Analyzing Pseudophosphatase Function.

PubMed

Hinton, Shantá D

2016-01-01

Pseudophosphatases regulate signal transduction cascades, but their mechanisms of action remain enigmatic. Reflecting this mystery, the prototypical pseudophosphatase STYX (phospho-serine-threonine/tyrosine-binding protein) was named with allusion to the river of the dead in Greek mythology to emphasize that these molecules are "dead" phosphatases. Although proteins with STYX domains do not catalyze dephosphorylation, this in no way precludes their having other functions as integral elements of signaling networks. Thus, understanding their roles in signaling pathways may mark them as potential novel drug targets. This chapter outlines common strategies used to characterize the functions of pseudophosphatases, using as an example MK-STYX [mitogen-activated protein kinase (MAPK) phospho-serine-threonine/tyrosine binding], which has been linked to tumorigenesis, apoptosis, and neuronal differentiation. We start with the importance of "restoring" (when possible) phosphatase activity in a pseudophosphatase so that the active mutant may be used as a comparison control throughout immunoprecipitation and mass spectrometry analyses. To this end, we provide protocols for site-directed mutagenesis, mammalian cell transfection, co-immunoprecipitation, phosphatase activity assays, and immunoblotting that we have used to investigate MK-STYX and the active mutant MK-STYXactive. We also highlight the importance of utilizing RNA interference (RNAi) "knockdown" technology to determine a cellular phenotype in various cell lines. Therefore, we outline our protocols for introducing short hairpin RNA (shRNA) expression plasmids into mammalians cells and quantifying knockdown of gene expression with real-time quantitative PCR (qPCR). A combination of cellular, molecular, biochemical, and proteomic techniques has served as powerful tools in identifying novel functions of the pseudophosphatase MK-STYX. Likewise, the information provided here should be a helpful guide to elucidating the function of other pseudophosphatases.

High intraocular pressure produces learning and memory impairments in rats.

PubMed

Yuan, Yuxiang; Chen, Zhiqi; Li, Lu; Li, Xing; Xia, Qian; Zhang, Hong; Duan, Qiming; Zhao, Yin

2017-11-15

Primary open angle glaucoma (POAG) is a leading cause of irreversible blindness worldwide. Previous MRI studies have revealed that POAG can be associated with alterations in hippocampal function. Thus, the aim of this study was to investigate a relationship between chronic high intraocular pressure (IOP) and hippocampal changes in a rat model. We used behavioural tests to assess learning and memory ability, and additionally investigated the hippocampal expression of pathological amyloid beta (Aβ), phospho-tau, and related pathway proteins. Chronic high IOP impaired learning and memory in rats and concurrently increased Aβ and phospho-tau expression in the hippocampus by altering the activation of different kinase (GSK-3β, BACE1) and phosphatase (PP2A) proteins in the hippocampus. This study provides novel evidence for the relationship between high IOP and hippocampal alterations, especially in the context of learning and memory. Copyright © 2017 Elsevier B.V. All rights reserved.

Glucose deprivation activates a metabolic and signaling amplification loop leading to cell death

PubMed Central

Graham, Nicholas A; Tahmasian, Martik; Kohli, Bitika; Komisopoulou, Evangelia; Zhu, Maggie; Vivanco, Igor; Teitell, Michael A; Wu, Hong; Ribas, Antoni; Lo, Roger S; Mellinghoff, Ingo K; Mischel, Paul S; Graeber, Thomas G

2012-01-01

The altered metabolism of cancer can render cells dependent on the availability of metabolic substrates for viability. Investigating the signaling mechanisms underlying cell death in cells dependent upon glucose for survival, we demonstrate that glucose withdrawal rapidly induces supra-physiological levels of phospho-tyrosine signaling, even in cells expressing constitutively active tyrosine kinases. Using unbiased mass spectrometry-based phospho-proteomics, we show that glucose withdrawal initiates a unique signature of phospho-tyrosine activation that is associated with focal adhesions. Building upon this observation, we demonstrate that glucose withdrawal activates a positive feedback loop involving generation of reactive oxygen species (ROS) by NADPH oxidase and mitochondria, inhibition of protein tyrosine phosphatases by oxidation, and increased tyrosine kinase signaling. In cells dependent on glucose for survival, glucose withdrawal-induced ROS generation and tyrosine kinase signaling synergize to amplify ROS levels, ultimately resulting in ROS-mediated cell death. Taken together, these findings illustrate the systems-level cross-talk between metabolism and signaling in the maintenance of cancer cell homeostasis. PMID:22735335

Unlimited multistability in multisite phosphorylation systems.

PubMed

Thomson, Matthew; Gunawardena, Jeremy

2009-07-09

Reversible phosphorylation on serine, threonine and tyrosine is the most widely studied posttranslational modification of proteins. The number of phosphorylated sites on a protein (n) shows a significant increase from prokaryotes, with n /= 150 sites. Multisite phosphorylation has many roles and site conservation indicates that increasing numbers of sites cannot be due merely to promiscuous phosphorylation. A substrate with n sites has an exponential number (2(n)) of phospho-forms and individual phospho-forms may have distinct biological effects. The distribution of these phospho-forms and how this distribution is regulated have remained unknown. Here we show that, when kinase and phosphatase act in opposition on a multisite substrate, the system can exhibit distinct stable phospho-form distributions at steady state and that the maximum number of such distributions increases with n. Whereas some stable distributions are focused on a single phospho-form, others are more diffuse, giving the phospho-proteome the potential to behave as a fluid regulatory network able to encode information and flexibly respond to varying demands. Such plasticity may underlie complex information processing in eukaryotic cells and suggests a functional advantage in having many sites. Our results follow from the unusual geometry of the steady-state phospho-form concentrations, which we show to constitute a rational algebraic curve, irrespective of n. We thereby reduce the complexity of calculating steady states from simulating 3 x 2(n) differential equations to solving two algebraic equations, while treating parameters symbolically. We anticipate that these methods can be extended to systems with multiple substrates and multiple enzymes catalysing different modifications, as found in posttranslational modification 'codes' such as the histone code. Whereas simulations struggle with exponentially increasing molecular complexity, mathematical methods of the kind developed here can provide a new language in which to articulate the principles of cellular information processing.

