline M.; Brandner, Sebastian; Tybulewicz, Victor L.J.; Fisher, Elizabeth M.C.; Wiseman, Frances K.

    2012-01-01

    Down syndrome (DS) results from trisomy of human chromosome 21 (Hsa21) and is associated with an increased risk of Alzheimer's disease (AD). Here, using the unique transchromosomic Tc1 mouse model of DS we investigate the influence of trisomy of Hsa21 on the protein tau, which is hyperphosphorylated in Alzheimer's disease. We show that in old, but not young, Tc1 mice increased phosphorylation of tau occurs at a site suggested to be targeted by the Hsa21 encoded kinase, dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A). We show that DYRK1A is upregulated in young and old Tc1 mice, but that young trisomic mice may be protected from accumulating aberrantly phosphorylated tau. We observe that the key tau kinase, glycogen synthase kinase3-β (GSK-3β) is aberrantly phosphorylated at an inhibitory site in the aged Tc1 brain which may reduce total glycogen synthase kinase3-β activity. It is possible that a similar mechanism may also occur in people with DS. PMID:21843906

  10. The Plastid Casein Kinase 2 Phosphorylates Rubisco Activase at the Thr-78 Site but Is Not Essential for Regulation of Rubisco Activation State

    PubMed Central

    Kim, Sang Y.; Bender, Kyle W.; Walker, Berkley J.; Zielinski, Raymond E.; Spalding, Martin H.; Ort, Donald R.; Huber, Steven C.

    2016-01-01

    Rubisco activase (RCA) is essential for the activation of Rubisco, the carboxylating enzyme of photosynthesis. In Arabidopsis, RCA is composed of a large RCAα and small RCAβ isoform that are formed by alternative splicing of a single gene (At2g39730). The activity of Rubisco is controlled in response to changes in irradiance by regulation of RCA activity, which is known to involve a redox-sensitive disulfide bond located in the carboxy-terminal extension of the RCAα subunit. Additionally, phosphorylation of RCA threonine-78 (Thr-78) has been reported to occur in the dark suggesting that phosphorylation may also be associated with dark-inactivation of RCA and deactivation of Rubisco. In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and used non-reducing SDS-PAGE to monitor the redox status of the RCAα subunit. By immunoblotting, phosphorylation of both RCA isoforms occurred at low light and in the dark and feeding peroxide or DTT to leaf segments indicated that redox status of the chloroplast stroma was a critical factor controlling RCA phosphorylation. Use of a knockout mutant identified the plastid-targeted casein kinase 2 (cpCK2α) as the major protein kinase involved in RCA phosphorylation. Studies with recombinant cpCK2α and synthetic peptide substrates identified acidic residues at the –1, +2, and +3 positions surrounding Thr-78 as strong positive recognition elements. The cpck2 knockout mutant had strongly reduced phosphorylation at the Thr-78 site but was similar to wild type plants in terms of induction kinetics of photosynthesis following transfer from darkness or low light to high light, suggesting that if phosphorylation of RCA Thr-78 plays a direct role it would be redundant to redox regulation for control of Rubisco activation state under normal conditions. PMID:27064346

  11. G Protein-coupled Receptor Kinase-mediated Phosphorylation Regulates Post-endocytic Trafficking of the D2 Dopamine Receptor*S⃞

    PubMed Central

    Namkung, Yoon; Dipace, Concetta; Javitch, Jonathan A.; Sibley, David R.

    2009-01-01

    We investigated the role of G protein-coupled receptor kinase (GRK)-mediated phosphorylation in agonist-induced desensitization, arrestin association, endocytosis, and intracellular trafficking of the D2 dopamine receptor (DAR). Agonist activation of D2 DARs results in rapid and sustained receptor phosphorylation that is solely mediated by GRKs. A survey of GRKs revealed that only GRK2 or GRK3 promotes D2 DAR phosphorylation. Mutational analyses resulted in the identification of eight serine/threonine residues within the third cytoplasmic loop of the receptor that are phosphorylated by GRK2/3. Simultaneous mutation of these eight residues results in a receptor construct, GRK(-), that is completely devoid of agonist-promoted GRK-mediated receptor phosphorylation. We found that both wild-type (WT) and GRK(-) receptors underwent a similar degree of agonist-induced desensitization as assessed using [35S]GTPγS binding assays. Similarly, both receptor constructs internalized to the same extent in response to agonist treatment. Furthermore, using bioluminescence resonance energy transfer assays to directly assess receptor association with arrestin3, we found no differences between the WT and GRK(-) receptors. Thus, phosphorylation is not required for arrestin-receptor association or agonist-induced desensitization or internalization. In contrast, when we examined recycling of the D2 DARs to the cell surface, subsequent to agonist-induced endocytosis, the GRK(-) construct exhibited less recycling in comparison with the WT receptor. This impairment appears to be due to a greater propensity of the GRK(-) receptors to down-regulate once internalized. In contrast, if the receptor is highly phosphorylated, then receptor recycling is promoted. These results reveal a novel role for GRK-mediated phosphorylation in regulating the post-endocytic trafficking of a G protein-coupled receptor. PMID:19332542

  12. SUMOylation of DNA topoisomerase IIα regulates histone H3 kinase Haspin and H3 phosphorylation in mitosis.

    PubMed

    Yoshida, Makoto M; Ting, Lily; Gygi, Steven P; Azuma, Yoshiaki

    2016-06-20

    DNA topoisomerase II (TOP2) plays a pivotal role in faithful chromosome separation through its strand-passaging activity that resolves tangled genomic DNA during mitosis. Additionally, TOP2 controls progression of mitosis by activating cell cycle checkpoints. Recent work showed that the enzymatically inert C-terminal domain (CTD) of TOP2 and its posttranslational modification are critical to this checkpoint regulation. However, the molecular mechanism has not yet been determined. By using Xenopus laevis egg extract, we found that SUMOylation of DNA topoisomerase IIα (TOP2A) CTD regulates the localization of the histone H3 kinase Haspin and phosphorylation of histone H3 at threonine 3 at the centromere, two steps known to be involved in the recruitment of the chromosomal passenger complex (CPC) to kinetochores in mitosis. Robust centromeric Haspin localization requires SUMOylated TOP2A CTD binding activity through SUMO-interaction motifs and the phosphorylation of Haspin. We propose a novel mechanism through which the TOP2 CTD regulates the CPC via direct interaction with Haspin at mitotic centromeres. PMID:27325792

  13. Dual-site phosphorylation of the control of virulence regulator impacts group a streptococcal global gene expression and pathogenesis.

    PubMed

    Horstmann, Nicola; Saldaña, Miguel; Sahasrabhojane, Pranoti; Yao, Hui; Su, Xiaoping; Thompson, Erika; Koller, Antonius; Shelburne, Samuel A

    2014-05-01

    Phosphorylation relays are a major mechanism by which bacteria alter transcription in response to environmental signals, but understanding of the functional consequences of bacterial response regulator phosphorylation is limited. We sought to characterize how phosphorylation of the control of virulence regulator (CovR) protein from the major human pathogen group A Streptococcus (GAS) influences GAS global gene expression and pathogenesis. CovR mainly serves to repress GAS virulence factor-encoding genes and has been shown to homodimerize following phosphorylation on aspartate-53 (D53) in vitro. We discovered that CovR is phosphorylated in vivo and that such phosphorylation is partially heat-stable, suggesting additional phosphorylation at non-aspartate residues. Using mass spectroscopy along with targeted mutagenesis, we identified threonine-65 (T65) as an additional CovR phosphorylation site under control of the serine/threonine kinase (Stk). Phosphorylation on T65, as mimicked by the recombinant CovR T65E variant, abolished in vitro CovR D53 phosphorylation. Similarly, isoallelic GAS strains that were either unable to be phosphorylated at D53 (CovR-D53A) or had functional constitutive phosphorylation at T65 (CovR-T65E) had essentially an identical gene repression profile to each other and to a CovR-inactivated strain. However, the CovR-D53A and CovR-T65E isoallelic strains retained the ability to positively influence gene expression that was abolished in the CovR-inactivated strain. Consistent with these observations, the CovR-D53A and CovR-T65E strains were hypervirulent compared to the CovR-inactivated strain in a mouse model of invasive GAS disease. Surprisingly, an isoalleic strain unable to be phosphorylated at CovR T65 (CovR-T65A) was hypervirulent compared to the wild-type strain, as auto-regulation of covR gene expression resulted in lower covR gene transcript and CovR protein levels in the CovR-T65A strain. Taken together, these data establish that Cov

  14. Dual-Site Phosphorylation of the Control of Virulence Regulator Impacts Group A Streptococcal Global Gene Expression and Pathogenesis

    PubMed Central

    Horstmann, Nicola; Saldaña, Miguel; Sahasrabhojane, Pranoti; Yao, Hui; Su, Xiaoping; Thompson, Erika; Koller, Antonius; Shelburne, Samuel A.

    2014-01-01

    Phosphorylation relays are a major mechanism by which bacteria alter transcription in response to environmental signals, but understanding of the functional consequences of bacterial response regulator phosphorylation is limited. We sought to characterize how phosphorylation of the control of virulence regulator (CovR) protein from the major human pathogen group A Streptococcus (GAS) influences GAS global gene expression and pathogenesis. CovR mainly serves to repress GAS virulence factor-encoding genes and has been shown to homodimerize following phosphorylation on aspartate-53 (D53) in vitro. We discovered that CovR is phosphorylated in vivo and that such phosphorylation is partially heat-stable, suggesting additional phosphorylation at non-aspartate residues. Using mass spectroscopy along with targeted mutagenesis, we identified threonine-65 (T65) as an additional CovR phosphorylation site under control of the serine/threonine kinase (Stk). Phosphorylation on T65, as mimicked by the recombinant CovR T65E variant, abolished in vitro CovR D53 phosphorylation. Similarly, isoallelic GAS strains that were either unable to be phosphorylated at D53 (CovR-D53A) or had functional constitutive phosphorylation at T65 (CovR-T65E) had essentially an identical gene repression profile to each other and to a CovR-inactivated strain. However, the CovR-D53A and CovR-T65E isoallelic strains retained the ability to positively influence gene expression that was abolished in the CovR-inactivated strain. Consistent with these observations, the CovR-D53A and CovR-T65E strains were hypervirulent compared to the CovR-inactivated strain in a mouse model of invasive GAS disease. Surprisingly, an isoalleic strain unable to be phosphorylated at CovR T65 (CovR-T65A) was hypervirulent compared to the wild-type strain, as auto-regulation of covR gene expression resulted in lower covR gene transcript and CovR protein levels in the CovR-T65A strain. Taken together, these data establish that Cov

  15. Phosphorylation of Rab11-FIP2 regulates polarity in MDCK cells

    PubMed Central

    Lapierre, Lynne A.; Avant, Kenya M.; Caldwell, Cathy M.; Oztan, Asli; Apodaca, Gerard; Knowles, Byron C.; Roland, Joseph T.; Ducharme, Nicole A.; Goldenring, James R.

    2012-01-01

    The Rab11 effector Rab11-family interacting protein 2 (Rab11-FIP2) regulates transcytosis through its interactions with Rab11a and myosin Vb. Previous studies implicated Rab11-FIP2 in the establishment of polarity in Madin–Darby canine kidney (MDCK) cells through phosphorylation of Ser-227 by MARK2. Here we examine the dynamic role of Rab11-FIP2 phosphorylation on MDCK cell polarity. Endogenous Rab11-FIP2 phosphorylated on Ser-227 coalesces on vesicular plaques during the reestablishment of polarity after either monolayer wounding or calcium switch. Whereas expression of the nonphosphorylatable Rab11-FIP2(S227A) elicits a loss in lumen formation in MDCK cell cysts grown in Matrigel, the putative pseudophosphorylated Rab11-FIP2(S227E) mutant induces the formation of cysts with multiple lumens. On permeable filters, Rab11-FIP2(S227E)–expressing cells exhibit alterations in the composition of both the adherens and tight junctions. At the adherens junction, p120 catenin and K-cadherin are retained, whereas the majority of the E-cadherin is lost. Although ZO-1 is retained at the tight junction, occludin is lost and the claudin composition is altered. Of interest, the effects of Rab11-FIP2 on cellular polarity did not involve myosin Vb or Rab11a. These results indicate that Ser-227 phosphorylation of Rab11-FIP2 regulates the composition of both adherens and tight junctions and is intimately involved in the regulation of polarity in epithelial cells. PMID:22553350

  16. Three-dimensional Reconstruction of Tarantula Myosin Filaments Suggests How Phosphorylation May Regulate Myosin Activity

    PubMed Central

    Alamo, Lorenzo; Wriggers, Willy; Pinto, Antonio; Bártoli, Fulvia; Salazar, Leiría; Zhao, Fa-Qing; Craig, Roger; Padrón, Raúl

    2008-01-01

    Summary Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLC). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that myosin is switched off by intramolecular interactions between its two heads, the free-head and the blocked head. Three-dimensional reconstruction of frozen-hydrated specimens reveals that this asymmetric head interaction is also present in native thick filaments of tarantula striated muscle. Our goal here has been to elucidate the structural features of the tarantula filament involved in phosphorylation-based regulation. A new reconstruction reveals intra- and intermolecular myosin interactions in addition to those seen previously. To help interpret the interactions, we sequenced the tarantula RLC, and fitted to the reconstruction an atomic model of the myosin head that included the predicted RLC atomic structure and an S2 crystal structure. The fitting suggests an intramolecular interaction between the cardiomyopathy loop of the free-head and its own S2 and two intermolecular interactions—between the cardio-loop of the free head and the ELC of the blocked head, and between the Leu-305 - Gln-327 “interaction loop” (loop I) of the free-head and the N-terminal fragment of the RLC of the blocked-head. These interactions, added to those previously described, would help to switch off the thick filament. Molecular dynamics simulations suggest how phosphorylation could increase the helical content of the RLC N-terminus, weakening these interactions, thus releasing both heads and activating the thick filament. PMID:18951904

  17. Regulation of gap junction channels and hemichannels by phosphorylation and redox changes: a revision.

    PubMed

    Pogoda, Kristin; Kameritsch, Petra; Retamal, Mauricio A; Vega, José L

    2016-01-01

    Post-translational modifications of connexins play an important role in the regulation of gap junction and hemichannel permeability. The prerequisite for the formation of functional gap junction channels is the assembly of connexin proteins into hemichannels and their insertion into the membrane. Hemichannels can affect cellular processes by enabling the passage of signaling molecules between the intracellular and extracellular space. For the intercellular communication hemichannels from one cell have to dock to its counterparts on the opposing membrane of an adjacent cell to allow the transmission of signals via gap junctions from one cell to the other. The controlled opening of hemichannels and gating properties of complete gap junctions can be regulated via post-translational modifications of connexins. Not only channel gating, but also connexin trafficking and assembly into hemichannels can be affected by post-translational changes. Recent investigations have shown that connexins can be modified by phosphorylation/dephosphorylation, redox-related changes including effects of nitric oxide (NO), hydrogen sulfide (H2S) or carbon monoxide (CO), acetylation, methylation or ubiquitination. Most of the connexin isoforms are known to be phosphorylated, e.g. Cx43, one of the most studied connexin at all, has 21 reported phosphorylation sites. In this review, we provide an overview about the current knowledge and relevant research of responsible kinases, connexin phosphorylation sites and reported effects on gap junction and hemichannel regulation. Regarding the effects of oxidants we discuss the role of NO in different cell types and tissues and recent studies about modifications of connexins by CO and H2S. PMID:27229925

  18. Hexokinase 2 from Saccharomyces cerevisiae: regulation of oligomeric structure by in vivo phosphorylation at serine-14.

    PubMed

    Behlke, J; Heidrich, K; Naumann, M; Müller, E C; Otto, A; Reuter, R; Kriegel, T

    1998-08-25

    Homodimeric hexokinase 2 from Saccharomyces cerevisiae is known to have two sites of phosphorylation: for serine-14 the modification in vivo increases with glucose exhaustion [Kriegel et al. (1994) Biochemistry 33, 148-152], while for serine-157 it occurs in vitro with ATP in the presence of nonphosphorylateable five-carbon analogues of glucose [Heidrich et al. (1997) Biochemistry 36, 1960-1964]. We show now by site-directed mutagenesis and sedimentation analysis that serine-14 phosphorylation affects the oligomeric state of hexokinase, its substitution by glutamate causing complete dissociation; glutamate exchange for serine-157 does not. Phosphorylation of wild-type hexokinase at serine-14 likewise causes dissociation in vitro. In view of the higher glucose affinity of monomeric hexokinase and the high hexokinase concentration in yeast [Womack, F., and Colowick, S. P. (1978) Arch. Biochem. Biophys. 191, 742-747; Mayes, E. L., Hoggett, J. G., and Kellett, G. L. (1983) Eur. J. Biochem. 133, 127-134], we speculate that the in vivo phosphorylation at serine-14 as transiently occurring in glucose derepression might provide a mechanism to improve glucose utilization from low level and/or that nuclear localization of the monomer might be involved in the signal transduction whereby glucose causes catabolite repression. PMID:9718324

  19. KIF5C S176 Phosphorylation Regulates Microtubule Binding and Transport Efficiency in Mammalian Neurons

    PubMed Central

    Padzik, Artur; Deshpande, Prasannakumar; Hollos, Patrik; Franker, Mariella; Rannikko, Emmy H.; Cai, Dawen; Prus, Piotr; Mågård, Mats; Westerlund, Nina; Verhey, Kristen J.; James, Peter; Hoogenraad, Casper C.; Coffey, Eleanor T.

    2016-01-01

    Increased phosphorylation of the KIF5 anterograde motor is associated with impaired axonal transport and neurodegeneration, but paradoxically also with normal transport, though the details are not fully defined. JNK phosphorylates KIF5C on S176 in the motor domain; a site that we show is phosphorylated in brain. Microtubule pelleting assays demonstrate that phosphomimetic KIF5C(1-560)S176D associates weakly with microtubules compared to KIF5C(1-560)WT. Consistent with this, 50% of KIF5C(1-560)S176D shows diffuse movement in neurons. However, the remaining 50% remains microtubule bound and displays decreased pausing and increased bidirectional movement. The same directionality switching is observed with KIF5C(1-560)WT in the presence of an active JNK chimera, MKK7-JNK. Yet, in cargo trafficking assays where peroxisome cargo is bound, KIF5C(1-560)S176D-GFP-FRB transports normally to microtubule plus ends. We also find that JNK increases the ATP hydrolysis of KIF5C in vitro. These data suggest that phosphorylation of KIF5C-S176 primes the motor to either disengage entirely from microtubule tracks as previously observed in response to stress, or to display improved efficiency. The final outcome may depend on cargo load and motor ensembles. PMID:27013971

  20. Somatic cell-oocyte interactions in mouse oogenesis: stage-specific regulation of mouse oocyte protein phosphorylation by granulosa cells.

    PubMed

    Colonna, R; Cecconi, S; Tatone, C; Mangia, F; Buccione, R

    1989-05-01

    The relative rate of synthesis of a number of proteins and the protein phosphorylation pattern of growing and fully grown oocytes were influenced by the presence of granulosa cells. In particular, a 74-kDa phosphorylated protein was detected only in granulosa cell-enclosed growing mouse oocytes. When reaggregated with granulosa cells, the growing oocyte displayed the phosphorylated form of the 74-kDa protein but when oocytes were cultured on Sertoli cell monolayers or in granulosa cell-conditioned medium the 74-kDa protein was not phosphorylated. We propose that (1) granulosa cells regulate protein phosphorylation in mouse oocytes; (2) a 74-kDa protein is phosphorylated only in growing oocytes when surrounded by granulosa cells; and (3) granulosa cells, but not Sertoli cells, are competent to send the appropriate "signal" to the growing oocyte. PMID:2707483

  1. RpoS proteolysis is regulated by a mechanism that does not require the SprE (RssB) response regulator phosphorylation site.

    PubMed

    Peterson, Celeste N; Ruiz, Natividad; Silhavy, Thomas J

    2004-11-01

    In Escherichia coli the response regulator SprE (RssB) facilitates degradation of the sigma factor RpoS by delivering it to the ClpXP protease. This process is regulated: RpoS is degraded in logarithmic phase but becomes stable upon carbon starvation, resulting in its accumulation. Because SprE contains a CheY domain with a conserved phosphorylation site (D58), the prevailing model posits that this control is mediated by phosphorylation. To test this model, we mutated the conserved response regulator phosphorylation site (D58A) of the chromosomal allele of sprE and monitored RpoS levels in response to carbon starvation. Though phosphorylation contributed to the SprE basal activity, we found that RpoS proteolysis was still regulated upon carbon starvation. Furthermore, our results indicate that phosphorylation of wild-type SprE occurs by a mechanism that is independent of acetyl phosphate. PMID:15489452

  2. Cell-cycle-regulated activation of Akt kinase by phosphorylation at its carboxyl terminus

    PubMed Central

    Liu, Pengda; Begley, Michael; Michowski, Wojciech; Inuzuka, Hiroyuki; Ginzberg, Miriam; Gao, Daming; Tsou, Peiling; Gan, Wenjian; Papa, Antonella; Kim, Byeong Mo; Wan, Lixin; Singh, Amrik; Zhai, Bo; Yuan, Min; Wang, Zhiwei; Gygi, Steven P.; Lee, Tae Ho; Lu, Kun-Ping; Toker, Alex; Pandolfi, Pier Paolo; Asara, John M.; Kirschner, Marc W.; Sicinski, Piotr; Cantley, Lewis; Wei, Wenyi

    2014-01-01

    Akt, also known as protein kinase B, plays key roles in cell proliferation, survival and metabolism. Akt hyperactivation contributes to many pathophysiological conditions, including human cancers1–3, and is closely associated with poor prognosis and chemo- or radio-therapeutic resistance4. Phosphorylation of Akt at S473 (ref. 5) and T308 (ref. 6) activates Akt. However, it remains unclear whether further mechanisms account for full Akt activation, and whether Akt hyperactivation is linked to misregulated cell cycle progression, another cancer hallmark7. Here we report that Akt activity fluctuates across the cell cycle, mirroring cyclin A expression. Mechanistically, phosphorylation of S477 and T479 at the Akt extreme carboxy terminus by cyclin-dependent kinase 2 (Cdk2)/cyclin A or mTORC2, under distinct physiological conditions, promotes Akt activation through facilitating, or functionally compensating for, S473 phosphorylation. Furthermore, deletion of the cyclin A2 allele in the mouse olfactory bulb leads to reduced S477/T479 phosphorylation and elevated cellular apoptosis. Notably, cyclin A2-deletion-induced cellular apoptosis in mouse embryonic stem cells is partly rescued by S477D/T479E-Akt1, supporting a physiological role for cyclin A2 in governing Akt activation. Together, the results of our study show Akt S477/T479 phosphorylation to be an essential layer of the Akt activation mechanism to regulate its physiological functions, thereby providing a new mechanistic link between aberrant cell cycle progression and Akt hyperactivation in cancer. PMID:24670654

  3. A mutation of the fission yeast EB1 overcomes negative regulation by phosphorylation and stabilizes microtubules

    SciTech Connect

    Iimori, Makoto; Ozaki, Kanako; Chikashige, Yuji; Habu, Toshiyuki; Hiraoka, Yasushi; Maki, Takahisa; Hayashi, Ikuko; Obuse, Chikashi; Matsumoto, Tomohiro

    2012-02-01

    Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+ TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly. The mutant Mal3 is no longer a + TIP, but binds strongly the microtubule lattice. Live cell imaging has revealed that while the wild type Mal3 proteins dissociate from the tip of the growing microtubules before the onset of shrinkage, the mutant Mal3 proteins persist on microtubules and reduces a rate of shrinkage after a longer pausing period. Consequently, the mutant Mal3 proteins cause abnormal elongation of microtubules composing the spindle and aster. Mal3 is phosphorylated at a cluster of serine/threonine residues in the linker connecting the CH and EB1-like C-terminal motif domains. The phosphorylation occurs in a microtubule-dependent manner and reduces the affinity of Mal3 to microtubules. We propose that because the 89R mutation is resistant to the effect of phosphorylation, it can associate persistently with microtubules and confers a stronger stability of microtubules likely by reinforcing the cylindrical structure. -- Highlights: Black-Right-Pointing-Pointer We characterize a mutation (mal3-89R) in fission yeast homolog of EB1. Black-Right-Pointing-Pointer The mutation enhances the activity to assemble microtubules. Black-Right-Pointing-Pointer Mal3 is phosphorylated in a microtubule-dependent manner. Black-Right-Pointing-Pointer The phosphorylation negatively regulates the Mal3 activity.

  4. Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro

    SciTech Connect

    Kaiserman, H.B.; Ingebritsen, T.S.; Benbow, R.M.

    1988-05-03

    DNA topoisomerase I has been purified to electrophoretic homogeneity from ovaries of the frog Xenopus laevis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction revealed a single major band at 110 kDa and less abundant minor bands centered at 62 kDa. Incubation of the most purified fraction with immobilized calf intestinal alkaline phosphatase abolished all DNA topoisomerase enzymatic activity in a time-dependent reaction. Treatment of the dephosphorylated X. laevis DNA topoisomerase I with a X. laevis casein kinase type II activity and ATP restored DNA topoisomerase activity to a level higher than that observed in the most purified fraction. In vitro labeling experiments which employed the most purified DNA topoisomerase I fraction, (..gamma..-/sup 32/P)ATP, and the casein kinase type II enzyme showed that both the 110- and 62-kDa bands became phosphorylated in approximately molar proportions. Phosphoamino acid analysis showed that only serine residues became phosphorylated. Phosphorylation was accompanied by an increase in DNA topoisomerase activity in vitro. Dephosphorylation of DNA topoisomerase I appears to block formation of the initial enzyme-substrate complex on the basis of the failure of the dephosphorylated enzyme to nick DNA in the presence of camptothecin. The authors conclude that X. laevis DNA topoisomerase I is partially phosphorylated as isolated and that this phosphorylation is essential for expression of enzymatic activity in vitro. On the basis of the ability of the casein kinase type II activity to reactivate dephosphorylated DNA topoisomerase I, they speculate that this kinase may contribute to the physiological regulation of DNA topoisomerase I activity.

  5. Phosphorylation on TRPV4 Serine 824 Regulates Interaction with STIM1.

    PubMed

    Shin, Sung H; Lee, Eun J; Chun, Jaesun; Hyun, Sunghee; Kang, Sang S

    2015-01-01

    The TRPV4 cation channel, a member of the TRP vanilloid subfamily, is expressed in a broad range of tissues where it participates in the generation of a Ca2+ signal and/or depolarization of membrane potential. Here, we identified stromal interaction molecule 1 precursor (STIM1) as an auxiliary protein of this epithelial Ca2+channel using confocal microscopy analysis and GST pull-down assay. The STIM1 protein associates specifically with the C-terminal tail of TRPV4 to form a complex. In previous reports, we demonstrated that the serine824 residue of TRPV4 is one of the target phosphorylation sites of serum/glucocorticoid regulated kinase 1 (SGK1). In this report we further identified the role of serine 824 phosphorylation. The TRPV4 mutant S824D (not S824A) exhibited a diminished capacity to bind STIM1. Using GST pull-down and co-immunoprecipitation assays, we demonstrated that STIM1 is part of the TRPV4 protein complex. Our observations clearly suggest that the formation of a complex between TRPV4 and STIM1 and its plasma membrane localization are regulated through phosphorylation of serine824 of TRPV4, and that the STIM1-TRPV4 complex plays crucial roles in routing TRPV4 to the plasma membrane from the endoplasmic reticulum and in maintaining its function. PMID:25972993

  6. Regulation of TDP-43 aggregation by phosphorylation and p62/SQSTM1.

    PubMed

    Brady, Owen A; Meng, Peter; Zheng, Yanqiu; Mao, Yuxin; Hu, Fenghua

    2011-01-01

    TAR DNA-binding protein-43 (TDP-43) proteinopathy has been linked to several neurodegenerative diseases, such as frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis. Phosphorylated and ubiquitinated TDP-43 C-terminal fragments have been found in cytoplasmic inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis patients. However, the factors and pathways that regulate TDP-43 aggregation are still not clear. We found that the C-terminal 15 kDa fragment of TDP-43 is sufficient to induce aggregation but the aggregation phenotype is modified by additional sequences. Aggregation is accompanied by phosphorylation at serine residues 409/410. Mutation of 409/410 to phosphomimetic aspartic acid residues significantly reduces aggregation. Inhibition of either proteasome or autophagy dramatically increases TDP-43 aggregation. Furthermore, TDP-43 aggregates colocalize with markers of autophagy and the adaptor protein p62/SQSTM1. Over-expression of p62/SQSTM1 reduces TDP-43 aggregation in an autophagy and proteasome-dependent manner. These studies suggest that aggregation of TDP-43 C-terminal fragments is regulated by phosphorylation events and both the autophagy and proteasome-mediated degradation pathways. PMID:21062285

  7. Casein Kinase 1 α Phosphorylates the Wnt Regulator Jade-1 and Modulates Its Activity*

    PubMed Central

    Borgal, Lori; Rinschen, Markus M.; Dafinger, Claudia; Hoff, Sylvia; Reinert, Matthäus J.; Lamkemeyer, Tobias; Lienkamp, Soeren S.; Benzing, Thomas; Schermer, Bernhard

    2014-01-01

    Tight regulation of Wnt/β-catenin signaling is critical for vertebrate development and tissue maintenance, and deregulation can lead to a host of disease phenotypes, including developmental disorders and cancer. Proteins associated with primary cilia and centrosomes have been demonstrated to negatively regulate canonical Wnt signaling in interphase cells. The plant homeodomain zinc finger protein Jade-1 can act as an E3 ubiquitin ligase-targeting β-catenin for proteasomal degradation and concentrates at the centrosome and ciliary basal body in addition to the nucleus in interphase cells. We demonstrate that the destruction complex component casein kinase 1α (CK1α) phosphorylates Jade-1 at a conserved SLS motif and reduces the ability of Jade-1 to inhibit β-catenin signaling. Consistently, Jade-1 lacking the SLS motif is more effective than wild-type Jade-1 in reducing β-catenin-induced secondary axis formation in Xenopus laevis embryos in vivo. Interestingly, CK1α also phosphorylates β-catenin and the destruction complex component adenomatous polyposis coli at a similar SLS motif to the effect that β-catenin is targeted for degradation. The opposing effect of Jade-1 phosphorylation by CK1α suggests a novel example of the dual functions of CK1α activity to either oppose or promote canonical Wnt signaling in a context-dependent manner. PMID:25100726

  8. Casein kinase 2 dependent phosphorylation of neprilysin regulates receptor tyrosine kinase signaling to Akt.

    PubMed

    Siepmann, Martin; Kumar, Sathish; Mayer, Günter; Walter, Jochen

    2010-01-01

    Neprilysin (NEP) is a type II membrane metalloproteinase that cleaves physiologically active peptides at the cell surface thus regulating the local concentration of these peptides available for receptor binding and signal transduction. In addition, the cytoplasmic N-terminal domain of NEP interacts with the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) thereby regulating intracellular signaling via Akt. Thus, NEP serves dual functions in extracellular and intracellular signal transduction. Here, we show that NEP undergoes phosphorylation at serine residue 6 within the N-terminal cytoplasmic domain. In vitro and cell culture experiments demonstrate that Ser 6 is efficiently phosphorylated by protein kinase CK2. The phosphorylation of the cytoplasmic domain of NEP inhibits its interaction with PTEN. Interestingly, expression of a pseudophosphorylated NEP variant (Ser6Asp) abrogates the inhibitory effect of NEP on insulin/insulin-like growth factor-1 (IGF-1) stimulated activation of Akt. Thus, our data demonstrate a regulatory role of CK2 in the interaction of NEP with PTEN and insulin/IGF-1 signaling. PMID:20957047

  9. Juvenile hormone prevents 20-hydroxyecdysone-induced metamorphosis by regulating the phosphorylation of a newly identified broad protein.

    PubMed

    Cai, Mei-Juan; Liu, Wen; Pei, Xu-Yang; Li, Xiang-Ru; He, Hong-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

    2014-09-19

    The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis. By contrast, juvenile hormone (JH) prevents metamorphosis. However, the mechanism by which JH inhibits metamorphosis remains unclear. In this study, we propose that JH induces the phosphorylation of Broad isoform Z7 (BrZ7), a newly identified protein, to inhibit 20E-mediated metamorphosis in the lepidopteran insect Helicoverpa armigera. The knockdown of BrZ7 in larvae inhibited metamorphosis by repressing the expression of the 20E response gene. BrZ7 was weakly expressed and phosphorylated during larval growth but highly expressed and non-phosphorylated during metamorphosis. JH regulated the rapid phosphorylation of BrZ7 via a G-protein-coupled receptor-, phospholipase C-, and protein kinase C-triggered pathway. The phosphorylated BrZ7 bound to the 5'-regulatory region of calponin to regulate its expression in the JH pathway. Exogenous JH induced BrZ7 phosphorylation to prevent metamorphosis by suppressing 20E-related gene transcription. JH promoted non-phosphorylated calponin interacting with ultraspiracle protein to activate the JH pathway and antagonize the 20E pathway. This study reveals one of the possible mechanisms by which JH counteracts 20E-regulated metamorphosis by inducing the phosphorylation of BrZ7. PMID:25096576

  10. Region-specific tolerance to cocaine-regulated cAMP-dependent protein phosphorylation following chronic self-administration.

    PubMed

    Edwards, Scott; Graham, Danielle L; Bachtell, Ryan K; Self, David W

    2007-04-01

    Chronic cocaine self-administration can produce either tolerance or sensitization to certain cocaine-regulated behaviours, but whether differential alterations develop in the biochemical response to cocaine is less clear. We measured cocaine-induced phosphorylation of multiple cAMP-dependent and -independent protein substrates in mesolimbic dopamine terminal regions following chronic self-administration. Changes in self-administering rats were compared to changes produced by passive yoked injection to identify reinforcement-related regulation, whereas acute and chronic yoked groups were compared to identify the development tolerance or sensitization in the biochemical response to cocaine. Microwave-fixed brain tissue was collected immediately following 4 h of intravenous cocaine administration, and subjected to Western blot analysis of phosphorylated and total protein substrates. Chronic cocaine produced region- and substrate-specific tolerance to cAMP-dependent protein phosphorylation, including GluR1(S845) phosphorylation in striatal and amygdala subregions and NR1(S897) phosphorylation in the CA1 subregion of the hippocampus. Tolerance also developed to cAMP-independent GluR1(S831) phosphorylation in the prefrontal cortex. In contrast, sensitization to presynaptic regulation of synapsin(S9) phosphorylation developed in the hippocampal CA3 subregion while cAMP-dependent tyrosine hydroxylase(S40) phosphorylation decreased in striatal dopamine terminals. Cocaine-induced ERK and CREB(S133) phosphorylation were dissociated in many brain regions and failed to develop either tolerance or sensitization with chronic administration. Positive reinforcement-related correlations between cocaine intake and protein phosphorylation were found only in self-administering animals, while negative dose-related correlations were found primarily with yoked administration. These regional- and substrate-specific adaptations in cocaine-induced protein phosphorylation are discussed in

  11. Plasmodium falciparum STEVOR phosphorylation regulates host erythrocyte deformability enabling malaria parasite transmission.

    PubMed

    Naissant, Bernina; Dupuy, Florian; Duffier, Yoann; Lorthiois, Audrey; Duez, Julien; Scholz, Judith; Buffet, Pierre; Merckx, Anais; Bachmann, Anna; Lavazec, Catherine

    2016-06-16

    Deformability of Plasmodium falciparum gametocyte-infected erythrocytes (GIEs) allows them to persist for several days in blood circulation and to ensure transmission to mosquitoes. Here, we investigate the mechanism by which the parasite proteins STEVOR (SubTElomeric Variable Open Reading frame) exert changes on GIE deformability. Using the microsphiltration method, immunoprecipitation, and mass spectrometry, we produce evidence that GIE stiffness is dependent on the cytoplasmic domain of STEVOR that interacts with ankyrin complex at the erythrocyte skeleton. Moreover, we show that GIE deformability is regulated by protein kinase A (PKA)-mediated phosphorylation of the STEVOR C-terminal domain at a specific serine residue (S324). Finally, we show that the increase of GIE stiffness induced by sildenafil (Viagra) is dependent on STEVOR phosphorylation status and on another independent mechanism. These data provide new insights into mechanisms by which phosphodiesterase inhibitors may block malaria parasite transmission. PMID:27136945

  12. Mitosis-specific phosphorylation of PML at T409 regulates spindle checkpoint.

    PubMed

    Jin, J; Liu, J

    2016-01-01

    During mitosis, Promyelocytic leukemia nuclear bodies (PML NBs) change dramatically in morphology and composition, but little is known about function of PML in mitosis. Here, we show that PML is phosphorylated at T409 (PML p409) in a mitosis-specific manner. More importantly, PML p409 contributes to maintain the duration of pro-metaphase and regulates spindle checkpoint. Deficient PML p409 caused a shortening of pro-metaphase and challenged the nocodazole-triggered mitotic arrest. T409A mutation led to a higher frequency of misaligned chromosomes on metaphase plate, and subsequently death in late mitosis. In addition, inhibition of PML p409 repressed growth of tumor cells, suggesting that PML p409 is a potential target for cancer therapy. Collectively, our study demonstrated an important phosphorylated site of PML, which contributed to explore the role of PML in mitosis. PMID:27609478

  13. Silica nanoparticles inhibit brown adipocyte differentiation via regulation of p38 phosphorylation

    NASA Astrophysics Data System (ADS)

    Son, Min Jeong; Kim, Won Kon; Kwak, Minjeong; Oh, Kyoung-Jin; Chang, Won Seok; Min, Jeong-Ki; Lee, Sang Chul; Song, Nam Woong; Bae, Kwang-Hee

    2015-10-01

    Nanoparticles are of great interest due to their wide variety of biomedical and bioengineering applications. However, they affect cellular differentiation and/or intracellular signaling when applied and exposed to target organisms or cells. The brown adipocyte is a cell type important in energy homeostasis and thus closely related to obesity. In this study, we assessed the effects of silica nanoparticles (SNPs) on brown adipocyte differentiation. The results clearly showed that brown adipocyte differentiation was significantly repressed by exposure to SNPs. The brown adipocyte-specific genes as well as mitochondrial content were also markedly reduced. Additionally, SNPs led to suppressed p38 phosphorylation during brown adipocyte differentiation. These effects depend on the size of SNPs. Taken together, these results lead us to suggest that SNP has anti-brown adipogenic effect in a size-dependent manner via regulation of p38 phosphorylation.

  14. Protein phosphorylation as a mechanism for regulation of spinach leaf sucrose-phosphate synthase activity

    SciTech Connect

    Huber, J.L.A.; Huber, S.C. )

    1989-04-01

    Protein phosphorylation has been identified as a mechanism for the light-dark regulation of spinach sucrose-phosphate synthase (SPS) activity, previously shown to involve some type of covalent modification of the enzyme. The 120 kD subunit of SPS in extracts of light-treated leaves was labeled with {sup 32}P in the presence of ({gamma}-{sup 32}P) ATP. In this in vitro system, {sup 32}P incorporation into light-activated SPS was dependent upon ATP and magnesium concentrations as well as time, and was closely paralleled by inactivation of the enzyme. The soluble protein kinase involved in the interconversion of SPS between activated and deactivated forms may be specific for SPS as it co-purifies with SPS during partial purification of the enzyme. The kinase appears not to be calcium activated and no evidence has been obtained for metabolite control of SPS phosphorylation/inactivation.