PHOSPHO1 is essential for mechanically competent mineralization and the avoidance of spontaneous fractures

PubMed Central

Huesa, Carmen; Yadav, Manisha C.; Finnilä, Mikko A.J.; Goodyear, Simon R.; Robins, Simon P.; Tanner, K. Elizabeth; Aspden, Richard M.; Millán, José Luis; Farquharson, Colin

2011-01-01

Phosphatases are essential for the mineralization of the extracellular matrix within the skeleton. Their precise identities and functions however remain unclear. PHOSPHO1 is a phosphoethanolamine/phosphocholine phosphatase involved in the generation of inorganic phosphate for bone mineralization. It is highly expressed at sites of mineralization in bone and cartilage. The bones of Phospho1−/− mice are hypomineralized, bowed and present with spontaneous greenstick fractures at birth. In this study we show that PHOSPHO1 is essential for mechanically competent mineralization that is able to withstand habitual load. Long bones from Phospho1−/− mice did not fracture during 3- point bending but deformed plastically. With dynamic loading nanoindentation the elastic modulus and hardness of Phospho1−/− tibiae were significantly lower than wild-type tibia. Raman microscopy revealed significantly lower mineral:matrix ratios and lower carbonate substitutions in Phospho1−/− tibia. The altered dihydroxylysinonorleucine/hydroxyllysinonorleucine and pyridoline/deoxypyridinoline collagen crosslink ratios indicated possible changes in lysyl hydroxylase-1 activity and/or bone mineralization status. The bone formation and resorption markers, N-terminal propeptide and C-terminal telopeptide of Type I collagen, were both increased in Phospho1−/− mice and this we associated with increased bone remodelling during fracture repair or an attempt to remodel a mechanically competent bone capable of withstanding physiological load. In summary these data indicate that Phospho1−/− bones are hypomineralized and, consequently, are softer and more flexible. An inability to withstand physiological loading may explain the deformations noted. We hypothesize that this phenotype is due to the reduced availability of inorganic phosphate to form hydroxyapatite during mineralization, creating an undermineralized yet active bone. PMID:21272676

Dermatophytes activate skin keratinocytes via mitogen-activated protein kinase signaling and induce immune responses.

PubMed

Achterman, Rebecca R; Moyes, David L; Thavaraj, Selvam; Smith, Adam R; Blair, Kris M; White, Theodore C; Naglik, Julian R

2015-04-01

Dermatophytes cause superficial and cutaneous fungal infections in immunocompetent hosts and invasive disease in immunocompromised hosts. However, the host mechanisms that regulate innate immune responses against these fungi are largely unknown. Here, we utilized commercially available epidermal tissues and primary keratinocytes to assess (i) damage induction by anthropophilic, geophilic, and zoophilic dermatophyte strains and (ii) the keratinocyte signaling pathways, transcription factors, and proinflammatory responses induced by a representative dermatophyte, Trichophyton equinum. Initially, five dermatophyte species were tested for their ability to invade, cause tissue damage, and induce cytokines, with Microsporum gypseum inducing the greatest level of damage and cytokine release. Using T. equinum as a representative dermatophyte, we found that the mitogen-activated protein kinase (MAPK) pathways were predominantly affected, with increased levels of phospho-p38 and phospho-Jun N-terminal protein kinase (JNK) but decreased levels of phospho-extracellular signal-regulated kinases 1 and 2 (ERK1/2). Notably, the NF-κB and PI3K pathways were largely unaffected. T. equinum also significantly increased expression of the AP-1-associated transcription factor, c-Fos, and the MAPK regulatory phosphatase, MKP1. Importantly, the ability of T. equinum to invade, cause tissue damage, activate signaling and transcription factors, and induce proinflammatory responses correlated with germination, indicating that germination may be important for dermatophyte virulence and host immune activation. Copyright © 2015, Achterman et al.

Characterization and 1.57 Å resolution structure of the key fire blight phosphatase AmsI from Erwinia amylovora.

PubMed

Salomone-Stagni, Marco; Musiani, Francesco; Benini, Stefano

2016-12-01

AmsI is a low-molecular-weight protein tyrosine phosphatase that regulates the production of amylovoran in the Gram-negative bacterium Erwinia amylovora, a specific pathogen of rosaceous plants such as apple, pear and quince. Amylovoran is an exopolysaccharide that is necessary for successful infection. In order to shed light on AmsI, its structure was solved at 1.57 Å resolution at the same pH as its highest measured activity (pH 5.5). In the active site, a water molecule, bridging between the catalytic Arg15 and the reaction-product analogue sulfate, might be representative of the water molecule attacking the phospho-cysteine intermediate in the second step of the reaction mechanism.