  15. S-nitrosylation regulates VE-cadherin phosphorylation and internalization in microvascular permeability.

    PubMed

    Guequén, Anita; Carrasco, Rodrigo; Zamorano, Patricia; Rebolledo, Lorena; Burboa, Pia; Sarmiento, José; Boric, Mauricio P; Korayem, Adam; Durán, Walter N; Sánchez, Fabiola A

    2016-04-15

    The adherens junction complex, composed mainly of vascular endothelial (VE)-cadherin, β-catenin, p120, and γ-catenin, is the main element of the endothelial barrier in postcapillary venules.S-nitrosylation of β-catenin and p120 is an important step in proinflammatory agents-induced hyperpermeability. We investigated in vitro and in vivo whether or not VE-cadherin isS-nitrosylated using platelet-activating factor (PAF) as agonist. We report that PAF-stimulatesS-nitrosylation of VE-cadherin, which disrupts its association with β-catenin. In addition, based on inhibition of nitric oxide production, our results strongly suggest thatS-nitrosylation is required for VE-cadherin phosphorylation on tyrosine and for its internalization. Our results unveil an important mechanism to regulate phosphorylation of junctional proteins in association withS-nitrosylation. PMID:26921435

  16. ULK3 regulates cytokinetic abscission by phosphorylating ESCRT-III proteins.

    PubMed

    Caballe, Anna; Wenzel, Dawn M; Agromayor, Monica; Alam, Steven L; Skalicky, Jack J; Kloc, Magdalena; Carlton, Jeremy G; Labrador, Leticia; Sundquist, Wesley I; Martin-Serrano, Juan

    2015-01-01

    The endosomal sorting complexes required for transport (ESCRT) machinery mediates the physical separation between daughter cells during cytokinetic abscission. This process is regulated by the abscission checkpoint, a genome protection mechanism that relies on Aurora B and the ESCRT-III subunit CHMP4C to delay abscission in response to chromosome missegregation. In this study, we show that Unc-51-like kinase 3 (ULK3) phosphorylates and binds ESCRT-III subunits via tandem MIT domains, and thereby, delays abscission in response to lagging chromosomes, nuclear pore defects, and tension forces at the midbody. Our structural and biochemical studies reveal an unusually tight interaction between ULK3 and IST1, an ESCRT-III subunit required for abscission. We also demonstrate that IST1 phosphorylation by ULK3 is an essential signal required to sustain the abscission checkpoint and that ULK3 and CHMP4C are functionally linked components of the timer that controls abscission in multiple physiological situations. PMID:26011858

  17. CDK-mediated RNF4 phosphorylation regulates homologous recombination in S-phase

    PubMed Central

    Luo, Kuntian; Deng, Min; Li, Yunhui; Wu, Chenming; Xu, Ziwen; Yuan, Jian; Lou, Zhenkun

    2015-01-01

    There are the two major pathways responsible for the repair of DNA double-strand breaks (DSBs): non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ operates throughout the cell-cycle, while HR is primarily active in the S/G2 phases suggesting that there are cell cycle-specific mechanisms that regulate the balance between NHEJ and HR. Here we reported that CDK2 could phosphorylate RNF4 on T26 and T112 and enhance RNF4 E3 ligase activity, which is important for MDC1 degradation and proper HR repair during S phase. Mutation of the RNF4 phosphorylation sites results in MDC1 stabilization, which in turn compromised HR during S-phase. These results suggest that in addition to drive cell cycle progression, CDK also targets RNF4, which is involved in the regulatory network of DSBs repair. PMID:25948581

  18. CDK-mediated RNF4 phosphorylation regulates homologous recombination in S-phase.

    PubMed

    Luo, Kuntian; Deng, Min; Li, Yunhui; Wu, Chenming; Xu, Ziwen; Yuan, Jian; Lou, Zhenkun

    2015-06-23

    There are the two major pathways responsible for the repair of DNA double-strand breaks (DSBs): non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ operates throughout the cell-cycle, while HR is primarily active in the S/G2 phases suggesting that there are cell cycle-specific mechanisms that regulate the balance between NHEJ and HR. Here we reported that CDK2 could phosphorylate RNF4 on T26 and T112 and enhance RNF4 E3 ligase activity, which is important for MDC1 degradation and proper HR repair during S phase. Mutation of the RNF4 phosphorylation sites results in MDC1 stabilization, which in turn compromised HR during S-phase. These results suggest that in addition to drive cell cycle progression, CDK also targets RNF4, which is involved in the regulatory network of DSBs repair. PMID:25948581

  19. Diacylglycerol kinase-zeta localization in skeletal muscle is regulated by phosphorylation and interaction with syntrophins.

    PubMed

    Abramovici, Hanan; Hogan, Angela B; Obagi, Christopher; Topham, Matthew K; Gee, Stephen H

    2003-11-01

    Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-zeta (DGK-zeta), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-zeta form a complex in skeletal muscle whose translocation from the cytosol to the plasma membrane is regulated by protein kinase C-dependent phosphorylation of the DGK-zeta MARCKS domain. DGK-zeta mutants that do not bind syntrophins were mislocalized, and an activated mutant of this sort induced atypical changes in the actin cytoskeleton, indicating syntrophins are important for localizing DGK-zeta and regulating its activity. Consistent with a role in actin organization, DGK-zeta and syntrophins were colocalized with filamentous (F)-actin and Rac in lamellipodia and ruffles. Moreover, extracellular signal-related kinase-dependent phosphorylation of DGK-zeta regulated its association with the cytoskeleton. In adult muscle, DGK-zeta was colocalized with syntrophins on the sarcolemma and was concentrated at neuromuscular junctions (NMJs), whereas in type IIB fibers it was found exclusively at NMJs. DGK-zeta was reduced at the sarcolemma of dystrophin-deficient mdx mouse myofibers but was specifically retained at NMJs, indicating that dystrophin is important for the sarcolemmal but not synaptic localization of DGK-zeta. Together, our findings suggest syntrophins localize DGK-zeta signaling complexes at specialized domains of muscle cells, which may be critical for the proper control of lipid-signaling pathways regulating actin organization. In dystrophic muscle, mislocalized DGK-zeta may cause abnormal cytoskeletal changes that contribute to disease pathogenesis. PMID:14551255

  20. Rapid estrogen signaling negatively regulates PTEN activity through phosphorylation in endometrial cancer cells

    PubMed Central

    Scully, Melanie M.; Palacios-Helgeson, Leslie K.; Wah, Lah S.; Jackson, Twila A.

    2014-01-01

    Hyperestrogenicity is a risk factor for endometrial cancer. 17β-estradiol (E2) is known to stimulate both genomic and nongenomic estrogen receptor-α (ERα) actions in a number of reproductive tissues. However, the contributions of transcription-independent ERα signaling on normal and malignant endometrium are not fully understood. Phosphatase and tensin homolog (PTEN) is a tumor suppressor that decreases cellular mitosis primarily through negative regulation of the phosphoinositide 3-kinase/AKT signaling axis. PTEN levels are elevated during the E2 dominated, mitotically active, proliferative phase of the menstrual cycle, indicating possible hormonal regulation of PTEN in the uterus. In order to determine if rapid E2 signaling regulates PTEN, we used ERα positive, PTEN positive, endometrial cells. We show that cytosolic E2/ERα signaling leads to increased phosphorylation of PTEN at key regulatory residues. Importantly, E2 stimulation decreased PTEN lipid phosphatase activity and caused consequent increases in phospho-AKT. We further demonstrate that cytosolic ERα forms a complex with PTEN in an E2-dependent manner, and that ERα constitutively complexes with protein kinase2-α (CK2α), a kinase previously shown to phosphorylate the C-terminal tail of PTEN. These results provide mechanistic support for an E2-dependent, ERα cytosolic signaling complex that negatively regulates PTEN activity through carboxy terminus phosphorylation. Using an animal model, we show that sustained E2 signaling results in increased phospho-PTEN (S380, T382, T383), total PTEN and phospho-AKT (S473). Taken together, we provide a novel mechanism in which transcription-independent E2/ERα signaling may promote a pro-tumorigenic environment in the endometrium. PMID:24844349

  1. Tgf-beta induced Erk phosphorylation of smad linker region regulates smad signaling.

    PubMed

    Hough, Chris; Radu, Maria; Doré, Jules J E

    2012-01-01

    The Transforming Growth Factor-Beta (TGF-β) family is involved in regulating a variety of cellular processes such as apoptosis, differentiation, and proliferation. TGF-β binding to a Serine/Threonine kinase receptor complex causes the recruitment and subsequent activation of transcription factors known as smad2 and smad3. These proteins subsequently translocate into the nucleus to negatively or positively regulate gene expression. In this study, we define a second signaling pathway leading to TGF-β receptor activation of Extracellular Signal Regulated Kinase (Erk) in a cell-type dependent manner. TGF-β induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells. By activating phosphotidylinositol 3-kinase (PI3K), TGF-β stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation. Activation of Erk was necessary for TGF-β induced fibroblast replication. In addition, Erk phosphorylated the linker region of nuclear localized smads, resulting in increased half-life of C-terminal phospho-smad 2 and 3 and increased duration of smad target gene transcription. Together, these data show that in mesenchymal cell types the TGF-β/PI3K/Pak2/Raf/MEK/Erk pathway regulates smad signaling, is critical for TGF-β-induced growth and is part of an integrated signaling web containing multiple interacting pathways rather than discrete smad/non-smad pathways. PMID:22880011

  2. 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

  3. Identification of Light-Sensitive Phosphorylation Sites on PERIOD That Regulate the Pace of Circadian Rhythms in Drosophila.

    PubMed

    Yildirim, Evrim; Chiu, Joanna C; Edery, Isaac

    2016-03-01

    The main components regulating the pace of circadian (≅24 h) clocks in animals are PERIOD (PER) proteins, transcriptional regulators that undergo daily changes in levels and nuclear accumulation by means of complex multisite phosphorylation programs. In the present study, we investigated the function of two phosphorylation sites, at Ser826 and Ser828, located in a putative nuclear localization signal (NLS) on the Drosophila melanogaster PER protein. These sites are phosphorylated by DOUBLETIME (DBT; Drosophila homolog of CK1δ/ε), the key circadian kinase regulating the daily changes in PER stability and phosphorylation. Mutant flies in which phosphorylation at Ser826/Ser828 is blocked manifest behavioral rhythms with periods slightly longer than 1 h and with altered temperature compensation properties. Intriguingly, although phosphorylation at these sites does not influence PER stability, timing of nuclear entry, or transcriptional autoinhibition, the phospho-occupancy at Ser826/Ser828 is rapidly stimulated by light and blocked by TIMELESS (TIM), the major photosensitive clock component in Drosophila and a crucial binding partner of PER. Our findings identify the first phosphorylation sites on core clock proteins that are acutely regulated by photic cues and suggest that some phosphosites on PER proteins can modulate the pace of downstream behavioral rhythms without altering central aspects of the clock mechanism. PMID:26711257

  4. mTORC1 directly phosphorylates and activates ERα upon estrogen stimulation.

    PubMed

    Alayev, A; Salamon, R S; Berger, S M; Schwartz, N S; Cuesta, R; Snyder, R B; Holz, M K

    2016-07-01

    Breast cancer is the leading cause of cancer-related deaths among women. Approximately 75% of breast cancers are estrogen receptor-α (ERα) positive, underscoring the dependence of cancer cells on estrogen for growth and survival. Patients treated with endocrine therapy often develop resistance, either de novo or acquired, which in some cases is caused by aberrations within the growth factor signaling pathways. The mechanistic target of rapamycin complex 1 (mTORC1) has emerged as a critical node in estrogenic signaling. We have previously shown that mTORC1 can phosphorylate and activate ERα on S167 via its effector-the 40S ribosomal S6 kinase 1 (S6K1). Presently, we have uncovered a direct link between mTORC1 and ERα. We found that ERα binds to regulatory-associated protein of mTOR (Raptor) and causes it to translocate to the nucleus upon estrogen stimulation. In addition, we identified mTOR as the kinase that phosphorylates ERα on S104/106 and activates transcription of ER target genes. Our findings show a direct link between mTORC1 and ERα, which further implicates mTORC1 signaling in the pathogenesis of ER-positive breast cancer and provides rationale for FDA-approved use of mTORC1 inhibitors in combination with endocrine agents for treatment of this disease. PMID:26522726

  5. CDK5 interacts with Slo and affects its surface expression and kinetics through direct phosphorylation

    PubMed Central

    Bai, Jun-Ping; Surguchev, Alexei; Joshi, Powrnima; Gross, Liza

    2012-01-01

    Large-conductance calcium-activated potassium (BK) channels are ubiquitous and play an important role in a number of diseases. In hair cells of the ear, they play a critical role in electrical tuning, a mechanism of frequency discrimination. These channels show variable kinetics and expression along the tonotopic axis. Although the molecular underpinnings to its function in hair cells are poorly understood, it is established that BK channels consist of a pore-forming α-subunit (Slo) and a number of accessory subunits. Here we identify CDK5, a member of the cyclin-dependent kinase family, as an interacting partner of Slo. We show CDK5 to be present in hair cells and expressed in high concentrations in the cuticular plate and in the circumferential zone. In human embryonic kidney cells, we show that CDK5 inhibits surface expression of Slo by direct phosphorylation of Slo. Similarly, we note that CDK5 affects Slo voltage activation and deactivation kinetics, by a direct phosphorylation of T847. Taken together with its increasing expression along the tonotopic axis, these data suggest that CDK5 likely plays a critical role in electrical tuning and surface expression of Slo in hair cells. PMID:22094329

  6. The phosphorylation of serine 492 of perilipin a directs lipid droplet fragmentation and dispersion.

    PubMed

    Marcinkiewicz, Amy; Gauthier, Denise; Garcia, Anne; Brasaemle, Dawn L

    2006-04-28

    Perilipin A is a key regulator of triacylglycerol storage and hydrolysis in adipocytes; phosphorylation of perilipin A by protein kinase A facilitates maximal lipolysis. Chronic stimulation of lipolysis in 3T3-L1 adipocytes causes large perinuclear lipid droplets to fragment into myriad dispersed perilipin A-covered microlipid droplets. In cultured fibroblasts stably expressing ectopic perilipin A, clustered lipid droplets disperse throughout the cytoplasm upon incubation of the cells with forskolin and isobutylmethylxanthine (IBMX) to elevate levels of cAMP and activate protein kinase A, mirroring events observed in adipocytes. Furthermore, diethylum-belliferyl phosphate inhibits stimulated lipolysis but not the dispersion of lipid droplets, suggesting that products of lipolysis are not required for this remodeling process. We hypothesized that protein kinase A-mediated phosphorylation of perilipin A triggers the remodeling of lipid droplets. The mutation of serine 492 of perilipin A to alanine prevented the dispersion of clustered lipid droplets in fibroblasts stably expressing the mutated perilipin upon incubation with forskolin and IBMX. In contrast, the substitution of serines 81, 222, 276, or 433 with alanine, either singly or in combinations, did not affect the protein kinase A-mediated remodeling of lipid droplets. Interestingly, substitution of serines 433, 492, and 517 of perilipin A with glutamic acid residues blocked the dispersion of clustered lipid droplets in cells incubated with forskolin and IBMX, indicating that the addition of a negative charge does not mimic a phosphate group. We conclude that protein kinase A-mediated phosphorylation of serine 492 of perilipin A drives the fragmentation and dispersion of lipid droplets. PMID:16488886

  7. Novel RNA chaperone domain of RNA-binding protein La is regulated by AKT phosphorylation

    PubMed Central

    Kuehnert, Julia; Sommer, Gunhild; Zierk, Avery W.; Fedarovich, Alena; Brock, Alexander; Fedarovich, Dzmitry; Heise, Tilman

    2015-01-01

    The cellular function of the cancer-associated RNA-binding protein La has been linked to translation of viral and cellular mRNAs. Recently, we have shown that the human La protein stimulates IRES-mediated translation of the cooperative oncogene CCND1 in cervical cancer cells. However, there is little known about the underlying molecular mechanism by which La stimulates CCND1 IRES-mediated translation, and we propose that its RNA chaperone activity is required. Herein, we show that La binds close to the CCND1 start codon and demonstrate that La's RNA chaperone activity can change the folding of its binding site. We map the RNA chaperone domain (RCD) within the C-terminal region of La in close proximity to a novel AKT phosphorylation site (T389). Phosphorylation at T389 by AKT-1 strongly impairs its RNA chaperone activity. Furthermore, we demonstrate that the RCD as well as T389 is required to stimulate CCND1 IRES-mediated translation in cells. In summary, we provide a model whereby a novel interplay between RNA-binding, RNA chaperoning and AKT phosphorylation of La protein regulates CCND1 IRES-mediated translation. PMID:25520193

  8. Phosphorylation of the Yeast γ-Tubulin Tub4 Regulates Microtubule Function

    PubMed Central

    Lin, Tien-chen; Gombos, Linda; Neuner, Annett; Sebastian, Dominik; Olsen, Jesper V.; Hrle, Ajla; Benda, Christian; Schiebel, Elmar

    2011-01-01

    The yeast γ-tubulin Tub4 is assembled with Spc97 and Spc98 into the small Tub4 complex. The Tub4 complex binds via the receptor proteins Spc72 and Spc110 to the spindle pole body (SPB), the functional equivalent of the mammalian centrosome, where the Tub4 complex organizes cytoplasmic and nuclear microtubules. Little is known about the regulation of the Tub4 complex. Here, we isolated the Tub4 complex with the bound receptors from yeast cells. Analysis of the purified Tub4 complex by mass spectrometry identified more than 50 phosphorylation sites in Spc72, Spc97, Spc98, Spc110 and Tub4. To examine the functional relevance of the phosphorylation sites, phospho-mimicking and non-phosphorylatable mutations in Tub4, Spc97 and Spc98 were analyzed. Three phosphorylation sites in Tub4 were found to be critical for Tub4 stability and microtubule organization. One of the sites is highly conserved in γ-tubulins from yeast to human. PMID:21573187

  9. Regulated phosphorylation of secretory granule membrane proteins of the rat parotid gland

    SciTech Connect

    Marino, C.R.; Castle, J.D.; Gorelick, F.S. )

    1990-07-01

    An antiserum raised against purified rat parotid secretory granule membrane proteins has been used to identify organelle-specific protein phosphorylation events following stimulation of intact cells from the rat parotid gland. After lobules were prelabeled with ({sup 32}P)orthophosphate and exposed to secretagogues, phosphoproteins were immunoprecipitated with the granule membrane protein antiserum, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. Parallel studies of stimulated amylase release were performed. Isoproterenol treatment of parotid lobules resulted in an increase in the phosphate content of immunoprecipitable 60- and 72-kDa proteins that correlated with amylase release in a time-dependent manner. Forskolin addition mimicked these effects, but only the isoproterenol effects were reversed by propranolol treatment. To confirm the specificity of the antiserum to the secretory granule membrane fraction, subcellular isolation techniques were employed following in situ phosphorylation. The 60- and 72-kDa phosphoproteins were immunoprecipitated from both a particulate fraction and a purified secretory granule fraction. Furthermore, the extraction properties of both species suggest that they are integral membrane proteins. These findings support the possibility that stimulus-regulated secretion may involve phosphorylation of integral membrane proteins of the exocrine secretory granule.

  10. Ceramide inhibits PKCθ by regulating its phosphorylation and translocation to lipid rafts in Jurkat cells.

    PubMed

    Hage-Sleiman, Rouba; Hamze, Asmaa B; El-Hed, Aimée F; Attieh, Randa; Kozhaya, Lina; Kabbani, Sarah; Dbaibo, Ghassan

    2016-08-01

    Protein kinase C theta (PKCθ) is a novel, calcium-independent member of the PKC family of kinases that was identified as a central player in T cell signaling and proliferation. Upon T cell activation by antigen-presenting cells, PKCθ gets phosphorylated and activated prior to its translocation to the immunological synapse where it couples with downstream effectors. PKCθ may be regulated by ceramide, a crucial sphingolipid that is known to promote differentiation, growth arrest, and apoptosis. To further investigate the mechanism, we stimulated human Jurkat T cells with either PMA or anti-CD3/anti-CD28 antibodies following induction of ceramide accumulation by adding exogenous ceramide, bacterial sphingomyelinase, or Fas ligation. Our results suggest that ceramide regulates the PKCθ pathway through preventing its critical threonine 538 (Thr538) phosphorylation and subsequent activation, thereby inhibiting the kinase's translocation to lipid rafts. Moreover, this inhibition is not likely to be a generic effect of ceramide on membrane reorganization. Other lipids, namely dihydroceramide, palmitate, and sphingosine, did not produce similar effects on PKCθ. Addition of the phosphatase inhibitors okadaic acid and calyculin A reversed the inhibition exerted by ceramide, and this suggests involvement of a ceramide-activated protein phosphatase. Such previously undescribed mechanism of regulation of PKCθ raises the possibility that ceramide, or one of its derivatives, and may prove valuable in novel therapeutic approaches for disorders involving autoimmunity or excessive inflammation-where PKCθ plays a critical role. PMID:26798039

  11. Regulation of oxidative phosphorylation: the flexible respiratory network of Paracoccus denitrificans.

    PubMed

    Van Spanning, R J; de Boer, A P; Reijnders, W N; De Gier, J W; Delorme, C O; Stouthamer, A H; Westerhoff, H V; Harms, N; van der Oost, J

    1995-10-01

    Paracoccus denitrificans is a facultative anaerobic bacterium that has the capacity to adjust its metabolic infrastructure, quantitatively and/or qualitatively, to the prevailing growth condition. In this bacterium the relative activity of distinct catabolic pathways is subject to a hierarchical control. In the presence of oxygen the aerobic respiration, the most efficient way of electron transfer-linked phosphorylation, has priority. At high oxygen tensions P. denitrificans synthesizes an oxidase with a relatively low affinity for oxygen, whereas under oxygen limitation a high-affinity oxidase appears specifically induced. During anaerobiosis, the pathways with lower free energy-transducing efficiency are induced. In the presence of nitrate, the expression of a number of dehydrogenases ensures the continuation of oxidative phosphorylation via denitrification. After identification of the structural components that are involved in both the aerobic and the anaerobic respiratory networks of P. denitrificans, the intriguing next challenge is to get insight in its regulation. Two transcription regulators have recently been demonstrated to be involved in the expression of a number of aerobic and/or anaerobic respiratory complexes in P. denitrificans. Understanding of the regulation machinery is beginning to emerge and promises much excitement in discovery. PMID:8718455

  12. Phosphorylation-dependent down-regulation of apolipoprotein A5 by insulin

    SciTech Connect

    Nowak, Maxine; Helleboid-Chapman, Audrey; Jakel, Heidelinde; Rommens, Corinne; Martin, Genevieve; Duran-Sandoval, Daniel; Staels, Bart; Rubin, Edward M.; Pennacchio, Len A.; Taskinen, Marja-Riitta; Fruchart-Najib, Jamila; Fruchart, Jean-Charles

    2004-02-15

    The apolipoprotein A5 (APOA5) gene has been shown to be important in lowering plasma triglyceride levels. Since several studies have shown that hyperinsulinemia is associated with hypertriglyceridemia, we sought to determine whether APOA5 gene is regulated by insulin. We show here that cell and mouse treatments with insulin down-regulated APOA5 expression in a dose-dependent manner. Furthermore, we determined that insulin decreases APOA5 promoter activity and subsequent deletion analyses revealed an E-box-containing fragment. We showed that Upstream Stimulatory Factors, USF1/USF2, bind to the identified E-box in the APOA5 promoter. Moreover, in cotransfection studies, USF1 stimulates APOA5 promoter activity. The treatment with insulin reduces the binding of USF1/USF2 to APOA5 promoter. The inhibition of PI3K pathway with wortmannin abolished the insulin s effect on APOA5 gene transcription. Using oligoprecipitation method of USF from nuclear extracts, we demonstrated that phosphorylated USF1 failed to bind to APOA5 promoter. This indicates that the APOA5 gene transrepression by insulin involves a phosphorylation of USF through PI3K, that modulate their binding to APOA5 promoter and results in APOA5 down-regulation. The effect of exogenous hyperinsulinemia in healthy men shows a decrease of the plasma ApoAV level. These data suggest a potential mechanism involving APOA5 gene in hypertriglyceridemia associated with hyperinsulinemia.

  13. TORC1 and TORC2 work together to regulate ribosomal protein S6 phosphorylation in Saccharomyces cerevisiae.

    PubMed

    Yerlikaya, Seda; Meusburger, Madeleine; Kumari, Romika; Huber, Alexandre; Anrather, Dorothea; Costanzo, Michael; Boone, Charles; Ammerer, Gustav; Baranov, Pavel V; Loewith, Robbie

    2016-01-15

    Nutrient-sensitive phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome is highly conserved. However, despite four decades of research, the functional consequences of this modification remain unknown. Revisiting this enigma in Saccharomyces cerevisiae, we found that the regulation of Rps6 phosphorylation on Ser-232 and Ser-233 is mediated by both TOR complex 1 (TORC1) and TORC2. TORC1 regulates phosphorylation of both sites via the poorly characterized AGC-family kinase Ypk3 and the PP1 phosphatase Glc7, whereas TORC2 regulates phosphorylation of only the N-terminal phosphosite via Ypk1. Cells expressing a nonphosphorylatable variant of Rps6 display a reduced growth rate and a 40S biogenesis defect, but these phenotypes are not observed in cells in which Rps6 kinase activity is compromised. Furthermore, using polysome profiling and ribosome profiling, we failed to uncover a role of Rps6 phosphorylation in either global translation or translation of individual mRNAs. Taking the results together, this work depicts the signaling cascades orchestrating Rps6 phosphorylation in budding yeast, challenges the notion that Rps6 phosphorylation plays a role in translation, and demonstrates that observations made with Rps6 knock-ins must be interpreted cautiously. PMID:26582391

  14. Novel Phosphorylation Sites in the S. cerevisiae Cdc13 Protein Reveal New Targets for Telomere Length Regulation

    PubMed Central

    Wu, Yun; DiMaggio, Peter A.; Perlman, David H.; Zakian, Virginia A.; Garcia, Benjamin A.

    2013-01-01

    The S. cerevisiae Cdc13 is a multifunctional protein with key roles in regulation of telomerase, telomere end protection, and conventional telomere replication, all of which are cell cycle-regulated processes. Given that phosphorylation is a key mechanism for regulating protein function, we identified sites of phosphorylation using nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). We also determined phosphorylation abundance on both wild type (WT) and a telomerase deficient form of Cdc13, encoded by the cdc13-2 allele, in both G1 phase cells, when telomerase is not active, and G2/M phase cells, when it is. We identified 21 sites of in vivo phosphorylation, of which only five had been reported previously. In contrast, phosphorylation of two in vitro targets of the ATM-like Tel1 kinase, S249 and S255, was not detected. This result helps resolve conflicting data on the importance of phosphorylation of these residues in telomerase recruitment. multiple residues showed differences in their cell cycle pattern of modification. For example, phosphorylation of S314 was significantly higher in the G2/M compared to the G1 phase and in WT versus mutant Cdc13, and a S314D mutation negatively affected telomere length. Our findings provide new targets in a key telomerase regulatory protein for modulation of telomere dynamics. PMID:23181431

  15. Apigenin up-regulates transgelin and inhibits invasion and migration of colorectal cancer through decreased phosphorylation of AKT.

    PubMed

    Chunhua, Li; Donglan, Lin; Xiuqiong, Fu; Lihua, Zhang; Qin, Fan; Yawei, Liu; Liang, Zhao; Ge, Wen; Linlin, Jing; Ping, Zeng; Kun, Li; Xuegang, Sun

    2013-10-01

    Colorectal cancer (CRC) is a major cause of morbidity and mortality throughout the world. Apigenin is a flavonoid that possesses various clinically relevant properties such as anti-tumour, anti-platelet and anti-inflammatory activities. Our results showed that apigenin has anti-proliferation, anti-invasion and anti-migration effects in three kinds of colorectal adenocarcinoma cell lines, namely SW480, DLD-1 and LS174T. Proteomic analysis with SW480 indicated that apigenin up-regulated the expression of transgelin (TAGLN) in mitochondria to exert its anti-tumour growth and anti-metastasis effects. Real-time quantitative polymerase chain reaction (RQ-PCR) and western blot confirm the up-regulation in all the three colorectal adenocarcinoma cells. An inverse correlation was observed between TAGLN expression and CRC metastasis in tissue microarray staining. TAGLN siRNA increased the viability of SW480. Apigenin decreased the expression of MMP-9 in a dose-dependent manner. Transfection of three truncated forms of TAGLN and wild type has identified TAGLN as a repressor of MMP-9 expression. A synergetic effect was observed in overexpression of TAGLN wild type and apigenin treatment which manifested as lowered phosphorylation of AKT Ser473 and ATK Thr308. In an orthotopic CRC model, apigenin inhibited tumour growth and metastasis to liver and lung. In conclusion, our research provided direct evidence that apigenin inhibited tumour growth and metastasis both in vitro and in vivo. Apigenin up-regulated TAGLN and hence down-regulated MMP-9 expression through decreasing phosphorylation of Akt at Ser473 and in particular Thr308 to prevent cell proliferation and migration. PMID:23773626

  16. Regulation of Ci-SCFSlimb binding, Ci proteolysis and Hedgehog pathway activity by Ci phosphorylation

    PubMed Central

    Smelkinson, Margery G.; Zhou, Qianhe; Kalderon, Daniel

    2007-01-01

    SUMMARY Hedgehog (Hh) proteins signal by inhibiting the proteolytic processing of Ci/Gli family transcription factors and by increasing Ci/Gli specific activity. In the absence of Hh, phosphorylation of Ci/Gli triggers binding to SCF ubiquitin ligase complexes and consequent proteolysis. Here we define the principal SCFSlimb binding site in Ci as an extended variant of a canonical Slimb/β-TRCP binding motif that can be created by PKA-priming of five successive CK1 sites. GSK3 enhances binding primarily through a nearby region of Ci, which may contact an SCF component other than Slimb. Studies of Ci variants with altered CK1 and GSK3 sites suggest that the large number of phosphorylation sites that direct SCFSlimb binding confers a Hh response that is both sensitive and graded, and that in the Drosophila wing disc, morphogenetic responses involve changes in both the level and specific activity of Ci. We also show that when Ci proteolysis is compromised, its specific activity is limited principally by Su(fu) and not by Cos2 cytoplasmic tethering or PKA phosphorylation. PMID:17925225

  17. Structural dynamics and topology of phosphorylated phospholamban homopentamer reveal its role in the regulation of calcium transport in sarcoplasmic reticulum

    PubMed Central

    Vostrikov, Vitaly V.; Mote, Kaustubh R.; Verardi, Raffaello; Veglia, Gianluigi

    2013-01-01

    Phospholamban (PLN) inhibits the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), thereby regulating cardiac diastole. In membranes, PLN assembles into homopentamers that in both the phosphorylated and non-phosphorylated states have been proposed to form ion-selective channels. Here, we determined the structure of the phosphorylated pentamer using a combination of solution and solid-state nuclear magnetic resonance methods. We found that the pinwheel architecture of the homopentamer is preserved upon phosphorylation, with each monomer having an L-shaped conformation of each monomer. The TM domains form a hydrophobic pore of approximately 24 Å long, and 2 Å in diameter, which is inconsistent with canonical Ca2+ selective channels. Phosphorylation, however, enhances the conformational dynamics of the cytoplasmic region of PLN, causing the partial unwinding of the amphipathic helix. We propose that PLN oligomers act as storage for active monomers, keeping SERCA function within a physiological window. PMID:24207128

  18. THE CHANNEL-KINASE TRPM7 REGULATES PHOSPHORYLATION OF THE TRANSLATIONAL FACTOR eEF2 via eEF2-k

    PubMed Central

    Perraud, Anne-Laure; Zhao, Xiaoyun; Ryazanov, Alexey G.; Schmitz, Carsten

    2010-01-01

    Protein translation is an essential but energetically expensive process, which is carefully regulated in accordance to the cellular nutritional and energy status. Eukaryotic elongation factor 2 (eEF2) is a central regulation point since it mediates ribosomal translocation, and can be inhibited by phosphorylation at Thr56. TRPM7 is the unique fusion of an ion channel with a functional Ser/Thr-kinase. While TRPM7’s channel function has been implicated in regulating vertebrate Mg2+-uptake required for cell growth, the function of its kinase domain remains unclear. Here, we show that under conditions where cell growth is limited by Mg2+-availability, TRPM7 via its kinase mediates enhanced Thr56-phosphorylation of eEF2. TRPM7-kinase does not appear to directly phosphorylate eEF2, but rather to influence the amount of eEF2’s cognate kinase eEF2-k, involving its phosphorylation at Ser77. These findings suggest that TRPM7’s structural duality ensures ideal positioning of its kinase in close proximity to channel-mediated Mg2+ uptake, allowing for the adjustment of protein translational rates to the availability of Mg2+. PMID:21112387

  19. Evolution of nuclear retinoic acid receptor alpha (RARα) phosphorylation sites. Serine gain provides fine-tuned regulation.

    PubMed

    Samarut, Eric; Amal, Ismail; Markov, Gabriel V; Stote, Roland; Dejaegere, Annick; Laudet, Vincent; Rochette-Egly, Cécile

    2011-07-01

    The human nuclear retinoic acid (RA) receptor alpha (hRARα) is a ligand-dependent transcriptional regulator, which is controlled by a phosphorylation cascade. The cascade starts with the RA-induced phosphorylation of a serine residue located in the ligand-binding domain, S(LBD), allowing the recruitment of the cdk7/cyclin H/MAT1 subcomplex of TFIIH through the docking of cyclin H. It ends by the subsequent phosphorylation by cdk7 of an other serine located in the N-terminal domain, S(NTD). Here, we show that this cascade relies on an increase in the flexibility of the domain involved in cyclin H binding, subsequently to the phosphorylation of S(LBD). Owing to the functional importance of RARα in several vertebrate species, we investigated whether the phosphorylation cascade was conserved in zebrafish (Danio rerio), which expresses two RARα genes: RARα-A and RARα-B. We found that in zebrafish RARαs, S(LBD) is absent, whereas S(NTD) is conserved and phosphorylated. Therefore, we analyzed the pattern of conservation of the phosphorylation sites and traced back their evolution. We found that S(LBD) is most often absent outside mammalian RARα and appears late during vertebrate evolution. In contrast, S(NTD) is conserved, indicating that the phosphorylation of this functional site has been under ancient high selection constraint. This suggests that, during evolution, different regulatory circuits control RARα activity. PMID:21297158

  20. Proteotoxic Stress Induces Phosphorylation of p62/SQSTM1 by ULK1 to Regulate Selective Autophagic Clearance of Protein Aggregates

    PubMed Central

    Lim, Junghyun; Lachenmayer, M. Lenard; Wu, Shuai; Liu, Wenchao; Kundu, Mondira; Wang, Rong; Komatsu, Masaaki; Oh, Young J.; Zhao, Yanxiang; Yue, Zhenyu

    2015-01-01

    Disruption of proteostasis, or protein homeostasis, is often associated with aberrant accumulation of misfolded proteins or protein aggregates. Autophagy offers protection to cells by removing toxic protein aggregates and injured organelles in response to proteotoxic stress. However, the exact mechanism whereby autophagy recognizes and degrades misfolded or aggregated proteins has yet to be elucidated. Mounting evidence demonstrates the selectivity of autophagy, which is mediated through autophagy receptor proteins (e.g. p62/SQSTM1) linking autophagy cargos and autophagosomes. Here we report that proteotoxic stress imposed by the proteasome inhibition or expression of polyglutamine expanded huntingtin (polyQ-Htt) induces p62 phosphorylation at its ubiquitin-association (UBA) domain that regulates its binding to ubiquitinated proteins. We find that autophagy-related kinase ULK1 phosphorylates p62 at a novel phosphorylation site S409 in UBA domain. Interestingly, phosphorylation of p62 by ULK1 does not occur upon nutrient starvation, in spite of its role in canonical autophagy signaling. ULK1 also phosphorylates S405, while S409 phosphorylation critically regulates S405 phosphorylation. We find that S409 phosphorylation destabilizes the UBA dimer interface, and increases binding affinity of p62 to ubiquitin. Furthermore, lack of S409 phosphorylation causes accumulation of p62, aberrant localization of autophagy proteins and inhibition of the clearance of ubiquitinated proteins or polyQ-Htt. Therefore, our data provide mechanistic insights into the regulation of selective autophagy by ULK1 and p62 upon proteotoxic stress. Our study suggests a potential novel drug target in developing autophagy-based therapeutics for the treatment of proteinopathies including Huntington’s disease. PMID:25723488

  1. Light-regulated translational control of circadian behavior by eIF4E phosphorylation.

    PubMed

    Cao, Ruifeng; Gkogkas, Christos G; de Zavalia, Nuria; Blum, Ian D; Yanagiya, Akiko; Tsukumo, Yoshinori; Xu, Haiyan; Lee, Choogon; Storch, Kai-Florian; Liu, Andrew C; Amir, Shimon; Sonenberg, Nahum

    2015-06-01

    The circadian (∼24 h) clock is continuously entrained (reset) by ambient light so that endogenous rhythms are synchronized with daily changes in the environment. Light-induced gene expression is thought to be the molecular mechanism underlying clock entrainment. mRNA translation is a key step of gene expression, but the manner in which clock entrainment is controlled at the level of mRNA translation is not well understood. We found that a light- and circadian clock-regulated MAPK/MNK pathway led to phosphorylation of the cap-binding protein eIF4E in the mouse suprachiasmatic nucleus of the hypothalamus, the locus of the master circadian clock in mammals. Phosphorylation of eIF4E specifically promoted translation of Period 1 (Per1) and Period 2 (Per2) mRNAs and increased the abundance of basal and inducible PER proteins, which facilitated circadian clock resetting and precise timekeeping. Together, these results highlight a critical role for light-regulated translational control in the physiology of the circadian clock. PMID:25915475

  2. 14-3-3 Proteins regulate Akt Thr308 phosphorylation in intestinal epithelial cells.

    PubMed

    Gómez-Suárez, M; Gutiérrez-Martínez, I Z; Hernández-Trejo, J A; Hernández-Ruiz, M; Suárez-Pérez, D; Candelario, A; Kamekura, R; Medina-Contreras, O; Schnoor, M; Ortiz-Navarrete, V; Villegas-Sepúlveda, N; Parkos, C; Nusrat, A; Nava, P

    2016-06-01

    Akt activation has been associated with proliferation, differentiation, survival and death of epithelial cells. Phosphorylation of Thr308 of Akt by phosphoinositide-dependent kinase 1 (PDK1) is critical for optimal stimulation of its kinase activity. However, the mechanism(s) regulating this process remain elusive. Here, we report that 14-3-3 proteins control Akt Thr308 phosphorylation during intestinal inflammation. Mechanistically, we found that IFNγ and TNFα treatment induce degradation of the PDK1 inhibitor, 14-3-3η, in intestinal epithelial cells. This mechanism requires association of 14-3-3ζ with raptor in a process that triggers autophagy and leads to 14-3-3η degradation. Notably, inhibition of 14-3-3 function by the chemical inhibitor BV02 induces uncontrolled Akt activation, nuclear Akt accumulation and ultimately intestinal epithelial cell death. Our results suggest that 14-3-3 proteins control Akt activation and regulate its biological functions, thereby providing a new mechanistic link between cell survival and apoptosis of intestinal epithelial cells during inflammation. PMID:26846144

  3. The LAMMER Kinase Homolog, Lkh1, Regulates Tup Transcriptional Repressors through Phosphorylation in Schizosaccharomyces pombe*

    PubMed Central

    Kang, Won-Hwa; Park, Yun-Hee; Park, Hee-Moon

    2010-01-01

    Disruption of the fission yeast LAMMER kinase, Lkh1, gene resulted in diverse phenotypes, including adhesive filamentous growth and oxidative stress sensitivity, but an exact cellular function had not been assigned to Lkh1. Through an in vitro pull-down approach, a transcriptional repressor, Tup12, was identified as an Lkh1 binding partner. Interactions between Lkh1 and Tup11 or Tup12 were confirmed by in vitro and in vivo binding assays. Tup proteins were phosphorylated by Lkh1 in a LAMMER motif-dependent manner. The LAMMER motif was also necessary for substrate recognition in vitro and cellular function in vivo. Transcriptional activity assays using promoters negatively regulated by Tup11 and Tup12 showed 6 or 2 times higher activity in the Δlkh1 mutant than the wild type, respectively. Northern analysis revealed derepressed expression of the fbp1+ mRNA in Δlkh1 and in Δtup11Δtup12 mutant cells under repressed conditions. Δlkh1 and Δtup11Δtup12 mutant cells showed flocculation, which was reversed by co-expression of Tup11 and -12 with Ssn6. Here, we presented a new aspect of the LAMMER kinase by demonstrating that the activities of global transcriptional repressors, Tup11 and Tup12, were positively regulated by Lkh1-mediated phosphorylation. PMID:20200159

  4. Regulation of yeast pyruvate kinase by ultrasensitive allostery independent of phosphorylation

    PubMed Central

    Xu, Yi-Fan; Zhao, Xin; Glass, David S.; Absalan, Farnaz; Perlman, David H.; Broach, James R.; Rabinowitz, Joshua D.

    2012-01-01

    Summary Allostery and covalent modification are major means of fast-acting metabolic regulation. Their relative roles in responding to environmental changes remain, however, unclear. Here we examine this issue, using as a case study the rapid decrease in pyruvate kinase flux in yeast upon glucose removal. The main pyruvate kinase isozyme (Cdc19) is phosphorylated in response to environmental cues. It also exhibits positively-cooperative (ultrasensitive) allosteric activation by fructose-1,6-bisphosphate (FBP). Glucose removal causes accumulation of Cdc19’s substrate, phosphoenolpyruvate. This response is retained in strains with altered protein-kinase-A or AMP-activated-protein-kinase activity or with CDC19 carrying mutated phosphorylation sites. In contrast, yeast engineered with a CDC19 point mutation that ablates FBP-based regulation fail to accumulate phosphoenolpyruvate. They also fail to grow on ethanol and slowly resume growth upon glucose upshift. Thus, while yeast pyruvate kinase is covalently modified in response to glucose availability, its activity is controlled almost exclusively by ultrasensitive allostery. PMID:22902555

  5. WAVE1 regulates Bcl-2 localization and phosphorylation in leukemia cells

    PubMed Central

    Rui, Kang; Daolin, Tang; Yan, Yu; Zhuo, Wang; Ting, Hu; Haichao, Wang; Lizhi, Cao

    2010-01-01

    Bcl-2 proteins are over-expressed in many tumors, and are critically important for cell survival. Their anti-apoptotic activities are determined by intracellular localization and post-translational modifications (such as phosphorylation). Here we showed that WAVE1, a member of the Wiskott-Aldrich syndrome protein family, was over-expressed in blood cancer cell lines, and functioned as a negative regulator of apoptosis. Further enhanced expression of WAVE1 by gene transfection rendered leukemia cells more resistant to anti-cancer drug-induced apoptosis; whereas suppression of WAVE1 expression by RNA interference restored leukemia cells' sensitivity to anti-drug-induced apoptosis. WAVE1 was found to be associated with mitochondrial Bcl-2, and its depletion led to mitochondrial release of Bcl-2, and phosphorylation of ASK1/JNK and Bcl-2. Furthermore, depletion of WAVE1 expression increased anti-cancer drug-induced production of reactive oxygen species in leukemia cells. Taken together, these results suggest WAVE1 as a novel regulator of apoptosis, and potential drug target for therapeutic intervention of leukemia. PMID:19890377

  6. Glycogen Synthase Kinase 3β Is Positively Regulated by Protein Kinase Cζ-Mediated Phosphorylation Induced by Wnt Agonists

    PubMed Central

    Tejeda-Muñoz, Nydia; González-Aguilar, Héctor; Santoyo-Ramos, Paula; Castañeda-Patlán, M. Cristina

    2015-01-01

    The molecular events that drive Wnt-induced regulation of glycogen synthase kinase 3β (GSK-3β) activity are poorly defined. In this study, we found that protein kinase Cζ (PKCζ) and GSK-3β interact mainly in colon cancer cells. Wnt stimulation induced a rapid GSK-3β redistribution from the cytoplasm to the nuclei in malignant cells and a transient PKC-mediated phosphorylation of GSK-3β at a different site from serine 9. In addition, while Wnt treatment induced a decrease in PKC-mediated phosphorylation of GSK-3β in nonmalignant cells, in malignant cells, this phosphorylation was increased. Pharmacological inhibition and small interfering RNA (siRNA)-mediated silencing of PKCζ abolished all of these effects, but unexpectedly, it also abolished the constitutive basal activity of GSK-3β. In vitro activity assays demonstrated that GSK-3β phosphorylation mediated by PKCζ enhanced GSK-3β activity. We mapped Ser147 of GSK-3β as the site phosphorylated by PKCζ, i.e., its mutation into alanine abolished GSK-3β activity, resulting in β-catenin stabilization and increased transcriptional activity, whereas phosphomimetic replacement of Ser147 by glutamic acid maintained GSK-3β basal activity. Thus, we found that PKCζ phosphorylates GSK-3β at Ser147 to maintain its constitutive activity in resting cells and that Wnt stimulation modifies the phosphorylation of Ser147 to regulate GSK-3β activity in opposite manners in normal and malignant colon cells. PMID:26711256

  7. Mechanism of SNARE protein binding and regulation of Cav2 channels by phosphorylation of the synaptic protein interaction site.

    PubMed

    Yokoyama, Charles T; Myers, Scott J; Fu, Jian; Mockus, Susan M; Scheuer, Todd; Catterall, William A

    2005-01-01

    Ca(v)2.1 and Ca(v)2.2 channels conduct P/Q-type and N-type Ca(2+) currents that initiate neurotransmission and bind SNARE proteins through a synaptic protein interaction (synprint) site. PKC and CaMKII phosphorylate the synprint site and inhibit SNARE protein binding in vitro. Here we identify two separate microdomains that each bind syntaxin 1A and SNAP-25 in vitro and are regulated by PKC phosphorylation at serines 774 and 898 and CaMKII phosphorylation at serines 784 and 896. Activation of PKC resulted in its recruitment to and phosphorylation of Ca(V)2.2 channels, but PKC phosphorylation did not dissociate Ca(V)2.2 channel/syntaxin 1A complexes. Chimeric Ca(V)2.1a channels containing the synprint site of Ca(v)2.2 gain modulation by syntaxin 1A, which is blocked by PKC phosphorylation at the sites identified above. Our results support a bipartite model for the synprint site in which each SNARE-binding microdomain is controlled by a separate PKC and CaMKII phosphorylation site that regulates channel modulation by SNARE proteins. PMID:15607937

  8. Regulation of WT1 by phosphorylation: inhibition of DNA binding, alteration of transcriptional activity and cellular translocation.

    PubMed Central

    Ye, Y; Raychaudhuri, B; Gurney, A; Campbell, C E; Williams, B R

    1996-01-01

    Phosphorylation is one of the major post-translational mechanisms by which the activity of transcription factors is regulated. We have investigated the role of phosphorylation in the regulation of nucleic acid binding activity and the nuclear translocation of WT1. Two recombinant WT1 proteins containing the DNA binding domain with or without a three amino acid (KTS) insertion (WT1ZF + KTS and WT1ZF - KTS) were strongly phosphorylated by protein kinase A (PKA) and protein kinase C (PKC) in vitro. Both PKA and PKC phosphorylation inhibited the ability of WT1ZF + KTS or WT1ZF - KTS to bind to a sequence derived from the WT1 promoter region in gel mobility shift assays. The binding of WT1ZF - KTS to an EGR1 consensus binding site was also inhibited by prior PKA and PKC phosphorylation. We also demonstrate the RNA binding activity of WT1, but this was not altered by phosphorylation. PKA activation by dibutyryl cAMP in WT1-transfected cells resulted in the reversal of WT1 suppression of a reporter construct. Although WT1 protein is predominantly localized to the nucleus, this expression pattern is altered upon PKA activation, resulting in the cytoplasmic retention of WT1. Accordingly, phosphorylation may play a role in modulating the transcriptional regulatory activity of WT1 through interference with nuclear translocation, as well as by inhibition of WT1 DNA binding. Images PMID:8896454

  9. WISp39 binds phosphorylated Coronin 1B to regulate Arp2/3 localization and Cofilin-dependent motility.

    PubMed

    Howell, Michael; Brickner, Howard; Delorme-Walker, Violaine D; Choi, Justin; Saffin, Jean-Michel; Miller, Daniel; Panopoulos, Andreas; DerMardirossian, Céline; Fotedar, Arun; Margolis, Robert L; Fotedar, Rati

    2015-03-30

    We previously identified Waf1 Cip1 stabilizing protein 39 (WISp39) as a binding partner for heat shock protein 90 (Hsp90). We now report that WISp39 has an essential function in the control of directed cell migration, which requires WISp39 interaction with Hsp90. WISp39 knockdown (KD) resulted in the loss of directional motility of mammalian cells and profound changes in cell morphology, including the loss of a single leading edge. WISp39 binds Coronin 1B, known to regulate the Arp2/3 complex and Cofilin at the leading edge. WISp39 preferentially interacts with phosphorylated Coronin 1B, allowing it to complex with Slingshot phosphatase (SSH) to dephosphorylate and activate Cofilin. WISp39 also regulates Arp2/3 complex localization at the leading edge. WISp39 KD-induced morphological changes could be rescued by overexpression of Coronin 1B together with a constitutively active Cofilin mutant. We conclude that WISp39 associates with Hsp90, Coronin 1B, and SSH to regulate Cofilin activation and Arp2/3 complex localization at the leading edge. PMID:25800056

  10. CASEIN KINASE1-LIKE PROTEIN2 Regulates Actin Filament Stability and Stomatal Closure via Phosphorylation of Actin Depolymerizing Factor.

    PubMed

    Zhao, Shuangshuang; Jiang, Yuxiang; Zhao, Yang; Huang, Shanjin; Yuan, Ming; Zhao, Yanxiu; Guo, Yan

    2016-06-01

    The opening and closing of stomata are crucial for plant photosynthesis and transpiration. Actin filaments undergo dynamic reorganization during stomatal closure, but the underlying mechanism for this cytoskeletal reorganization remains largely unclear. In this study, we identified and characterized Arabidopsis thaliana casein kinase 1-like protein 2 (CKL2), which responds to abscisic acid (ABA) treatment and participates in ABA- and drought-induced stomatal closure. Although CKL2 does not bind to actin filaments directly and has no effect on actin assembly in vitro, it colocalizes with and stabilizes actin filaments in guard cells. Further investigation revealed that CKL2 physically interacts with and phosphorylates actin depolymerizing factor 4 (ADF4) and inhibits its activity in actin filament disassembly. During ABA-induced stomatal closure, deletion of CKL2 in Arabidopsis alters actin reorganization in stomata and renders stomatal closure less sensitive to ABA, whereas deletion of ADF4 impairs the disassembly of actin filaments and causes stomatal closure to be more sensitive to ABA Deletion of ADF4 in the ckl2 mutant partially recues its ABA-insensitive stomatal closure phenotype. Moreover, Arabidopsis ADFs from subclass I are targets of CKL2 in vitro. Thus, our results suggest that CKL2 regulates actin filament reorganization and stomatal closure mainly through phosphorylation of ADF. PMID:27268429

  11. Protein phosphorylation changes reveal new candidates in the regulation of egg activation and early embryogenesis in D. melanogaster

    PubMed Central

    Krauchunas, Amber R.; Horner, Vanessa L.; Wolfner, Mariana F.

    2012-01-01

    Egg activation is the series of events that must occur for a mature oocyte to become capable of supporting embryogenesis. These events include changes to the egg’s outer coverings, the resumption and completion of meiosis, the translation of new proteins, and the degradation of specific maternal mRNAs. While we know some of the molecules that direct the initial events of egg activation, it remains unclear how multiple pathways are coordinated to change the cellular state from mature oocyte to activated egg. Using a proteomic approach we have identified new candidates for the regulation and progression of egg activation. Reasoning that phosphorylation can simultaneously and rapidly modulate the activity of many proteins, we identified proteins that are post-translationally modified during the transition from oocyte to activated egg in Drosophila melanogaster. We find that at least 311 proteins change in phosphorylation state between mature oocytes and activated eggs. These proteins fall into various functional classes related to the events of egg activation including calcium binding, proteolysis, and protein translation. Our set of candidates includes genes already associated with egg activation, as well as many genes not previously studied during this developmental period. RNAi knockdown of a subset of these genes revealed a new gene, mrityu, necessary for embryonic development past the first mitosis. Thus, by identifying phospho-modulated proteins we have produced a focused candidate set for future genetic studies to test their roles in egg activation and the initiation of embryogenesis. PMID:22884528

  12. Differential phosphorylation of DNA-PKcs regulates the interplay between end-processing and end-ligation during non-homologous end-joining

    PubMed Central

    Jiang, Wenxia; Crowe, Jennifer L.; Liu, Xiangyu; Nakajima, Satoshi; Wang, Yunyue; Li, Chen; Lee, Brian J.; Dubois, Richard L.; Liu, Chao; Yu, Xiaochun; Lan, Li; Zha, Shan

    2015-01-01

    SUMMARY Non-homologous end-joining (NHEJ) is a major DNA double strand break repair pathway that is conserved in eukaryotes. In vertebrates, NHEJ further acquires end-processing capacities (e.g., hairpin opening) in addition to direct end-ligation. The catalytic subunit of DNA-PK (DNA-PKcs) is a vertebrate specific NHEJ factor that can be auto-phosphorylated or trans-phosphorylated by ATM kinase. Using a mouse model expressing a kinase-dead (KD) DNA-PKcs protein, we show that ATM-mediated trans-phosphorylation of DNA-PKcs regulates end-processing at the level of Artemis recruitment, while strictly auto-phosphorylation of DNA-PKcs is necessary to relieve the physical blockage on end-ligation imposed by the DNA-PKcs protein itself. Accordingly, DNA-PKcsKD/KD mice and cells show severe end-ligation defects and p53- and Ku-dependent embryonic lethality, but open hairpin-sealed ends normally in the presence of ATM kinase activity. Together, our findings identify DNA-PKcs as the molecular switch that coordinates end-processing and end-ligation at the DNA ends through differential phosphorylations. PMID:25818648

  13. Regulation of secretory transport by protein kinase D-mediated phosphorylation of the ceramide transfer protein.

    PubMed

    Fugmann, Tim; Hausser, Angelika; Schöffler, Patrik; Schmid, Simone; Pfizenmaier, Klaus; Olayioye, Monilola A

    2007-07-01

    Protein kinase D (PKD) has been identified as a crucial regulator of secretory transport at the trans-Golgi network (TGN). Recruitment and activation of PKD at the TGN is mediated by the lipid diacylglycerol, a pool of which is generated by sphingomyelin synthase from ceramide and phosphatidylcholine. The nonvesicular transfer of ceramide from the endoplasmic reticulum to the Golgi complex is mediated by the lipid transfer protein CERT (ceramide transport). In this study, we identify CERT as a novel in vivo PKD substrate. Phosphorylation on serine 132 by PKD decreases the affinity of CERT toward its lipid target phosphatidylinositol 4-phosphate at Golgi membranes and reduces ceramide transfer activity, identifying PKD as a regulator of lipid homeostasis. We also show that CERT, in turn, is critical for PKD activation and PKD-dependent protein cargo transport to the plasma membrane. Thus, the interdependence of PKD and CERT is key to the maintenance of Golgi membrane integrity and secretory transport. PMID:17591919

  14. Regulation of secretory transport by protein kinase D–mediated phosphorylation of the ceramide transfer protein

    PubMed Central

    Fugmann, Tim; Hausser, Angelika; Schöffler, Patrik; Schmid, Simone; Pfizenmaier, Klaus; Olayioye, Monilola A.

    2007-01-01

    Protein kinase D (PKD) has been identified as a crucial regulator of secretory transport at the trans-Golgi network (TGN). Recruitment and activation of PKD at the TGN is mediated by the lipid diacylglycerol, a pool of which is generated by sphingomyelin synthase from ceramide and phosphatidylcholine. The nonvesicular transfer of ceramide from the endoplasmic reticulum to the Golgi complex is mediated by the lipid transfer protein CERT (ceramide transport). In this study, we identify CERT as a novel in vivo PKD substrate. Phosphorylation on serine 132 by PKD decreases the affinity of CERT toward its lipid target phosphatidylinositol 4-phosphate at Golgi membranes and reduces ceramide transfer activity, identifying PKD as a regulator of lipid homeostasis. We also show that CERT, in turn, is critical for PKD activation and PKD-dependent protein cargo transport to the plasma membrane. Thus, the interdependence of PKD and CERT is key to the maintenance of Golgi membrane integrity and secretory transport. PMID:17591919

  15. Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

    NASA Technical Reports Server (NTRS)

    Morfini, Gerardo; Szebenyi, Gyorgyi; Elluru, Ravindhra; Ratner, Nancy; Brady, Scott T.

    2002-01-01

    Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

  16. PFKL/miR-128 axis regulates glycolysis by inhibiting AKT phosphorylation and predicts poor survival in lung cancer.

    PubMed

    Yang, Jie; Li, Jingqiu; Le, Yanping; Zhou, Chengwei; Zhang, Shun; Gong, Zhaohui

    2016-01-01

    MicroRNAs (miRNAs) affect cancer cell glucose metabolism by targeting mRNAs of diverse enzymes that have been implicated in oxidative phosphorylation (OXPHOS) and glycolytic pathways. However, the mechanisms that underlie miRNA-mediated regulation of phosphofructokinase (PFK), a key rate-limiting enzyme in glycolysis, remain largely unknown. Here, we show that miR-128 directly targets PFK liver type (PFKL) in lung cancer cells and regulates endogenous expression of PFKL at both the mRNA and protein levels. In line with this, overexpression of miR-128 decreased glucose uptake and lactate production, as well as increased cellular ATP content. Interestingly, knockdown of miR-128 was shown to promote lung cancer cell growth and colony formation. Moreover, we observed that miR-128 expression inversely correlated with PFKL mRNA levels in clinic lung cancer samples and that increased PFKL expression predicted poor overall survival in lung cancer patients. Mechanistically, we showed that miR-128 regulates PFKL via a feedback loop that involves inhibition of the AKT signaling pathway. Together, our results suggest that miR-128 acts as a metabolic regulator in lung cancer cells that may be therapeutically exploited. PMID:27186417

  17. PFKL/miR-128 axis regulates glycolysis by inhibiting AKT phosphorylation and predicts poor survival in lung cancer

    PubMed Central

    Yang, Jie; Li, Jingqiu; Le, Yanping; Zhou, Chengwei; Zhang, Shun; Gong, Zhaohui

    2016-01-01

    MicroRNAs (miRNAs) affect cancer cell glucose metabolism by targeting mRNAs of diverse enzymes that have been implicated in oxidative phosphorylation (OXPHOS) and glycolytic pathways. However, the mechanisms that underlie miRNA-mediated regulation of phosphofructokinase (PFK), a key rate-limiting enzyme in glycolysis, remain largely unknown. Here, we show that miR-128 directly targets PFK liver type (PFKL) in lung cancer cells and regulates endogenous expression of PFKL at both the mRNA and protein levels. In line with this, overexpression of miR-128 decreased glucose uptake and lactate production, as well as increased cellular ATP content. Interestingly, knockdown of miR-128 was shown to promote lung cancer cell growth and colony formation. Moreover, we observed that miR-128 expression inversely correlated with PFKL mRNA levels in clinic lung cancer samples and that increased PFKL expression predicted poor overall survival in lung cancer patients. Mechanistically, we showed that miR-128 regulates PFKL via a feedback loop that involves inhibition of the AKT signaling pathway. Together, our results suggest that miR-128 acts as a metabolic regulator in lung cancer cells that may be therapeutically exploited. PMID:27186417

  18. Regulation of Molecular Chaperone Gene Transcription Involves the Serine Phosphorylation, 14-3-3ɛ Binding, and Cytoplasmic Sequestration of Heat Shock Factor 1

    PubMed Central

    Wang, XiaoZhe; Grammatikakis, Nicholas; Siganou, Aliki; Calderwood, Stuart K.

    2003-01-01

    Heat shock factor 1 (HSF1) regulates the transcription of molecular chaperone hsp genes. However, the cellular control mechanisms that regulate HSF1 activity are not well understood. In this study, we have demonstrated for the first time that human HSF1 binds to the essential cell signaling protein 14-3-3ɛ. Binding of HSF1 to 14-3-3ɛ occurs in cells in which extracellular signal regulated kinase (ERK) is activated and blockade of the ERK pathway by treatment with the specific ERK pathway inhibitor PD98059 in vivo strongly suppresses the binding. We previously showed that ERK1 phosphorylates HSF1 on serine 307 and leads to secondary phosphorylation by glycogen synthase kinase 3 (GSK3) on serine 303 within the regulatory domain and that these phosphorylation events repress HSF1. We show here that HSF1 binding to 14-3-3ɛ requires HSF1 phosphorylation on serines 303 and 307. Furthermore, the serine phosphorylation-dependent binding of HSF1 to 14-3-3ɛ results in the transcriptional repression of HSF1 and its sequestration in the cytoplasm. Leptomycin B, a specific inhibitor of nuclear export receptor CRM1, was found to reverse the cytoplasmic sequestration of HSF1 mediated by 14-3-3ɛ, suggesting that CRM1/14-3-3ɛ directed nuclear export plays a major role in repression of HSF1 by the ERK/GSK3/14-3-3ɛ pathway. Our experiments indicate a novel pathway for HSF1 regulation and suggest a mechanism for suppression of its activity during cellular proliferation. PMID:12917326

  19. TORC1 inhibits GSK3-mediated Elo2 phosphorylation to regulate very long chain fatty acid synthesis and autophagy.

    PubMed

    Zimmermann, Christine; Santos, Aline; Gable, Kenneth; Epstein, Sharon; Gururaj, Charulatha; Chymkowitch, Pierre; Pultz, Dennis; Rødkær, Steven V; Clay, Lorena; Bjørås, Magnar; Barral, Yves; Chang, Amy; Færgeman, Nils J; Dunn, Teresa M; Riezman, Howard; Enserink, Jorrit M

    2013-11-27

    Very long chain fatty acids (VLCFAs) are essential fatty acids with multiple functions, including ceramide synthesis. Although the components of the VLCFA biosynthetic machinery have been elucidated, how their activity is regulated to meet the cell's metabolic demand remains unknown. The goal of this study was to identify mechanisms that regulate the rate of VLCFA synthesis, and we discovered that the fatty acid elongase Elo2 is regulated by phosphorylation. Elo2 phosphorylation is induced upon inhibition of TORC1 and requires GSK3. Expression of nonphosphorylatable Elo2 profoundly alters the ceramide spectrum, reflecting aberrant VLCFA synthesis. Furthermore, VLCFA depletion results in constitutive activation of autophagy, which requires sphingoid base phosphorylation. This constitutive activation of autophagy diminishes cell survival, indicating that VLCFAs serve to dampen the amplitude of autophagy. Together, our data reveal a function for TORC1 and GSK3 in the regulation of VLCFA synthesis that has important implications for autophagy and cell homeostasis. PMID:24239358

  20. Phosphorylation of CHO1 by Lats1/2 regulates the centrosomal activation of LIMK1 during cytokinesis

    PubMed Central

    Okamoto, Ayumi; Yabuta, Norikazu; Mukai, Satomi; Torigata, Kosuke; Nojima, Hiroshi

    2015-01-01

    Large tumor suppressor 1 and 2 (Lats1/2) regulate centrosomal integrity, chromosome segregation and cytokinesis. As components of the centralspindlin complex, the kinesin-like protein CHO1 and its splicing variant MKLP1 colocalize with chromosome passenger proteins and GTPases and regulate the formation of the contractile ring and cytokinesis; however, the regulatory mechanisms of CHO1/MKLP1 remain elusive. Here, we show that Lats1/2 phosphorylate Ser716 in the F-actin-interacting region of CHO1, which is absent in MKLP1. Phosphorylated CHO1 localized to the centrosomes and midbody, and the actin polymerization factor LIM-kinase 1 (LIMK1) was identified as its binding partner. Overexpression of constitutively phosphorylated and non-phosphorylated CHO1 altered the mitotic localization and activation of LIMK1 at the centrosomes in HeLa cells, leading to the inhibition of cytokinesis through excessive phosphorylation of Cofilin and mislocalization of Ect2. These results suggest that Lats1/2 stringently control cytokinesis by regulating CHO1 phosphorylation and the mitotic activation of LIMK1 on centrosomes. PMID:25786116

  1. Role of Protein Phosphorylation in the Regulation of Cell Cycle and DNA-Related Processes in Bacteria

    PubMed Central

    Garcia-Garcia, Transito; Poncet, Sandrine; Derouiche, Abderahmane; Shi, Lei; Mijakovic, Ivan; Noirot-Gros, Marie-Françoise

    2016-01-01

    In all living organisms, the phosphorylation of proteins modulates various aspects of their functionalities. In eukaryotes, protein phosphorylation plays a key role in cell signaling, gene expression, and differentiation. Protein phosphorylation is also involved in the global control of DNA replication during the cell cycle, as well as in the mechanisms that cope with stress-induced replication blocks. Similar to eukaryotes, bacteria use Hanks-type kinases and phosphatases for signal transduction, and protein phosphorylation is involved in numerous cellular processes. However, it remains unclear whether protein phosphorylation in bacteria can also regulate the activity of proteins involved in DNA-mediated processes such as DNA replication or repair. Accumulating evidence supported by functional and biochemical studies suggests that phospho-regulatory mechanisms also take place during the bacterial cell cycle. Recent phosphoproteomics and interactomics studies identified numerous phosphoproteins involved in various aspect of DNA metabolism strongly supporting the existence of such level of regulation in bacteria. Similar to eukaryotes, bacterial scaffolding-like proteins emerged as platforms for kinase activation and signaling. This review reports the current knowledge on the phosphorylation of proteins involved in the maintenance of genome integrity and the regulation of cell cycle in bacteria that reveals surprising similarities to eukaryotes. PMID:26909079

  2. A Computational Model for the AMPA Receptor Phosphorylation Master Switch Regulating Cerebellar Long-Term Depression

    PubMed Central

    Gallimore, Andrew R.; Aricescu, A. Radu; Yuzaki, Michisuke; Calinescu, Radu

    2016-01-01

    The expression of long-term depression (LTD) in cerebellar Purkinje cells results from the internalisation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) from the postsynaptic membrane. This process is regulated by a complex signalling pathway involving sustained protein kinase C (PKC) activation, inhibition of serine/threonine phosphatase, and an active protein tyrosine phosphatase, PTPMEG. In addition, two AMPAR-interacting proteins–glutamate receptor-interacting protein (GRIP) and protein interacting with C kinase 1 (PICK1)–regulate the availability of AMPARs for trafficking between the postsynaptic membrane and the endosome. Here we present a new computational model of these overlapping signalling pathways. The model reveals how PTPMEG cooperates with PKC to drive LTD expression by facilitating the effect of PKC on the dissociation of AMPARs from GRIP and thus their availability for trafficking. Model simulations show that LTD expression is increased by serine/threonine phosphatase inhibition, and negatively regulated by Src-family tyrosine kinase activity, which restricts the dissociation of AMPARs from GRIP under basal conditions. We use the model to expose the dynamic balance between AMPAR internalisation and reinsertion, and the phosphorylation switch responsible for the perturbation of this balance and for the rapid plasticity initiation and regulation. Our model advances the understanding of PF-PC LTD regulation and induction, and provides a validated extensible platform for more detailed studies of this fundamental synaptic process. PMID:26807999

  3. A Computational Model for the AMPA Receptor Phosphorylation Master Switch Regulating Cerebellar Long-Term Depression.

    PubMed

    Gallimore, Andrew R; Aricescu, A Radu; Yuzaki, Michisuke; Calinescu, Radu

    2016-01-01

    The expression of long-term depression (LTD) in cerebellar Purkinje cells results from the internalisation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) from the postsynaptic membrane. This process is regulated by a complex signalling pathway involving sustained protein kinase C (PKC) activation, inhibition of serine/threonine phosphatase, and an active protein tyrosine phosphatase, PTPMEG. In addition, two AMPAR-interacting proteins-glutamate receptor-interacting protein (GRIP) and protein interacting with C kinase 1 (PICK1)-regulate the availability of AMPARs for trafficking between the postsynaptic membrane and the endosome. Here we present a new computational model of these overlapping signalling pathways. The model reveals how PTPMEG cooperates with PKC to drive LTD expression by facilitating the effect of PKC on the dissociation of AMPARs from GRIP and thus their availability for trafficking. Model simulations show that LTD expression is increased by serine/threonine phosphatase inhibition, and negatively regulated by Src-family tyrosine kinase activity, which restricts the dissociation of AMPARs from GRIP under basal conditions. We use the model to expose the dynamic balance between AMPAR internalisation and reinsertion, and the phosphorylation switch responsible for the perturbation of this balance and for the rapid plasticity initiation and regulation. Our model advances the understanding of PF-PC LTD regulation and induction, and provides a validated extensible platform for more detailed studies of this fundamental synaptic process. PMID:26807999

  4. Regulation by phosphorylation of Xenopus laevis poly(ADP-ribose) polymerase enzyme activity during oocyte maturation.

    PubMed Central

    Aoufouchi, S; Shall, S

    1997-01-01

    Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme that is dependent on DNA breaks and nicks for its enzyme activity. These DNA nicks and breaks function as allosteric effectors of the enzyme activity. This reaction is important for efficient DNA base excision repair, although it is not a component of the elementary repair pathway itself. The physiological relevance of this reaction might be to ensure correct and efficient DNA repair. We have examined the enzyme activity of PARP in oocytes and eggs of Xenopus laevis. Although both oocytes and eggs contain approximately the same amounts of enzyme protein, there is no detectable enzyme activity in the oocytes, whereas in the eggs the enzyme is active. Enzyme activity appears during oocyte maturation, approx. 4 h after induction by progesterone. This enzyme activation coincides with the appearance of active maturation-promoting factor. Enzyme activation is accompanied by a shift in the electrophoretic mobility of the polypeptide, from an apparent molecular mass of 116 kDa to 125 kDa. Treatment with either bacterial or potato phosphatase reverses the mobility shift and abolishes enzyme activity. Incubation of maturing X. laevis eggs with radioactive inorganic phosphate and subsequent immunoprecipitation demonstrate that the PARP protein is phosphorylated in vivo. We show that maturation-promoting factor (Cyclin B/cdc2) cannot itself be responsible for the phosphorylation and activation of PARP in maturing X. laevis eggs. Together, these results demonstrate that the enzyme activity of PARP in X. laevis oocytes and eggs is regulated by post-translational, covalent phosphorylation. PMID:9230139

  5. (S)-Lacosamide Binding to Collapsin Response Mediator Protein 2 (CRMP2) Regulates CaV2.2 Activity by Subverting Its Phosphorylation by Cdk5.

    PubMed

    Moutal, Aubin; François-Moutal, Liberty; Perez-Miller, Samantha; Cottier, Karissa; Chew, Lindsey Anne; Yeon, Seul Ki; Dai, Jixun; Park, Ki Duk; Khanna, May; Khanna, Rajesh

    2016-04-01

    The neuronal circuit remodels during development as well as in human neuropathologies such as epilepsy. Neurite outgrowth is an obligatory step in these events. We recently reported that alterations in the phosphorylation state of an axon specification/guidance protein, the collapsin response mediator protein 2 (CRMP2), play a major role in the activity-dependent regulation of neurite outgrowth. We also identified (S)-LCM, an inactive stereoisomer of the clinically used antiepileptic drug (R)-LCM (Vimpat®), as a novel tool for preferentially targeting CRMP2-mediated neurite outgrowth. Here, we investigated the mechanism by which (S)-LCM affects CRMP2 phosphorylation by two key kinases, cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3β (GSK-3β). (S)-LCM application to embryonic cortical neurons resulted in reduced levels of Cdk5- and GSK-3β-phosphorylated CRMP2. Mechanistically, (S)-LCM increased CRMP2 binding to both Cdk5- and GSK-3β without affecting binding of CRMP2 to its canonical partner tubulin. Saturation transfer difference nuclear magnetic resonance (STD NMR) and differential scanning fluorimetry (DSF) experiments demonstrated direct binding of (S)-LCM to CRMP2. Using an in vitro luminescent kinase assay, we observed that (S)-LCM specifically inhibited Cdk5-mediated phosphorylation of CRMP2. Cross-linking experiments and analytical ultracentrifugation showed no effect of (S)-LCM on the oligomerization state of CRMP2. The increased association between Cdk5-phosphorylated CRMP2 and CaV2.2 was reduced by (S)-LCM in vitro and in vivo. This reduction translated into a decrease of calcium influx via CaV2.2 in (S)-LCM-treated neurons compared to controls. (S)-LCM, to our knowledge, is the first molecule described to directly inhibit CRMP2 phosphorylation and may be useful for delineating CRMP2-facilitated functions. PMID:25846820

  6. ULK3 regulates cytokinetic abscission by phosphorylating ESCRT-III proteins

    PubMed Central

    Caballe, Anna; Wenzel, Dawn M; Agromayor, Monica; Alam, Steven L; Skalicky, Jack J; Kloc, Magdalena; Carlton, Jeremy G; Labrador, Leticia; Sundquist, Wesley I; Martin-Serrano, Juan

    2015-01-01

    The endosomal sorting complexes required for transport (ESCRT) machinery mediates the physical separation between daughter cells during cytokinetic abscission. This process is regulated by the abscission checkpoint, a genome protection mechanism that relies on Aurora B and the ESCRT-III subunit CHMP4C to delay abscission in response to chromosome missegregation. In this study, we show that Unc-51-like kinase 3 (ULK3) phosphorylates and binds ESCRT-III subunits via tandem MIT domains, and thereby, delays abscission in response to lagging chromosomes, nuclear pore defects, and tension forces at the midbody. Our structural and biochemical studies reveal an unusually tight interaction between ULK3 and IST1, an ESCRT-III subunit required for abscission. We also demonstrate that IST1 phosphorylation by ULK3 is an essential signal required to sustain the abscission checkpoint and that ULK3 and CHMP4C are functionally linked components of the timer that controls abscission in multiple physiological situations. DOI: http://dx.doi.org/10.7554/eLife.06547.001 PMID:26011858

  7. LIMK Regulates Tumor-Cell Invasion and Matrix Degradation Through Tyrosine Phosphorylation of MT1-MMP

    PubMed Central

    Lagoutte, Emilie; Villeneuve, Clémentine; Lafanechère, Laurence; Wells, Claire M.; Jones, Gareth E.; Chavrier, Philippe; Rossé, Carine

    2016-01-01

    During their metastatic spread, cancer cells need to remodel the extracellular matrix in order to migrate through stromal compartments adjacent to the primary tumor. Dissemination of breast carcinoma cells is mediated by membrane type 1-matrix metalloproteinase (MT1-MMP/MMP14), the main invadopodial matrix degradative component. Here, we identify MT1-MMP as a novel interacting partner of dual-specificity LIM Kinase-1 and -2 (LIMK1/2), and provide several evidence for phosphorylation of tyrosine Y573 in the cytoplasmic domain of MT1-MMP by LIMK. Phosphorylation of Y573 influences association of F-actin binding protein cortactin to MT1-MMP-positive endosomes and invadopodia formation and matrix degradation. Moreover, we show that LIMK1 regulates cortactin association to MT1-MMP-positive endosomes, while LIMK2 controls invadopodia-associated cortactin. In turn, LIMK1 and LIMK2 are required for MT1-MMP-dependent matrix degradation and cell invasion in a three-dimensional type I collagen environment. This novel link between LIMK1/2 and MT1-MMP may have important consequences for therapeutic control of breast cancer cell invasion. PMID:27116935

  8. Phosphorylation-dependent regulation of amidotransferase during the development of Blastocladiella emersonii.

    PubMed

    Etchebehere, L C; Maia, J C

    1989-08-01

    The enzyme amidotransferase [2-amino-2-deoxy-D-glucose-6-phosphate ketol isomerase (amino-transferring); EC 2.6.1.16] catalyzes the first step in the hexosamine biosynthetic pathway. In Blastocladiella emersonii the sensitivity of the enzyme to the inhibitor uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) is developmentally regulated. The inhibitable form of amidotransferase activity present in the zoospore is converted to a noninhibitable form during germination. The latter form is present throughout the growth phase and sensitivity to UDP-GlcNAc gradually returns to the zoospore level during sporulation [C.P. Selitrennikoff, N.E. Dalley, and D.R. Sonneborn (1980) Proc. Natl. Acad. Sci. USA 77, 5998-6002]. The following evidence suggests that a phosphorylation/dephosphorylation mechanism underlies this interconversion: (i) Both the vegetative and zoospore enzymes have the same molecular weight of 140,000, but the vegetative enzyme elutes significantly earlier on a DEAE-cellulose column than does the zoospore enzyme. (ii) The increased sensitivity to UDP-GlcNAc occurring in vivo and in vitro correlates with increased phosphorylation of a polypeptide of apparent Mr 76,000. This component copurifies with amidotransferase activity through ion-exchange chromatography and sucrose density gradient centrifugation. (iii) Desensitization and concurrent dephosphorylation of sensitive amidotransferase can be observed in vitro after treatment with a partially purified magnesium-dependent phosphoprotein phosphatase from zoospores. PMID:2546495

  9. Opiate receptor agonists regulate phosphorylation of synapsin I in cocultures of rat spinal cord and dorsal root ganglion.

    PubMed Central

    Nah, S Y; Saya, D; Barg, J; Vogel, Z

    1993-01-01

    Kappa opiate receptor agonists applied to cocultures of spinal cord and dorsal root ganglion neurons have been previously shown to inhibit voltage-dependent Ca2+ influx and adenylate cyclase activity. Here we describe the effect of kappa opiate receptor agonists on phosphorylation of synapsin I, a synaptic-vesicle-associated protein whose phosphorylation was shown to be regulated by cAMP and Ca2+ concentrations. Depolarization of spinal cord-dorsal root ganglion cocultured cells (by high K+ or veratridine) and the addition of forskolin (which activates adenylate cyclase) led to increased phosphorylation of synapsin I. Addition of kappa opiate agonists attenuated both the depolarization- and the forskolin-induced phosphorylation of synapsin I. This attenuation was blocked by the opiate antagonist naloxone. mu and delta opiate receptor agonists had much weaker effects on the depolarization-induced phosphorylation of synapsin I. Similarly, kappa opiate agonists decreased (by 40-60%) the high-K+- or veratridine-induced phosphorylation of synapsin I in spinal cord synaptosomes. These results show that opiate ligands modulate synapsin I phosphorylation. Moreover, the data could explain the reduction in synaptic efficacy observed after opiate treatment. Images Fig. 1 Fig. 2 Fig. 3 Fig. 5 Fig. 7 PMID:8097883

  10. Tyrosine phosphorylation/dephosphorylation of myosin II essential light chains of Entamoeba histolytica trophozoites regulates their motility.

    PubMed

    Bonilla-Moreno, Raúl; Pérez-Yépez, Eloy-Andrés; Villegas-Sepúlveda, Nicolás; Morales, Fernando O; Meza, Isaura

    2016-08-01

    Entamoeba histolytica trophozoites dwell in the human intestine as comensals although under still unclear circumstances become invasive and destroy the host tissues. For these activities, trophozoites relay on remarkable motility provided by the cytoskeleton organization. Amebic actin and some of its actin-associated proteins are well known, while components of the myosin II molecule, although predicted from the E. histolytica genome, need biochemical and functional characterization. Recently, an amebic essential light myosin II chain, named EhMLCI, was identified and reported to be phosphorylated in tyrosines. The phosphorylated form of the protein was associated with the soluble assembly incompetent conformation of the heavy myosin chains, while the non-phosphorylated protein was identified with filamentous heavy chains, organized in an assembly competent conformation. It was postulated that EhMLCI tyrosine phosphorylation could act as a negative regulator of myosin II activity by its phosphorylation/dephosphorylation cycles. To test this hypothesis, we constructed an expression vector containing an EhMLCI DNA sequence where two tyrosine residues, with strong probability of phosphorylation and fall within the single EF-hand domain that interacts with the N-terminus of myosin II heavy chains, were replaced by phenylalanines. Transfected trophozoites, expressing the mutant MutEhMLCI protein cannot process it, thereby not incorporated into the phosphorylation/dephosphorylation cycles required for myosin II activity, results in motility defective trophozoites. PMID:27318258

  11. Phosphorylation-Independent Regulation of the Diguanylate Cyclase WspR

    PubMed Central

    De, Nabanita; Pirruccello, Michelle; Krasteva, Petya Violinova; Bae, Narae; Raghavan, Rahul Veera; Sondermann, Holger

    2008-01-01

    Environmental signals that trigger bacterial pathogenesis and biofilm formation are mediated by changes in the level of cyclic dimeric guanosine monophosphate (c-di-GMP), a unique eubacterial second messenger. Tight regulation of cellular c-di-GMP concentration is governed by diguanylate cyclases and phosphodiesterases, which are responsible for its production and degradation, respectively. Here, we present the crystal structure of the diguanylate cyclase WspR, a conserved GGDEF domain-containing response regulator in Gram-negative bacteria, bound to c-di-GMP at an inhibitory site. Biochemical analyses revealed that feedback regulation involves the formation of at least three distinct oligomeric states. By switching from an active to a product-inhibited dimer via a tetrameric assembly, WspR utilizes a novel mechanism for modulation of its activity through oligomerization. Moreover, our data suggest that these enzymes can be activated by phosphodiesterases. Thus, in addition to the canonical pathways via phosphorylation of the regulatory domains, both product and enzyme concentration contribute to the coordination of c-di-GMP signaling. A structural comparison reveals resemblance of the oligomeric states to assemblies of GAF domains, widely used regulatory domains in signaling molecules conserved from archaea to mammals, suggesting a similar mechanism of regulation. PMID:18366254

  12. A Computational Screen for Regulators of Oxidative Phosphorylation Implicates SLIRP in Mitochondrial RNA Homeostasis

    PubMed Central

    Baughman, Joshua M.; Nilsson, Roland; Gohil, Vishal M.; Arlow, Daniel H.; Gauhar, Zareen; Mootha, Vamsi K.