Differential Regulation of MAPK Phosphorylation in the Dorsal Hippocampus in Response to Prolonged Morphine Withdrawal-Induced Depressive-Like Symptoms in Mice

PubMed Central

Shi, Jianguo; Wu, Bin; Dang, Wei; Du, Ying; Zhou, Qiong; Wang, Jianhua; Zhang, Rui

2013-01-01

Depression is one of the most frequent neuropsychiatric comorbidities associated with opiate addiction. Mitogen activated protein kinase (MAPK) and MAPK phosphatase (MKP) are involved in drug addiction and depression. However, the potential role of MAPK and MKP in depression caused by morphine withdrawal remains unclear. We utilized a mouse model of repeated morphine administration to examine the molecular mechanisms that contribute to prolonged withdrawal induced depressive-like behaviors. Depressive-like behaviors were significant at 1 week after withdrawal and worsened over time. Phospho-ERK (extracellular signal-regulated protein kinase) was decreased and MKP-1 was elevated in the hippocampus, and JNK (c-Jun N-terminal protein kinase), p38 (p38 protein kinase) and MKP-3 were unaffected. A pharmacological blockade of MKP-1 by intra-hippocampal sanguinarine (SA) infusion prevented the development of depressive-like behaviors and resulted in relatively normal levels of MKP-1 and phospho-ERK after withdrawal. Our findings support the association between hippocampal MAPK phosphorylation and prolonged morphine withdrawal-induced depression, and emphasize the MKP-1 as an negative regulator of the ERK phosphorylation that contributes to depression. PMID:23823128

Ectopic ERK Expression Induces Phenotypic Conversion of C10 Cells and Alters DNA Methyltransferase Expression

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

Sontag, Ryan L.; Weber, Thomas J.

2012-05-04

In some model systems constitutive extracellular signal regulated kinase (ERK) activation is sufficient to promote an oncogenic phenotype. Here we investigate whether constitutive ERK expression influences phenotypic conversion in murine C10 type II alveolar epithelial cells. C10 cells were stably transduced with an ERK1-green fluorescent protein (ERK1-GFP) chimera or empty vector and ectopic ERK expression was associated with the acquisition of soft agar focus-forming potential in late passage, but not early passage cells. Late passage ERK1-GFP cells exhibited a significant increase in the expression of DNA methyl transferases (DNMT1 and 3b) and a marked increase in sensitivity to 5-azacytidine (5-azaC)-mediatedmore » toxicity, relative to early passage ERK1-GFP cells and vector controls. The expression of xeroderma pigmentosum complementation group A (XPA) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were significantly increased in late passage cells, suggesting enhanced DNA damage recognition and repair activity which we interpret as a reflection of genomic instability. Phospho-ERK levels were dramatically decreased in late passage ERK1-GFP cells, relative to early passage and vector controls, and phospho-ERK levels were restored by treatment with sodium orthovanadate, indicating a role for phosphatase activity in this response. Collectively these observations suggest that ectopic ERK expression promotes phenotypic conversion of C10 cells that is associated with latent effects on epigenetic programming and phosphatase activities.« less

Mover Is a Homomeric Phospho-Protein Present on Synaptic Vesicles

PubMed Central

Kremer, Thomas; Hoeber, Jan; Kiran Akula, Asha; Urlaub, Henning; Islinger, Markus; Kirsch, Joachim; Dean, Camin; Dresbach, Thomas

2013-01-01

With remarkably few exceptions, the molecules mediating synaptic vesicle exocytosis at active zones are structurally and functionally conserved between vertebrates and invertebrates. Mover was found in a yeast-2-hybrid assay using the vertebrate-specific active zone scaffolding protein bassoon as a bait. Peptides of Mover have been reported in proteomics screens for self-interacting proteins, phosphorylated proteins, and synaptic vesicle proteins, respectively. Here, we tested the predictions arising from these screens. Using flotation assays, carbonate stripping of peripheral membrane proteins, mass spectrometry, immunogold labelling of purified synaptic vesicles, and immuno-organelle isolation, we found that Mover is indeed a peripheral synaptic vesicle membrane protein. In addition, by generating an antibody against phosphorylated Mover and Western blot analysis of fractionated rat brain, we found that Mover is a bona fide phospho-protein. The localization of Mover to synaptic vesicles is phosphorylation dependent; treatment with a phosphatase caused Mover to dissociate from synaptic vesicles. A yeast-2-hybrid screen, co-immunoprecipitation and cell-based optical assays of homomerization revealed that Mover undergoes homophilic interaction, and regions within both the N- and C- terminus of the protein are required for this interaction. Deleting a region required for homomeric interaction abolished presynaptic targeting of recombinant Mover in cultured neurons. Together, these data prove that Mover is associated with synaptic vesicles, and implicate phosphorylation and multimerization in targeting of Mover to synaptic vesicles and presynaptic sites. PMID:23723986

Cooperative Interaction between Phosphorylation Sites on PERIOD Maintains Circadian Period in Drosophila

PubMed Central

Garbe, David S.; Fang, Yanshan; Zheng, Xiangzhong; Sowcik, Mallory; Anjum, Rana; Gygi, Steven P.; Sehgal, Amita

2013-01-01

Circadian rhythms in Drosophila rely on cyclic regulation of the period (per) and timeless (tim) clock genes. The molecular cycle requires rhythmic phosphorylation of PER and TIM proteins, which is mediated by several kinases and phosphatases such as Protein Phosphatase-2A (PP2A) and Protein Phosphatase-1 (PP1). Here, we used mass spectrometry to identify 35 “phospho-occupied” serine/threonine residues within PER, 24 of which are specifically regulated by PP1/PP2A. We found that cell culture assays were not good predictors of protein function in flies and so we generated per transgenes carrying phosphorylation site mutations and tested for rescue of the per01 arrhythmic phenotype. Surprisingly, most transgenes restore wild type rhythms despite carrying mutations in several phosphorylation sites. One particular transgene, in which T610 and S613 are mutated to alanine, restores daily rhythmicity, but dramatically lengthens the period to ∼30 hrs. Interestingly, the single S613A mutation extends the period by 2–3 hours, while the single T610A mutation has a minimal effect, suggesting these phospho-residues cooperate to control period length. Conservation of S613 from flies to humans suggests that it possesses a critical clock function, and mutational analysis of residues surrounding T610/S613 implicates the entire region in determining circadian period. Biochemical and immunohistochemical data indicate defects in overall phosphorylation and altered timely degradation of PER carrying the double or single S613A mutation(s). The PER-T610A/S613A mutant also alters CLK phosphorylation and CLK-mediated output. Lastly, we show that a mutation at a previously identified site, S596, is largely epistatic to S613A, suggesting that S613 negatively regulates phosphorylation at S596. Together these data establish functional significance for a new domain of PER, demonstrate that cooperativity between phosphorylation sites maintains PER function, and support a model in which specific phosphorylated regions regulate others to control circadian period. PMID:24086144

Downregulation of protein tyrosine phosphatase PTPL1 alters cell cycle and upregulates invasion-related genes in prostate cancer cells.

PubMed

Castilla, Carolina; Flores, M Luz; Conde, José M; Medina, Rafael; Torrubia, Francisco J; Japón, Miguel A; Sáez, Carmen

2012-04-01

PTPL1, a non-receptor type protein tyrosine phosphatase, has been involved in the regulation of apoptosis and invasiveness of various tumour cell types, but its role in prostate cancer remained to be investigated. We report here that downregulation of PTPL1 by small interfering RNA in PC3 cells decreases cell proliferation and concomitantly reduces the expression of cell cycle-related proteins such as cyclins E and B1, PCNA, PTTG1 and phospho-histone H3. PTPL1 downregulation also increases the invasion ability of PC3 cells through Matrigel coated membranes. cDNA array of PTPL1-silenced PC3 cells versus control cells showed an upregulation of invasion-related genes such as uPA, uPAR, tPA, PAI-1, integrin α6 and osteopontin. This increased expression was also confirmed in PTPL1-silenced DU145 prostate cancer cells by quantitative real time PCR and western blot. These findings suggest that PTPL1 is an important mediator of central cellular processes such as proliferation and invasion.

Estrogen regulates energy metabolic pathway and upstream adenosine 5'-monophosphate-activated protein kinase and phosphatase enzyme expression in dorsal vagal complex metabolosensory neurons during glucostasis and hypoglycemia.

PubMed

Tamrakar, Pratistha; Ibrahim, Baher A; Gujar, Amit D; Briski, Karen P

2015-02-01

The ability of estrogen to shield the brain from the bioenergetic insult hypoglycemia is unclear. Estradiol (E) prevents hypoglycemic activation of the energy deficit sensor adenosine 5'-monophosphate-activated protein kinase (AMPK) in hindbrain metabolosensory A2 noradrenergic neurons. This study investigates the hypothesis that estrogen regulates A2 AMPK through control of fuel metabolism and/or upstream protein kinase/phosphatase enzyme expression. A2 cells were harvested by laser microdissection after insulin or vehicle (V) injection of E- or oil (O)-implanted ovariectomized female rats. Cell lysates were evaluated by immunoblot for glycolytic, tricarboxylic acid cycle, respiratory chain, and acetyl-CoA-malonyl-CoA pathway enzymes. A2 phosphofructokinase (PFKL), isocitrate dehydrogenase, pyruvate dehydrogenase, and ATP synthase subunit profiles were elevated in E/V vs. O/V; hypoglycemia augmented PFKL and α-ketoglutarate dehydrogenase expression in E only. Hypoglycemia increased A2 Ca(2+) /calmodulin-dependent protein kinase-β in O and reduced protein phosphatase in both groups. A2 phospho-AMPK levels were equivalent in O/V vs. E/V but elevated during hypoglycemia in O only. These results implicate E in compensatory upregulation of substrate catabolism and corresponding maintenance of energy stability of A2 metabolosensory neurons during hypoglycemia, outcomes that support the potential viability of molecular substrates for hormone action as targets for therapies alleviating hypoglycemic brain injury. © 2014 Wiley Periodicals, Inc.

Increased amount of phosphorylated proinflammatory osteopontin in rheumatoid arthritis synovia is associated to decreased tartrate-resistant acid phosphatase 5B/5A ratio.

PubMed

Luukkonen, Jani; Pascual, Laia Mira; Patlaka, Christina; Lång, Pernilla; Turunen, Sanna; Halleen, Jussi; Nousiainen, Tomi; Valkealahti, Maarit; Tuukkanen, Juha; Andersson, Göran; Lehenkari, Petri