    2009-01-01

    The human oxidative phosphorylation (OxPhos) system consists of approximately 90 proteins encoded by nuclear and mitochondrial genomes and serves as the primary cellular pathway for ATP biosynthesis. While the core protein machinery for OxPhos is well characterized, many of its assembly, maturation, and regulatory factors remain unknown. We exploited the tight transcriptional control of the genes encoding the core OxPhos machinery to identify novel regulators. We developed a computational procedure, which we call expression screening, which integrates information from thousands of microarray data sets in a principled manner to identify genes that are consistently co-expressed with a target pathway across biological contexts. We applied expression screening to predict dozens of novel regulators of OxPhos. For two candidate genes, CHCHD2 and SLIRP, we show that silencing with RNAi results in destabilization of OxPhos complexes and a marked loss of OxPhos enzymatic activity. Moreover, we show that SLIRP plays an essential role in maintaining mitochondrial-localized mRNA transcripts that encode OxPhos protein subunits. Our findings provide a catalogue of potential novel OxPhos regulators that advance our understanding of the coordination between nuclear and mitochondrial genomes for the regulation of cellular energy metabolism. PMID:19680543

  13. Regulation of µ-Opioid Receptors: Desensitization, Phosphorylation, Internalization, and Tolerance

    PubMed Central

    Williams, John T.; Ingram, Susan L.; Henderson, Graeme; Chavkin, Charles; von Zastrow, Mark; Schulz, Stefan; Koch, Thomas; Evans, Christopher J.

    2013-01-01

    Morphine and related µ-opioid receptor (MOR) agonists remain among the most effective drugs known for acute relief of severe pain. A major problem in treating painful conditions is that tolerance limits the long-term utility of opioid agonists. Considerable effort has been expended on developing an understanding of the molecular and cellular processes that underlie acute MOR signaling, short-term receptor regulation, and the progression of events that lead to tolerance for different MOR agonists. Although great progress has been made in the past decade, many points of contention and controversy cloud the realization of this progress. This review attempts to clarify some confusion by clearly defining terms, such as desensitization and tolerance, and addressing optimal pharmacological analyses for discerning relative importance of these cellular mechanisms. Cellular and molecular mechanisms regulating MOR function by phosphorylation relative to receptor desensitization and endocytosis are comprehensively reviewed, with an emphasis on agonist-biased regulation and areas where knowledge is lacking or controversial. The implications of these mechanisms for understanding the substantial contribution of MOR signaling to opioid tolerance are then considered in detail. While some functional MOR regulatory mechanisms contributing to tolerance are clearly understood, there are large gaps in understanding the molecular processes responsible for loss of MOR function after chronic exposure to opioids. Further elucidation of the cellular mechanisms that are regulated by opioids will be necessary for the successful development of MOR-based approaches to new pain therapeutics that limit the development of tolerance. PMID:23321159

  14. Phosphorylation-dependent regulation of cyclin D1 nuclear export and cyclin D1–dependent cellular transformation

    PubMed Central

    Alt, Jodi R.; Cleveland, John L.; Hannink, Mark; Diehl, J. Alan

    2000-01-01

    GSK-3β-dependent phosphorylation of cyclin D1 at Thr-286 promotes the nuclear-to-cytoplasmic redistribution of cyclin D1 during S phase of the cell cycle, but how phosphorylation regulates redistribution has not been resolved. For example, phosphorylation of nuclear cyclin D1 could increase its rate of nuclear export relative to nuclear import; alternatively, phosphorylation of cytoplasmic cyclin D1 by GSK-3β could inhibit nuclear import. Here, we report that GSK-3β-dependent phosphorylation promotes cyclin D1 nuclear export by facilitating the association of cyclin D1 with the nuclear exportin CRM1. D1-T286A, a cyclin D1 mutant that cannot be phosphorylated by GSK-3β, remains nuclear throughout the cell cycle, a consequence of its reduced binding to CRM1. Constitutive overexpression of the nuclear cyclin D1-T286A in murine fibroblasts results in cellular transformation and promotes tumor growth in immune compromised mice. Thus, removal of cyclin D1 from the nucleus during S phase appears essential for regulated cell division. PMID:11124803

  15. G Protein-coupled Receptor Kinase 5 Phosphorylates Nucleophosmin and Regulates Cell Sensitivity to Polo-like Kinase 1 Inhibition*

    PubMed Central

    So, Christopher H.; Michal, Allison M.; Mashayekhi, Rouzbeh; Benovic, Jeffrey L.

    2012-01-01

    G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, leading to their desensitization and endocytosis. GRKs have also been implicated in phosphorylating other classes of proteins and can localize in a variety of cellular compartments, including the nucleus. Here, we attempted to identify potential nuclear substrates for GRK5. Our studies reveal that GRK5 is able to interact with and phosphorylate nucleophosmin (NPM1) both in vitro and in intact cells. NPM1 is a nuclear protein that regulates a variety of cell functions including centrosomal duplication, cell cycle control, and apoptosis. GRK5 interaction with NPM1 is mediated by the N-terminal domain of each protein, and GRK5 primarily phosphorylates NPM1 at Ser-4, a site shared with polo-like kinase 1 (PLK1). NPM1 phosphorylation by GRK5 and PLK1 correlates with the sensitivity of cells to undergo apoptosis with cells having higher GRK5 levels being less sensitive and cells with lower GRK5 being more sensitive to PLK1 inhibitor-induced apoptosis. Taken together, our results demonstrate that GRK5 phosphorylates Ser-4 in nucleophosmin and regulates the sensitivity of cells to PLK1 inhibition. PMID:22467873

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

    PubMed

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

    2015-12-11

    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. Ser(858) 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

  17. Architecture of the cystic fibrosis transmembrane conductance regulator protein and structural changes associated with phosphorylation and nucleotide binding.

    PubMed

    Zhang, Liang; Aleksandrov, Luba A; Zhao, Zhefeng; Birtley, James R; Riordan, John R; Ford, Robert C

    2009-09-01

    We describe biochemical and structural studies of the isolated cystic fibrosis transmembrane conductance regulator (CFTR) protein. Using electron cryomicroscopy, low resolution three-dimensional structures have been obtained for the non-phosphorylated protein in the absence of nucleotide and for the phosphorylated protein with ATP. In the latter state, the cytosolic nucleotide-binding domains move closer together, forming a more compact packing arrangement. Associated with this is a reorganization within the cylindrical transmembrane domains, consistent with a shift from an inward-facing to outward-facing configuration. A region of density in the non-phosphorylated protein that extends from the bottom of the cytosolic regions up to the transmembrane domains is hypothesised to represent the unique regulatory region of CFTR. These data offer insights into the architecture of this ATP-binding cassette protein, and shed light on the global motions associated with nucleotide binding and priming of the chloride channel via phosphorylation of the regulatory region. PMID:19524678

  18. Histone phosphorylation

    PubMed Central

    Rossetto, Dorine; Avvakumov, Nikita; Côté, Jacques

    2012-01-01

    Histone posttranslational modifications are key components of diverse processes that modulate chromatin structure. These marks function as signals during various chromatin-based events, and act as platforms for recruitment, assembly or retention of chromatin-associated factors. The best-known function of histone phosphorylation takes place during cellular response to DNA damage, when phosphorylated histone H2A(X) demarcates large chromatin domains around the site of DNA breakage. However, multiple studies have also shown that histone phosphorylation plays crucial roles in chromatin remodeling linked to other nuclear processes. In this review, we summarize the current knowledge of histone phosphorylation and describe the many kinases and phosphatases that regulate it. We discuss the key roles played by this histone mark in DNA repair, transcription and chromatin compaction during cell division and apoptosis. Additionally, we describe the intricate crosstalk that occurs between phosphorylation and other histone modifications and allows for sophisticated control over the chromatin remodeling processes. PMID:22948226

  19. Regulation by the neuropeptide cholecystokinin (CCK-8S) of protein phosphorylation in the neostriatum.

    PubMed Central

    Snyder, G L; Fisone, G; Morino, P; Gundersen, V; Ottersen, O P; Hökfelt, T; Greengard, P

    1993-01-01

    Despite physiological evidence that cholecystokinin (CCK) is an excitatory neurotransmitter in the brain, little is known about its mechanism of action. CCK immunoreactivity in the brain, including projections to the striatum, is primarily attributable to the sulfated octapeptide CCK-8S. We report here that CCK-8S abolishes cAMP-dependent phosphorylation of a dopamine- and cAMP-regulated 32-kDa phosphoprotein (DARPP-32) in striatal neurons. The effect of CCK-8S is prevented by antagonists of CCKB and N-methyl-D-aspartate receptors. Our results support a model in which CCK-8S, originating from CCK or CCK/glutamate corticostriatal neurons, promotes the release of an excitatory neurotransmitter that causes the dephosphorylation and inactivation of DARPP-32, a potent protein phosphatase inhibitor, thereby modulating neuronal excitability. Images Fig. 1 Fig. 2 Fig. 3 PMID:8248241

  20. AND-34/BCAR3 Regulates Adhesion-Dependent p130Cas Serine Phosphorylation and Breast Cancer Cell Growth Pattern

    PubMed Central

    Makkinje, Anthony; Near, Richard I.; Infusini, Giuseppe; Borre, Pierre Vanden; Bloom, Alexander; Cai, Dongpo; Costello, Catherine E.; Lerner, Adam

    2009-01-01

    NSP protein family members associate with p130Cas, a focal adhesion adapter protein best known as a Src substrate that integrates adhesion-related signaling. Over-expression of AND-34/BCAR3/NSP2 (BCAR3), but not NSP1 or NSP3, induces anti-estrogen resistance in human breast cancer cell lines. BCAR3 over-expression in epithelial MCF-7 cells augments levels of a phosphorylated p130Cas species that migrates more slowly on SDS PAGE while NSP-1 and NSP3 induce modest or no phosphorylation, respectively. Conversely, reduction in BCAR3 expression in mesenchymal MDA-231 cells by inducible shRNA results in loss of such p130Cas phosphorylation. Replacement of NSP3's serine/proline-rich domain with that of AND-34/BCAR3 instills the ability to induce p130Cas phosphorylation. Phospho-amino acid analysis demonstrates that BCAR3 induces p130Cas serine phosphorylation. Mass spectrometry identified phosphorylation at p130Cas serines 139, 437 and 639. p130Cas serine phosphorylation accumulates for several hours after adhesion of MDA-231 cells to fibronectin and is dependent upon BCAR3 expression. BCAR3 knockdown alters p130Cas localization and converts MDA-231 growth to an epithelioid pattern characterized by striking cohesiveness and lack of cellular projections at colony borders. These studies demonstrate that BCAR3 regulates p130Cas serine phosphorylation that is adhesion-dependent, temporally distinct from previously well-characterized rapid Fak and Src kinase-mediated p130Cas tyrosine phosphorylation and that correlates with invasive phenotype. PMID:19454314

  1. Roles of Phosphorylation of the Nucleocapsid Protein of Mumps Virus in Regulating Viral RNA Transcription and Replication

    PubMed Central

    Zengel, James; Pickar, Adrian; Xu, Pei; Lin, Alita

    2015-01-01

    ABSTRACT Mumps virus (MuV) is a paramyxovirus with a negative-sense nonsegmented RNA genome. The viral RNA genome is encapsidated by the nucleocapsid protein (NP) to form the ribonucleoprotein (RNP), which serves as a template for transcription and replication. In this study, we investigated the roles of phosphorylation sites of NP in MuV RNA synthesis. Using radioactive labeling, we first demonstrated that NP was phosphorylated in MuV-infected cells. Using both liquid chromatography-mass spectrometry (LC-MS) and in silico modeling, we identified nine putative phosphorylated residues within NP. We mutated these nine residues to alanine. Mutation of the serine residue at position 439 to alanine (S439A) was found to reduce the phosphorylation of NP in transfected cells by over 90%. The effects of these mutations on the MuV minigenome system were examined. The S439A mutant was found to have higher activity, four mutants had lower activity, and four mutants had similar activity compared to wild-type NP. MuV containing the S439A mutation had 90% reduced phosphorylation of NP and enhanced viral RNA synthesis and viral protein expression at early time points after infection, indicating that S439 is the major phosphorylation site of NP and its phosphorylation plays an important role in downregulating viral RNA synthesis. IMPORTANCE Mumps virus (MuV), a paramyxovirus, is an important human pathogen that is reemerging in human populations. Nucleocapsid protein (NP) of MuV is essential for viral RNA synthesis. We have identified the major phosphorylation site of NP. We have found that phosphorylation of NP plays a critical role in regulating viral RNA synthesis. The work will lead to a better understanding of viral RNA synthesis and possible novel targets for antiviral drug development. PMID:25948749

  2. The Rho-GTPase effector ROCK regulates meiotic maturation of the bovine oocyte via myosin light chain phosphorylation and cofilin phosphorylation.

    PubMed

    Lee, So-Rim; Xu, Yong-Nan; Jo, Yu-Jin; Namgoong, Suk; Kim, Nam-Hyung

    2015-11-01

    Oocyte meiosis involves a unique asymmetric division involving spindle movement from the central cytoplasm to the cortex, followed by polar body extrusion. ROCK is a Rho-GTPase effector involved in various cellular functions in somatic cells as well as oocyte meiosis. ROCK was previously shown to promote actin organization by phosphorylating several downstream targets, including LIM domain kinase (LIMK), phosphorylated cofilin (p-cofilin), and myosin light chain (MLC). In this study, we investigated the roles of ROCK and MLC during bovine oocyte meiosis. We found that ROCK was localized around the nucleus at the oocyte's germinal-vesicle (GV) stage, but spreads to the rest of the cytoplasm in later developmental stages. On the other hand, phosphorylated MLC (p-MLC) localized at the cortex, and its abundance decreased by the metaphase-II stage. Disrupting ROCK activity, via RNAi or the chemical inhibitor Y-27632, blocked both cell cycle progression and polar body extrusion. ROCK inhibition also resulted in decreased cortical actin, p-cofilin, and p-MLC levels. Similar to the phenotype associated with inhibition of ROCK activity, inhibition of MLC kinase by the chemical inhibitor ML-7 caused defects in polar body extrusion. Collectively, our results suggest that the ROCK/MLC/actomyosin as well as ROCK/LIMK/cofilin pathways regulate meiotic spindle migration and cytokinesis during bovine oocyte maturation. PMID:26175189

  3. Mitochondrial oxidative phosphorylation: tissue oxygen sensor for regulation of coronary flow.

    PubMed

    Nuutinen, E M; Wilson, D F; Erecińska, M

    1984-01-01

    The observation that mitochondrial oxidative phosphorylation in vivo is dependent on oxygen tension throughout the physiological range (Wilson et al., 1979a , 1979b ) has made this metabolic pathway the most probable candidate for the tissue oxygen sensor in the regulation of local blood flow. We have utilized the oxygen dependent regulatory system for coronary blood flow to examine this possibility. Alterations in coronary flow were induced by: 1. Varied work load; 2. Infusion of Amytal (an inhibitor of mitochondrial respiration); 3. Infusion of DNP; 4. Hypoxia. Increased work load caused increased coronary flow with no decrease in effluent oxygen tension while Amytal infusion and hypoxia caused vasodilation with increased and decreased O2 tension respectively. This indicates that oxygen tension per se cannot be responsible for the observed vasodilation. Tissue energy metabolism was evaluated by measuring metabolite levels in hearts which were freeze-clamped in each state of perfusion. In all four methods of vasodilation, a decrease in cellular energy state ratio ([ATP]f/[ADP]f[Pi]) expressed as the calculated ratio of free adenine nucleotides, was observed for conditions which increased flow. Systematic variation of work load, Amytal or DNP concentration resulted in quantitatively the same correlation between tissue [ATP]f/[ADP]f[Pi] and coronary flow. It is concluded that mitochondrial oxidative phosphorylation is the oxygen sensor for the regulation of coronary blood flow by tissue oxygen tension. Infusion of adenosine, a known coronary vasodilator, induced vasodilation which was completely blocked by theophylline.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:6731096

  4. Hematopoietic Stem Cell Activity Is Regulated by Pten Phosphorylation Through a Niche-Dependent Mechanism.

    PubMed

    Li, Jing; Zhang, Jun; Tang, Minghui; Xin, Junping; Xu, Yan; Volk, Andrew; Hao, Caiqin; Hu, Chenglong; Sun, Jiewen; Wei, Wei; Cao, Quichan; Breslin, Peter; Zhang, Jiwang

    2016-08-01

    The phosphorylated form of Pten (p-Pten) is highly expressed in >70% of acute myeloid leukemia samples. However, the role of p-Pten in normal and abnormal hematopoiesis has not been studied. We found that Pten protein levels are comparable among long-term (LT) hematopoietic stem cells (HSCs), short-term (ST) HSCs, and multipotent progenitors (MPPs); however, the levels of p-Pten are elevated during the HSC-to-MPP transition. To study whether p-Pten is involved in regulating self-renewal and differentiation in HSCs, we compared the effects of overexpression of p-Pten and nonphosphorylated Pten (non-p-Pten) on the hematopoietic reconstitutive capacity (HRC) of HSCs. We found that overexpression of non-p-Pten enhances the LT-HRC of HSCs, whereas overexpression of p-Pten promotes myeloid differentiation and compromises the LT-HRC of HSCs. Such phosphorylation-regulated Pten functioning is mediated by repressing the cell:cell contact-induced activation of Fak/p38 signaling independent of Pten's lipid phosphatase activity because both p-Pten and non-p-Pten have comparable activity in repressing PI3K/Akt signaling. Our studies suggest that, in addition to repressing PI3K/Akt/mTor signaling, non-p-Pten maintains HSCs in bone marrow niches via a cell-contact inhibitory mechanism by inhibiting Fak/p38 signaling-mediated proliferation and differentiation. In contrast, p-Pten promotes the proliferation and differentiation of HSCs by enhancing the cell contact-dependent activation of Src/Fak/p38 signaling. Stem Cells 2016;34:2130-2144. PMID:27096933

  5. Cyclin-dependent kinase 5 modulates nociceptive signaling through direct phosphorylation of transient receptor potential vanilloid 1

    PubMed Central

    Pareek, Tej K.; Keller, Jason; Kesavapany, Sashi; Agarwal, Nitin; Kuner, Rohini; Pant, Harish C.; Iadarola, Michael J.; Brady, Roscoe O.; Kulkarni, Ashok B.

    2007-01-01

    Transient receptor potential vanilloid 1 (TRPV1), a ligand-gated cation channel highly expressed in small-diameter sensory neurons, is activated by heat, protons, and capsaicin. The phosphorylation of TRPV1 provides a versatile regulation of intracellular calcium levels and is critical for TRPV1 function in responding to a pain stimulus. We have previously reported that cyclin-dependent kinase 5 (Cdk5) activity regulates nociceptive signaling. In this article we report that the Cdk5-mediated phosphorylation of TRPV1 at threonine-407 can modulate agonist-induced calcium influx. Inhibition of Cdk5 activity in cultured dorsal root ganglia neurons resulted in a significant reduction of TRPV1-mediated calcium influx, and this effect could be reversed by restoring Cdk5 activity. Primary nociceptor-specific Cdk5 conditional-knockout mice showed reduced TRPV1 phosphorylation, resulting in significant hypoalgesia. Thus, the present study indicates that Cdk5-mediated TRPV1 phosphorylation is important in the regulation of pain signaling. PMID:17194758

  6. Raf Kinase Inhibitory Protein Function Is Regulated via a Flexible Pocket and Novel Phosphorylation-Dependent Mechanism▿ †

    PubMed Central

    Granovsky, Alexey E.; Clark, Matthew C.; McElheny, Dan; Heil, Gary; Hong, Jia; Liu, Xuedong; Kim, Youngchang; Joachimiak, Grazyna; Joachimiak, Andrzej; Koide, Shohei; Rosner, Marsha Rich

    2009-01-01

    Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics. PMID:19103740

  7. Molecular dynamics simulation of phosphorylation-induced conformational transitions in the mycobacterium tuberculosis response regulator PrrA

    SciTech Connect

    Chen, Guo; Mcmahon, Benjamin H; Tung, Chang - Shung

    2008-01-01

    Phosphorylation-activated modulation of response regulators (RR) is predominantly used by bacteria as a strategy in regulating their two-component signaling (TCS) systems, the underlying molecular mechanisms are however far from fully understood. In this work we have conducted a molecular dynamics (MD) simulation of the phosphorylation-induced conformational transitions of RRs with the Mycobacterium Tuberculosis PrrA as a particular example. Starting from the full-length inactive structure of PrrA we introduced a local disturbance by phosphorylating the conserved aspartic acid residue, Asp-58, in the regulatory domain. A Go-model-type algorithm packaged with AMBER force fields was then applied to simulate the dynamics upon phosphorylation. The MD simulation shows that the phosphorylation of Asp-58 facilitates PrrA, whose inactive state has a compact conformation with a closed interdomain interface, to open up with its interdomain separation being increased by an average of about 1.5 {angstrom} for a simulation of 20 ns. The trans-activation loop, which is completely buried within the interdomain interface in the inactive PrrA, is found to become more exposed with the phosphorylated structure as well. These results provide more structural details of how the phosphorylation of a local aspartate activates PrrA to undergo a global conformational rearrangement toward its extended active state. This work also indicates that MD simulations can serve as a fast tool to unravel the regulation mechanisms of all RRs, which is especially valuable when the structures of full-length active RRs are currently unavailable.

  8. Mdm2 Phosphorylation Regulates Its Stability and Has Contrasting Effects on Oncogene and Radiation-Induced Tumorigenesis.

    PubMed

    Carr, Michael I; Roderick, Justine E; Gannon, Hugh S; Kelliher, Michelle A; Jones, Stephen N

    2016-09-01

    ATM phosphorylation of Mdm2-S394 is required for robust p53 stabilization and activation in DNA-damaged cells. We have now utilized Mdm2(S394A) knockin mice to determine that phosphorylation of Mdm2-S394 regulates p53 activity and the DNA damage response in lymphatic tissues in vivo by modulating Mdm2 stability. Mdm2-S394 phosphorylation delays lymphomagenesis in Eμ-myc transgenic mice, and preventing Mdm2-S394 phosphorylation obviates the need for p53 mutation in Myc-driven tumorigenesis. However, irradiated Mdm2(S394A) mice also have increased hematopoietic stem and progenitor cell functions, and we observed decreased lymphomagenesis in sub-lethally irradiated Mdm2(S394A) mice. These findings document contrasting effects of ATM-Mdm2 signaling on p53 tumor suppression and reveal that destabilizing Mdm2 by promoting its phosphorylation by ATM would be effective in treating oncogene-induced malignancies, while inhibiting Mdm2-S394 phosphorylation during radiation exposure or chemotherapy would ameliorate bone marrow failure and prevent the development of secondary hematological malignancies. PMID:27568562

  9. ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1

    PubMed Central

    Kijas, Amanda W.; Lim, Yi Chieh; Bolderson, Emma; Cerosaletti, Karen; Gatei, Magtouf; Jakob, Burkhard; Tobias, Frank; Taucher-Scholz, Gisela; Gueven, Nuri; Oakley, Greg; Concannon, Patrick; Wolvetang, Ernst; Khanna, Kum Kum; Wiesmüller, Lisa; Lavin, Martin F.

    2015-01-01

    The MRE11/RAD50/NBS1 (MRN) complex plays a central role as a sensor of DNA double strand breaks (DSB) and is responsible for the efficient activation of ataxia-telangiectasia mutated (ATM) kinase. Once activated ATM in turn phosphorylates RAD50 and NBS1, important for cell cycle control, DNA repair and cell survival. We report here that MRE11 is also phosphorylated by ATM at S676 and S678 in response to agents that induce DNA DSB, is dependent on the presence of NBS1, and does not affect the association of members of the complex or ATM activation. A phosphosite mutant (MRE11S676AS678A) cell line showed decreased cell survival and increased chromosomal aberrations after radiation exposure indicating a defect in DNA repair. Use of GFP-based DNA repair reporter substrates in MRE11S676AS678A cells revealed a defect in homology directed repair (HDR) but single strand annealing was not affected. More detailed investigation revealed that MRE11S676AS678A cells resected DNA ends to a greater extent at sites undergoing HDR. Furthermore, while ATM-dependent phosphorylation of Kap1 and SMC1 was normal in MRE11S676AS678A cells, there was no phosphorylation of Exonuclease 1 consistent with the defect in HDR. These results describe a novel role for ATM-dependent phosphorylation of MRE11 in limiting the extent of resection mediated through Exonuclease 1. PMID:26240375

  10. mTOR regulates tau phosphorylation and degradation: implications for Alzheimer's disease and other tauopathies.

    PubMed

    Caccamo, Antonella; Magrì, Andrea; Medina, David X; Wisely, Elena V; López-Aranda, Manuel F; Silva, Alcino J; Oddo, Salvatore

    2013-06-01

    Accumulation of tau is a critical event in several neurodegenerative disorders, collectively known as tauopathies, which include Alzheimer's disease and frontotemporal dementia. Pathological tau is hyperphosphorylated and aggregates to form neurofibrillary tangles. The molecular mechanisms leading to tau accumulation remain unclear and more needs to be done to elucidate them. Age is a major risk factor for all tauopathies, suggesting that molecular changes contributing to the aging process may facilitate tau accumulation and represent common mechanisms across different tauopathies. Here, we use multiple animal models and complementary genetic and pharmacological approaches to show that the mammalian target of rapamycin (mTOR) regulates tau phosphorylation and degradation. Specifically, we show that genetically increasing mTOR activity elevates endogenous mouse tau levels and phosphorylation. Complementary to it, we further demonstrate that pharmacologically reducing mTOR signaling with rapamycin ameliorates tau pathology and the associated behavioral deficits in a mouse model overexpressing mutant human tau. Mechanistically, we provide compelling evidence that the association between mTOR and tau is linked to GSK3β and autophagy function. In summary, we show that increasing mTOR signaling facilitates tau pathology, while reducing mTOR signaling ameliorates tau pathology. Given the overwhelming evidence that reducing mTOR signaling increases lifespan and healthspan, the data presented here have profound clinical implications for aging and tauopathies and provide the molecular basis for how aging may contribute to tau pathology. Additionally, these results provide preclinical data indicating that reducing mTOR signaling may be a valid therapeutic approach for tauopathies. PMID:23425014

  11. TAB4 stimulates TAK1-TAB1 phosphorylation and binds polyubiquitin to direct signaling to NF-kappaB.

    PubMed

    Prickett, Todd D; Ninomiya-Tsuji, Jun; Broglie, Peter; Muratore-Schroeder, Tara L; Shabanowitz, Jeffrey; Hunt, Donald F; Brautigan, David L

    2008-07-11

    Responses to transforming growth factor beta and multiple cytokines involve activation of transforming growth factor beta-activated kinase-1 (TAK1) kinase, which activates kinases IkappaB kinase (IKK) and MKK3/6, leading to the parallel activation of NF-kappaB and p38 MAPK. Activation of TAK1 by autophosphorylation is known to involve three different TAK1-binding proteins (TABs). Here we report a protein phosphatase subunit known as type 2A phosphatase-interacting protein (TIP) that also acts as a TAB because it co-precipitates with and directly binds to TAK1, enhances TAK1 autophosphorylation at unique sites, and promotes TAK1 phosphorylation of IKKbeta and signaling to NF-kappaB. Mass spectrometry demonstrated that co-expression of TAB4 protein significantly increased phosphorylation of four sites in TAK1, in a linker region between the kinase and TAB2/3 binding domains, and two sites in TAB1. Recombinant GST-TAB4 bound in an overlay assay directly to inactive TAK1 and activated TAK1 but not TAK1 phosphorylated in the linker sites, suggesting a bind and release mechanism. In kinase assays using TAK1 immune complexes, added GST-TAB4 selectively stimulated IKK phosphorylation. TAB4 co-precipitated polyubiquitinated proteins dependent on a Phe-Pro motif that was required to enhance phosphorylation of TAK1. TAB4 mutated at Phe-Pro dominantly interfered with IL-1beta activation of NF-kappaB involving IKK-dependent but not p38 MAPK-dependent signaling. The results show that TAB4 binds TAK1 and polyubiquitin chains to promote specific sites of phosphorylation in TAK1-TAB1, which activates IKK signaling to NF-kappaB. PMID:18456659

  12. Bordetella pertussis fim3 gene regulation by BvgA: phosphorylation controls the formation of inactive vs. active transcription complexes.

    PubMed

    Boulanger, Alice; Moon, Kyung; Decker, Kimberly B; Chen, Qing; Knipling, Leslie; Stibitz, Scott; Hinton, Deborah M

    2015-02-10

    Two-component systems [sensor kinase/response regulator (RR)] are major tools used by microorganisms to adapt to environmental conditions. RR phosphorylation is typically required for gene activation, but few studies have addressed how and if phosphorylation affects specific steps during transcription initiation. We characterized transcription complexes made with RNA polymerase and the Bordetella pertussis RR, BvgA, in its nonphosphorylated or phosphorylated (BvgA∼P) state at P(fim3), the promoter for the virulence gene fim3 (fimbrial subunit), using gel retardation, potassium permanganate and DNase I footprinting, cleavage reactions with protein conjugated with iron bromoacetamidobenzyl-EDTA, and in vitro transcription. Previous work has shown that the level of nonphosphorylated BvgA remains high in vivo under conditions in which BvgA is phosphorylated. Our results here indicate that surprisingly both BvgA and BvgA∼P form open and initiating complexes with RNA polymerase at P(fim3). However, phosphorylation of BvgA is needed to generate the correct conformation that can transition to competent elongation. Footprints obtained with the complexes made with nonphosphorylated BvgA are atypical; while the initiating complex with BvgA synthesizes short RNA, it does not generate full-length transcripts. Extended incubation of the BvgA/RNA polymerase initiated complex in the presence of heparin generates a stable, but defective species that depends on the initial transcribed sequence of fim3. We suggest that the presence of nonphosphorylated BvgA down-regulates P(fim3) activity when phosphorylated BvgA is present and may allow the bacterium to quickly adapt to the loss of inducing conditions by rapidly eliminating P(fim3) activation once the signal for BvgA phosphorylation is removed. PMID:25624471

  13. Phosphorylation of EXO1 by CDKs 1 and 2 regulates DNA end resection and repair pathway choice

    PubMed Central

    Tomimatsu, Nozomi; Mukherjee, Bipasha; Hardebeck, Molly Catherine; Ilcheva, Mariya; Camacho, Cristel Vanessa; Harris, Janelle Louise; Porteus, Matthew; Llorente, Bertrand; Khanna, Kum Kum; Burma, Sandeep

    2014-01-01

    Resection of DNA double-strand breaks (DSBs) is a pivotal step during which the choice between NHEJ and HR DNA repair pathways is made. While CDKs are known to control initiation of resection, their role in regulating long-range resection remains elusive. Here we show that CDKs 1/2 phosphorylate the long-range resection nuclease EXO1 at four C-terminal S/TP sites during S/G2 phases of the cell cycle. Impairment of EXO1 phosphorylation attenuates resection, chromosomal integrity, cell survival, and HR, but augments NHEJ upon DNA damage. In contrast, cells expressing phospho-mimic EXO1 are proficient in resection even after CDK inhibition and favor HR over NHEJ. Mutation of cyclin-binding sites on EXO1 attenuates CDK binding and EXO1 phosphorylation, causing a resection defect that can be rescued by phospho-mimic mutations. Mechanistically, phosphorylation of EXO1 augments its recruitment to DNA breaks possibly via interactions with BRCA1. In sum, phosphorylation of EXO1 by CDKs is a novel mechanism regulating repair pathway choice. PMID:24705021

  14. Cyclin-dependent kinase 4 phosphorylates and positively regulates PAX3-FOXO1 in human alveolar rhabdomyosarcoma cells.

    PubMed

    Liu, Lingling; Wu, Jing; Ong, Su Sien; Chen, Taosheng

    2013-01-01

    Alveolar rhabdomyosarcoma (ARMS) is an aggressive childhood muscle sarcoma with a 5-year survival rate of less than 30%. More than 80% of ARMSs harbor a PAX3-FOXO1 fusion transcription factor. However, expression of PAX3-FOXO1 in muscle cells alone is not sufficient and requires the loss of function of Ink4a/ARF to promote malignant proliferation of muscle cells in vitro or initiate ARMS tumor formation in vivo. This prompted us to examine the signaling pathways required to activate the function of PAX3-FOXO1 and to explore the functional interaction between the Ink4a/ARF and PAX3-FOXO1 signaling pathways. Here we report that inhibition of cyclin-dependent kinase 4 (Cdk4) by fascaplysin (a small molecule selective inhibitor of Cdk4/cyclin D1 that we identified in a screen for compounds that inhibit PAX3-FOXO1) led to inhibition of the transcriptional activity of PAX3-FOXO1 in ARMS cell line Rh30. Consistent with this finding, activation of Cdk4 enhanced the activity of PAX3-FOXO1. In vitro kinase assays revealed that Cdk4 directly phosphorylated PAX3-FOXO1 at Ser(430). Whereas fascaplysin did not affect the protein level of PAX3-FOXO1, it did increase the cytoplasmic level of PAX3-FOXO1 in a portion of cells. Our findings indicate that Cdk4 phosphorylates and positively regulates PAX3-FOXO1 and suggest that inhibition of Cdk4 activity should be explored as a promising avenue for developing therapy for ARMS. PMID:23469153

  15. CIP2A regulates cancer metabolism and CREB phosphorylation in non-small cell lung cancer.

    PubMed

    Peng, Bo; Lei, Ningjing; Chai, Yurong; Chan, Edward K L; Zhang, Jian-Ying

    2015-01-01

    The cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently characterized endogenous inhibitor of the phosphatase activity of protein phosphatase 2A (PP2A), which extends the half-life of oncogenic protein c-myc and promotes in vivo tumor growth. The function of CIP2A in cancer progression is still poorly understood. To uncover the underlying mechanism of CIP2A-mediated cell proliferation, we implemented a two-dimensional electrophoresis (2DE)-based proteomic approach to examine lung cancer cell H1299 with and without CIP2A. We found 47 proteins differentially expressed where 19 proteins were upregulated and 28 proteins were downregulated. These were categorized into functional groups such as metabolism (25%), transcriptional and translational control (23%), and the signaling pathway and protein degradation (20%). On one hand, we validate our proteomic work by measuring the metabolic change. The knockdown of CIP2A decreased the expression of LDH-A as well as the enzymatic activity, accompanying with a decreased lactate production, an increased NADH/NAD+ ratio and ROS production. On the other hand, we found that CIP2A may regulate CREB activity through bioinformatics analysis. Our following experiments showed that, CIP2A positively regulated the phosphorylation of CREB in response to the serum treatment. Therefore, our proteomic study suggested that CIP2A mediates cancer progression through the metabolic pathway and intracellular signaling cascade. PMID:25325377

  16. The Late S-Phase Transcription Factor Hcm1 Is Regulated through Phosphorylation by the Cell Wall Integrity Checkpoint

    PubMed Central

    Negishi, Takahiro; Veis, Jiri; Hollenstein, David; Sekiya, Mizuho; Ammerer, Gustav

    2016-01-01

    The cell wall integrity (CWI) checkpoint in the budding yeast Saccharomyces cerevisiae coordinates cell wall construction and cell cycle progression. In this study, we showed that the regulation of Hcm1, a late-S-phase transcription factor, arrests the cell cycle via the cell wall integrity checkpoint. Although the HCM1 mRNA level remained unaffected when the cell wall integrity checkpoint was induced, the protein level decreased. The overproduction of Hcm1 resulted in the failure of the cell wall integrity checkpoint. We identified 39 Hcm1 phosphorylation sites, including 26 novel sites, by tandem mass spectrometry analysis. A mutational analysis revealed that phosphorylation of Hcm1 at S61, S65, and S66 is required for the proper onset of the cell wall integrity checkpoint by regulating the timely decrease in its protein level. Hyperactivation of the CWI mitogen-activated protein kinase (MAPK) signaling pathway significantly reduced the Hcm1 protein level, and the deletion of CWI MAPK Slt2 resulted in a failure to decrease Hcm1 protein levels in response to stress, suggesting that phosphorylation is regulated by CWI MAPK. In conclusion, we suggest that Hcm1 is regulated negatively by the cell wall integrity checkpoint through timely phosphorylation and degradation under stress to properly control budding yeast proliferation. PMID:26729465

  17. Bacillus subtilis Two-Component System Sensory Kinase DegS Is Regulated by Serine Phosphorylation in Its Input Domain

    PubMed Central

    Jers, Carsten; Kobir, Ahasanul; Søndergaard, Elsebeth Oline; Jensen, Peter Ruhdal; Mijakovic, Ivan

    2011-01-01

    Bacillus subtilis two-component system DegS/U is well known for the complexity of its regulation. The cytosolic sensory kinase DegS does not receive a single predominant input signal like most two-component kinases, instead it integrates a wide array of metabolic inputs that modulate its activity. The phosphorylation state of the response regulator DegU also does not confer a straightforward “on/off” response; it is fine-tuned and at different levels triggers different sub-regulons. Here we describe serine phosphorylation of the DegS sensing domain, which stimulates its kinase activity. We demonstrate that DegS phosphorylation can be carried out by at least two B. subtilis Hanks-type kinases in vitro, and this stimulates the phosphate transfer towards DegU. The consequences of this process were studied in vivo, using phosphomimetic (Ser76Asp) and non-phosphorylatable (Ser76Ala) mutants of DegS. In a number of physiological assays focused on different processes regulated by DegU, DegS S76D phosphomimetic mutant behaved like a strain with intermediate levels of DegU phosphorylation, whereas DegS S76A behaved like a strain with lower levels of DegU phophorylation. These findings suggest a link between DegS phosphorylation at serine 76 and the level of DegU phosphorylation, establishing this post-translational modification as an additional trigger for this two-component system. PMID:21304896

  18. PKC{delta}-mediated IRS-1 Ser24 phosphorylation negatively regulates IRS-1 function

    SciTech Connect

    Greene, Michael W. . E-mail: michael.greene@bassett.org; Ruhoff, Mary S.; Roth, Richard A.; Kim, Jeong-a; Quon, Michael J.; Krause, Jean A.

    2006-10-27

    The IRS-1 PH and PTB domains are essential for insulin-stimulated IRS-1 Tyr phosphorylation and insulin signaling, while Ser/Thr phosphorylation of IRS-1 disrupts these signaling events. To investigate consensus PKC phosphorylation sites in the PH-PTB domains of human IRS-1, we changed Ser24, Ser58, and Thr191 to Ala (3A) or Glu (3E), to block or mimic phosphorylation, respectively. The 3A mutant abrogated the inhibitory effect of PKC{delta} on insulin-stimulated IRS-1 Tyr phosphorylation, while reductions in insulin-stimulated IRS-1 Tyr phosphorylation, cellular proliferation, and Akt activation were observed with the 3E mutant. When single Glu mutants were tested, the Ser24 to Glu mutant had the greatest inhibitory effect on insulin-stimulated IRS-1 Tyr phosphorylation. PKC{delta}-mediated IRS-1 Ser24 phosphorylation was confirmed in cells with PKC{delta} catalytic domain mutants and by an RNAi method. Mechanistic studies revealed that IRS-1 with Ala and Glu point mutations at Ser24 impaired phosphatidylinositol-4,5-bisphosphate binding. In summary, our data are consistent with the hypothesis that Ser24 is a negative regulatory phosphorylation site in IRS-1.

  19. Tissue Strain Reorganizes Collagen With a Switchlike Response That Regulates Neuronal Extracellular Signal-Regulated Kinase Phosphorylation In Vitro: Implications for Ligamentous Injury and Mechanotransduction.

    PubMed

    Zhang, Sijia; Cao, Xuan; Stablow, Alec M; Shenoy, Vivek B; Winkelstein, Beth A

    2016-02-01

    Excessive loading of ligaments can activate the neural afferents that innervate the collagenous tissue, leading to a host of pathologies including pain. An integrated experimental and modeling approach was used to define the responses of neurons and the surrounding collagen fibers to the ligamentous matrix loading and to begin to understand how macroscopic deformation is translated to neuronal loading and signaling. A neuron-collagen construct (NCC) developed to mimic innervation of collagenous tissue underwent tension to strains simulating nonpainful (8%) or painful ligament loading (16%). Both neuronal phosphorylation of extracellular signal-regulated kinase (ERK), which is related to neuroplasticity (R2 ≥ 0.041; p ≤ 0.0171) and neuronal aspect ratio (AR) (R2 ≥ 0.250; p < 0.0001), were significantly correlated with tissue-level strains. As NCC strains increased during a slowly applied loading (1%/s), a "switchlike" fiber realignment response was detected with collagen reorganization occurring only above a transition point of 11.3% strain. A finite-element based discrete fiber network (DFN) model predicted that at bulk strains above the transition point, heterogeneous fiber strains were both tensile and compressive and increased, with strains in some fibers along the loading direction exceeding the applied bulk strain. The transition point identified for changes in collagen fiber realignment was consistent with the measured strain threshold (11.7% with a 95% confidence interval of 10.2-13.4%) for elevating ERK phosphorylation after loading. As with collagen fiber realignment, the greatest degree of neuronal reorientation toward the loading direction was observed at the NCC distraction corresponding to painful loading. Because activation of neuronal ERK occurred only at strains that produced evident collagen fiber realignment, findings suggest that tissue strain-induced changes in the micromechanical environment, especially altered local

  20. Threonine-408 Regulates the Stability of Human Pregnane X Receptor through Its Phosphorylation and the CHIP/Chaperone-Autophagy Pathway.

    PubMed

    Sugatani, Junko; Noguchi, Yuji; Hattori, Yoshiki; Yamaguchi, Masahiko; Yamazaki, Yasuhiro; Ikari, Akira

    2016-01-01

    The human pregnane X receptor (hPXR) is a xenobiotic-sensing nuclear receptor that transcriptionally regulates drug metabolism-related genes. The aim of the present study was to elucidate the mechanism by which hPXR is regulated through threonine-408. A phosphomimetic mutation at threonine-408 (T408D) reduced the transcriptional activity of hPXR and its protein stability in HepG2 and SW480 cells in vitro and mouse livers in vivo. Proteasome inhibitors (calpain inhibitor I and MG132) and Hsp90 inhibitor geldanamycin, but not Hsp70 inhibitor pifithrin-μ, increased wild-type (WT) hPXR in the nucleus. The translocation of the T408D mutant to the nucleus was significantly reduced even in the presence of proteasome inhibitors, whereas the complex of yellow fluorescent protein (YFP)-hPXR T408D mutant with heat shock cognate protein 70/heat shock protein 70 and carboxy terminus Hsp70-interacting protein (CHIP; E3 ligase) was similar to that of the WT in the cytoplasm. Treatment with pifithrin-μ and transfection with anti-CHIP small-interfering RNA reduced the levels of CYP3A4 mRNA induced by rifampicin, suggesting the contribution of Hsp70 and CHIP to the transactivation of hPXR. Autophagy inhibitor 3-methyladenine accumulated YFP-hPXR T408D mutant more efficiently than the WT in the presence of proteasome inhibitor lactacystin, and the T408D mutant colocalized with the autophagy markers, microtubule-associated protein 1 light chain 3 and p62, which were contained in the autophagic cargo. Lysosomal inhibitor chloroquine caused the marked accumulation of the T408D mutant in the cytoplasm. Protein kinase C (PKC) directly phosphorylated the threonine-408 of hPXR. These results suggest that hPXR is regulated through its phosphorylation at threonine-408 by PKC, CHIP/chaperone-dependent stability check, and autophagic degradation pathway. PMID:26534988

  1. Protein kinase D negatively regulates hepatitis C virus secretion through phosphorylation of oxysterol-binding protein and ceramide transfer protein.

    PubMed

    Amako, Yutaka; Syed, Gulam H; Siddiqui, Aleem

    2011-04-01

    Hepatitis C virus (HCV) RNA replicates its genome on specialized endoplasmic reticulum modified membranes termed membranous web and utilizes lipid droplets for initiating the viral nucleocapsid assembly. HCV maturation and/or the egress pathway requires host sphingolipid synthesis, which occur in the Golgi. Ceramide transfer protein (CERT) and oxysterol-binding protein (OSBP) play a crucial role in sphingolipid biosynthesis. Protein kinase D (PKD), a serine/threonine kinase, is recruited to the trans-Golgi network where it influences vesicular trafficking to the plasma membrane by regulation of several important mediators via phosphorylation. PKD attenuates the function of both CERT and OSBP by phosphorylation at their respective Ser(132) and Ser(240) residues (phosphorylation inhibition). Here, we investigated the functional role of PKD in HCV secretion. Our studies show that HCV gene expression down-regulated PKD activation. PKD depletion by shRNA or inhibition by pharmacological inhibitor Gö6976 enhanced HCV secretion. Overexpression of a constitutively active form of PKD suppressed HCV secretion. The suppression by PKD was subverted by the ectopic expression of nonphosphorylatable serine mutant CERT S132A or OSBP S240A. These observations imply that PKD negatively regulates HCV secretion/release by attenuating OSBP and CERT functions by phosphorylation inhibition. This study identifies the key role of the Golgi components in the HCV maturation process. PMID:21285358

  2. Caveolin-1 binding to endoplasmic reticulum membranes and entry into the regulated secretory pathway are regulated by serine phosphorylation. Protein sorting at the level of the endoplasmic reticulum.

    PubMed

    Schlegel, A; Arvan, P; Lisanti, M P

    2001-02-01

    Caveolin-1 serves as the main coat protein of caveolae membranes, as an intracellular cholesterol shuttle, and as a regulator of diverse signaling molecules. Of the 12 residues conserved across all caveolin isoforms from all species examined to date, only Ser(80) and Ser(168) could serve as phosphorylation sites. We show here that mimicking chronic phosphorylation of Ser(80) by mutation to Glu (i.e. Cav-1(S80E)), blocks phosphate incorporation. However, Cav-1(S168E) is phosphorylated to the same extent as wild-type caveolin-1. Cav-1(S80E) targets to the endoplasmic reticulum membrane, remains oligomeric, and maintains normal membrane topology. In contrast, Cav-1(S80A), which cannot be phosphorylated, targets to caveolae membranes. Some exocrine cells secrete caveolin-1 in a regulated manner. Cav-1(S80A) is not secreted by AR42J pancreatic adenocarcinoma cells even in the presence of dexamethasone, an agent that induces the secretory phenotype. Conversely, Cav-1(S80E) is secreted to a greater extent than wild-type caveolin-1 following dexamethasone treatment. We conclude that caveolin-1 phosphorylation on invariant serine residue 80 is required for endoplasmic reticulum retention and entry into the regulated secretory pathway. PMID:11078729

  3. PI3K/mTORC2 regulates TGF-β/Activin signalling by modulating Smad2/3 activity via linker phosphorylation

    PubMed Central

    Yu, Jason S. L.; Ramasamy, Thamil Selvee; Murphy, Nick; Holt, Marie K.; Czapiewski, Rafal; Wei, Shi-Khai; Cui, Wei

    2015-01-01

    Crosstalk between the phosphatidylinositol 3-kinase (PI3K) and the transforming growth factor-β signalling pathways play an important role in regulating many cellular functions. However, the molecular mechanisms underpinning this crosstalk remain unclear. Here, we report that PI3K signalling antagonizes the Activin-induced definitive endoderm (DE) differentiation of human embryonic stem cells by attenuating the duration of Smad2/3 activation via the mechanistic target of rapamycin complex 2 (mTORC2). Activation of mTORC2 regulates the phosphorylation of the Smad2/3-T220/T179 linker residue independent of Akt, CDK and Erk activity. This phosphorylation primes receptor-activated Smad2/3 for recruitment of the E3 ubiquitin ligase Nedd4L, which in turn leads to their degradation. Inhibition of PI3K/mTORC2 reduces this phosphorylation and increases the duration of Smad2/3 activity, promoting a more robust mesendoderm and endoderm differentiation. These findings present a new and direct crosstalk mechanism between these two pathways in which mTORC2 functions as a novel and critical mediator. PMID:25998442

  4. PI3K/mTORC2 regulates TGF-β/Activin signalling by modulating Smad2/3 activity via linker phosphorylation.

    PubMed

    Yu, Jason S L; Ramasamy, Thamil Selvee; Murphy, Nick; Holt, Marie K; Czapiewski, Rafal; Wei, Shi-Khai; Cui, Wei

    2015-01-01

    Crosstalk between the phosphatidylinositol 3-kinase (PI3K) and the transforming growth factor-β signalling pathways play an important role in regulating many cellular functions. However, the molecular mechanisms underpinning this crosstalk remain unclear. Here, we report that PI3K signalling antagonizes the Activin-induced definitive endoderm (DE) differentiation of human embryonic stem cells by attenuating the duration of Smad2/3 activation via the mechanistic target of rapamycin complex 2 (mTORC2). Activation of mTORC2 regulates the phosphorylation of the Smad2/3-T220/T179 linker residue independent of Akt, CDK and Erk activity. This phosphorylation primes receptor-activated Smad2/3 for recruitment of the E3 ubiquitin ligase Nedd4L, which in turn leads to their degradation. Inhibition of PI3K/mTORC2 reduces this phosphorylation and increases the duration of Smad2/3 activity, promoting a more robust mesendoderm and endoderm differentiation. These findings present a new and direct crosstalk mechanism between these two pathways in which mTORC2 functions as a novel and critical mediator. PMID:25998442

  5. CFTR Cl– channel functional regulation by phosphorylation of focal adhesion kinase at tyrosine 407 in osmosensitive ion transporting mitochondria rich cells of euryhaline killifish

    PubMed Central

    Marshall, William S.; Watters, Kaitlyn D.; Hovdestad, Leah R.; Cozzi, Regina R. F.; Katoh, Fumi

    2009-01-01

    Summary Cystic fibrosis transmembrane conductance regulator (CFTR) anion channels are the regulated exit pathway in Cl– secretion by teleost mitochondria rich salt secreting (MR) cells of the gill and opercular epithelia of euryhaline teleosts. By confocal light immunocytochemistry, immunogold transmission electron microscopy (TEM), and co-immunoprecipitation, using regular and phospho-antibodies directed against conserved sites, we found that killifish CFTR (kfCFTR) and the tyrosine kinase focal adhesion kinase (FAK) phosphorylated at Y407 (FAK pY407) are colocalized in the apical membrane and in subjacent membrane vesicles of MR cells. We showed previously that basolateral FAK pY407, unlike other FAK phosphorylation sites, is osmosensitive and dephosphorylates during hypotonic shock of epithelial cells (Marshall et al., 2008). In the present study, we found that hypotonic shock and the α2-adrenergic agonist clonidine (neither of which affects cAMP levels) rapidly and reversibly inhibit Cl– secretion by isolated opercular membranes, simultaneous with dephosphorylation of FAK pY407, located in the apical membrane. FAK pY407 is rephosphorylated and Cl– secretion rapidly restored by hypertonic shock as well as by forskolin and isoproterenol, which operate via cAMP and protein kinase A. We conclude that hormone mediated, cAMP dependent and osmotically mediated, cAMP independent pathways converge on a mechanism to activate CFTR and Cl– secretion, possibly through tyrosine phosphorylation of CFTR by FAK. PMID:19617429

  6. Cardiac β2-Adrenergic Receptor Phosphorylation at Ser355/356 Regulates Receptor Internalization and Functional Resensitization.

    PubMed

    Fan, Xiaofang; Gu, Xuejiang; Zhao, Ru; Zheng, Qingqing; Li, Lan; Yang, Wenbing; Ding, Lu; Xue, Feng; Fan, Junming; Gong, Yongsheng; Wang, Yongyu

    2016-01-01

    Previous studies have demonstrated that β2-adrenergic receptors (β2ARs) can be phosphorylated by G protein-coupled receptor kinases (GRKs) and protein kinase A (PKA), affecting β2AR internalization and desensitization. However, the exact physiological function of β2ARs in cardiomyocytes is unknown. In this study, we showed that neonatal mouse cardiomyocytes had different contraction and internalization responses to sustained or repeated, transient agonist stimulation. Specifically, short-time stimulation (10 min) with epinephrine or norepinephrine increased the cardiomyocyte contraction rate, reaching a maximum at 5 min, followed by a slow decline. When the agonist was re-added after a 60-min wash-out period, the increase in the cardiomyocyte contraction rate was similar to the initial response. In contrast, when cardiomyocytes were exposed continuously to epinephrine or norepinephrine for 60 min, the second agonist stimulation did not increase the contraction response. These results indicated that continuous β2AR stimulation caused functional desensitization. Phosphorylation of β2ARs at serine (Ser)355/356 GRK phosphorylation sites, but not at Ser345/346 PKA phosphorylation sites increased with continuous epinephrine stimulation for 60 min. Accordingly, β2AR internalization increased. Interestingly, β2AR internalization was blocked by mutations at the GRK phosphorylation sites, but not by mutations at the PKA phosphorylation sites. Furthermore, inhibition of β2AR dephosphorylation by okadaic acid, a phosphatase 2A inhibitor, impaired the recovery of internalized β2ARs and reduced the cardiomyocyte contraction rate in response to epinephrine. Finally, epinephrine treatment induced the physical interaction of β-arrestin with internalized β2ARs in cardiomyocytes. Together, these data revealed the essential role of the Ser355/356 phosphorylation status of β2ARs in regulating receptor internalization and physiological resensitization in neonatal

  7. Characterization of the Effect of the Histidine Kinase CovS on Response Regulator Phosphorylation in Group A Streptococcus

    PubMed Central

    Horstmann, Nicola; Sahasrabhojane, Pranoti; Saldaña, Miguel; Ajami, Nadim J.; Flores, Anthony R.; Sumby, Paul; Liu, Chang-Gong; Yao, Hui; Su, Xiaoping; Thompson, Erika

    2015-01-01

    Two-component gene regulatory systems (TCSs) are a major mechanism by which bacteria respond to environmental stimuli and thus are critical to infectivity. For example, the control of virulence regulator/sensor kinase (CovRS) TCS is central to the virulence of the major human pathogen group A Streptococcus (GAS). Here, we used a combination of quantitative in vivo phosphorylation assays, isoallelic strains that varied by only a single amino acid in CovS, and transcriptome analyses to characterize the impact of CovS on CovR phosphorylation and GAS global gene expression. We discovered that CovS primarily serves to phosphorylate CovR, thereby resulting in the repression of virulence factor-encoding genes. However, a GAS strain selectively deficient in CovS phosphatase activity had a distinct transcriptome relative to that of its parental strain, indicating that both CovS kinase and phosphatase activities influence the CovR phosphorylation status. Surprisingly, compared to a serotype M3 strain, serotype M1 GAS strains had high levels of phosphorylated CovR, low transcript levels of CovR-repressed genes, and strikingly different responses to environmental cues. Moreover, the inactivation of CovS in the serotype M1 background resulted in a greater decrease in phosphorylated CovR levels and a greater increase in the transcript levels of CovR-repressed genes than did CovS inactivation in a serotype M3 strain. These data clarify the influence of CovS on the CovR phosphorylation status and provide insight into why serotype M1 GAS strains have high rates of spontaneous mutations in covS during invasive GAS infection, thus providing a link between TCS molecular function and the epidemiology of deadly bacterial infections. PMID:25561708

  8. Characterization of the effect of the histidine kinase CovS on response regulator phosphorylation in group A Streptococcus.

    PubMed

    Horstmann, Nicola; Sahasrabhojane, Pranoti; Saldaña, Miguel; Ajami, Nadim J; Flores, Anthony R; Sumby, Paul; Liu, Chang-Gong; Yao, Hui; Su, Xiaoping; Thompson, Erika; Shelburne, Samuel A

    2015-03-01

    Two-component gene regulatory systems (TCSs) are a major mechanism by which bacteria respond to environmental stimuli and thus are critical to infectivity. For example, the control of virulence regulator/sensor kinase (CovRS) TCS is central to the virulence of the major human pathogen group A Streptococcus (GAS). Here, we used a combination of quantitative in vivo phosphorylation assays, isoallelic strains that varied by only a single amino acid in CovS, and transcriptome analyses to characterize the impact of CovS on CovR phosphorylation and GAS global gene expression. We discovered that CovS primarily serves to phosphorylate CovR, thereby resulting in the repression of virulence factor-encoding genes. However, a GAS strain selectively deficient in CovS phosphatase activity had a distinct transcriptome relative to that of its parental strain, indicating that both CovS kinase and phosphatase activities influence the CovR phosphorylation status. Surprisingly, compared to a serotype M3 strain, serotype M1 GAS strains had high levels of phosphorylated CovR, low transcript levels of CovR-repressed genes, and strikingly different responses to environmental cues. Moreover, the inactivation of CovS in the serotype M1 background resulted in a greater decrease in phosphorylated CovR levels and a greater increase in the transcript levels of CovR-repressed genes than did CovS inactivation in a serotype M3 strain. These data clarify the influence of CovS on the CovR phosphorylation status and provide insight into why serotype M1 GAS strains have high rates of spontaneous mutations in covS during invasive GAS infection, thus providing a link between TCS molecular function and the epidemiology of deadly bacterial infections. PMID:25561708

  9. Papaverine Prevents Vasospasm by Regulation of Myosin Light Chain Phosphorylation and Actin Polymerization in Human Saphenous Vein

    PubMed Central

    Hocking, Kyle M.; Putumbaka, Gowthami; Wise, Eric S.; Cheung-Flynn, Joyce; Brophy, Colleen M.; Komalavilas, Padmini

    2016-01-01

    Objective Papaverine is used to prevent vasospasm in human saphenous veins (HSV) during vein graft preparation prior to implantation as a bypass conduit. Papaverine is a nonspecific inhibitor of phosphodiesterases, leading to increases in both intracellular cGMP and cAMP. We hypothesized that papaverine reduces force by decreasing intracellular calcium concentrations ([Ca2+]i) and myosin light chain phosphorylation, and increasing actin depolymerization via regulation of actin regulatory protein phosphorylation. Approach and Results HSV was equilibrated in a muscle bath, pre-treated with 1 mM papaverine followed by 5 μM norepinephrine, and force along with [Ca2+]i levels were concurrently measured. Filamentous actin (F-actin) level was measured by an in vitro actin assay. Tissue was snap frozen to measure myosin light chain and actin regulatory protein phosphorylation. Pre-treatment with papaverine completely inhibited norepinephrine-induced force generation, blocked increases in [Ca2+]i and led to a decrease in the phosphorylation of myosin light chain. Papaverine pre-treatment also led to increased phosphorylation of the heat shock-related protein 20 (HSPB6) and the vasodilator stimulated phosphoprotein (VASP), as well as decreased filamentous actin (F-actin) levels suggesting depolymerization of actin. Conclusions These results suggest that papaverine-induced force inhibition of HSV involves [Ca2+]i-mediated inhibition of myosin light chain phosphorylation and actin regulatory protein phosphorylation-mediated actin depolymerization. Thus, papaverine induces sustained inhibition of contraction of HSV by the modulation of both myosin cross-bridge formation and actin cytoskeletal dynamics and is a pharmacological alternative to high pressure distention to prevent vasospasm. PMID:27136356

  10. Cardiac β2-Adrenergic Receptor Phosphorylation at Ser355/356 Regulates Receptor Internalization and Functional Resensitization

    PubMed Central

    Zhao, Ru; Zheng, Qingqing; Li, Lan; Yang, Wenbing; Ding, Lu; Xue, Feng; Fan, Junming; Gong, Yongsheng

    2016-01-01

    Previous studies have demonstrated that β2-adrenergic receptors (β2ARs) can be phosphorylated by G protein-coupled receptor kinases (GRKs) and protein kinase A (PKA), affecting β2AR internalization and desensitization. However, the exact physiological function of β2ARs in cardiomyocytes is unknown. In this study, we showed that neonatal mouse cardiomyocytes had different contraction and internalization responses to sustained or repeated, transient agonist stimulation. Specifically, short-time stimulation (10 min) with epinephrine or norepinephrine increased the cardiomyocyte contraction rate, reaching a maximum at 5 min, followed by a slow decline. When the agonist was re-added after a 60-min wash-out period, the increase in the cardiomyocyte contraction rate was similar to the initial response. In contrast, when cardiomyocytes were exposed continuously to epinephrine or norepinephrine for 60 min, the second agonist stimulation did not increase the contraction response. These results indicated that continuous β2AR stimulation caused functional desensitization. Phosphorylation of β2ARs at serine (Ser)355/356 GRK phosphorylation sites, but not at Ser345/346 PKA phosphorylation sites increased with continuous epinephrine stimulation for 60 min. Accordingly, β2AR internalization increased. Interestingly, β2AR internalization was blocked by mutations at the GRK phosphorylation sites, but not by mutations at the PKA phosphorylation sites. Furthermore, inhibition of β2AR dephosphorylation by okadaic acid, a phosphatase 2A inhibitor, impaired the recovery of internalized β2ARs and reduced the cardiomyocyte contraction rate in response to epinephrine. Finally, epinephrine treatment induced the physical interaction of β-arrestin with internalized β2ARs in cardiomyocytes. Together, these data revealed the essential role of the Ser355/356 phosphorylation status of β2ARs in regulating receptor internalization and physiological resensitization in neonatal

  11. Phosphorylation of LRRK2 by casein kinase 1α regulates trans-Golgi clustering via differential interaction with ARHGEF7

    PubMed Central

    Chia, Ruth; Haddock, Sara; Beilina, Alexandra; Rudenko, Iakov N; Mamais, Adamantios; Kaganovich, Alice; Li, Yan; Kumaran, Ravindran; Nalls, Michael A; Cookson, Mark R

    2014-01-01

    LRRK2, a gene relevant to Parkinson's disease, encodes a scaffolding protein with both GTPase and kinase activities. LRRK2 protein is itself phosphorylated and therefore subject to regulation by cell signaling but the kinase(s) responsible for this event have not been definitively identified. Here, using an unbiased siRNA kinome screen, we identify and validate casein kinase 1α (CK1α) as being responsible for LRRK2 phosphorylation, including in the adult mouse striatum. We further show that LRRK2 recruitment to TGN46-positive Golgi-derived vesicles is modulated by constitutive LRRK2 phosphorylation by CK1α. These effects are mediated by differential protein interactions of LRRK2 with a guanine nucleotide exchange factor, ARHGEF7. These pathways are therefore likely involved in the physiological maintenance of the Golgi in cells, which may play a role in the pathogenesis of Parkinson's disease. PMID:25500533

  12. Calcium-regulated in vivo protein phosphorylation in Zea mays L. root tips

    NASA Technical Reports Server (NTRS)

    Raghothama, K. G.; Reddy, A. S.; Friedmann, M.; Poovaiah, B. W.

    1987-01-01

    Calcium dependent protein phosphorylation was studied in corn (Zea mays L.) root tips. Prior to in vivo protein phosphorylation experiments, the effect of calcium, ethyleneglycol-bis-(beta-aminoethyl ether)-N-N' -tetraacetic acid (EGTA) and calcium ionophore (A-23187) on phosphorus uptake was studied. Calcium increased phosphorus uptake, whereas EGTA and A-23187 decreased it. Consequently, phosphorus concentration in the media was adjusted so as to attain similar uptake in different treatments. Phosphoproteins were analyzed by two-dimensional gel electrophoresis. Distinct changes in phosphorylation were observed following altered calcium levels. Calcium depletion in root tips with EGTA and A-23187 decreased protein phosphorylation. However, replenishment of calcium following EGTA and ionophore pretreatment enhanced phosphorylation of proteins. Preloading of the root tips with 32P in the presence of EGTA and A-23187 followed by a ten minute calcium treatment, resulted in increased phosphorylation indicating the involvement of calcium, calcium and calmodulin-dependent kinases. Calmodulin antagonist W-7 was effective in inhibiting calcium-promoted phosphorylation. These studies suggest a physiological role for calcium-dependent phosphorylation in calcium-mediated processes in plants.

  13. Phosphorylation-regulated Nucleocytoplasmic Trafficking of Internalized Fibroblast Growth Factor-1

    PubMed Central

    Więdłocha, Antoni; Nilsen, Trine; Wesche, Jørgen; Sørensen, Vigdis; Małecki, Jędrzej; Marcinkowska, Ewa; Olsnes, Sjur

    2005-01-01

    Fibroblast growth factor-1 (FGF-1), which stimulates cell growth, differentiation, and migration, is capable of crossing cellular membranes to reach the cytosol and the nucleus in cells containing specific FGF receptors. The cell entry process can be monitored by phosphorylation of the translocated FGF-1. We present evidence that phosphorylation of FGF-1 occurs in the nucleus by protein kinase C (PKC)δ. The phosphorylated FGF-1 is subsequently exported to the cytosol. A mutant growth factor where serine at the phosphorylation site is exchanged with glutamic acid, to mimic phosphorylated FGF-1, is constitutively transported to the cytosol, whereas a mutant containing alanine at this site remains in the nucleus. The export can be blocked by leptomycin B, indicating active and receptor-mediated nuclear export of FGF-1. Thapsigargin, but not leptomycin B, prevents the appearance of active PKCδ in the nucleus, and FGF-1 is in this case phosphorylated in the cytosol. Leptomycin B increases the amount of phosphorylated FGF-1 in the cells by preventing dephosphorylation of the growth factor, which seems to occur more rapidly in the cytoplasm than in the nucleus. The nucleocytoplasmic trafficking of the phosphorylated growth factor is likely to play a role in the activity of internalized FGF-1. PMID:15574884

  14. Phosphorylation of the Synaptonemal Complex Protein Zip1 Regulates the Crossover/Noncrossover Decision during Yeast Meiosis

    PubMed Central

    Chen, Xiangyu; Suhandynata, Ray T.; Sandhu, Rima; Rockmill, Beth; Mohibullah, Neeman; Niu, Hengyao; Liang, Jason; Lo, Hsiao-Chi; Miller, Danny E.; Zhou, Huilin; Börner, G. Valentin; Hollingsworth, Nancy M.

    2015-01-01

    Interhomolog crossovers promote proper chromosome segregation during meiosis and are formed by the regulated repair of programmed double-strand breaks. This regulation requires components of the synaptonemal complex (SC), a proteinaceous structure formed between homologous chromosomes. In yeast, SC formation requires the “ZMM” genes, which encode a functionally diverse set of proteins, including the transverse filament protein, Zip1. In wild-type meiosis, Zmm proteins promote the biased resolution of recombination intermediates into crossovers that are distributed throughout the genome by interference. In contrast, noncrossovers are formed primarily through synthesis-dependent strand annealing mediated by the Sgs1 helicase. This work identifies a conserved region on the C terminus of Zip1 (called Zip1 4S), whose phosphorylation is required for the ZMM pathway of crossover formation. Zip1 4S phosphorylation is promoted both by double-strand breaks (DSBs) and the meiosis-specific kinase, MEK1/MRE4, demonstrating a role for MEK1 in the regulation of interhomolog crossover formation, as well as interhomolog bias. Failure to phosphorylate Zip1 4S results in meiotic prophase arrest, specifically in the absence of SGS1. This gain of function meiotic arrest phenotype is suppressed by spo11Δ, suggesting that it is due to unrepaired breaks triggering the meiotic recombination checkpoint. Epistasis experiments combining deletions of individual ZMM genes with sgs1-md zip1-4A indicate that Zip1 4S phosphorylation functions prior to the other ZMMs. These results suggest that phosphorylation of Zip1 at DSBs commits those breaks to repair via the ZMM pathway and provides a mechanism by which the crossover/noncrossover decision can be dynamically regulated during yeast meiosis. PMID:26682552

  15. CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor i with chromatin

    PubMed Central

    Jeffery, Daniel CB; Kakusho, Naoko; You, Zhiying; Gharib, Marlene; Wyse, Brandon; Drury, Erin; Weinreich, Michael; Thibault, Pierre; Verreault, Alain; Masai, Hisao; Yankulov, Krassimir

    2015-01-01

    Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase CDC7. Here we show that another kinase, CDC28, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of CDC28 but not of CDC7 substantially reduce the in vivo phosphorylation of Cac1p. However, mutations in the putative CDC7 target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a cdc28–1 mutant and to a lesser extent in a cdc7–1 mutant. In addition, mutations in the Cac1p-phosphorylation sites by both CDC28 and CDC7 reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate CDC28 in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly. PMID:25602519

  16. CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor I with chromatin.

    PubMed

    Jeffery, Daniel C B; Kakusho, Naoko; You, Zhiying; Gharib, Marlene; Wyse, Brandon; Drury, Erin; Weinreich, Michael; Thibault, Pierre; Verreault, Alain; Masai, Hisao; Yankulov, Krassimir

    2015-01-01

    Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase CDC7. Here we show that another kinase, CDC28, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of CDC28 but not of CDC7 substantially reduce the in vivo phosphorylation of Cac1p. However, mutations in the putative CDC7 target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a cdc28-1 mutant and to a lesser extent in a cdc7-1 mutant. In addition, mutations in the Cac1p-phosphorylation sites by both CDC28 and CDC7 reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate CDC28 in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly. PMID:25602519

  17. Cystic fibrosis transmembrane conductance regulator Cl- channels with R domain deletions and translocations show phosphorylation-dependent and -independent activity.

    PubMed

    Baldursson, O; Ostedgaard, L S; Rokhlina, T; Cotten, J F; Welsh, M J

    2001-01-19

    Phosphorylation of the R domain regulates cystic fibrosis transmembrane conductance regulator Cl- channel activity. Earlier studies suggested that the R domain controls activity via more than one mechanism; a phosphorylated R domain may stimulate activity, and an unphosphorylated R domain may prevent constitutive activity, i.e. opening with ATP alone. However, the mechanisms responsible for these two regulatory properties are not understood. In this study we asked whether the two effects are dependent on its position in the protein and whether smaller regions from the R domain mediate the effects. We found that several portions of the R domain conferred phosphorylation-stimulated activity. This was true whether the R domain sequences were present in their normal location or were translocated to the C terminus. We also found that some parts of the R domain could be deleted without inducing constitutive activity. However, when residues 760-783 were deleted, channels opened without phosphorylation. Translocation of the R domain to the C terminus did not prevent constitutive activity. These results suggest that different parts of the phosphorylated R domain can stimulate activity and that their location within the protein is not critical. In contrast, prevention of constitutive activity required a short specific sequence that could not be moved to the C terminus. These results are consistent with a recent model of an R domain composed primarily of random coil in which more than one phosphorylation site is capable of stimulating channel activity, and net activity reflects interactions between multiple sites in the R domain and the rest of the channel. PMID:11038358

  18. Phosphorylation of Serine Residues in the C-terminal Cytoplasmic Tail of Connexin43 Regulates Proliferation of Ovarian Granulosa Cells

    PubMed Central

    Dyce, Paul W.; Norris, Rachael P.; Lampe, Paul D.; Kidder, Gerald M.

    2013-01-01

    Connexin43 (Cx43) forms 22 s gap junctions that couple the granulosa cells of ovarian follicles. In Cx43 knockout mice, follicle growth is restricted due to impaired granulosa cell proliferation. We have used these mice to examine the importance of specific Cx43 phosphorylation sites in follicle growth. Serines at residues 255, 262, 279 and 282 are MAP kinase substrates that, when phosphorylated, reduce junctional conductance. Mutant forms of Cx43 were constructed with these serines replaced with amino acids that cannot be phosphorylated. These mutants were transduced into Cx43 knockout ovarian somatic cells which were combined with wildtype oocytes and grafted into immunocompromised female mice permitting follicle growth in vivo. Despite residues 255 or 262 being mutated to prevent their being phosphorylated, recombinant ovaries constructed with these mutants were able to rescue the null phenotype, restoring complete folliculogenesis. In contrast, Cx43 with serine to alanine mutations at both residues 279 and 282 or at all four residues failed to rescue folliculogenesis; the mutant molecules were largely confined to intracellular sites, with few gap junctions. Using an in vitro proliferation assay, we confirmed a decrease in proliferation of granulosa cells expressing the double mutant construct. These results indicate that Cx43 phosphorylation by MAP kinase at serines 279 and 282 occurs in granulosa cells of early follicles and that this is involved in regulating follicle development. PMID:22729691

  19. MYC Phosphorylation, Activation, and Tumorigenic Potential in Hepatocellular Carcinoma are Regulated by HMG-CoA Reductase

    PubMed Central

    Cao, Zhongwei; Fan-Minogue, Hua; Bellovin, David I.; Yevtodiyenko, Aleksey; Arzeno, Julia; Yang, Qiwei; Gambhir, Sanjiv Sam; Felsher, Dean W.

    2011-01-01

    MYC is a potential target for many cancers but is not amenable to existing pharmacological approaches. Inhibition of HMG-CoA reductase by statins has shown potential efficacy against a number of cancers. Here, we demonstrate that inhibition of HMG-CoA reductase by atorvastatin blocks both MYC phosphorylation and activation, suppressing tumor initiation and growth in vivo in a transgenic model of MYC-induced HCC as well as in human HCC-derived cell lines. To confirm specificity, we show that the anti-tumor effects of atorvastatin are blocked by co-treatment with the HMG-CoA reductase product, Mevalonate. Moreover, by using a novel molecular imaging sensor, we confirm that inhibition of HMG-CoA reductase blocks MYC phosphorylation in vivo. Importantly, the introduction of phosphorylation mutants of MYC at Ser62 or Thr58 into tumors blocks their sensitivity to inhibition of HMG-CoA reductase. Finally, we demonstrate that inhibition of HMG-CoA reductase suppresses MYC phosphorylation through Rac GTPase. Therefore, HMG-CoA reductase is a critical regulator of MYC phosphorylation, activation, and tumorigenic properties. The inhibition of HMG-CoA reductase may be a useful target for the treatment of MYC-associated HCC as well as other tumors. PMID:21262914

  20. The condensing activities of the Mycobacterium tuberculosis type II fatty acid synthase are differentially regulated by phosphorylation.

    PubMed

    Molle, Virginie; Brown, Alistair K; Besra, Gurdyal S; Cozzone, Alain J; Kremer, Laurent

    2006-10-01

    Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently become of major physiological importance because of its possible involvement in virulence of bacterial pathogens. Although Mycobacterium tuberculosis has eleven STPKs, the nature and function of the substrates of these enzymes remain largely unknown. In this work, we have identified for the first time STPK substrates in M. tuberculosis forming part of the type II fatty acid synthase (FAS-II) system involved in mycolic acid biosynthesis: the malonyl-CoA::AcpM transacylase mtFabD, and the beta-ketoacyl AcpM synthases KasA and KasB. All three enzymes were phosphorylated in vitro by different kinases, suggesting a complex network of interactions between STPKs and these substrates. In addition, both KasA and KasB were efficiently phosphorylated in M. bovis BCG each at different sites and could be dephosphorylated by the M. tuberculosis Ser/Thr phosphatase PstP. Enzymatic studies revealed that, whereas phosphorylation decreases the activity of KasA in the elongation process of long chain fatty acids synthesis, this modification enhances that of KasB. Such a differential effect of phosphorylation may represent an unusual mechanism of FAS-II system regulation, allowing pathogenic mycobacteria to produce full-length mycolates, which are required for adaptation and intracellular survival in macrophages. PMID:16873379

  1. Cytoplasmic cyclin D1 regulates cell invasion and metastasis through the phosphorylation of paxillin.

    PubMed

    Fusté, Noel P; Fernández-Hernández, Rita; Cemeli, Tània; Mirantes, Cristina; Pedraza, Neus; Rafel, Marta; Torres-Rosell, Jordi; Colomina, Neus; Ferrezuelo, Francisco; Dolcet, Xavier; Garí, Eloi

    2016-01-01

    Cyclin D1 (Ccnd1) together with its binding partner Cdk4 act as a transcriptional regulator to control cell proliferation and migration, and abnormal Ccnd1·Cdk4 expression promotes tumour growth and metastasis. While different nuclear Ccnd1·Cdk4 targets participating in cell proliferation and tissue development have been identified, little is known about how Ccnd1·Cdk4 controls cell adherence and invasion. Here, we show that the focal adhesion component paxillin is a cytoplasmic substrate of Ccnd1·Cdk4. This complex phosphorylates a fraction of paxillin specifically associated to the cell membrane, and promotes Rac1 activation, thereby triggering membrane ruffling and cell invasion in both normal fibroblasts and tumour cells. Our results demonstrate that localization of Ccnd1·Cdk4 to the cytoplasm does not simply act to restrain cell proliferation, but constitutes a functionally relevant mechanism operating under normal and pathological conditions to control cell adhesion, migration and metastasis through activation of a Ccnd1·Cdk4-paxillin-Rac1 axis. PMID:27181366

  2. Cytoplasmic cyclin D1 regulates cell invasion and metastasis through the phosphorylation of paxillin

    PubMed Central

    Fusté, Noel P.; Fernández-Hernández, Rita; Cemeli, Tània; Mirantes, Cristina; Pedraza, Neus; Rafel, Marta; Torres-Rosell, Jordi; Colomina, Neus; Ferrezuelo, Francisco; Dolcet, Xavier; Garí, Eloi

    2016-01-01

    Cyclin D1 (Ccnd1) together with its binding partner Cdk4 act as a transcriptional regulator to control cell proliferation and migration, and abnormal Ccnd1·Cdk4 expression promotes tumour growth and metastasis. While different nuclear Ccnd1·Cdk4 targets participating in cell proliferation and tissue development have been identified, little is known about how Ccnd1·Cdk4 controls cell adherence and invasion. Here, we show that the focal adhesion component paxillin is a cytoplasmic substrate of Ccnd1·Cdk4. This complex phosphorylates a fraction of paxillin specifically associated to the cell membrane, and promotes Rac1 activation, thereby triggering membrane ruffling and cell invasion in both normal fibroblasts and tumour cells. Our results demonstrate that localization of Ccnd1·Cdk4 to the cytoplasm does not simply act to restrain cell proliferation, but constitutes a functionally relevant mechanism operating under normal and pathological conditions to control cell adhesion, migration and metastasis through activation of a Ccnd1·Cdk4-paxillin-Rac1 axis. PMID:27181366

  3. Cell-cycle dependent phosphorylation of yeast pericentrin regulates γ-TuSC-mediated microtubule nucleation.

    PubMed

    Lin, Tien-Chen; Neuner, Annett; Schlosser, Yvonne T; Scharf, Annette N D; Weber, Lisa; Schiebel, Elmar

    2014-01-01

    Budding yeast Spc110, a member of γ-tubulin complex receptor family (γ-TuCR), recruits γ-tubulin complexes to microtubule (MT) organizing centers (MTOCs). Biochemical studies suggest that Spc110 facilitates higher-order γ-tubulin complex assembly (Kollman et al., 2010). Nevertheless the molecular basis for this activity and the regulation are unclear. Here we show that Spc110 phosphorylated by Mps1 and Cdk1 activates γ-TuSC oligomerization and MT nucleation in a cell cycle dependent manner. Interaction between the N-terminus of the γ-TuSC subunit Spc98 and Spc110 is important for this activity. Besides the conserved CM1 motif in γ-TuCRs (Sawin et al., 2004), a second motif that we named Spc110/Pcp1 motif (SPM) is also important for MT nucleation. The activating Mps1 and Cdk1 sites lie between SPM and CM1 motifs. Most organisms have both SPM-CM1 (Spc110/Pcp1/PCNT) and CM1-only (Spc72/Mto1/Cnn/CDK5RAP2/myomegalin) types of γ-TuCRs. The two types of γ-TuCRs contain distinct but conserved C-terminal MTOC targeting domains.DOI: http://dx.doi.org/10.7554/eLife.02208.001. PMID:24842996

  4. ATR-mediated phosphorylation of FANCI regulates dormant origin firing in response to replication stress

    PubMed Central

    Chen, Yu-Hung; Jones, Mathew J. K.; Yin, Yandong; Crist, Sarah B.; Colnaghi, Luca; Sims, Robert J.; Rothenberg, Eli; Jallepalli, Prasad V.; Huang, Tony T.