2017-01-01

Osteopontin (OPN) is an immunoregulatory protein which production increases in both rheumatoid arthritis (RA) and osteoarthritis (OA). Phosphorylated osteopontin (Phospho-OPN) is known to increase macrophage and osteoclast activation, this process is controlled by extracellular tartrate-resistant acid phosphatase (TRAcP), also a biomarker for RA. Here, we evaluated the phosphorylation status of OPN in RA and OA synovia, as well as its correlation with TRAcP isoforms. Synovial tissue and fluid were obtained from 24 RA (14 seropositive and 10 seronegative) and 24 OA patients. Western blotting was used to analyze the extent of OPN phosphorylation. TRAcP isoforms were measured in synovial fluid using ELISA; immunohistochemistry assessed the distribution of OPN and TRAcP expressing cells in the synovial tissue, especially distinguishing between the TRAcP isoforms. Full-length OPN was more phosphorylated in RA than in OA (p<0.05). The thrombin cleaved C-terminal end of OPN was also more phosphorylated in RA (p<0.05). RA patients had a lower concentration of TRAcP 5B and higher concentration of less active 5A in their synovial fluid compared to OA patients. The TRAcP 5B/5A ratio was decreased in RA and correlated negatively with the amount of phospho-OPN (p<0.05). TRAcP positive cells for both isoforms were found all along the synovial lining; OPN antibody staining was localized in the extracellular matrix. Our data suggests that in RA the synovial fluid contains insufficient amounts of TRAcP 5B which increase levels of the proinflammatory phospho-OPN. This may lead to increased macrophage and osteoclast activation, resulting in the increased local inflammation and bone resorption present in RA joints.

A distinctive patchy osteomalacia characterises Phospho1-deficient mice.

PubMed

Boyde, Alan; Staines, Katherine A; Javaheri, Behzad; Millan, Jose Luis; Pitsillides, Andrew A; Farquharson, Colin

2017-08-01

The phosphatase PHOSPHO1 is involved in the initiation of biomineralisation. Bones in Phospho1 knockout (KO) mice show histological osteomalacia with frequent bowing of long bones and spontaneous fractures: they contain less mineral, with smaller mineral crystals. However, the consequences of Phospho1 ablation on the microscale structure of bone are not yet fully elucidated. Tibias and femurs obtained from wild-type and Phospho1 null (KO) mice (25-32 weeks old) were embedded in PMMA, cut and polished to produce near longitudinal sections. Block surfaces were studied using 20 kV backscattered-electron (BSE) imaging, and again after iodine staining to reveal non-mineralised matrix and cellular components. For 3D characterisation, we used X-ray micro-tomography. Bones opened with carbide milling tools to expose endosteal surfaces were macerated using an alkaline bacterial pronase enzyme detergent, 5% hydrogen peroxide and 7% sodium hypochlorite solutions to produce 3D surfaces for study with 3D BSE scanning electron microscopy (SEM). Extensive regions of both compact cortical and trabecular bone matrix in Phospho1 KO mice contained no significant mineral and/or showed arrested mineralisation fronts, characterised by a failure in the fusion of the calcospherite-like, separately mineralising, individual micro-volumes within bone. Osteoclastic resorption of the uncalcified matrix in Phospho1 KO mice was attenuated compared with surrounding normally mineralised bone. The extent and position of this aberrant biomineralisation varied considerably between animals, contralateral limbs and anatomical sites. The most frequent manifestation lay, however, in the nearly complete failure of mineralisation in the bone surrounding the numerous transverse blood vessel canals in the cortices. In conclusion, SEM disclosed defective mineralising fronts and extensive patchy osteomalacia, which has previously not been recognised. These data further confirm the role of this phosphatase in physiological skeletal mineralisation. © 2017 Anatomical Society.

Isoflurane unveils a critical role of glutamate transporter type 3 in regulating hippocampal GluR1 trafficking and context-related learning and memory in mice.

PubMed

Cao, J; Wang, Z; Mi, W; Zuo, Z

2014-07-11

Glutamate transporter type 3 (EAAT3) may play a role in cognition. Isoflurane enhances EAAT3 trafficking to the plasma membrane. Thus, we used isoflurane to determine how EAAT3 might regulate learning and memory and the trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, such as GluR1, to the plasma membrane, a fundamental biochemical process for learning and memory. Here, isoflurane increased EAAT3 but did not change GluR1 levels in the plasma membrane of wild-type mouse hippocampus. Isoflurane increased protein phosphatase activity in the wild-type and EAAT3(-/-) mouse hippocampus. Also, isoflurane reduced GluR1 in the plasma membrane and decreased phospho-GluR1 in EAAT3(-/-) mice. The phosphatase inhibitor okadaic acid attenuated these effects. Finally, isoflurane inhibited context-related fear conditioning in EAAT3(-/-) mice but not in wild-type mice. Thus, isoflurane may increase GluR1 trafficking to the plasma membrane via EAAT3 and inhibit GluR1 trafficking via protein phosphatase. Lack of EAAT3 effects leads to decreased GluR1 trafficking and impaired cognition after isoflurane exposure in EAAT3(-/-) mice. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Switching of the substrate specificity of protein tyrosine phosphatase N12 by cyclin-dependent kinase 2 phosphorylation orchestrating 2 oncogenic pathways.

PubMed

Li, Hui; Yang, Duxiao; Ning, Shanglei; Xu, Yinghui; Yang, Fan; Yin, Rusha; Feng, Taihu; Han, Shouqing; Guo, Lu; Zhang, Pengju; Qu, Wenjie; Guo, Renbo; Song, Chen; Xiao, Peng; Zhou, Chengjun; Xu, Zhigang; Sun, Jin-Peng; Yu, Xiao