    2015-01-01

    SUMMARY Excess dormant origins bound by the minichromosome maintenance (MCM) replicative helicase complex play a critical role in preventing replication stress, chromosome instability and tumorigenesis. In response to DNA damage, replicating cells must coordinate DNA repair and dormant origin firing to ensure complete and timely replication of the genome; how cells regulate this process remains elusive. Herein, we identify a member of the Fanconi Anemia (FA) DNA repair pathway, FANCI, as a key effector of dormant origin firing in response to replication stress. Cells lacking FANCI have reduced number of origins, increased inter-origin distances and slowed proliferation rates. Intriguingly, ATR-mediated FANCI phosphorylation inhibits dormant origin firing while promoting replication fork restart/DNA repair. Using super-resolution microscopy, we show that FANCI co-localizes with MCM-bound chromatin in response to replication stress. These data reveal a unique role for FANCI as a modulator of dormant origin firing and links timely genome replication to DNA repair. PMID:25843623

  5. PKD2 and RSK1 Regulate Integrin β4 Phosphorylation at Threonine 1736

    PubMed Central

    te Molder, Lisa; Sonnenberg, Arnoud

    2015-01-01

    The integrin α6β4, a major component of hemidesmosomes (HDs), stabilizes keratinocyte cell adhesion to the epidermal basement membrane through binding to the cytoskeletal linker protein plectin and association with keratin filaments. Disruption of the α6β4-plectin interaction through phosphorylation of the β4 subunit results in a reduction in adhesive strength of keratinocytes to laminin-332 and the dissolution of HDs. Previously, we have demonstrated that phosphorylation of T1736 in the C-terminal end of the β4 cytoplasmic domain disrupts the interaction of β4 with the plakin domain of plectin. Furthermore, we showed that β4-T1736 can be phosphorylated by PKD1 in vitro, and although both PMA and EGF induced T1736 phosphorylation, only PMA was able to activate PKD1. Here, we show that depletion of [Ca2+]i augments PMA- and EGF-induced phosphorylation of β4-T1736 and that this is caused by inhibition of the calcium-sensitive protein phosphatase calcineurin and augmentation of ERK1/2 activation. We also show that in keratinocytes the PMA-stimulated phosphorylation of β4-T1736 primarily is mediated by PKD2 activation downstream of PKCδ. On the other hand, both the EGF-stimulated phosphorylation of T1736 and the EGF-induced dissolution of HDs are dependent on a functional MAPK signaling pathway, and treatment with the RSK inhibitor BI-D1870 prevented EGF-stimulated phosphorylation of β4-T1736. Moreover, phosphorylation of β4-T1736 is enhanced by overexpression of wild-type RSK1, while it is reduced by the expression of kinase-inactive RSK1 or by siRNA-mediated depletion of RSK1. In summary, our data indicate that different stimuli can lead to the phosphorylation of β4-T1736 by either PKD2 or RSK1. PMID:26580203

  6. Binding to Syntenin-1 Protein Defines a New Mode of Ubiquitin-based Interactions Regulated by Phosphorylation*

    PubMed Central

    Rajesh, Sundaresan; Bago, Ružica; Odintsova, Elena; Muratov, Gayrat; Baldwin, Gouri; Sridhar, Pooja; Rajesh, Sandya; Overduin, Michael; Berditchevski, Fedor

    2011-01-01

    Syntenin-1 is a PDZ domain-containing adaptor that controls trafficking of transmembrane proteins including those associated with tetraspanin-enriched microdomains. We describe the interaction of syntenin-1 with ubiquitin through a novel binding site spanning the C terminus of ubiquitin, centered on Arg72, Leu73, and Arg74. A conserved LYPSL sequence in the N terminus, as well as the C-terminal region of syntenin-1, are essential for binding to ubiquitin. We present evidence for the regulation of this interaction through syntenin-1 dimerization. We have also established that syntenin-1 is phosphorylated downstream of Ulk1, a serine/threonine kinase that plays a critical role in autophagy and regulates endocytic trafficking. Importantly, Ulk1-dependent phosphorylation of Ser6 in the LYPSL prevents the interaction of syntenin-1 with ubiquitin. These results define an unprecedented ubiquitin-dependent pathway involving syntenin-1 that is regulated by Ulk1. PMID:21949238

  7. Rad9 BRCT domain interaction with phosphorylated H2AX regulates the G1 checkpoint in budding yeast.

    PubMed

    Hammet, Andrew; Magill, Christine; Heierhorst, Jörg; Jackson, Stephen P

    2007-09-01

    Phosphorylation of histone H2A or H2AX is an early and sensitive marker of DNA damage in eukaryotic cells, although mutation of the conserved damage-dependent phosphorylation site is well tolerated. Here, we show that H2A phosphorylation is required for cell-cycle arrest in response to DNA damage at the G1/S transition in budding yeast. Furthermore, we show that the tandem BRCT domain of Rad9 interacts directly with phosphorylated H2A in vitro and that a rad9 point mutation that abolishes this interaction results in in vivo phenotypes that are similar to those caused by an H2A phosphorylation site mutation. Remarkably, similar checkpoint defects are also caused by a Rad9 Tudor domain mutation that impairs Rad9 chromatin association already in undamaged cells. These findings indicate that constitutive Tudor domain-mediated and damage-specific BRCT domain-phospho-H2A-dependent interactions of Rad9 with chromatin cooperate to establish G1 checkpoint arrest. PMID:17721446

  8. Rad9 BRCT domain interaction with phosphorylated H2AX regulates the G1 checkpoint in budding yeast

    PubMed Central

    Hammet, Andrew; Magill, Christine; Heierhorst, Jörg; Jackson, Stephen P

    2007-01-01

    Phosphorylation of histone H2A or H2AX is an early and sensitive marker of DNA damage in eukaryotic cells, although mutation of the conserved damage-dependent phosphorylation site is well tolerated. Here, we show that H2A phosphorylation is required for cell-cycle arrest in response to DNA damage at the G1/S transition in budding yeast. Furthermore, we show that the tandem BRCT domain of Rad9 interacts directly with phosphorylated H2A in vitro and that a rad9 point mutation that abolishes this interaction results in in vivo phenotypes that are similar to those caused by an H2A phosphorylation site mutation. Remarkably, similar checkpoint defects are also caused by a Rad9 Tudor domain mutation that impairs Rad9 chromatin association already in undamaged cells. These findings indicate that constitutive Tudor domain-mediated and damage-specific BRCT domain–phospho-H2A-dependent interactions of Rad9 with chromatin cooperate to establish G1 checkpoint arrest. PMID:17721446

  9. Yeast GSK-3 kinase regulates astral microtubule function through phosphorylation of the microtubule-stabilizing kinesin Kip2.

    PubMed

    Drechsler, Hauke; Tan, Ann Na; Liakopoulos, Dimitris

    2015-11-01

    The S. cerevisiae kinesin Kip2 stabilises astral microtubules (MTs) and facilitates spindle positioning through transport of MT-associated proteins, such as the yeast CLIP-170 homologue Bik1, dynein and the adenomatous-polyposis-coli-related protein Kar9 to the plus ends of astral MTs. Here, we show that Kip2 associates with its processivity factor Bim1, the yeast homologue of the plus-end-tracking protein EB1. This interaction requires an EB1-binding motif in the N-terminal extension of Kip2 and is negatively regulated by phosphorylation through Mck1, the yeast glycogen synthase kinase 3. In addition, Mck1-dependent phosphorylation decreases the intrinsic MT affinity of Kip2. Reduction in Kip2 phosphorylation leads to stabilisation of astral MTs, and accumulation of Kip2, dynein and Kar9 at MT plus ends, whereas loss of Mck1 function leads to defects in spindle positioning. Furthermore, we provide evidence that a subpopulation of Mck1 at the bud-cortex phosphorylates Kip2. We propose that yeast GSK-3 spatially controls astral MT dynamics and the loading of dynein and Kar9 on astral MT plus ends by regulating Kip2 interactions with Bim1 and MTs. PMID:26395399

  10. Phosphorylation of Ser-180 of rat aquaporin-4 shows marginal affect on regulation of water permeability: molecular dynamics study.

    PubMed

    Sachdeva, Ruchi; Singh, Balvinder

    2014-04-01

    Water permeation through rat aquaporin-4 (rAQP4), predominantly found in mammalian brain is regulated by phosphorylation of Ser-180. The present study has been carried out to understand the structural mechanism of regulation of water permeability across the channel. Molecular dynamics (MD) simulations have been carried out to investigate the structural changes caused due to phosphorylation of Ser-180 in the tetrameric assembly of rAQP4 along with predicted C-terminal region (255-323). The interactions involving opposite charges are observed between cytoplasmic loops and the C-terminal region during MD simulations. This results in movement of C-terminal region of rAQP4 towards the cytoplasmic mouth of water channel. Despite this movement, there was a gap between C-terminal region and cytoplasmic mouth of the channel through which water molecules were able to gain entry into the channel. The interactions between C-terminus and loop D of neighboring monomers in a tetrameric assembly appear to prevent the complete closure of cytoplasmic mouth of the water channel. Further, the rates of water permeation through phosphorylated and unphosphorylated rAQP4 have also been compared. The simulation studies showed a continuous movement of water in a single file across pore of unphosphorylated as well as phosphorylated rAQP4. PMID:23651078

  11. Brefeldin A-inhibited guanine exchange factor 2 regulates filamin A phosphorylation and neuronal migration.

    PubMed

    Zhang, Jingping; Neal, Jason; Lian, Gewei; Shi, Bingxing; Ferland, Russell J; Sheen, Volney

    2012-09-01

    Periventricular heterotopia (PH) is a human malformation of cortical development associated with gene mutations in ADP-ribosylation factor guanine exchange factor 2 (ARFGEF2 encodes for Big2 protein) and Filamin A (FLNA). PH is thought to derive from neuroependymal disruption, but the extent to which neuronal migration contributes to this phenotype is unknown. Here, we show that Arfgef2 null mice develop PH and exhibit impaired neural migration with increased protein expression for both FlnA and phosphoFlnA at Ser2152. Big2 physically interacts with FlnA and overexpression of phosphomimetic Ser2512 FLNA impairs neuronal migration. FlnA phosphorylation directs FlnA localization toward the cell cytoplasm, diminishes its binding affinity to actin skeleton, and alters the number and size of paxillin focal adhesions. Collectively, our results demonstrate a molecular mechanism whereby Big2 inhibition promotes phosphoFlnA (Ser2152) expression, and increased phosphoFlnA impairs its actin binding affinity and the distribution of focal adhesions, thereby disrupting cell intrinsic neuronal migration. PMID:22956851

  12. Negative control of circadian clock regulator E4BP4 by casein kinase Iepsilon-mediated phosphorylation.

    PubMed

    Doi, Masao; Okano, Toshiyuki; Yujnovsky, Irene; Sassone-Corsi, Paolo; Fukada, Yoshitaka

    2004-06-01

    Light-dependent transcriptional regulation of clock genes is a crucial step in the entrainment of the circadian clock. E4bp4 is a light-inducible gene in the chick pineal gland, and it encodes a bZIP protein that represses transcription of cPer2, a chick pineal clock gene. Here, we demonstrate that prolonged light period-dependent accumulation of E4BP4 protein is temporally coordinated with a delay of the rising phase of cPer2 in the morning. E4BP4 was phosphorylated progressively and then disappeared in parallel with induced cPer2 expression. Characterization of E4BP4 revealed Ser182, a phosphoacceptor site located at the amino-terminal border of the Ser/Thr cluster, which forms the phosphorylation motifs for casein kinase 1epsilon (CK1epsilon). CK1epsilon physically associated with E4BP4 and phosphorylated it. CK1epsilon-catalyzed phosphorylation of E4BP4 resulted in proteasomal proteolysis-dependent decrease of E4BP4 levels, while E4BP4 nuclear accumulation was attenuated by CK1epsilon in a kinase activity-independent manner. CK1epsilon-mediated posttranslational regulation was accompanied by reduction of the transcriptional repression executed by E4BP4. These results not only demonstrate a phosphorylation-dependent regulatory mechanism for E4BP4 function but also highlight the role of CK1epsilon as a negative regulator for E4BP4-mediated repression of cPer2. PMID:15182670

  13. Profilin-1 Phosphorylation Directs Angiocrine Expression and Glioblastoma Progression through HIF-1α Accumulation

    PubMed Central

    Fan, Yi; Potdar, Alka A.; Gong, Yanqing; Eswarappa, Sandeepa M.; Donnola, Shannon; Lathia, Justin D.; Hambardzumyan, Dolores; Rich, Jeremy N.; Fox, Paul L.

    2014-01-01

    SUMMARY The tumor vascular microenvironment supports tumorigenesis by supplying not only oxygen and diffusible nutrients but also by secreting soluble factors that promote tumorigenesis. Here we identify a feed-forward mechanism in which endothelial cells (EC), in response to tumor-derived mediators, release angiocrines driving aberrant vascularization and glioblastoma multiforme (GBM) progression through a hypoxia-independent induction of hypoxia-inducible factor (HIF)-1α. Phosphorylation of profilin-1 (Pfn-1) at Tyr129 in EC induces binding to tumor suppressor protein von Hippel-Lindau (VHL), prevents VHL-mediated degradation of prolyl-hydroxylated HIF-1α, culminating in HIF-1α accumulation even in normoxia. Elevated HIF-1α induces expression of multiple angiogenic factors, leading to vascular abnormality and tumor progression. In a genetic model of GBM, mice with an EC-specific defect in Pfn-1 phosphorylation exhibit reduced tumor angiogenesis, normalized vasculature, and improved survival. Moreover, EC-specific Pfn-1 phosphorylation is associated with tumor aggressiveness in human glioma. These findings suggest that targeting Pfn-1 phosphorylation may offer a selective strategy for therapeutic intervention of malignant solid tumors. PMID:24747440

  14. Policies for regulation of direct broadcast satellites

    NASA Astrophysics Data System (ADS)

    Setzer, F. O.; Franca, B. A.; Cornell, N. W.

    1980-09-01

    The potential market for satellite to home television broadcasting was examined and recommendations were made to the FCC concerning appropriate regulatory policies for direct broadcast satellites (DBS) for the period following the 1983 Regional Administrative Radio Conference (RARC). It is concluded that many substitutes for DBS will be available in the market for subscription video programming. Either conventional broadcast regulation, which assumes scarcity of channels, nor common carrier regulation, which assumes monopoly power will be appropriate. The report recommends several functions the Commission should perform because of its spectrum allocation responsibilities, but recommends that the Commission make no rules concerning compatibility, signal quality, ownership of receiving equipment, program content, prices, service offerings, or control of channels.

  15. Identification of PECAM-1 association with sphingosine kinase 1 and its regulation by agonist-induced phosphorylation.

    PubMed

    Fukuda, Yu; Aoyama, Yuki; Wada, Atsushi; Igarashi, Yasuyuki

    2004-02-27

    Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator generated from sphingosine by sphingosine kinase (SPHK). S1P acts both extracellularly and intracellularly as a signaling molecule, although its intracellular targets are still undefined. Intracellular level of S1P is under strict regulatory control of SPHK regulation, S1P degradation, and S1P dephosphorylation. Therefore, clarifying the mechanisms regulating SPHK activity may help us understand when and where S1P is generated. In this study, we performed yeast two-hybrid screening to search for SPHK1a-binding molecules that may be involved in the regulation of the kinase localization or activity. Platelet endothelial cell adhesion molecule-1 (PECAM-1) was identified as a protein potentially associating with SPHK1a. Their association was confirmed by co-immunoprecipitation analysis using HEK293 cells overexpressing PECAM-1 and SPHK1a. Moreover, the kinase activity appeared to be reduced in stable PECAM-1-expressing cells. PECAM-1 is expressed on the cell surface of vascular cells, and several stimuli are known to induce phosphorylation of its tyrosine residues. We found that such phosphorylation attenuated its association with SPHK1a. This association/dissociation of SPHK with PECAM-1, regulated by the phosphorylated state of the membrane protein, may be involved in the control of localized kinase activity in certain cell types. PMID:14984734

  16. Phosphorylation-Regulated Transitions in an Oligomeric State Control the Activity of the Sae2 DNA Repair Enzyme

    PubMed Central

    Fu, Qiong; Chow, Julia; Bernstein, Kara A.; Makharashvili, Nodar; Arora, Sucheta; Lee, Chia-Fang; Person, Maria D.; Rothstein, Rodney

    2014-01-01

    In the DNA damage response, many repair and signaling molecules mobilize rapidly at the sites of DNA double-strand breaks. This network of immediate responses is regulated at the level of posttranslational modifications that control the activation of DNA processing enzymes, protein kinases, and scaffold proteins to coordinate DNA repair and checkpoint signaling. Here we investigated the DNA damage-induced oligomeric transitions of the Sae2 protein, an important enzyme in the initiation of DNA double-strand break repair. Sae2 is a target of multiple phosphorylation events, which we identified and characterized in vivo in the budding yeast Saccharomyces cerevisiae. Both cell cycle-dependent and DNA damage-dependent phosphorylation sites in Sae2 are important for the survival of DNA damage, and the cell cycle-regulated modifications are required to prime the damage-dependent events. We found that Sae2 exists in the form of inactive oligomers that are transiently released into smaller active units by this series of phosphorylations. DNA damage also triggers removal of Sae2 through autophagy and proteasomal degradation, ensuring that active Sae2 is present only transiently in cells. Overall, this analysis provides evidence for a novel type of protein regulation where the activity of an enzyme is controlled dynamically by posttranslational modifications that regulate its solubility and oligomeric state. PMID:24344201

  17. Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

    PubMed Central

    Xu, Daichao; Shan, Bing; Lee, Byung-Hoon; Zhu, Kezhou; Zhang, Tao; Sun, Huawang; Liu, Min; Shi, Linyu; Liang, Wei; Qian, Lihui; Xiao, Juan; Wang, Lili; Pan, Lifeng; Finley, Daniel; Yuan, Junying

    2015-01-01

    Regulation of ubiquitin-proteasome system (UPS), which controls the turnover of short-lived proteins in eukaryotic cells, is critical in maintaining cellular proteostasis. Here we show that USP14, a major deubiquitinating enzyme that regulates the UPS, is a substrate of Akt, a serine/threonine-specific protein kinase critical in mediating intracellular signaling transducer for growth factors. We report that Akt-mediated phosphorylation of USP14 at Ser432, which normally blocks its catalytic site in the inactive conformation, activates its deubiquitinating activity in vitro and in cells. We also demonstrate that phosphorylation of USP14 is critical for Akt to regulate proteasome activity and consequently global protein degradation. Since Akt can be activated by a wide range of growth factors and is under negative control by phosphoinosotide phosphatase PTEN, we suggest that regulation of UPS by Akt-mediated phosphorylation of USP14 may provide a common mechanism for growth factors to control global proteostasis and for promoting tumorigenesis in PTEN-negative cancer cells. DOI: http://dx.doi.org/10.7554/eLife.10510.001 PMID:26523394

  18. H-2Dd engagement of Ly49A leads directly to Ly49A phosphorylation and recruitment of SHP1

    PubMed Central

    Daws, M R; Eriksson, M; Öberg, L; Ullén, A; Sentman, C L

    1999-01-01

    We have used a number of in vitro and in vivo techniques to identify the molecules that can bind to the cytoplasmic tail of the Ly49A receptor. Affinity chromatography using peptides corresponding to the N-terminal 18 amino acids of Ly49A allowed the recovery of a number of proteins that bound preferentially to the tyrosine-phosphorylated peptide, including SH2-containing phosphatase-1 (SHP1) and the SH2-containing inositol 5′ phosphatase (SHIP). In another approach, using the entire cytoplasmic domain of the Ly49A receptor, we found that SHP2 also interacted with the tyrosine-phosphorylated form of the Ly49A cytoplasmic tail. Using BIACORE®2000 analysis, we determined that both SHP1 and SHP2 bound to the tyrosine-phosphorylated cytoplasmic tail of Ly49A with affinities in the nanomolar range, whilst SHIP showed no binding. Mutation of tyrosine-36 to phenylalanine did not significantly affect the affinities of these proteins for the tyrosine-phosphorylated cytoplasmic tail of Ly49A. In addition, using a whole-cell system with T-cell lymphoma cell lines that expressed the Ly49A receptor or its H-2Dd ligand, we determined that engagement of Ly49A by its major histocompatibility complex (MHC) ligand leads to tyrosine-phosphorylation events and recruitment of SHP1. Recruitment of SHP1 was rapid and transient, reaching a maximum after 5 min. These data suggest that mechanisms for the inhibitory signal are generated following receptor engagement. They also provide direct evidence that ligand engagement of the Ly49A receptor is responsible for recruitment of downstream signalling molecules. PMID:10457220

  19. Modulation of LSD1 phosphorylation by CK2/WIP1 regulates RNF168-dependent 53BP1 recruitment in response to DNA damage

    PubMed Central

    Peng, Bin; Wang, Jing; Hu, Yuan; Zhao, Hongli; Hou, Wenya; Zhao, Hongchang; Wang, Hailong; Liao, Ji; Xu, Xingzhi

    2015-01-01

    Proper DNA damage response is essential for the maintenance of genome integrity. The E3 ligase RNF168 deficiency fully prevents both the initial recruitment and retention of 53BP1 at sites of DNA damage. In response to DNA damage, RNF168-dependent recruitment of the lysine-specific demethylase LSD1 to the site of DNA damage promotes local H3K4me2 demethylation and ubiquitination of H2A/H2AX, facilitating 53BP1 recruitment to sites of DNA damage. Alternatively, RNF168-mediated K63-linked ubiquitylation of 53BP1 is required for the initial recruitment of 53BP1 to sites of DNA damage and for its function in repair. We demonstrated here that phosphorylation and dephosphorylation of LSD1 at S131 and S137 was mediated by casein kinase 2 (CK2) and wild-type p53-induced phosphatase 1 (WIP1), respectively. LSD1, RNF168 and 53BP1 interacted with each other directly. CK2-mediated phosphorylation of LSD1 exhibited no impact on its interaction with 53BP1, but promoted its interaction with RNF168 and RNF168-dependent 53BP1 ubiquitination and subsequent recruitment to the DNA damage sites. Furthermore, overexpression of phosphorylation-defective mutants failed to restore LSD1 depletion-induced cellular sensitivity to DNA damage. Taken together, our results suggest that LSD1 phosphorylation modulated by CK2/WIP1 regulates RNF168-dependent 53BP1 recruitment directly in response to DNA damage and cellular sensitivity to DNA damaging agents. PMID:25999347

  20. Phosphorylation of the Antiviral Protein Interferon-inducible Transmembrane Protein 3 (IFITM3) Dually Regulates Its Endocytosis and Ubiquitination*

    PubMed Central

    Chesarino, Nicholas M.; McMichael, Temet M.; Hach, Jocelyn C.; Yount, Jacob S.

    2014-01-01

    Interferon-inducible transmembrane protein 3 (IFITM3) is essential for innate defense against influenza virus in mice and humans. IFITM3 localizes to endolysosomes where it prevents virus fusion, although mechanisms controlling its trafficking to this cellular compartment are not fully understood. We determined that both mouse and human IFITM3 are phosphorylated by the protein-tyrosine kinase FYN on tyrosine 20 (Tyr20) and that mouse IFITM3 is also phosphorylated on the non-conserved Tyr27. Phosphorylation led to a cellular redistribution of IFITM3, including plasma membrane accumulation. Mutation of Tyr20 caused a similar redistribution of IFITM3 and resulted in decreased antiviral activity against influenza virus, whereas Tyr27 mutation of mouse IFITM3 showed minimal effects on localization or activity. Using FYN knockout cells, we also found that IFITM3 phosphorylation is not a requirement for its antiviral activity. Together, these results indicate that Tyr20 is part of an endocytosis signal that can be blocked by phosphorylation or by mutation of this residue. Further mutagenesis narrowed this endocytosis-controlling region to four residues conforming to a YXXΦ (where X is any amino acid and Φ is Val, Leu, or Ile) endocytic motif that, when transferred to CD4, resulted in its internalization from the cell surface. Additionally, we found that phosphorylation of IFITM3 by FYN and mutagenesis of Tyr20 both resulted in decreased IFITM3 ubiquitination. Overall, these results suggest that modification of Tyr20 may serve in a cellular checkpoint controlling IFITM3 trafficking and degradation and demonstrate the complexity of posttranslational regulation of IFITM3. PMID:24627473

  1. RIP1 maintains DNA integrity and cell proliferation by regulating PGC-1α-mediated mitochondrial oxidative phosphorylation and glycolysis

    PubMed Central

    Chen, W; Wang, Q; Bai, L; Chen, W; Wang, X; Tellez, C S; Leng, S; Padilla, M T; Nyunoya, T; Belinsky, S A; Lin, Y

    2014-01-01

    Aerobic glycolysis or the Warburg effect contributes to cancer cell proliferation; however, how this glucose metabolism pathway is precisely regulated remains elusive. Here we show that receptor-interacting protein 1 (RIP1), a cell death and survival signaling factor, regulates mitochondrial oxidative phosphorylation and aerobic glycolysis. Loss of RIP1 in lung cancer cells suppressed peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) expression, impairing mitochondrial oxidative phosphorylation and accelerating glycolysis, resulting in spontaneous DNA damage and p53-mediated cell proliferation inhibition. Thus, although aerobic glycolysis within a certain range favors cancer cell proliferation, excessive glycolysis causes cytostasis. Our data suggest that maintenance of glycolysis by RIP1 is pivotal to cancer cell energy homeostasis and DNA integrity and may be exploited for use in anticancer therapy. PMID:24583643

  2. Akt phosphorylation and regulation of transketolase is a nodal point for amino acid control of purine synthesis.

    PubMed

    Saha, Arindam; Connelly, Stephen; Jiang, Jingjing; Zhuang, Shunhui; Amador, Deron T; Phan, Tony; Pilz, Renate B; Boss, Gerry R

    2014-07-17

    The phosphatidylinositol 3-kinase (PI3K)/Akt pathway integrates environmental clues to regulate cell growth and survival. We showed previously that depriving cells of a single essential amino acid rapidly and reversibly arrests purine synthesis. Here we demonstrate that amino acids via mammalian target of rapamycin 2 and IκB kinase regulate Akt activity and Akt association and phosphorylation of transketolase (TKT), a key enzyme of the nonoxidative pentose phosphate pathway (PPP). Akt phosphorylates TKT on Thr382, markedly enhancing enzyme activity and increasing carbon flow through the nonoxidative PPP, thereby increasing purine synthesis. Mice fed a lysine-deficient diet for 2 days show decreased Akt activity, TKT activity, and purine synthesis in multiple organs. These results provide a mechanism whereby Akt coordinates amino acid availability with glucose utilization, purine synthesis, and RNA and DNA synthesis. PMID:24981175

  3. Vinculin phosphorylation differentially regulates mechanotransduction at cell–cell and cell–matrix adhesions

    PubMed Central

    Bays, Jennifer L.; Peng, Xiao; Tolbert, Catlin E.; Guilluy, Christophe; Angell, Ashley E.; Pan, Yuan; Superfine, Richard; Burridge, Keith

    2014-01-01

    Cells experience mechanical forces throughout their lifetimes. Vinculin is critical for transmitting these forces, yet how it achieves its distinct functions at cell–cell and cell–matrix adhesions remains unanswered. Here, we show vinculin is phosphorylated at Y822 in cell–cell, but not cell–matrix, adhesions. Phosphorylation at Y822 was elevated when forces were applied to E-cadherin and was required for vinculin to integrate into the cadherin complex. The mutation Y822F ablated these activities and prevented cells from stiffening in response to forces on E-cadherin. In contrast, Y822 phosphorylation was not required for vinculin functions in cell–matrix adhesions, including integrin-induced cell stiffening. Finally, forces applied to E-cadherin activated Abelson (Abl) tyrosine kinase to phosphorylate vinculin; Abl inhibition mimicked the loss of vinculin phosphorylation. These data reveal an unexpected regulatory mechanism in which vinculin Y822 phosphorylation determines whether cadherins transmit force and provides a paradigm for how a shared component of adhesions can produce biologically distinct functions. PMID:24751539

  4. Identification of a Novel Human LAP1 Isoform That Is Regulated by Protein Phosphorylation

    PubMed Central

    Santos, Mariana; Domingues, Sara C.; Costa, Patrícia; Muller, Thorsten; Galozzi, Sara; Marcus, Katrin; da Cruz e Silva, Edgar F.; da Cruz e Silva, Odete A.; Rebelo, Sandra

    2014-01-01

    Lamina associated polypeptide 1 (LAP1) is an integral protein of the inner nuclear membrane that is ubiquitously expressed. LAP1 binds to lamins and chromatin, probably contributing to the maintenance of the nuclear envelope architecture. Moreover, LAP1 also interacts with torsinA and emerin, proteins involved in DYT1 dystonia and X-linked Emery-Dreifuss muscular dystrophy disorder, respectively. Given its relevance to human pathological conditions, it is important to better understand the functional diversity of LAP1 proteins. In rat, the LAP1 gene (TOR1AIP1) undergoes alternative splicing to originate three LAP1 isoforms (LAP1A, B and C). However, it remains unclear if the same occurs with the human TOR1AIP1 gene, since only the LAP1B isoform had thus far been identified in human cells. In silico analysis suggested that, across different species, potential new LAP1 isoforms could be generated by alternative splicing. Using shRNA to induce LAP1 knockdown and HPLC-mass spectrometry analysis the presence of two isoforms in human cells was described and validated: LAP1B and LAP1C; the latter is putatively N-terminal truncated. LAP1B and LAP1C expression profiles appear to be dependent on the specific tissues analyzed and in cultured cells LAP1C was the major isoform detected. Moreover, LAP1B and LAP1C expression increased during neuronal maturation, suggesting that LAP1 is relevant in this process. Both isoforms were found to be post-translationally modified by phosphorylation and methionine oxidation and two LAP1B/LAP1C residues were shown to be dephosphorylated by PP1. This study permitted the identification of the novel human LAP1C isoform and partially unraveled the molecular basis of LAP1 regulation. PMID:25461922

  5. The PI3K/Akt Pathway Regulates Oxygen Metabolism via Pyruvate Dehydrogenase (PDH)-E1α Phosphorylation.

    PubMed

    Cerniglia, George J; Dey, Souvik; Gallagher-Colombo, Shannon M; Daurio, Natalie A; Tuttle, Stephen; Busch, Theresa M; Lin, Alexander; Sun, Ramon; Esipova, Tatiana V; Vinogradov, Sergei A; Denko, Nicholas; Koumenis, Constantinos; Maity, Amit

    2015-08-01

    Inhibition of the PI3K/Akt pathway decreases hypoxia within SQ20B human head and neck cancer xenografts. We set out to understand the molecular mechanism underlying this observation. We measured oxygen consumption using both a Clark electrode and an extracellular flux analyzer. We made these measurements after various pharmacologic and genetic manipulations. Pharmacologic inhibition of the PI3K/mTOR pathway or genetic inhibition of Akt/PI3K decreased the oxygen consumption rate (OCR) in vitro in SQ20B and other cell lines by 30% to 40%. Pharmacologic inhibition of this pathway increased phosphorylation of the E1α subunit of the pyruvate dehydrogenase (PDH) complex on Ser293, which inhibits activity of this critical gatekeeper of mitochondrial respiration. Expressing wild-type PTEN in a doxycycline-inducible manner in a cell line with mutant PTEN led to an increase in PDH-E1α phosphorylation and a decrease in OCR. Pretreatment of SQ20B cells with dichloroacetate (DCA), which inhibits PDH-E1α phosphorylation by inhibiting dehydrogenase kinases (PDK), reversed the decrease in OCR in response to PI3K/Akt/mTOR inhibition. Likewise, introduction of exogenous PDH-E1α that contains serine to alanine mutations, which can no longer be regulated by phosphorylation, also blunted the decrease in OCR seen with PI3K/mTOR inhibition. Our findings highlight an association between the PI3K/mTOR pathway and tumor cell oxygen consumption that is regulated in part by PDH phosphorylation. These results have important implications for understanding the effects of PI3K pathway activation in tumor metabolism and also in designing cancer therapy trials that use inhibitors of this pathway. PMID:25995437

  6. Dynamics of re-constitution of the human nuclear proteome after cell division is regulated by NLS-adjacent phosphorylation

    PubMed Central

    Róna, Gergely; Borsos, Máté; Ellis, Jonathan J; Mehdi, Ahmed M; Christie, Mary; Környei, Zsuzsanna; Neubrandt, Máté; Tóth, Judit; Bozóky, Zoltán; Buday, László; Madarász, Emília; Bodén, Mikael; Kobe, Bostjan; Vértessy, Beáta G

    2014-01-01

    Phosphorylation by the cyclin-dependent kinase 1 (Cdk1) adjacent to nuclear localization signals (NLSs) is an important mechanism of regulation of nucleocytoplasmic transport. However, no systematic survey has yet been performed in human cells to analyze this regulatory process, and the corresponding cell-cycle dynamics have not yet been investigated. Here, we focused on the human proteome and found that numerous proteins, previously not identified in this context, are associated with Cdk1-dependent phosphorylation sites adjacent to their NLSs. Interestingly, these proteins are involved in key regulatory events of DNA repair, epigenetics, or RNA editing and splicing. This finding indicates that cell-cycle dependent events of genome editing and gene expression profiling may be controlled by nucleocytoplasmic trafficking. For in-depth investigations, we selected a number of these proteins and analyzed how point mutations, expected to modify the phosphorylation ability of the NLS segments, perturb nucleocytoplasmic localization. In each case, we found that mutations mimicking hyper-phosphorylation abolish nuclear import processes. To understand the mechanism underlying these phenomena, we performed a video microscopy-based kinetic analysis to obtain information on cell-cycle dynamics on a model protein, dUTPase. We show that the NLS-adjacent phosphorylation by Cdk1 of human dUTPase, an enzyme essential for genomic integrity, results in dynamic cell cycle-dependent distribution of the protein. Non-phosphorylatable mutants have drastically altered protein re-import characteristics into the nucleus during the G1 phase. Our results suggest a dynamic Cdk1-driven mechanism of regulation of the nuclear proteome composition during the cell cycle. PMID:25483092

  7. Curcumin attenuates high glucose-induced podocyte apoptosis by regulating functional connections between caveolin-1 phosphorylation and ROS

    PubMed Central

    Sun, Li-na; Liu, Xiang-chun; Chen, Xiang-jun; Guan, Guang-ju; Liu, Gang

    2016-01-01

    Aim: Caveolin-1 (cav-1) is a major multifunctional scaffolding protein of caveolae. Cav-1 is primarily expressed in mesangial cells, renal proximal tubule cells and podocytes in kidneys. Recent evidence shows that the functional connections between cav-1 and ROS play a key role in many diseases. In this study we investigated whether regulating the functional connections between cav-1 and ROS in kidneys contributed to the beneficial effects of curcumin in treating diabetic nephropathy in vitro and in vivo. Methods: Cultured mouse podocytes (mpc5) were incubated in a high glucose (HG, 30 mmol/L) medium for 24, 48 or 72 h. Male rats were injected with STZ (60 mg/kg, ip) to induce diabetes. ROS generation, SOD activity, MDA content and caspase-3 activity in the cultured cells and kidney cortex homogenate were determined. Apoptotic proteins and cav-1 phosphorylation were analyzed using Western blot analyses. Results: Incubation in HG-containing medium time-dependently increased ROS production, oxidative stress, apoptosis, and cav-1 phosphorylation in podocytes. Pretreatment with curcumin (1, 5, and 10 μmol/L) dose-dependently attenuated these abnormalities in HG-treated podocytes. Furthermore, in HG-containing medium, the podocytes transfected with a recombinant plasmid GFP-cav-1 Y14F (mutation at a cav-1 phosphorylation site) exhibited significantly decreased ROS production and apoptosis compared with the cells transfected with empty vector. In diabetic rats, administration of curcumin (100 or 200 mg/kg body weight per day, ig, for 8 weeks) not only significantly improved the renal function, but also suppressed ROS levels, oxidative stress, apoptosis and cav-1 phosphorylation in the kidneys. Conclusion: Curcumin attenuates high glucose-induced podocyte apoptosis in vitro and diabetic nephropathy in vivo partly through regulating the functional connections between cav-1 phosphorylation and ROS. PMID:26838071

  8. Calcium regulates motility and protein phosphorylation by changing cAMP and ATP concentrations in boar sperm in vitro.

    PubMed

    Li, Xinhong; Wang, Lirui; Li, Yuhua; Zhao, Na; Zhen, Linqing; Fu, Jieli; Yang, Qiangzhen

    2016-09-01

    Considering the importance of calcium (Ca(2+)) in regulating sperm capacitation, hyperactivation and acrosome reaction, little is known about the molecular mechanism of action of this ion in this process. In the present study, assessment of the molecular mechanism from the perspective of energy metabolism occurred. Sperm motility variables were determined using computer-assisted sperm analysis (CASA) and the phosphorylation of PKA substrates, tyrosine residues and AMP-activated protein kinase (AMPK) were analyzed by Western blot. Moreover, intracellular sperm-specific glyceraldehyde 3-phosphatedehydrogenase (GAPDH) activity, 3'-5'-cyclic adenosine monophosphate (cAMP) and adenosine 5'-triphosphate (ATP) concentrations were assessed in boar sperm treated with Ca(2+). Results of the present study indicated that, under greater extracellular Ca(2+)concentrations (≥3.0mM), sperm motility and protein phosphorylation were inhibited. Interestingly, these changes were correlated with that of GAPDH activity, AMPK phosphorylation, cAMP and ATP concentrations. The negative effects of Ca(2+) on these intracellular processes were attenuated by addition of the calmodulin (CaM) inhibitor W7 and the inhibitor of calmodulin-dependent protein kinase (CaMK), KN-93. In the presence of greater extracellular Ca(2+), however, the phosphorylation pathway was suppressed by H-89. Taken together, these results suggested that Ca(2+) had a dual role in regulating boar sperm motility and protein phosphorylation due to the changes of cAMP and ATP concentrations, in response to cAMP-mediated signal transduction and the Ca(2+) signaling cascade. The present study provided some novel insights into the molecular mechanism underlying the effects of Ca(2+) on boar sperm as well as the involvement of energy metabolism in this mechanism. PMID:27423488

  9. The viral transactivator HBx protein exhibits a high potential for regulation via phosphorylation through an evolutionarily conserved mechanism

    PubMed Central

    2012-01-01

    Background Hepatitis B virus (HBV) encodes an oncogenic factor, HBx, which is a multifunctional protein that can induce dysfunctional regulation of signaling pathways, transcription, and cell cycle progression, among other processes, through interactions with target host factors. The subcellular localization of HBx is both cytoplasmic and nuclear. This dynamic distribution of HBx could be essential to the multiple roles of the protein at different stages during HBV infection. Transactivational functions of HBx may be exerted both in the nucleus, via interaction with host DNA-binding proteins, and in the cytoplasm, via signaling pathways. Although there have been many studies describing different pathways altered by HBx, and its innumerable binding partners, the molecular mechanism that regulates its different roles has been difficult to elucidate. Methods In the current study, we took a bioinformatics approach to investigate whether the viral protein HBx might be regulated via phosphorylation by an evolutionarily conserved mechanism. Results We found that the phylogenetically conserved residues Ser25 and Ser41 (both within the negative regulatory domain), and Thr81 (in the transactivation domain) are predicted to be phosphorylated. By molecular 3D modeling of HBx, we further show these residues are all predicted to be exposed on the surface of the protein, making them easily accesible to these types of modifications. Furthermore, we have also identified Yin Yang sites that might have the potential to be phosphorylated and O-β-GlcNAc interplay at the same residues. Conclusions Thus, we propose that the different roles of HBx displayed in different subcellular locations might be regulated by an evolutionarily conserved mechanism of posttranslational modification, via phosphorylation. PMID:23079056

  10. Glucose Regulated Protein 78 Phosphorylation in Sperm Undergoes Dynamic Changes during Maturation

    PubMed Central

    Lobo, Vivian; Rao, Parimala; Gajbhiye, Rahul; Kulkarni, Vijay; Parte, Priyanka

    2015-01-01

    GRP78, a resident endoplasmic reticulum (ER) chaperone involved in protein transport, folding and assembly, has been reported in sperm. It is shown to be localized in the neck region of human sperm. We have previously reported GRP78 to be less phosphorylated in asthenozoosperm.The present study aimed to determine whether sperm GRP78 undergoes phosphorylation changes during epididymal maturation and whether there are any differences in GRP78 phosphoforms in asthenozoosperm vis-à-vis normozoosperm. Testicular- and cauda epididymal- sperm from adult male Holtzman rats, and semen ejaculates collected from normal and asthenozoospermic individuals were investigated. DIGE carried out to determine phosphorylation of GRP78 in asthenozoosperm and normal sperm reveals a shift in the location of GRP78 of asthenozoosperm towards the alkaline pH, indicative of reduced GRP78 phosphorylation. Immunoprecipitation studies using antibodies specific to GRP78, serine-, threonine-, and tyrosine phosphorylation and Pan phospho antibody demonstrates GRP78 to be phosphorylated at all three residues in rat spermatozoa. Phosphatase assays using Calf intestinal alkaline phosphatase and Lambda protein phosphatase followed by nanofluidic proteomic immunoassay (NIA) show that in rat, GP4.96, GP4.94 and GP4.85 are the three phosphoforms in mature (caudal) sperm as against two phosphoforms GP4.96and GP4.94in immature (testicular) sperm. In mature human sperm GP5.04, GP4.96, and GP4.94were the 3 phosphoforms observed. GP4.94[P = 0.014]andGP5.04 [P = 0.02] are significantly reduced in asthenozoosperm. Ours is the first report indicating GRP78 in sperm to be phosphorylated at serine, threonine and tyrosine residues contrary to published literature reporting GRP78 not to be tyrosine phosphorylated. We report the presence of GRP78 phosphoforms in rat- and human- sperm and our data suggest that GRP78 phosphorylation in sperm undergoes spatial reorganization during epididymal maturation. Significant

  11. Phosphorylation regulates proteolytic efficiency of TEV protease detected by a 5(6)-carboxyfluorescein-pyrene based fluorescent sensor.

    PubMed

    He, Yao-Hui; Li, Yan-Mei; Chen, Yong-Xiang

    2016-04-01

    TEV protease is of great importance for in vitro and in vivo site-specific cleavage of proteins. The proteolytic efficiency of TEV protease is often regulated by mutation of the substrate, which is irreversible and hard to be modulated. Herein, a facile and reversible method, based on phosphorylation in the substrate, is developed to regulate the cleavage capability of TEV protease. Phosphorylation at P3 tyrosine hinders the recognition of TEV protease to the substrate by using a robust fluorescent protease sensor. Moreover, the phosphate group can be easily removed by alkaline phosphatases for recovering the proteolytic efficiency of TEV protease. Additionally, 5(6)-carboxyfluorescein and pyrene have been used as high-efficiency mutual fluorophore-quencher pair in the peptide-based protease sensor for the first time, which provides a chance to simultaneously monitor the cleavage process in two respective fluorescence channels. Further studies indicated both dynamic and static components contributing to the mutual quenching system. The phosphorylation-regulated TEV protease proteolysis system can be used in conditional cleavage of protein or peptide tag. PMID:26838417

  12. 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. PMID:26739493

  13. Notch signaling regulates the phosphorylation of Akt and survival of lipopolysaccharide-activated macrophages via regulator of G protein signaling 19 (RGS19)

    PubMed Central

    Sangphech, Naunpun; Osborne, Barbara A.; Palaga, Tanapat

    2014-01-01

    Macrophages play critical roles in innate immune defense by sensing microbes using pattern-recognition receptors. Lipopolysaccharide (LPS) stimulates macrophages via TLR, which leads to activation of downstream signaling cascades. In this study, we investigated the roles of a conserved signaling pathway, Notch signaling, in regulating the downstream signaling cascades of the LPS/TLR4 pathways in macrophages. Using a phosphoproteomic approach and a gamma-secretase inhibitor (GSI) to suppress the processing and activation of Notch signaling, we identified regulator of G protein signaling 19 (RGS19) as a target protein whose phosphorylation was affected by GSI treatment. RGS19 is a guanosine triphosphatase (GTPase)-activating protein that functions to negatively regulate G protein-coupled receptors via Gαi/Gαq-linked signaling. Stimulation of RAW264.7 cells with LPS increased the level of the phosphorylated form of RGS19, while LPS stimulation in the presence of GSI decreased its level. GSI treatment did not alter the mRNA level of rgs19. Treatment with GSI or silencing of rgs19 in macrophages impaired the phosphorylation of Akt Thr308 upon LPS stimulation. Furthermore, targeted deletion of a DNA-binding protein and binding partner of the Notch receptor, RBP-Jκ/CSL, in macrophages resulted in delayed and decreased Akt phosphorylation. Because the PI3K/Akt pathway regulates cell survival in various cell types, the cell cycle and cell death were assayed upon GSI treatment, phosphatidylinositol 3 kinase (PI3K) inhibitor treatment or silencing of rgs19. GSI treatment resulted in decreased cell populations in the G1 and S phases, while it increased the cell population of cell death. Similarly, silencing of rgs19 resulted in a decreased cell population in the G1 phase and an increased cell population in the subG1 phase. Inhibition of Akt phosphorylation by PI3K inhibitor in LPS-stimulated macrophages increased cell population in G1 phase, suggesting a possible cell cycle

  14. A Pan-specific Antibody for Direct Detection of Protein Histidine Phosphorylation

    PubMed Central

    Kee, Jung-Min; Oslund, Rob C.; Perlman, David H.; Muir, Tom W.

    2013-01-01

    Despite its importance in central metabolism and bacterial cell signaling, protein histidine phosphorylation has remained elusive with respect to its extent and functional roles in biological systems due to the lack of adequate research tools. We report the development of the first pan-pHis antibody using a stable phosphohistidine (pHis) mimetic as the hapten. This antibody was successfully used in ELISA, Western blot, dot blot, immunoprecipitation, and in detection and identification of histidine-phosphorylated proteins from native cell lysates when coupled with mass spectrometric analysis. We also observed that protein pHis levels in E. coli lysates depend on carbon source and nitrogen availability in the growth media. In particular, we found that pHis levels on PpsA are sensitive to nitrogen availability in vivo and that α-ketoglutarate (α-KG) inhibits phosphotransfer from phosphorylated phosphoenolpyruvate synthase (PpsA) to pyruvate. We expect this antibody to open opportunities for investigating other pHis-proteins and their functions. PMID:23708076

  15. Identification of a novel phosphorylation site in adipose triglyceride lipase as a regulator of lipid droplet localization.

    PubMed

    Xie, Xitao; Langlais, Paul; Zhang, Xiaodong; Heckmann, Bradlee L; Saarinen, Alicia M; Mandarino, Lawrence J; Liu, Jun

    2014-06-15

    Adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triacylglycerol (TG) hydrolysis, has long been known to be a phosphoprotein. However, the potential phosphorylation events that are involved in the regulation of ATGL function remain incompletely defined. Here, using a combinatorial proteomics approach, we obtained evidence that at least eight different sites of ATGL can be phosphorylated in adipocytes. Among them, Thr³⁷² resides within the hydrophobic region known to mediate lipid droplet (LD) targeting. Although it had no impact on the TG hydrolase activity, substitution of phosphorylation-mimic Asp for Thr³⁷² eliminated LD localization and LD-degrading capacity of ATGL expressed in HeLa cells. In contrast, mutation of Thr³⁷² to Ala gave a protein that bound LDs and functioned the same as the wild-type protein. In nonstimulated adipocytes, the Asp mutation led to decreased LD association and basal lipolytic activity of ATGL, whereas the Ala mutation produced opposite effects. Moreover, the LD translocation of ATGL upon β-adrenergic stimulation was also compromised by the Asp mutation. In accord with these findings, the Ala mutation promoted and the Asp mutation attenuated the capacity of ATGL to mediate lipolysis in adipocytes under both basal and stimulated conditions. Collectively, these studies identified Thr³⁷² as a novel phosphorylation site that may play a critical role in determining subcellular distribution as well as lipolytic action of ATGL. PMID:24801391

  16. WIP1, a homeostatic regulator of the DNA damage response, is targeted by HIPK2 for phosphorylation and degradation.

    PubMed

    Choi, Dong Wook; Na, Wooju; Kabir, Mohammad Humayun; Yi, Eunbi; Kwon, Seonjeong; Yeom, Jeonghun; Ahn, Jang-Won; Choi, Hee-Hyun; Lee, Youngha; Seo, Kyoung Wan; Shin, Min Kyoo; Park, Se-Ho; Yoo, Hae Yong; Isono, Kyo-Ichi; Koseki, Haruhiko; Kim, Seong-Tae; Lee, Cheolju; Kwon, Yunhee Kim; Choi, Cheol Yong

    2013-08-01

    WIP1 (wild-type p53-induced phosphatase 1) functions as a homeostatic regulator of the ataxia telangiectasia mutated (ATM)-mediated signaling pathway in response to ionizing radiation (IR). Here we identify homeodomain-interacting protein kinase 2 (HIPK2) as a protein kinase that targets WIP1 for phosphorylation and proteasomal degradation. In unstressed cells, WIP1 is constitutively phosphorylated by HIPK2 and maintained at a low level by proteasomal degradation. In response to IR, ATM-dependent AMPKα2-mediated HIPK2 phosphorylation promotes inhibition of WIP1 phosphorylation through dissociation of WIP1 from HIPK2, followed by stabilization of WIP1 for termination of the ATM-mediated double-strand break (DSB) signaling cascade. Notably, HIPK2 depletion impairs IR-induced γ-H2AX foci formation, cell-cycle checkpoint activation, and DNA repair signaling, and the survival rate of hipk2+/- mice upon γ-irradiation is markedly reduced compared to wild-type mice. Taken together, HIPK2 plays a critical role in the initiation of DSB repair signaling by controlling WIP1 levels in response to IR. PMID:23871434

  17. Phosphorylation of Deinococcus radiodurans RecA Regulates Its Activity and May Contribute to Radioresistance.

    PubMed

    Rajpurohit, Yogendra S; Bihani, Subhash C; Waldor, Matthew K; Misra, Hari S

    2016-08-01

    Deinococcus radiodurans has a remarkable capacity to survive exposure to extreme levels of radiation that cause hundreds of DNA double strand breaks (DSBs). DSB repair in this bacterium depends on its recombinase A protein (DrRecA). DrRecA plays a pivotal role in both extended synthesis-dependent strand annealing and slow crossover events of DSB repair during the organism's recovery from DNA damage. The mechanisms that control DrRecA activity during the D. radiodurans response to γ radiation exposure are unknown. Here, we show that DrRecA undergoes phosphorylation at Tyr-77 and Thr-318 by a DNA damage-responsive serine threonine/tyrosine protein kinase (RqkA). Phosphorylation modifies the activity of DrRecA in several ways, including increasing its affinity for dsDNA and its preference for dATP over ATP. Strand exchange reactions catalyzed by phosphorylated versus unphosphorylated DrRecA also differ. In silico analysis of DrRecA structure support the idea that phosphorylation can modulate crucial functions of this protein. Collectively, our findings suggest that phosphorylation of DrRecA enables the recombinase to selectively use abundant dsDNA substrate present during post-irradiation recovery for efficient DSB repair, thereby promoting the extraordinary radioresistance of D. radiodurans. PMID:27255712

  18. Structural Mechanism for Regulation of Bcl-2 protein Noxa by phosphorylation

    NASA Astrophysics Data System (ADS)

    Karim, Christine B.; Michel Espinoza-Fonseca, L.; James, Zachary M.; Hanse, Eric A.; Gaynes, Jeffrey S.; Thomas, David D.; Kelekar, Ameeta

    2015-09-01

    We showed previously that phosphorylation of Noxa, a 54-residue Bcl-2 protein, at serine 13 (Ser13) inhibited its ability to promote apoptosis through interactions with canonical binding partner, Mcl-1. Using EPR spectroscopy, molecular dynamics (MD) simulations and binding assays, we offer evidence that a structural alteration caused by phosphorylation partially masks Noxa’s BH3 domain, inhibiting the Noxa-Mcl-1 interaction. EPR of unphosphorylated Noxa, with spin-labeled amino acid TOAC incorporated within the BH3 domain, revealed equilibrium between ordered and dynamically disordered states. Mcl-1 further restricted the ordered component for non-phosphorylated Noxa, but left the pSer13 Noxa profile unchanged. Microsecond MD simulations indicated that the BH3 domain of unphosphorylated Noxa is housed within a flexible loop connecting two antiparallel β-sheets, flanked by disordered N- and C-termini and Ser13 phosphorylation creates a network of salt-bridges that facilitate the interaction between the N-terminus and the BH3 domain. EPR showed that a spin label inserted near the N-terminus was weakly immobilized in unphosphorylated Noxa, consistent with a solvent-exposed helix/loop, but strongly constrained in pSer13 Noxa, indicating a more ordered peptide backbone, as predicted by MD simulations. Together these studies reveal a novel mechanism by which phosphorylation of a distal serine inhibits a pro-apoptotic BH3 domain and promotes cell survival.

  19. Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity

    SciTech Connect

    Hammerstrom, Troy G.; Horton, Lori B.; Swick, Michelle C.; Joachimiak, Andrzej; Osipiuk, Jerzy; Koehler, Theresa M.

    2014-12-30

    The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthesis operon. AtxA activity is elevated during growth in media containing glucose and CO2/bicarbonate, and there is a positive correlation between the CO2/bicarbonate signal, AtxA activity, and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His → Asp) and phosphoablative (His → Ala) amino acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (1) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (2) phosphorylation of H379 in PRD2 disrupts dimer formation. In conclusion, the AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.

  20. Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity

    DOE PAGESBeta

    Hammerstrom, Troy G.; Horton, Lori B.; Swick, Michelle C.; Joachimiak, Andrzej; Osipiuk, Jerzy; Koehler, Theresa M.

    2014-12-30

    The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthesis operon. AtxA activity is elevated during growth in media containing glucose and CO2/bicarbonate, and there is a positive correlation between the CO2/bicarbonate signal, AtxA activity, and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His → Asp) and phosphoablative (His → Ala) aminomore » acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (1) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (2) phosphorylation of H379 in PRD2 disrupts dimer formation. In conclusion, the AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.« less

  1. The Inhibition Mechanism of Non-phosphorylated Ser768 in the Regulatory Domain of Cystic Fibrosis Transmembrane Conductance Regulator*

    PubMed Central

    Wang, Guangyu

    2011-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette transporters but serves as a chloride channel dysfunctional in cystic fibrosis. The activity of CFTR is tightly controlled not only by ATP-driven dimerization of its nucleotide-binding domains but also by phosphorylation of a unique regulatory (R) domain by protein kinase A (PKA). The R domain has multiple excitatory phosphorylation sites, but Ser737 and Ser768 are inhibitory. The underlying mechanism is unclear. Here, sulfhydryl-specific cross-linking strategy was employed to demonstrate that Ser768 or Ser737 could interact with outwardly facing hydrophilic residues of cytoplasmic loop 3 regulating channel gating. Furthermore, mutation of these residues to alanines promoted channel opening by curcumin in an ATP-dependent manner even in the absence of PKA. However, mutation of Ser768 and His950 with different hydrogen bond donors or acceptors clearly changed ATP- and PKA-dependent channel activity no matter whether curcumin was present or not. More importantly, significant activation of a double mutant H950R/S768R needed only ATP. Finally, in vitro and in vivo single channel recordings suggest that Ser768 may form a putative hydrogen bond with His950 of cytoplasmic loop 3 to prevent channel opening by ATP in the non-phosphorylated state and by subsequent cAMP-dependent phosphorylation. These observations support an electron cryomicroscopy-based structural model on which the R domain is closed to cytoplasmic loops regulating channel gating. PMID:21059651

  2. The catalytic region and PEST domain of PTPN18 distinctly regulate the HER2 phosphorylation and ubiquitination barcodes.

    PubMed

    Wang, Hong-Mei; Xu, Yun-Fei; Ning, Shang-Lei; Yang, Du-Xiao; Li, Yi; Du, Yu-Jie; Yang, Fan; Zhang, Ya; Liang, Nan; Yao, Wei; Zhang, Ling-Li; Gu, Li-Chuan; Gao, Cheng-Jiang; Pang, Qi; Chen, Yu-Xin; Xiao, Kun-Hong; Ma, Rong; Yu, Xiao; Sun, Jin-Peng

    2014-09-01

    The tyrosine phosphorylation barcode encoded in C-terminus of HER2 and its ubiquitination regulate diverse HER2 functions. PTPN18 was reported as a HER2 phosphatase; however, the exact mechanism by which it defines HER2 signaling is not fully understood. Here, we demonstrate that PTPN18 regulates HER2-mediated cellular functions through defining both its phosphorylation and ubiquitination barcodes. Enzymologic characterization and three crystal structures of PTPN18 in complex with HER2 phospho-peptides revealed the molecular basis for the recognition between PTPN18 and specific HER2 phosphorylation sites, which assumes two distinct conformations. Unique structural properties of PTPN18 contribute to the regulation of sub-cellular phosphorylation networks downstream of HER2, which are required for inhibition of HER2-mediated cell growth and migration. Whereas the catalytic domain of PTPN18 blocks lysosomal routing and delays the degradation of HER2 by dephosphorylation of HER2 on pY(1112), the PEST domain of PTPN18 promotes K48-linked HER2 ubiquitination and its rapid destruction via the proteasome pathway and an HER2 negative feedback loop. In agreement with the negative regulatory role of PTPN18 in HER2 signaling, the HER2/PTPN18 ratio was correlated with breast cancer stage. Taken together, our study presents a structural basis for selective HER2 dephosphorylation, a previously uncharacterized mechanism for HER2 degradation and a novel function for the PTPN18 PEST domain. The new regulatory role of the PEST domain in the ubiquitination pathway will broaden our understanding of the functions of other important PEST domain-containing phosphatases, such as LYP and PTPN12. PMID:25081058

  3. The catalytic region and PEST domain of PTPN18 distinctly regulate the HER2 phosphorylation and ubiquitination barcodes

    PubMed Central

    Wang, Hong-Mei; Xu, Yun-Fei; Ning, Shang-Lei; Yang, Du-Xiao; Li, Yi; Du, Yu-Jie; Yang, Fan; Zhang, Ya; Liang, Nan; Yao, Wei; Zhang, Ling-Li; Gu, Li-Chuan; Gao, Cheng-Jiang; Pang, Qi; Chen, Yu-Xin; Xiao, Kun-Hong; Ma, Rong; Yu, Xiao; Sun, Jin-Peng

    2014-01-01

    The tyrosine phosphorylation barcode encoded in C-terminus of HER2 and its ubiquitination regulate diverse HER2 functions. PTPN18 was reported as a HER2 phosphatase; however, the exact mechanism by which it defines HER2 signaling is not fully understood. Here, we demonstrate that PTPN18 regulates HER2-mediated cellular functions through defining both its phosphorylation and ubiquitination barcodes. Enzymologic characterization and three crystal structures of PTPN18 in complex with HER2 phospho-peptides revealed the molecular basis for the recognition between PTPN18 and specific HER2 phosphorylation sites, which assumes two distinct conformations. Unique structural properties of PTPN18 contribute to the regulation of sub-cellular phosphorylation networks downstream of HER2, which are required for inhibition of HER2-mediated cell growth and migration. Whereas the catalytic domain of PTPN18 blocks lysosomal routing and delays the degradation of HER2 by dephosphorylation of HER2 on pY1112, the PEST domain of PTPN18 promotes K48-linked HER2 ubiquitination and its rapid destruction via the proteasome pathway and an HER2 negative feedback loop. In agreement with the negative regulatory role of PTPN18 in HER2 signaling, the HER2/PTPN18 ratio was correlated with breast cancer stage. Taken together, our study presents a structural basis for selective HER2 dephosphorylation, a previously uncharacterized mechanism for HER2 degradation and a novel function for the PTPN18 PEST domain. The new regulatory role of the PEST domain in the ubiquitination pathway will broaden our understanding of the functions of other important PEST domain-containing phosphatases, such as LYP and PTPN12. PMID:25081058

  4. Cofactor Strap regulates oxidative phosphorylation and mitochondrial p53 activity through ATP synthase

    PubMed Central

    Maniam, S; Coutts, A S; Stratford, M R; McGouran, J; Kessler, B; La Thangue, N B

    2015-01-01

    Metabolic reprogramming is a hallmark of cancer cells. Strap (stress-responsive activator of p300) is a novel TPR motif OB-fold protein that contributes to p53 transcriptional activation. We show here that, in addition to its established transcriptional role, Strap is localised at mitochondria where one of its key interaction partners is ATP synthase. Significantly, the interaction between Strap and ATP synthase downregulates mitochondrial ATP production. Under glucose-limiting conditions, cancer cells are sensitised by mitochondrial Strap to apoptosis, which is rescued by supplementing cells with an extracellular source of ATP. Furthermore, Strap augments the apoptotic effects of mitochondrial p53. These findings define Strap as a dual regulator of cellular reprogramming: first as a nuclear transcription cofactor and second in the direct regulation of mitochondrial respiration. PMID:25168243

  5. Phosphorylation of Ser-204 and Tyr-405 in human malonyl-CoA decarboxylase expressed in silkworm Bombyx mori regulates catalytic decarboxylase activity.

    PubMed

    Hwang, In-Wook; Makishima, Yu; Suzuki, Tomohiro; Kato, Tatsuya; Park, Sungjo; Terzic, Andre; Chung, Shin-Kyo; Park, Enoch Y

    2015-11-01

    Decarboxylation of malonyl-CoA to acetyl-CoA by malonyl-CoA decarboxylase (MCD; EC 4.1.1.9) is a vital catalytic reaction of lipid metabolism. While it is established that phosphorylation of MCD modulates the enzymatic activity, the specific phosphorylation sites associated with the catalytic function have not been documented due to lack of sufficient production of MCD with proper post-translational modifications. Here, we used the silkworm-based Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system to express human MCD (hMCD) and mapped phosphorylation effects on enzymatic function. Purified MCD from silkworm displayed post-translational phosphorylation and demonstrated coherent enzymatic activity with high yield (-200 μg/silkworm). Point mutations in putative phosphorylation sites, Ser-204 or Tyr-405 of hMCD, identified by bioinformatics and proteomics analyses reduced the catalytic activity, underscoring the functional significance of phosphorylation in modulating decarboxylase-based catalysis. Identified phosphorylated residues are distinct from the decarboxylation catalytic site, implicating a phosphorylation-induced global conformational change of MCD as responsible in altering catalytic function. We conclude that phosphorylation of Ser-204 and Tyr-405 regulates the decarboxylase function of hMCD leveraging the silkworm-based BmNPV bacmid expression system that offers a fail-safe eukaryotic production platform implementing proper post-translational modification such as phosphorylation. PMID:26004805

  6. Phosphorylation by SR kinases regulates the binding of PTB-associated splicing factor (PSF) to the pre-mRNA polypyrimidine tract

    PubMed Central

    Huang, Ching-Jung; Tang, Zhaohua; Lin, Ren-Jang; Tucker, Philip W.

    2007-01-01

    PSF (PTB-associated splicing factor) is a multi-functional protein that participates in transcription and RNA processing. While phosphorylation of PSF has been shown to be important for some functions, the sites and the kinases involved are not well understood. Although PSF does not contain a typical RS domain, we report here that PSF is phosphorylated in vivo to generate an epitope(s) that can be recognized by a monoclonal antibody specific for phosphorylated RS motifs within SR proteins. PSF can be phosphorylated by human and yeast SR kinases in vivo and in vitro at two isolated RS motifs within its N terminus. A functional consequence of SR phosphorylation of PSF is to inhibit its binding to the 3’ polypyrimidine tract of pre-mRNA. These results indicate that PSF is a substrate of SR kinases whose phosphorylation regulates its RNA binding capacity and ultimate biological function. PMID:17188683

  7. Phosphorylation of the Drosophila Transient Receptor Potential Ion Channel Is Regulated by the Phototransduction Cascade and Involves Several Protein Kinases and Phosphatases

    PubMed Central

    Voolstra, Olaf; Bartels, Jonas-Peter; Oberegelsbacher, Claudia; Pfannstiel, Jens; Huber, Armin

    2013-01-01

    Protein phosphorylation plays a cardinal role in regulating cellular processes in eukaryotes. Phosphorylation of proteins is controlled by protein kinases and phosphatases. We previously reported the light-dependent phosphorylation of the Drosophila transient receptor potential (TRP) ion channel at multiple sites. TRP generates the receptor potential upon stimulation of the photoreceptor cell by light. An eye-enriched protein kinase C (eye-PKC) has been implicated in the phosphorylation of TRP by in vitro studies. Other kinases and phosphatases of TRP are elusive. Using phosphospecific antibodies and mass spectrometry, we here show that phosphorylation of most TRP sites depends on the phototransduction cascade and the activity of the TRP ion channel. A candidate screen to identify kinases and phosphatases provided in vivo evidence for an involvement of eye-PKC as well as other kinases and phosphatases in TRP phosphorylation. PMID:24040070

  8. DYRK1A-mediated phosphorylation of GluN2A at Ser1048 regulates the surface expression and channel activity of GluN1/GluN2A receptors

    PubMed Central

    Grau, Cristina; Arató, Krisztina; Fernández-Fernández, José M.; Valderrama, Aitana; Sindreu, Carlos; Fillat, Cristina; Ferrer, Isidre; de la Luna, Susana; Altafaj, Xavier

    2014-01-01

    N-methyl-D-aspartate glutamate receptors (NMDARs) play a pivotal role in neural development and synaptic plasticity, as well as in neurological disease. Since NMDARs exert their function at the cell surface, their density in the plasma membrane is finely tuned by a plethora of molecules that regulate their production, trafficking, docking and internalization in response to external stimuli. In addition to transcriptional regulation, the density of NMDARs is also influenced by post-translational mechanisms like phosphorylation, a modification that also affects their biophysical properties. We previously described the increased surface expression of GluN1/GluN2A receptors in transgenic mice overexpressing the Dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), suggesting that DYRK1A regulates NMDARs. Here we have further investigated whether the density and activity of NMDARs were modulated by DYRK1A phosphorylation. Accordingly, we show that endogenous DYRK1A is recruited to GluN2A-containing NMDARs in the adult mouse brain, and we identify a DYRK1A phosphorylation site at Ser1048 of GluN2A, within its intracellular C-terminal domain. Mechanistically, the DYRK1A-dependent phosphorylation of GluN2A at Ser1048 hinders the internalization of GluN1/GluN2A, causing an increase of surface GluN1/GluN2A in heterologous systems, as well as in primary cortical neurons. Furthermore, GluN2A phosphorylation at Ser1048 increases the current density and potentiates the gating of GluN1/GluN2A receptors. We conclude that DYRK1A is a direct regulator of NMDA receptors and we propose a novel mechanism for the control of NMDAR activity in neurons. PMID:25368549

  9. Mechanisms regulating phosphatase specificity and the removal of individual phosphorylation sites during mitotic exit.

    PubMed

    Rogers, Samuel; McCloy, Rachael; Watkins, D Neil; Burgess, Andrew

    2016-07-01

    Entry into mitosis is driven by the activity of kinases, which phosphorylate over 7000 proteins on multiple sites. For cells to exit mitosis and segregate their genome correctly, these phosphorylations must be removed in a specific temporal order. This raises a critical and important question: how are specific phosphorylation sites on an individual protein removed? Traditionally, the temporal order of dephosphorylation was attributed to decreasing kinase activity. However, recent evidence in human cells has identified unique patterns of dephosphorylation during mammalian mitotic exit that cannot be fully explained by the loss of kinase activity. This suggests that specificity is determined in part by phosphatases. In this review, we explore how the physicochemical properties of an individual phosphosite and its surrounding amino acids can affect interactions with a phosphatase. These positive and negative interactions in turn help determine the specific pattern of dephosphorylation required for correct mitotic exit. PMID:27417119

  10. Phosphorylation of CREB at Ser-133 induces complex formation with CREB-binding protein via a direct mechanism.

    PubMed Central

    Parker, D; Ferreri, K; Nakajima, T; LaMorte, V J; Evans, R; Koerber, S C; Hoeger, C; Montminy, M R

    1996-01-01

    We have characterized a phosphoserine binding domain in the coactivator CREB-binding protein (CBP) which interacts with the protein kinase A-phosphorylated, and hence activated, form of the cyclic AMP-responsive factor CREB. The CREB binding domain, referred to as KIX, is alpha helical and binds to an unstructured kinase-inducible domain in CREB following phosphorylation of CREB at Ser-133. Phospho-Ser-133 forms direct contacts with residues in KIX, and these contacts are further stabilized by hydrophobic residues in the kinase-inducible domain which flank phospho-Ser-133. Like the src homology 2 (SH2) domains which bind phosphotyrosine-containing peptides, phosphoserine 133 appears to coordinate with a single arginine residue (Arg-600) in KIX which is conserved in the CBP-related protein P300. Since mutagenesis of Arg-600 to Gln severely reduces CREB-CBP complex formation, our results demonstrate that, as in the case of tyrosine kinase pathways, signal transduction through serine/threonine kinase pathways may also require protein interaction motifs which are capable of recognizing phosphorylated amino acids. PMID:8552098

  11. Mitotic Phosphorylation of Histone H3: Spatio-Temporal Regulation by Mammalian Aurora Kinases

    PubMed Central

    Crosio, Claudia; Fimia, Gian Maria; Loury, Romain; Kimura, Masashi; Okano, Yukio; Zhou, Hongyi; Sen, Subrata; Allis, C. David; Sassone-Corsi, Paolo

    2002-01-01

    Phosphorylation at a highly conserved serine residue (Ser-10) in the histone H3 tail is considered to be a crucial event for the onset of mitosis. This modification appears early in the G2 phase within pericentromeric heterochromatin and spreads in an ordered fashion coincident with mitotic chromosome condensation. Mutation of Ser-10 is essential in Tetrahymena, since it results in abnormal chromosome segregation and extensive chromosome loss during mitosis and meiosis, establishing a strong link between signaling and chromosome dynamics. Although mitotic H3 phosphorylation has been long recognized, the transduction routes and the identity of the protein kinases involved have been elusive. Here we show that the expression of Aurora-A and Aurora-B, two kinases of the Aurora/AIK family, is tightly coordinated with H3 phosphorylation during the G2/M transition. During the G2 phase, the Aurora-A kinase is coexpressed while the Aurora-B kinase colocalizes with phosphorylated histone H3. At prophase and metaphase, Aurora-A is highly localized in the centrosomic region and in the spindle poles while Aurora-B is present in the centromeric region concurrent with H3 phosphorylation, to then translocate by cytokinesis to the midbody region. Both Aurora-A and Aurora-B proteins physically interact with the H3 tail and efficiently phosphorylate Ser10 both in vitro and in vivo, even if Aurora-A appears to be a better H3 kinase than Aurora-B. Since Aurora-A and Aurora-B are known to be overexpressed in a variety of human cancers, our findings provide an attractive link between cell transformation, chromatin modifications and a specific kinase system. PMID:11784863

  12. Phosphorylation on Ser-279 and Ser-282 of connexin43 regulates endocytosis and gap junction assembly in pancreatic cancer cells

    PubMed Central

    Johnson, Kristen E.; Mitra, Shalini; Katoch, Parul; Kelsey, Linda S.; Johnson, Keith R.; Mehta, Parmender P.

    2013-01-01

    The molecular mechanisms regulating the assembly of connexins (Cxs) into gap junctions are poorly understood. Using human pancreatic tumor cell lines BxPC3 and Capan-1, which express Cx26 and Cx43, we show that, upon arrival at the cell surface, the assembly of Cx43 is impaired. Connexin43 fails to assemble, because it is internalized by clathrin-mediated endocytosis. Assembly is restored upon expressing a sorting-motif mutant of Cx43, which does not interact with the AP2 complex, and by expressing mutants that cannot be phosphorylated on Ser-279 and Ser-282. The mutants restore assembly by preventing clathrin-mediated endocytosis of Cx43. Our results also document that the sorting-motif mutant is assembled into gap junctions in cells in which the expression of endogenous Cx43 has been knocked down. Remarkably, Cx43 mutants that cannot be phosphorylated on Ser-279 or Ser-282 are assembled into gap junctions only when connexons are composed of Cx43 forms that can be phosphorylated on these serines and forms in which phosphorylation on these serines is abolished. Based on the subcellular fate of Cx43 in single and contacting cells, our results document that the endocytic itinerary of Cx43 is altered upon cell–cell contact, which causes Cx43 to traffic by EEA1-negative endosomes en route to lysosomes. Our results further show that gap-junctional plaques formed of a sorting motif–deficient mutant of Cx43, which is unable to be internalized by the clathrin-mediated pathway, are predominantly endocytosed in the form of annular junctions. Thus the differential phosphorylation of Cx43 on Ser-279 and Ser-282 is fine-tuned to control Cx43’s endocytosis and assembly into gap junctions. PMID:23363606

  13. Regulation Dynamics of Leishmania Differentiation: Deconvoluting Signals and Identifying Phosphorylation Trends*

    PubMed Central

    Tsigankov, Polina; Gherardini, Pier Federico; Helmer-Citterich, Manuela; Späth, Gerald F.; Myler, Peter J.; Zilberstein, Dan

    2014-01-01

    Leishmania are obligatory intracellular parasitic protozoa that cause a wide range of diseases in humans, cycling between extracellular promastigotes in the mid-gut of sand flies and intracellular amastigotes in the phagolysosomes of mammalian macrophages. Although many of the molecular mechanisms of development inside macrophages remain a mystery, the development of a host-free system that simulates phagolysosome conditions (37 °C and pH 5.5) has provided new insights into these processes. The time course of promastigote-to-amastigote differentiation can be divided into four morphologically distinct phases: I, signal perception (0–5 h after exposure); II, movement cessation and aggregation (5–10 h); III, amastigote morphogenesis (10–24 h); and IV, maturation (24–120 h). Transcriptomic and proteomic analyses have indicated that differentiation is a coordinated process that results in adaptation to life inside phagolysosomes. Recent phosphoproteomic analysis revealed extensive differences in phosphorylation between promastigotes and amastigotes and identified stage-specific phosphorylation motifs. We hypothesized that the differentiation signal activates a phosphorylation pathway that initiates Leishmania transformation, and here we used isobaric tags for relative and absolute quantitation to interrogate the dynamics of changes in the phosphorylation profile during Leishmania donovani promastigote-to-amastigote differentiation. Analysis of 163 phosphopeptides (from 106 proteins) revealed six distinct kinetic profiles; with increases in phosphorylation predominated during phases I and III, whereas phases II and IV were characterized by greater dephosphorylation. Several proteins (including a protein kinase) were phosphorylated in phase I after exposure to the complete differentiation signal (i.e. signal-specific; 37 °C and pH 5.5), but not after either of the physical parameters separately. Several other protein kinases (including regulatory subunits) and

  14. WNK1-regulated inhibitory phosphorylation of the KCC2 cotransporter maintains the depolarizing action of GABA in immature neurons.

    PubMed

    Friedel, Perrine; Kahle, Kristopher T; Zhang, Jinwei; Hertz, Nicholas; Pisella, Lucie I; Buhler, Emmanuelle; Schaller, Fabienne; Duan, JingJing; Khanna, Arjun R; Bishop, Paul N; Shokat, Kevan M; Medina, Igor

    2015-06-30

    Activation of Cl(-)-permeable γ-aminobutyric acid type A (GABAA) receptors elicits synaptic inhibition in mature neurons but excitation in immature neurons. This developmental "switch" in the GABA function depends on a postnatal decrease in intraneuronal Cl(-) concentration mediated by KCC2, a Cl(-)-extruding K(+)-Cl(-) cotransporter. We showed that the serine-threonine kinase WNK1 [with no lysine (K)] forms a physical complex with KCC2 in the developing mouse brain. Dominant-negative mutation, genetic depletion, or chemical inhibition of WNK1 in immature neurons triggered a hyperpolarizing shift in GABA activity by enhancing KCC2-mediated Cl(-) extrusion. This increase in KCC2 activity resulted from reduced inhibitory phosphorylation of KCC2 at two C-terminal threonines, Thr(906) and Thr(1007). Phosphorylation of both Thr(906) and Thr(1007) was increased in immature versus mature neurons. Together, these data provide insight into the mechanism regulating Cl(-) homeostasis in immature neurons, and suggest that WNK1-regulated changes in KCC2 phosphorylation contribute to the developmental excitatory-to-inhibitory GABA sequence. PMID:26126716

  15. Phosphorylation of the Bin, Amphiphysin, and RSV161/167 (BAR) domain of ACAP4 regulates membrane tubulation.

    PubMed

    Zhao, Xuannv; Wang, Dongmei; Liu, Xing; Liu, Lifang; Song, Zhenwei; Zhu, Tongge; Adams, Gregory; Gao, Xinjiao; Tian, Ruijun; Huang, Yuejia; Chen, Runhua; Wang, Fengsong; Liu, Dong; Yu, Xue; Chen, Yong; Chen, Zhengjun; Teng, Maikun; Ding, Xia; Yao, Xuebiao

    2013-07-01

    ArfGAP With Coiled-Coil, Ankyrin Repeat And PH Domains 4 (ACAP4) is an ADP-ribosylation factor 6 (ARF6) GTPase-activating protein essential for EGF-elicited cell migration. However, how ACAP4 regulates membrane dynamics and curvature in response to EGF stimulation is unknown. Here, we show that phosphorylation of the N-terminal region of ACAP4, named the Bin, Amphiphysin, and RSV161/167 (BAR) domain, at Tyr34 is necessary for EGF-elicited membrane remodeling. Domain structure analysis demonstrates that the BAR domain regulates membrane curvature. EGF stimulation of cells causes phosphorylation of ACAP4 at Tyr34, which subsequently promotes ACAP4 homodimer curvature. The phospho-mimicking mutant of ACAP4 demonstrates lipid-binding activity and tubulation in vitro, and ARF6 enrichment at the membrane is associated with ruffles of EGF-stimulated cells. Expression of the phospho-mimicking ACAP4 mutant promotes ARF6-dependent cell migration. Thus, the results present a previously undefined mechanism by which EGF-elicited phosphorylation of the BAR domain controls ACAP4 molecular plasticity and plasma membrane dynamics during cell migration. PMID:23776207

  16. Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation.

    PubMed

    Feng, Yuchen; Backues, Steven K; Baba, Misuzu; Heo, Jin-Mi; Harper, J Wade; Klionsky, Daniel J

    2016-04-01

    Macroautophagy is primarily a degradative process that cells use to break down their own components to recycle macromolecules and provide energy under stress conditions, and defects in macroautophagy lead to a wide range of diseases. Atg9, conserved from yeast to mammals, is the only identified transmembrane protein in the yeast core macroautophagy machinery required for formation of the sequestering compartment termed the autophagosome. This protein undergoes dynamic movement between the phagophore assembly site (PAS), where the autophagosome precursor is nucleated, and peripheral sites that may provide donor membrane for expansion of the phagophore. Atg9 is a phosphoprotein that is regulated by the Atg1 kinase. We used stable isotope labeling by amino acids in cell culture (SILAC) to identify phosphorylation sites on this protein and identified an Atg1-independent phosphorylation site at serine 122. A nonphosphorylatable Atg9 mutant showed decreased autophagy activity, whereas the phosphomimetic mutant enhanced activity. Electron microscopy analysis suggests that the different levels of autophagy activity reflect differences in autophagosome formation, correlating with the delivery of Atg9 to the PAS. Finally, this phosphorylation regulates Atg9 interaction with Atg23 and Atg27. PMID:27050455

  17. Shaggy/glycogen synthase kinase 3β and phosphorylation of Sarah/regulator of calcineurin are essential for completion of Drosophila female meiosis

    PubMed Central

    Takeo, Satomi; Swanson, Selene K.; Nandanan, Kavyasree; Nakai, Yasuhiro; Aigaki, Toshiro; Washburn, Michael P.; Florens, Laurence; Hawley, R. Scott

    2012-01-01

    The Ca2+/Calmodulin-dependent phosphatase calcineurin is essential for exit from meiotic arrest at metaphases I and II in Drosophila and Xenopus oocytes. We previously found that Sarah, the Drosophila homolog of regulator of calcineurin, acts as a positive regulator of calcineurin and is required to complete anaphase I of female meiosis. Here, we undertook biochemical approaches, including MS and posttranslational modification analyses, to better understand the mechanism by which Sarah regulates calcineurin. A search for phosphorylated residues revealed that Sarah is highly phosphorylated at Ser100, Thr102, and Ser219 in both ovaries and activated eggs and that Ser215 is phosphorylated only in activated eggs. Functional analyses using mutant forms of Sarah showed that phosphorylation at Ser215, a consensus phosphorylation site for glycogen synthase kinase 3β (GSK-3β) and its priming kinase site Ser219, are essential for Sarah function. Furthermore, germ-line clones homozygous for a null allele of shaggy (Drosophila GSK-3β) both fail to complete meiosis and lack phosphorylation of Sarah at Ser215, suggesting that the phosphorylation of Sarah by Shaggy/GSK-3β is required to complete meiosis. Our findings suggest a mechanism in which Shaggy/GSK-3β activates calcineurin through Sarah phosphorylation on egg activation in Drosophila. PMID:22421435

  18. KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1

    SciTech Connect

    Goettel, Jeremy A.; Liang, Dongchun; Hilliard, Valda C.; Edelblum, Karen L.; Broadus, Matthew R.; Gould, Kathleen L.; Hanks, Steven K.; Polk, D. Brent

    2011-02-15

    The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway is a highly conserved signaling pathway that regulates diverse cellular processes including differentiation, proliferation, and survival. Kinase suppressor of Ras-1 (KSR1) binds each of the three ERK cascade components to facilitate pathway activation. Even though KSR1 contains a C-terminal kinase domain, evidence supporting the catalytic function of KSR1 remains controversial. In this study, we produced recombinant wild-type or kinase-inactive (D683A/D700A) KSR1 proteins in Escherichia coli to test the hypothesis that KSR1 is a functional protein kinase. Recombinant wild-type KSR1, but not recombinant kinase-inactive KSR1, underwent autophosphorylation on serine residue(s), phosphorylated myelin basic protein (MBP) as a generic substrate, and phosphorylated recombinant kinase-inactive MAPK/ERK kinase-1 (MEK1). Furthermore, FLAG immunoprecipitates from KSR1{sup -/-} colon epithelial cells stably expressing FLAG-tagged wild-type KSR1 (+KSR1), but not vector (+vector) or FLAG-tagged kinase-inactive KSR1 (+D683A/D700A), were able to phosphorylate kinase-inactive MEK1. Since TNF activates the ERK pathway in colon epithelial cells, we tested the biological effects of KSR1 in the survival response downstream of TNF. We found that +vector and +D683A/D700A cells underwent apoptosis when treated with TNF, whereas +KSR1 cells were resistant. However, +KSR1 cells were sensitized to TNF-induced cell loss in the absence of MEK kinase activity. These data provide clear evidence that KSR1 is a functional protein kinase, MEK1 is an in vitro substrate of KSR1, and the catalytic activities of both proteins are required for eliciting cell survival responses downstream of TNF.