2018-01-01

The protein tyrosine phosphatase nonreceptor type 12 (PTPN12) is a multifunctional protein and has elicited much research attention because its decreased protein level has been associated with poor prognosis of several types of cancers. Recently, we have solved the crystal structure of the phosphatase domain of PTPN12, which disclosed a specific PTPN12-insert-loop harboring a cyclin-dependent kinase 2 (CDK2) phosphorylation site. However, the functional significance of this phosphorylation is undefined. In the present study, we found that S19 site phosphorylation of PTPN12 by CDK2 discharged its antitumor activity by down-regulation of its inhibitory role in cell migration, but not affecting its other regulatory functions. Phosphorylation of PTPN12 at the S19 site changed its substrate interface, and by doing so, selectively decreased its activity toward the human epidermal growth factor receptor 2 (HER2)- pY 1196 site, but not other HER2 phosphorylation sites or other known PTPN12 substrates. A further in-depth mechanism study revealed that the phosphorylation of PTPN12 by CDK2 impaired recruitment of the serine/threonine-protein kinase 1 (PAK1) to HER2, resulted in the blockade of the HER2-pY 1196 -PAK1-T 423 signaling pathway, thus increased tumor cell motility. Taken together, our results identified a new phosphorylation-based substrate recognition mechanism of PTPN12 by CDK2, which orchestrated signaling crosstalk between the oncogenic CDK2 and HER2 pathways. The newly identified governing mechanism of the substrate selectivity of a particular phosphatase was previously unappreciated and exemplifies how a phospho-network is precisely controlled in different cellular contexts.-Li, H., Yang, D., Ning, S., Xu, Y., Yang, F., Yin, R., Feng, T., Han, S., Guo, L., Zhang, P., Qu, W., Guo, R., Song, C., Xiao, P., Zhou, C., Xu, Z., Sun, J.-P., Yu, X. Switching of the substrate specificity of protein tyrosine phosphatase N12 by cyclin-dependent kinase 2 phosphorylation orchestrating 2 oncogenic pathways. © FASEB.

Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C.

PubMed

Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar

2003-08-01

DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.

[Phosphatase activity in Amoeba proteus at low pH].

PubMed

Sopina, V A

2009-01-01

In free-living Amoeba proteus (strain B), three forms of tartrate-sensitive phosphatase were revealed using PAGE of the supernatant of ameba homogenates obtained with 1% Triton X-100 or distilled water and subsequent staining of gels with 2-naphthyl phosphate as substrate (pH 4.0). The form with the highest mobility in the ameba supernatant was sensitive to all tested phosphatase activity modulators. Two other forms with the lower mobilities were completely or significantly inactivated not only by sodium L-(+)-tartrate, but also by L-(+)-tartaric acid, sodium orthovanadate, ammonium molybdate, EDTA, EGTA, o-phospho-L-tyrosine, DL-dithiotreitol, H2O2, 2-mercaptoethanol, and ions of heavy metals - Fe2+, Fe3+, and Cu2+. Based on results of inhibitory analysis, lysosome location in the ameba cell, and wide substrate specificity of these two forms, it has been concluded that they belong to nonspecific acid phosphomonoesterases (AcP, EC 3.1.3.2). This AcP is suggested to have both phosphomonoesterase and phosphotyrosyl-protein phosphatase activitis. Two ecto-phosphatases were revealed in the culture medium, in which amebas were cultivated. One of them was inhibited by the same reagents as the ameba tartrate-sensitive AcP and seems to be the AcP released into the culture medium in the process of exocytosis of the content of food vacuoles. In the culture medium, apart from this AcP, another phosphatase was revealed, which was not inhibited by any tested inhibitors of AcP and alkaline phosphatase. It cannot be ruled out that this phosphatase belong to the ecto-ATPases found in many protists; however, its ability to hydrolyze ATP has not yet been proven.

Pea DNA Topoisomerase I Is Phosphorylated and Stimulated by Casein Kinase 2 and Protein Kinase C

PubMed Central

Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar

2003-01-01

DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg2+-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants. PMID:12913165

Skeletal mineralization deficits and impaired biogenesis and function of chondrocyte-derived matrix vesicles in Phospho1−/− and Phospho1/Pit1 double knockout mice

PubMed Central

Yadav, Manisha C.; Bottini, Massimo; Cory, Esther; Bhattacharya, Kunal; Kuss, Pia; Narisawa, Sonoko; Sah, Robert L.; Beck, Laurent; Fadeel, Bengt; Farquharson, Colin; Millán, José Luis

2016-01-01

We have previously shown that ablation of either the Phospho1 or Alpl gene, encoding PHOSPHO1 and tissue-nonspecific alkaline phosphatase (TNAP) respectively, lead to hyperosteoidosis but that their chondrocyte- and osteoblast-derived matrix vesicles (MVs) are able to initiate mineralization. In contrast, the double ablation of Phospho1 and Alpl completely abolish initiation and progression of skeletal mineralization. We argued that MVs initiate mineralization by a dual mechanism: PHOSPHO1-mediated intravesicular generation of Pi and phosphate transporter-mediated influx of Pi. To test this hypothesis, we generated mice with col2a1-driven cre-mediated ablation of Slc20a1, hereafter referred to as Pit1, alone or in combination with a Phospho1 gene deletion. Pit1col2/col2 mice did not show any major phenotypic abnormalities, while severe skeletal deformities were observed in the [Phospho1−/−; Pit1col2/col2] double knockout mice that were more pronounced than those observed in the Phospho1−/− mice. Histological analysis of [Phospho1−/−; Pit1col2/col2] bones showed growth plate abnormalities with a shorter hypertrophic chondrocyte zone and extensive hyperosteoidosis. The [Phospho1−/−; Pit1col2/col2] skeleton displayed significantly decreases in BV/TV%, trabecular number and bone mineral density, as well as decreased stiffness, decreased strength, and increased post-yield deflection compared to Phospho1−/− mice. Using atomic force microscopy we found that ~80% of [Phospho1−/−; Pit1col2/col2] MVs were devoid of mineral in comparison to ~50 % for the Phospho1−/− MVs and ~25% for the WT and Pit1col2/col2 MVs. We also found a significant decrease in the number of MVs produced by both Phospho1−/− and [Phospho1−/−; Pit1col2/col2] chondrocytes. These data support the involvement of PiT-1 in the initiation of skeletal mineralization and provide compelling evidence that PHOSPHO1 function is involved in MV biogenesis. PMID:26773408

Preparative resolution of D,L-threonine catalyzed by immobilized phosphatase.

PubMed

Scollar, M P; Sigal, G; Klibanov, A M

1985-03-01

Hydrolysis of L- and D-O-phosphothreonines catalyzed by four different phosphatases, alkaline phosphatases from calf intestine and E. coli and acid phosphatases from wheat germ and potato, has been kinetically studied. Alkaline phosphatases were found to have comparable reactivities towards the optical isomers. On the other hand, both acid phosphatases displayed a marked stereoselectivity, hydrolyzing the L-ester much faster than its D counterpart. Wheat germ acid phosphatase was the most stereoselective enzyme: V(L)/V(D) = 24 and K(m,L)/K(m,D) = 0.17. This enzyme was immobilized (in k-carrageenan gel, followed by crosslinking with glutaraldehyde) and used for the preparative resolution of D,L-threonine: the latter was first chemically O-phosphorylated and then asymmetrically hydrolyzed by the immobilized phosphatase. As a result, gram quantities of L-threonine of high optical purity and O-phospho-D-threonine were prepared. Immobilized wheat germ phosphatase has been tested for the resolution of other racemic alcohols: serine, 2-amino-1-butanol, 1-amino-2-propanol, 2-octanol, and menthol. In all those cases, the enzyme was either not sufficiently stereoselective or too slow for preparative resolutions.

RAS/ERK modulates TGFbeta-regulated PTEN expression in human pancreatic adenocarcinoma cells.

PubMed

Chow, Jimmy Y C; Quach, Khai T; Cabrera, Betty L; Cabral, Jennifer A; Beck, Stayce E; Carethers, John M

2007-11-01

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is rarely mutated in pancreatic cancers, but its regulation by transforming growth factor (TGF)-beta might mediate growth suppression and other oncogenic actions. Here, we examined the role of TGFbeta and the effects of oncogenic K-RAS/ERK upon PTEN expression in the absence of SMAD4. We utilized two SMAD4-null pancreatic cell lines, CAPAN-1 (K-RAS mutant) and BxPc-3 (WT-K-RAS), both of which express TGFbeta surface receptors. Cells were treated with TGFbeta1 and separated into cytosolic/nuclear fractions for western blotting with phospho-SMAD2, SMAD 2, 4 phospho-ATP-dependent tyrosine kinases (Akt), Akt and PTEN antibodies. PTEN mRNA levels were assessed by reverse transcriptase-polymerase chain reaction. The MEK1 inhibitor, PD98059, was used to block the downstream action of oncogenic K-RAS/ERK, as was a dominant-negative (DN) K-RAS construct. TGFbeta increased phospho-SMAD2 in both cytosolic and nuclear fractions. PD98059 treatment further increased phospho-SMAD2 in the nucleus of both pancreatic cell lines, and DN-K-RAS further improved SMAD translocation in K-RAS mutant CAPAN cells. TGFbeta treatment significantly suppressed PTEN protein levels concomitant with activation of Akt by 48 h through transcriptional reduction of PTEN mRNA that was evident by 6 h. TGFbeta-induced PTEN suppression was reversed by PD98059 and DN-K-RAS compared with treatments without TGFbeta. TGFbeta-induced PTEN expression was inversely related to cellular proliferation. Thus, oncogenic K-RAS/ERK in pancreatic adenocarcinoma facilitates TGFbeta-induced transcriptional down-regulation of the tumor suppressor PTEN in a SMAD4-independent manner and could constitute a signaling switch mechanism from growth suppression to growth promotion in pancreatic cancers.

PIK3CA mutations, phosphatase and tensin homolog, human epidermal growth factor receptor 2, and insulin-like growth factor 1 receptor and adjuvant tamoxifen resistance in postmenopausal breast cancer patients.

PubMed

Beelen, Karin; Opdam, Mark; Severson, Tesa M; Koornstra, Rutger H T; Vincent, Andrew D; Wesseling, Jelle; Muris, Jettie J; Berns, Els M J J; Vermorken, Jan B; van Diest, Paul J; Linn, Sabine C

2014-01-27

Inhibitors of the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway can overcome endocrine resistance in estrogen receptor (ER) α-positive breast cancer, but companion diagnostics indicating PI3K/AKT/mTOR activation and consequently endocrine resistance are lacking. PIK3CA mutations frequently occur in ERα-positive breast cancer and result in PI3K/AKT/mTOR activation in vitro. Nevertheless, the prognostic and treatment-predictive value of these mutations in ERα-positive breast cancer is contradictive. We tested the clinical validity of PIK3CA mutations and other canonic pathway drivers to predict intrinsic resistance to adjuvant tamoxifen. In addition, we tested the association between these drivers and downstream activated proteins. Primary tumors from 563 ERα-positive postmenopausal patients, randomized between adjuvant tamoxifen (1 to 3 years) versus observation were recollected. PIK3CA hotspot mutations in exon 9 and exon 20 were assessed with Sequenom Mass Spectometry. Immunohistochemistry was performed for human epidermal growth factor receptor 2 (HER2), phosphatase and tensin homolog (PTEN), and insulin-like growth factor 1 receptor (IGF-1R). We tested the association between these molecular alterations and downstream activated proteins (like phospho-protein kinase B (p-AKT), phospho-mammalian target of rapamycin (p-mTOR), p-ERK1/2, and p-p70S6K). Recurrence-free interval improvement with tamoxifen versus control was assessed according to the presence or absence of canonic pathway drivers, by using Cox proportional hazard models, including a test for interaction. PIK3CA mutations (both exon 9 and exon 20) were associated with low tumor grade. An enrichment of PIK3CA exon 20 mutations was observed in progesterone receptor- positive tumors. PIK3CA exon 20 mutations were not associated with downstream-activated proteins. No significant interaction between PIK3CA mutations or any of the other canonic pathway drivers and tamoxifen-treatment benefit was found. PIK3CA mutations do not have clinical validity to predict intrinsic resistance to adjuvant tamoxifen and may therefore be unsuitable as companion diagnostic for PI3K/AKT/mTOR inhibitors in ERα- positive, postmenopausal, early breast cancer patients.