  19. Cross-talk between Arg methylation and Ser phosphorylation modulates apoptosis signal–regulating kinase 1 activation in endothelial cells

    PubMed Central

    Chen, Ming; Qu, Xiaosheng; Zhang, Zhiqing; Wu, Huayu; Qin, Xia; Li, Fuji; Liu, Zhenfang; Tian, Liyuan; Miao, Jianhua; Shu, Wei

    2016-01-01

    We describe a novel functional interaction between ASK1 and PRMT5. We show that PRMT5 interacts with and methylates ASK1 at arginine residue 89 and thereby negatively regulates its activity by promoting the interaction between ASK1 and Akt and thus phosphorylating ASK1 at serine residue 83. Furthermore, the association between ASK1 and Akt is enhanced by VEGF stimulation, and PRMT5 is required for this association. Moreover, PRMT5-mediated ASK1 methylation impaired the H2O2-induced activity of ASK1, and this inhibitory effect of PRMT5 was abolished by replacement of arginine 89 with Trp or depletion of PRMT5 expression by RNA interference. Together the results demonstrate cross-talk between arginine methylation and serine phosphorylation in ASK1. PMID:26912789

  20. Novel mechanisms of Na+ retention in obesity: phosphorylation of NKCC2 and regulation of SPAK/OSR1 by AMPK.

    PubMed

    Davies, Matthew; Fraser, Scott A; Galic, Sandra; Choy, Suet-Wan; Katerelos, Marina; Gleich, Kurt; Kemp, Bruce E; Mount, Peter F; Power, David A

    2014-07-01

    Enhanced tubular reabsorption of salt is important in the pathogenesis of obesity-related hypertension, but the mechanisms remain poorly defined. To identify changes in the regulation of salt transporters in the kidney, C57BL/6 mice were fed a 40% fat diet [high-fat diet (HFD)] or a 12% fat diet (control diet) for 14 wk. Compared with control diet-fed mice, HFD-fed mice had significantly greater elevations in weight, blood pressure, and serum insulin and leptin levels. When we examined Na(+) transporter expression, Na(+)-K(+)-2Cl(-) cotransporter (NKCC2) was unchanged in whole kidney and reduced in the cortex, Na(+)-Cl(-) cotransporter (NCC) and α-epithelial Na(+) channel (ENaC) and γ-ENaC were unchanged, and β-ENaC was reduced. Phosphorylation of NCC was unaltered. Activating phosphorylation of NKCC2 at S126 was increased 2.5-fold. Activation of STE-20/SPS1-related proline-alanine-rich protein kinase (SPAK)/oxidative stress responsive 1 kinase (OSR1) was increased in kidneys from HFD-fed mice, and enhanced phosphorylation of NKCC2 at T96/T101 was evident in the cortex. Increased activity of NKCC2 in vivo was confirmed with diuretic experiments. HFD-fed mice had reduced activating phosphorylation of AMP-activated protein kinase (AMPK) in the renal cortex. In vitro, activation of AMPK led to a reduction in phospho-SPAK/phospho-OSR1 in AMPK(+/+) murine embryonic fibroblasts (MEFs), but no effect was seen in AMPK(-/-) MEFs, indicating an AMPK-mediated effect. Activation of the with no lysine kinase/SPAK/OSR1 pathway with low-NaCl solution invoked a greater elevation in phospho-SPAK/phospho-OSR1 in AMPK(-/-) MEFs than in AMPK(+/+) MEFs, consistent with a negative regulatory effect of AMPK on SPAK/OSR1 phosphorylation. In conclusion, this study identifies increased phosphorylation of NKCC2 on S126 as a hitherto-unrecognized mediator of enhanced Na(+) reabsorption in obesity and identifies a new role for AMPK in regulating the activity of SPAK/OSR1. PMID:24808538

  1. S6 kinase inactivation impairs growth and translational target phosphorylation in muscle cells maintaining proper regulation of protein turnover.

    PubMed

    Mieulet, Virginie; Roceri, Mila; Espeillac, Catherine; Sotiropoulos, Athanassia; Ohanna, Mickael; Oorschot, Viola; Klumperman, Judith; Sandri, Marco; Pende, Mario

    2007-08-01

    A defect in protein turnover underlies multiple forms of cell atrophy. Since S6 kinase (S6K)-deficient cells are small and display a blunted response to nutrient and growth factor availability, we have hypothesized that mutant cell atrophy may be triggered by a change in global protein synthesis. By using mouse genetics and pharmacological inhibitors targeting the mammalian target of rapamycin (mTOR)/S6K pathway, here we evaluate the control of translational target phosphorylation and protein turnover by the mTOR/S6K pathway in skeletal muscle and liver tissues. The phosphorylation of ribosomal protein S6 (rpS6), eukaryotic initiation factor-4B (eIF4B), and eukaryotic elongation factor-2 (eEF2) is predominantly regulated by mTOR in muscle cells. Conversely, in liver, the MAPK and phosphatidylinositol 3-kinase pathways also play an important role, suggesting a tissue-specific control. S6K deletion in muscle mimics the effect of the mTOR inhibitor rapamycin on rpS6 and eIF4B phosphorylation without affecting eEF2 phosphorylation. To gain insight on the functional consequences of these modifications, methionine incorporation and polysomal distribution were assessed in muscle cells. Rates and rapamycin sensitivity of global translation initiation are not altered in S6K-deficient muscle cells. In addition, two major pathways of protein degradation, autophagy and expression of the muscle-specific atrophy-related E3 ubiquitin ligases, are not affected by S6K deletion. Our results do not support a role for global translational control in the growth defect due to S6K deletion, suggesting specific modes of growth control and translational target regulation downstream of mTOR. PMID:17494629

  2. Phosphorylation Regulates the Endocytic Function of the Yeast Dynamin-Related Protein Vps1.

    PubMed

    Smaczynska-de Rooij, Iwona I; Marklew, Christopher J; Allwood, Ellen G; Palmer, Sarah E; Booth, Wesley I; Mishra, Ritu; Goldberg, Martin W; Ayscough, Kathryn R

    2016-03-01

    The family of dynamin proteins is known to function in many eukaryotic membrane fusion and fission events. The yeast dynamin-related protein Vps1 functions at several stages of membrane trafficking, including Golgi apparatus to endosome and vacuole, peroxisomal fission, and endocytic scission. We have previously shown that in its endocytic role, Vps1 functions with the amphiphysin heterodimer Rvs161/Rvs167 to facilitate scission and release of vesicles. Phosphoproteome studies of Saccharomyces cerevisiae have identified a phosphorylation site in Vps1 at serine 599. In this study, we confirmed this phosphorylation event, and we reveal that, like Rvs167, Vps1 can be phosphorylated by the yeast cyclin-associated kinase Pho85 in vivo and in vitro. The importance of this posttranslational modification was revealed when mutagenesis of S599 to a phosphomimetic or nonphosphorylatable form caused defects in endocytosis but not in other functions associated with Vps1. Mutation to nonphosphorylatable valine inhibited the Rvs167 interaction, while both S599V and S599D caused defects in vesicle scission, as shown by both live-cell imaging and electron microscopy of endocytic invaginations. Our data support a model in which phosphorylation and dephosphorylation of Vps1 promote distinct interactions and highlight the importance of such regulatory events in facilitating sequential progression of the endocytic process. PMID:26711254

  3. Regulation of TCF3 by Wnt-dependent phosphorylation during vertebrate axis specification.

    PubMed

    Hikasa, Hiroki; Ezan, Jerome; Itoh, Keiji; Li, Xiaotong; Klymkowsky, Michael W; Sokol, Sergei Y

    2010-10-19

    A commonly accepted model of Wnt/β-catenin signaling involves target gene activation by a complex of β-catenin with a T-cell factor (TCF) family member. TCF3 is a transcriptional repressor that has been implicated in Wnt signaling and plays key roles in embryonic axis specification and stem cell differentiation. Here we demonstrate that Wnt proteins stimulate TCF3 phosphorylation in gastrulating Xenopus embryos and mammalian cells. This phosphorylation event involves β-catenin-mediated recruitment of homeodomain-interacting protein kinase 2 (HIPK2) to TCF3 and culminates in the dissociation of TCF3 from a target gene promoter. Mutated TCF3 proteins resistant to Wnt-dependent phosphorylation function as constitutive inhibitors of Wnt-mediated activation of Vent2 and Cdx4 during anteroposterior axis specification. These findings reveal an alternative in vivo mechanism of Wnt signaling that involves TCF3 phosphorylation and subsequent derepression of target genes and link this molecular event to a specific developmental process. PMID:20951344

  4. Phosphorylation of leukocyte PECAM and its association with detergent-resistant membranes regulate transendothelial migration.

    PubMed

    Florey, Oliver; Durgan, Joanne; Muller, William

    2010-08-01

    Leukocyte migration across the endothelial lining is a critical step in the body's response to infection and inflammation. The homophilic interaction between endothelial PECAM and leukocyte PECAM is essential for this process. The molecular events that are triggered in the endothelial cell by PECAM engagement have been well characterized; however, the function of leukocyte PECAM remains to be elucidated. To study this, we first blocked leukocyte transmigration using anti-PECAM Ab and then specifically activated leukocyte PECAM. This was sufficient to overcome the block and promote transmigration, suggesting an active signaling role for leukocyte PECAM. Consistent with this, we found that ligation of leukocyte PECAM induces phosphorylation of two tyrosine residues on its cytoplasmic tail. By performing RNA interference-rescue experiments, we demonstrate that these phosphorylation events are indispensable for transendothelial migration. Finally, we show that leukocyte PECAM translocates to a detergent-resistant membrane (DRM) during transmigration. PECAM localized in DRMs displays reduced phosphorylation and does not support transmigration. Together, these data support a model whereby engagement of leukocyte PECAM induces its transient tyrosine phosphorylation and induction of downstream signals that drive transmigration. These signals are then downregulated following PECAM translocation to DRMs. PMID:20581150

  5. Vimentin expression influences flow dependent VASP phosphorylation and regulates cell migration and proliferation

    SciTech Connect

    Lund, Natalie; Henrion, Daniel; Tiede, Petra; Ziche, Marina; Schunkert, Heribert; Ito, Wulf D.

    2010-05-07

    The cytoskeleton plays a central role for the integration of biochemical and biomechanical signals across the cell required for complex cellular functions. Recent studies indicate that the intermediate filament vimentin is necessary for endothelial cell morphogenesis e.g. in the context of leukocyte transmigration. Here, we present evidence, that the scaffold provided by vimentin is essential for VASP localization and PKG mediated VASP phosphorylation and thus controls endothelial cell migration and proliferation. Vimentin suppression using siRNA technique significantly decreased migration velocity by 50% (videomicroscopy), diminished transmigration activity by 42.5% (Boyden chamber) and reduced proliferation by 43% (BrdU-incorporation). In confocal microscopy Vimentin colocalized with VASP and PKG in endothelial cells. Vimentin suppression was accompanied with a translocation of VASP from focal contacts to the perinuclear region. VASP/Vimentin and PKG/Vimentin colocalization appeared to be essential for proper PKG mediated VASP phosphorylation because we detected a diminished expression of PKG and p{sup Ser239}-VASP in vimentin-suppressed cells, Furthermore, the induction of VASP phosphorylation in perfused arteries was markedly decreased in vimentin knockout mice compared to wildtypes. A link is proposed between vimentin, VASP phosphorylation and actin dynamics that delivers an explanation for the important role of vimentin in controlling endothelial cell morphogenesis.

  6. Phosphorylation Regulates the Endocytic Function of the Yeast Dynamin-Related Protein Vps1

    PubMed Central

    Smaczynska-de Rooij, Iwona I.; Marklew, Christopher J.; Allwood, Ellen G.; Palmer, Sarah E.; Booth, Wesley I.; Mishra, Ritu; Goldberg, Martin W.

    2015-01-01

    The family of dynamin proteins is known to function in many eukaryotic membrane fusion and fission events. The yeast dynamin-related protein Vps1 functions at several stages of membrane trafficking, including Golgi apparatus to endosome and vacuole, peroxisomal fission, and endocytic scission. We have previously shown that in its endocytic role, Vps1 functions with the amphiphysin heterodimer Rvs161/Rvs167 to facilitate scission and release of vesicles. Phosphoproteome studies of Saccharomyces cerevisiae have identified a phosphorylation site in Vps1 at serine 599. In this study, we confirmed this phosphorylation event, and we reveal that, like Rvs167, Vps1 can be phosphorylated by the yeast cyclin-associated kinase Pho85 in vivo and in vitro. The importance of this posttranslational modification was revealed when mutagenesis of S599 to a phosphomimetic or nonphosphorylatable form caused defects in endocytosis but not in other functions associated with Vps1. Mutation to nonphosphorylatable valine inhibited the Rvs167 interaction, while both S599V and S599D caused defects in vesicle scission, as shown by both live-cell imaging and electron microscopy of endocytic invaginations. Our data support a model in which phosphorylation and dephosphorylation of Vps1 promote distinct interactions and highlight the importance of such regulatory events in facilitating sequential progression of the endocytic process. PMID:26711254

  7. Subcellular localization and Ser-137 phosphorylation regulate tumor-suppressive activity of profilin-1.

    PubMed

    Diamond, Marc I; Cai, Shirong; Boudreau, Aaron; Carey, Clifton J; Lyle, Nicholas; Pappu, Rohit V; Swamidass, S Joshua; Bissell, Mina; Piwnica-Worms, Helen; Shao, Jieya

    2015-04-01

    The actin-binding protein profilin-1 (Pfn1) inhibits tumor growth and yet is also required for cell proliferation and survival, an apparent paradox. We previously identified Ser-137 of Pfn1 as a phosphorylation site within the poly-l-proline (PLP) binding pocket. Here we confirm that Ser-137 phosphorylation disrupts Pfn1 binding to its PLP-containing ligands with little effect on actin binding. We find in mouse xenografts of breast cancer cells that mimicking Ser-137 phosphorylation abolishes cell cycle arrest and apoptotic sensitization by Pfn1 and confers a growth advantage to tumors. This indicates a previously unrecognized role of PLP binding in Pfn1 antitumor effects. Spatial restriction of Pfn1 to the nucleus or cytoplasm indicates that inhibition of tumor cell growth by Pfn1 requires its nuclear localization, and this activity is abolished by a phosphomimetic mutation on Ser-137. In contrast, cytoplasmic Pfn1 lacks inhibitory effects on tumor cell growth but rescues morphological and proliferative defects of PFN1 null mouse chondrocytes. These results help reconcile seemingly opposed cellular effects of Pfn1, provide new insights into the antitumor mechanism of Pfn1, and implicate Ser-137 phosphorylation as a potential therapeutic target for breast cancer. PMID:25681442

  8. Regulation of RNA Polymerase II Termination by Phosphorylation of Gdown1*

    PubMed Central

    Guo, Jiannan; Turek, Michael E.; Price, David H.

    2014-01-01

    Gdown1 is a substoichiometric subunit of RNA polymerase II (Pol II) that has been recently demonstrated to be involved in stabilizing promoter-proximal paused Pol II. It was shown to inhibit termination of Pol II by transcription termination factor 2 (TTF2) as well as block elongation stimulation by transcription factor IIF (TFIIF). Here, using in vitro transcription assays, we identified two functional domains in Gdown1. Although both are required to maintain a tight association with Pol II, the N- and C-terminal domains are responsible for blocking TTF2 and TFIIF, respectively. A highly conserved LPDKG motif found in the N-terminal domain of Gdown1 is also highly conserved in TTF2. Deletion of this motif eliminated the TTF2 inhibitory activity of Gdown1. We identified a phosphorylated form of Gdown1 with altered mobility in SDS-PAGE that appears during mitosis. A kinase in HeLa nuclear extract that caused the shift was partially purified. In vitro, Gdown1 phosphorylated by this kinase demonstrated reduced activity in blocking both TTF2 and TFIIF because of its reduced affinity for Pol II. Mass spectrometry identified Ser-270 as the site of this phosphorylation. An S270A mutation was not phosphorylated by the partially purified kinase, and an S270E mutation partially mimicked the properties of phospho-Gdown1. Gdown1 Ser-270 phosphorylation occurs predominately during mitosis, and we suggest that this would enable TTF2 to terminate all Pol II even if it is associated with Gdown1. PMID:24634214

  9. Algal dual-specificity tyrosine phosphorylation-regulated kinase, triacylglycerol accumulation regulator1, regulates accumulation of triacylglycerol in nitrogen or sulfur deficiency.

    PubMed

    Kajikawa, Masataka; Sawaragi, Yuri; Shinkawa, Haruka; Yamano, Takashi; Ando, Akira; Kato, Misako; Hirono, Masafumi; Sato, Naoki; Fukuzawa, Hideya

    2015-06-01

    Although microalgae accumulate triacylglycerol (TAG) and starch in response to nutrient-deficient conditions, the regulatory mechanisms are poorly understood. We report here the identification and characterization of a kinase, triacylglycerol accumulation regulator1 (TAR1), that is a member of the yeast (Saccharomyces cerevisiae) Yet another kinase1 (Yak1) subfamily in the dual-specificity tyrosine phosphorylation-regulated kinase family in a green alga (Chlamydomonas reinhardtii). The kinase domain of TAR1 showed auto- and transphosphorylation activities. A TAR1-defective mutant, tar1-1, accumulated TAG to levels 0.5- and 0.1-fold of those of a wild-type strain in sulfur (S)- and nitrogen (N)-deficient conditions, respectively. In N-deficient conditions, tar1-1 showed more pronounced arrest of cell division than the wild type, had increased cell size and cell dry weight, and maintained chlorophyll and photosynthetic activity, which were not observed in S-deficient conditions. In N-deficient conditions, global changes in expression levels of N deficiency-responsive genes in N assimilation and tetrapyrrole metabolism were noted between tar1-1 and wild-type cells. These results indicated that TAR1 is a regulator of TAG accumulation in S- and N-deficient conditions, and it functions in cell growth and repression of photosynthesis in conditions of N deficiency. PMID:25922058

  10. Algal Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase, Triacylglycerol Accumulation Regulator1, Regulates Accumulation of Triacylglycerol in Nitrogen or Sulfur Deficiency1[OPEN

    PubMed Central

    Kajikawa, Masataka; Sawaragi, Yuri; Shinkawa, Haruka; Yamano, Takashi; Ando, Akira; Kato, Misako; Hirono, Masafumi; Sato, Naoki

    2015-01-01

    Although microalgae accumulate triacylglycerol (TAG) and starch in response to nutrient-deficient conditions, the regulatory mechanisms are poorly understood. We report here the identification and characterization of a kinase, triacylglycerol accumulation regulator1 (TAR1), that is a member of the yeast (Saccharomyces cerevisiae) Yet another kinase1 (Yak1) subfamily in the dual-specificity tyrosine phosphorylation-regulated kinase family in a green alga (Chlamydomonas reinhardtii). The kinase domain of TAR1 showed auto- and transphosphorylation activities. A TAR1-defective mutant, tar1-1, accumulated TAG to levels 0.5- and 0.1-fold of those of a wild-type strain in sulfur (S)- and nitrogen (N)-deficient conditions, respectively. In N-deficient conditions, tar1-1 showed more pronounced arrest of cell division than the wild type, had increased cell size and cell dry weight, and maintained chlorophyll and photosynthetic activity, which were not observed in S-deficient conditions. In N-deficient conditions, global changes in expression levels of N deficiency-responsive genes in N assimilation and tetrapyrrole metabolism were noted between tar1-1 and wild-type cells. These results indicated that TAR1 is a regulator of TAG accumulation in S- and N-deficient conditions, and it functions in cell growth and repression of photosynthesis in conditions of N deficiency. PMID:25922058

  11. Myofibril growth during cardiac hypertrophy is regulated through dual phosphorylation and acetylation of the actin capping protein CapZ.

    PubMed

    Lin, Ying-Hsi; Warren, Chad M; Li, Jieli; McKinsey, Timothy A; Russell, Brenda

    2016-08-01

    The mechanotransduction signaling pathways initiated in heart muscle by increased mechanical loading are known to lead to long-term transcriptional changes and hypertrophy, but the rapid events for adaptation at the sarcomeric level are not fully understood. The goal of this study was to test the hypothesis that actin filament assembly during cardiomyocyte growth is regulated by post-translational modifications (PTMs) of CapZβ1. In rapidly hypertrophying neonatal rat ventricular myocytes (NRVMs) stimulated by phenylephrine (PE), two-dimensional gel electrophoresis (2DGE) of CapZβ1 revealed a shift toward more negative charge. Consistent with this, mass spectrometry identified CapZβ1 phosphorylation on serine-204 and acetylation on lysine-199, two residues which are near the actin binding surface of CapZβ1. Ectopic expression of dominant negative PKCɛ (dnPKCɛ) in NRVMs blunted the PE-induced increase in CapZ dynamics, as evidenced by the kinetic constant (Kfrap) of fluorescence recovery after photobleaching (FRAP), and concomitantly reduced phosphorylation and acetylation of CapZβ1. Furthermore, inhibition of class I histone deacetylases (HDACs) increased lysine-199 acetylation on CapZβ1, which increased Kfrap of CapZ and stimulated actin dynamics. Finally, we show that PE treatment of NRVMs results in decreased binding of HDAC3 to myofibrils, suggesting a signal-dependent mechanism for the regulation of sarcomere-associated CapZβ1 acetylation. Taken together, this dual regulation through phosphorylation and acetylation of CapZβ1 provides a novel model for the regulation of myofibril growth during cardiac hypertrophy. PMID:27185186

  12. Regulation of Nociceptive Plasticity Threshold and DARPP-32 Phosphorylation in Spinal Dorsal Horn Neurons by Convergent Dopamine and Glutamate Inputs.

    PubMed

    Buesa, Itsaso; Aira, Zigor; Azkue, Jon Jatsu

    2016-01-01

    Dopamine can influence NMDA receptor function and regulate glutamate-triggered long-term changes in synaptic strength in several regions of the CNS. In spinal cord, regulation of the threshold of synaptic plasticity may determine the proneness to undergo sensitization and hyperresponsiveness to noxious input. In the current study, we increased endogenous dopamine levels in the dorsal horn by using re-uptake inhibitor GBR 12935. During the so-induced hyperdopaminergic transmission, conditioning low-frequency (1 Hz) stimulation (LFS) to the sciatic nerve induced long-term potentiation (LTP) of C-fiber-evoked potentials in dorsal horn neurons. The magnitude of LTP was attenuated by blockade of either dopamine D1-like receptors (D1LRs) by with SCH 23390 or NMDA receptor subunit NR2B with antagonist Ro25-6981. Conditioning LFS during GBR 12935 administration increased phosphorylation of dopamine- and cAMP-regulated phosphoprotein of Mr 32kDa (DARPP-32) at threonine 34 residue in synaptosomal (P3) fraction of dorsal horn homogenates, as assessed by Western blot analysis, which was partially prevented by NR2B blockade prior to conditioning stimulation. Conditioning LFS also was followed by higher co-localization of phosphorylated form of NR2B at tyrosine 1472 and pDARPP-32Thr34- with postsynaptic marker PSD-95 in transverse L5 dorsal horn sections. Such increase could be significantly attenuated by D1LR blockade with SCH 23390. The current results support that coincidental endogenous recruitment of D1LRs and NR2B in dorsal horn synapses plays a role in regulating afferent-induced nociceptive plasticity. Parallel increases in DARPP-32 phosphorylation upon LTP induction suggests a role for this phosphoprotein as intracellular detector of convergent D1L- and NMDA receptor activation. PMID:27610622

  13. UV Damage-Induced Phosphorylation of HBO1 Triggers CRL4DDB2-Mediated Degradation To Regulate Cell Proliferation.

    PubMed

    Matsunuma, Ryoichi; Niida, Hiroyuki; Ohhata, Tatsuya; Kitagawa, Kyoko; Sakai, Satoshi; Uchida, Chiharu; Shiotani, Bunsyo; Matsumoto, Masaki; Nakayama, Keiichi I; Ogura, Hiroyuki; Shiiya, Norihiko; Kitagawa, Masatoshi

    2016-02-01

    Histone acetyltransferase binding to ORC-1 (HBO1) is a critically important histone acetyltransferase for forming the prereplicative complex (pre-RC) at the replication origin. Pre-RC formation is completed by loading of the MCM2-7 heterohexameric complex, which functions as a helicase in DNA replication. HBO1 recruited to the replication origin by CDT1 acetylates histone H4 to relax the chromatin conformation and facilitates loading of the MCM complex onto replication origins. However, the acetylation status and mechanism of regulation of histone H3 at replication origins remain elusive. HBO1 positively regulates cell proliferation under normal cell growth conditions. Whether HBO1 regulates proliferation in response to DNA damage is poorly understood. In this study, we demonstrated that HBO1 was degraded after DNA damage to suppress cell proliferation. Ser50 and Ser53 of HBO1 were phosphorylated in an ATM/ATR DNA damage sensor-dependent manner after UV treatment. ATM/ATR-dependently phosphorylated HBO1 preferentially interacted with DDB2 and was ubiquitylated by CRL4(DDB2). Replacement of endogenous HBO1 in Ser50/53Ala mutants maintained acetylation of histone H3K14 and impaired cell cycle regulation in response to UV irradiation. Our findings demonstrate that HBO1 is one of the targets in the DNA damage checkpoint. These results show that ubiquitin-dependent control of the HBO1 protein contributes to cell survival during UV irradiation. PMID:26572825

  14. TBL1 and TBLR1 Phosphorylation on Regulated Gene Promoters Overcomes Dual CtBP and NCoR/SMRT Transcriptional Repression Checkpoints

    PubMed Central

    Perissi, Valentina; Scafoglio, Claudio; Zhang, Jie; Ohgi, Kenneth A.; Rose, David W.; Glass, Christopher K.; Rosenfeld, Michael G.

    2008-01-01

    A key strategy to achieve regulated gene expression in higher eukaryotes is to prevent illegitimate signal-independent activation by imposing robust control on the dismissal of corepressors. Here, we report that many signaling pathways, including Notch, NFkB, and nuclear receptor ligands, are subjected to a dual repression “check point” based on distinct corepressor complexes. Gene activation requires the release of both CtBP1/2- and NCoR/SMRT-dependent repression, through the coordinate action of two highly related exchange factors, the transducer β-like proteins TBL1 and TBLR1, that license ubiquitylation and degradation of CtBP1/2 and NCoR/SMRT, respectively. Intriguingly, their function and differential specificity resides in only five specific Ser/Thr phosphorylation site differences, regulated by direct phosphorylation at the level of the promoter, as exemplified by the role of PKCδ in TBLR1-dependent dismissal of NCoR. Thus, our data reveal a strategy of dual- factors repression checkpoints, in which dedicated exchange factors serve as sensors for signal- specific dismissal of distinct corepressors, with specificity imposed by upstream signaling pathways. PMID:18374649

  15. Phosphorylation of intracellular proteins related to the multihormonal regulation of prolactin: comparison of normal anterior pituitary cells in culture with the tumor-derived GH cell lines

    SciTech Connect

    Beretta, L.; Boutterin, M.C.; Sobel, A.

    1988-01-01

    We have previously identified a group of cytoplasmic phosphoproteins (proteins 1-11) whose phosphorylation could be related, on a pharmacological basis, to the multihormonal regulation of PRL synthesis and release in the anterior pituitary tumor-derived GH cell lines. Phosphoproteins with identical migration properties on two-dimensional electrophoresis gels were also detectable in normal rat anterior pituitary cells in culture. We designed appropriate culture and (/sup 32/P) phosphate-labeling conditions allowing to analyze the regulation of the phosphorylation of these proteins in normal pituitary cells. TRH, 12-O-tetradecanoylphorbol-13-acetate, and vasoactive intestinal peptide induced the same qualitative changes in phosphorylation of proteins 1-11 in normal as in GH cells. Quantitative differences observed are most likely due to the heterogeneity of primary pituitary cultures. Phosphorylation changes affecting proteins 14-16, not previously detected in GH cells, were also observed with normal anterior pituitary cells. GH cell lines have lost the sensitivity of pituitary lactotrophs for dopamine, an important physiological inhibitor of PRL synthesis and release. In normal anterior pituitary cells in culture, dopamine inhibited also the TRH-stimulated phosphorylation of proteins 1-10, thus strengthening the correlation between phosphorylation of these proteins and multihormonal regulation of pituitary cell functions. Our results indicate: 1) that the same phosphoproteins as in GH cells are related to the multihormonal regulation of nontumoral, normal anterior pituitary cells in culture; 2) that dopamine acts by interfering with the phosphorylation of these proteins.

  16. Dopamine D2 receptors are involved in the regulation of Fyn and metabotropic glutamate receptor 5 phosphorylation in the rat striatum in vivo.

    PubMed

    Mao, Li-Min; Wang, John Q

    2016-04-01

    Fyn, a major Src family kinase (SFK) member that is densely expressed in striatal neurons, is actively involved in the regulation of cellular and synaptic activities in local neurons. This SFK member is likely regulated by dopamine signaling through a receptor mechanism involving dopamine D2 receptors (D2Rs). This study characterizes the D2R-dependent regulation of Fyn in the rat striatum in vivo. Moreover, we explore whether D2Rs regulate metabotropic glutamate receptor 5 (mGluR5) in its tyrosine phosphorylation and whether the D2R-SFK pathway modulates trafficking of mGluR5. We found that blockade of D2Rs by systemic administration of a D2R antagonist, eticlopride, substantially increased SFK phosphorylation in the striatum. This increase was a transient and reversible event. The eticlopride-induced SFK phosphorylation occurred predominantly in immunopurified Fyn but not in another SFK member, Src. Eticlopride also elevated tyrosine phosphorylation of mGluR5. In parallel, eticlopride enhanced synaptic delivery of active Fyn and mGluR5. Pretreatment with an SFK inhibitor blocked the eticlopride-induced tyrosine phosphorylation and synaptic trafficking of mGluR5. These results indicate that D2Rs inhibit SFK (mainly Fyn) phosphorylation in the striatum. D2Rs also inhibit tyrosine phosphorylation and synaptic recruitment of mGluR5 through a signaling mechanism likely involving Fyn. PMID:26777117

  17. Rad53-dependent phosphorylation of Swi6 and down-regulation of CLN1 and CLN2 transcription occur in response to DNA damage in Saccharomyces cerevisiae

    PubMed Central

    Sidorova, Julia M.; Breeden, Linda L.

    1997-01-01

    Budding yeast possesses a checkpoint-dependent mechanism of delaying G1 progression in response to UV and ionizing radiation DNA damage. We have shown that after a pulse of DNA damage in G1 with the alkylating agent MMS, there is also a MEC1-, RAD53-, and RAD9-dependent delay in G1. This delay occurs at or before Start, as the MMS-treated cells do not bud, remain sensitive to α-factor, and have low CLN1 and CLN2 transcript levels for a longer time than untreated cells. We further show that MMS directly and reversibly down-regulates CLN1 and CLN2 transcript levels. The initial drop in CLN transcript levels in MMS is not RAD53 dependent, but the kinetics of reaccumulation of CLN messages as cells recover from the damage is faster in rad53-11 cells than in wild type cells. This is not an indirect effect of faster progression through G1, because CLN transcripts reaccumulate faster in rad53-11 mutants arrested in G1 as well. In addition, the recovery of CLN mRNA levels can be also hastened by a SWI6 deletion or by overexpression of the truncated Swi4 (Swi4-t) that lacks the carboxy-terminal domain through which Swi4 associates with Swi6. This indicates that both Rad53 and Swi6 are negative regulators of CLN expression after DNA damage. Finally, Swi6 undergoes an MMS-inducible, RAD53-dependent phosphorylation in G1 cells, and Rad53, immunoprecipitated from MMS-treated cells, phosphorylates Swi6 in vitro. On the basis of these observations, we suggest that the Rad53-dependent phosphorylation of Swi6 may delay the transition to S phase by inhibiting CLN transcription. PMID:9367985

  18. Extracellular Signal-regulated Kinase Activation Is Required for Serine 727 Phosphorylation of STAT3 in Schwann Cells in vitro and in vivo

    PubMed Central

    Lee, Hyun Kyoung; Jung, Junyang; Lee, Sang Hwa; Seo, Su-Yeong; Suh, Duk Joon

    2009-01-01

    In the peripheral nerves, injury-induced cytokines and growth factors perform critical functions in the activation of both the MEK/ERK and JAK/STAT3 pathways. In this study, we determined that nerve injury-induced ERK activation was temporally correlated with STAT3 phosphorylation at the serine 727 residue. In cultured Schwann cells, we noted that ERK activation is required for the serine phosphorylation of STAT3 by neuropoietic cytokine interleukin-6 (IL-6). Serine phosphorylated STAT3 by IL-6 was transported into Schwann cell nuclei, thereby indicating that ERK may regulate the transcriptional activity of STAT3 via the induction of serine phosphorylation of STAT3. Neuregulin-1 (NRG) also induced the serine phosphorylation of STAT3 in an ERK-dependent fashion. In contrast with the IL-6 response, serine phosphorylated STAT3 induced by NRG was not detected in the nucleus, thus indicating the non-nuclear function of serine phosphorylated STAT3 in response to NRG. Finally, we determined that the inhibition of ERK prevented injury-induced serine phosphorylation of STAT3 in an ex-vivo explants culture of the sciatic nerves. Collectively, the results of this study show that ERK may be an upstream kinase for the serine phosphorylation of STAT3 induced by multiple stimuli in Schwann cells after peripheral nerve injury. PMID:19885032

  19. Extracellular Signal-regulated Kinase (ERK)-dependent Phosphorylation of Y-Box-binding Protein 1 (YB-1) Enhances Gene Expression in Granulosa Cells in Response to Follicle-stimulating Hormone (FSH).

    PubMed

    Donaubauer, Elyse M; Hunzicker-Dunn, Mary E

    2016-06-01

    Within the ovarian follicle, immature oocytes are surrounded and supported by granulosa cells (GCs). Stimulation of GCs by FSH leads to their proliferation and differentiation, events that are necessary for fertility. FSH activates multiple signaling pathways to regulate genes necessary for follicular maturation. Herein, we investigated the role of Y-box-binding protein-1 (YB-1) within GCs. YB-1 is a nucleic acid binding protein that regulates transcription and translation. Our results show that FSH promotes an increase in the phosphorylation of YB-1 on Ser(102) within 15 min that is maintained at significantly increased levels until ∼8 h post treatment. FSH-stimulated phosphorylation of YB-1(Ser(102)) is prevented by pretreatment of GCs with the PKA-selective inhibitor PKA inhibitor (PKI), the MEK inhibitor PD98059, or the ribosomal S6 kinase-2 (RSK-2) inhibitor BI-D1870. Thus, phosphorylation of YB-1 on Ser(102) is PKA-, ERK-, and RSK-2-dependent. However, pretreatment of GCs with the protein phosphatase 1 (PP1) inhibitor tautomycin increased phosphorylation of YB-1(Ser(102)) in the absence of FSH; FSH did not further increase YB-1(Ser(102)) phosphorylation. This result suggests that the major effect of RSK-2 is to inhibit PP1 rather than to directly phosphorylate YB-1 on Ser(102) YB-1 coimmunoprecipitated with PP1β catalytic subunit and RSK-2. Transduction of GCs with the dephospho-adenoviral-YB-1(S102A) mutant prevented the induction by FSH of Egfr, Cyp19a1, Inha, Lhcgr, Cyp11a1, Hsd17b1, and Pappa mRNAs and estradiol-17β production. Collectively, our results reveal that phosphorylation of YB-1 on Ser(102) via the ERK/RSK-2 signaling pathway is necessary for FSH-mediated expression of target genes required for maturation of follicles to a preovulatory phenotype. PMID:27080258

  20. Differential phosphorylation of translation initiation regulators 4EBP1, S6k1, and Erk 1/2 following inhibition of alcohol metabolism in mouse heart.

    PubMed

    Vary, Thomas C; Lang, Charles H

    2008-03-01

    Acute alcohol intoxication leads to an inhibition of protein synthesis in heart that results in part through altered phosphorylation of protein factors controlling mRNA translation initiation. The purpose of the present set of experiments was designed to examine the effects of inhibitors of ethanol metabolism on the phosphorylation of 4E-binding protein (4EBP1) and S6k1(Thr(389)), two factors regulating mRNA translation initiation. Phosphorylation of 4E-BP1, S6k1(Thr(389)), and Erk 1/2 was reduced 2 h following IP injection of alcohol. Pretreatment with 4-methylpyrazole (4-MP), an inhibitor of alcohol dehydrogenase (ADH), did not attenuate the ethanol-induced decrease in phosphorylation of 4EBP1 and S6k1(Thr(389)). In contrast, 4-MP prevented the decrease in Erk 1/2 phosphorylation observed with acute ethanol intoxication. Pretreatment with cyanamide, an inhibitor of aldehyde dehydrogenase, did not attenuate the ethanol-induced decrease in phosphorylation S6k1(Thr(389)), but partially prevented the ethanol-induced lowering of 4EBP1 phosphorylation. The studies indicate that modulation of ethanol metabolism through inhibition of ADH or aldehyde dehydrogenase leads to preferential modulation of the phosphorylation of distinct myocardial signaling systems involved in regulating protein synthesis. PMID:18317950

  1. Environmental controls: Market incentives v. direct regulation

    SciTech Connect

    Kosobud, R.F.; Atallah, D.S.

    1996-12-31

    Cap-and-trade environmental markets, where the commodities are tradable pollution rights, are being introduced in several closely watched applications as a potentially more cost-effective way of cleaning up the environment than direct or command-and-control (CAC) regulation. In this study, we examine the evidence on control cost savings provided by price and transactions data from the first few years of activity in two markets designed to reduce atmospheric pollution. Some observers of both markets have argued that prices for tradable permits lower than expected, and transactions fewer than expected, are evidence that the markets are not achieving the hoped for savings. It was found, on the contrary, that observed prices point toward more flexible and improved pollution control choices and that the number of transactions has been steadily increasing as market incentives are incorporated into enterprise decisions. These new markets during their first few years are generating, according to our estimates, control cost savings in the neighborhood of one to two billion dollars annually. However, there is evidence that the markets have not yet reached their full potential. In the course of this study, several obstacles to market performance were found that are worthy of attention by policy makers. 13 refs., 4 figs., 1 tab.

  2. Virus-Dependent Phosphorylation of the IRF-3 Transcription Factor Regulates Nuclear Translocation, Transactivation Potential, and Proteasome-Mediated Degradation

    PubMed Central

    Lin, Rongtuan; Heylbroeck, Christophe; Pitha, Paula M.; Hiscott, John

    1998-01-01

    The interferon regulatory factors (IRF) consist of a growing family of related transcription proteins first identified as regulators of the alpha beta interferon (IFN-α/β) gene promoters, as well as the interferon-stimulated response element (ISRE) of some IFN-stimulated genes. IRF-3 was originally identified as a member of the IRF family based on homology with other IRF family members and on binding to the ISRE of the ISG15 promoter. IRF-3 is expressed constitutively in a variety of tissues, and the relative levels of IRF-3 mRNA do not change in virus-infected or IFN-treated cells. In the present study, we demonstrate that following Sendai virus infection, IRF-3 is