A molecular switch on an arrestin-like protein relays glucose signaling to transporter endocytosis.

PubMed

Becuwe, Michel; Vieira, Neide; Lara, David; Gomes-Rezende, Jéssica; Soares-Cunha, Carina; Casal, Margarida; Haguenauer-Tsapis, Rosine; Vincent, Olivier; Paiva, Sandra; Léon, Sébastien

2012-01-23

Endocytosis regulates the plasma membrane protein landscape in response to environmental cues. In yeast, the endocytosis of transporters depends on their ubiquitylation by the Nedd4-like ubiquitin ligase Rsp5, but how extracellular signals trigger this ubiquitylation is unknown. Various carbon source transporters are known to be ubiquitylated and endocytosed when glucose-starved cells are exposed to glucose. We show that this required the conserved arrestin-related protein Rod1/Art4, which was activated in response to glucose addition. Indeed, Rod1 was a direct target of the glucose signaling pathway composed of the AMPK homologue Snf1 and the PP1 phosphatase Glc7/Reg1. Glucose promoted Rod1 dephosphorylation and its subsequent release from a phospho-dependent interaction with 14-3-3 proteins. Consequently, this allowed Rod1 ubiquitylation by Rsp5, which was a prerequisite for transporter endocytosis. This paper therefore demonstrates that the arrestin-related protein Rod1 relays glucose signaling to transporter endocytosis and provides the first molecular insights into the nutrient-induced activation of an arrestin-related protein through a switch in post-translational modifications.

Selective Effects of PDE10A Inhibitors on Striatopallidal Neurons Require Phosphatase Inhibition by DARPP-321,2,3

PubMed Central

Polito, Marina; Guiot, Elvire; Gangarossa, Giuseppe; Longueville, Sophie; Doulazmi, Mohamed; Valjent, Emmanuel; Hervé, Denis; Girault, Jean-Antoine

2015-01-01

Abstract Type 10A phosphodiesterase (PDE10A) is highly expressed in the striatum, in striatonigral and striatopallidal medium-sized spiny neurons (MSNs), which express D1 and D2 dopamine receptors, respectively. PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear. We analyzed the effects of PDE10A inhibition in vivo by immunohistochemistry, and imaged cAMP, cAMP-dependent protein kinase A (PKA), and cGMP signals with biosensors in mouse brain slices. PDE10A inhibition in mouse striatal slices produced a steady-state increase in intracellular cAMP concentration in D1 and D2 MSNs, demonstrating that PDE10A regulates basal cAMP levels. Surprisingly, the PKA-dependent AKAR3 phosphorylation signal was strong in D2 MSNs, whereas D1 MSNs remained unresponsive. This effect was also observed in adult mice in vivo since PDE10A inhibition increased phospho-histone H3 immunoreactivity selectively in D2 MSNs in the dorsomedial striatum. The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for protein phosphatase-1 inhibition. These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs. This study shows that PDE10A inhibitors share with antipsychotic medications the property of activating preferentially PKA-dependent signaling in D2 MSNs. PMID:26465004

A protein phosphatase feedback mechanism regulates the basal phosphorylation of Chk2 kinase in the absence of DNA damage.

PubMed

Carlessi, Luigi; Buscemi, Giacomo; Fontanella, Enrico; Delia, Domenico

2010-10-01

The checkpoint kinase Chk2 is an effector component of the ATM-dependent DNA damage response (DDR) pathway. The activation of Chk2 by genotoxic stress involves its phosphorylation on T68 by ATM and additional auto/transphosphorylations. Here we demonstrate that in unperturbed cells, chemical inhibition of Chk2 by VRX0466617 (VRX) enhances the phosphorylation of Chk2-T68 throughout the cell cycle phases. This event, dependent on the presence of ATM and catalytically functional Chk2, is not consequential to DNA damage, as neither gamma-H2AX nuclear foci nor increased ATM activation is detected in VRX-treated cells, suggesting the involvement of other regulatory proteins. As serine/threonine protein phosphatases (PPs) regulate the phosphorylation and deactivation of proteins of the DDR pathway, we analyzed their role in phospho-T68-Chk2 regulation. We found that intracellular inhibition of PP1 and PP2A-like activities by okadaic acid markedly raised the accumulation of Chk2-pT68 without DNA damage induction, and this phenomenon was also seen when PP1-C, PP2A-C, and Wip1/PPM1D were simultaneously knockdown by siRNA. Altogether, these data indicate a novel mechanism in undamaged cells where PPs function to maintain the balance between ATM and its direct substrate Chk2 through a regulatory circuit. Copyright © 2010 Elsevier B.V. All rights reserved.

Focused microwave irradiation-assisted immunohistochemistry to study effects of ketamine on phospho-ERK expression in the mouse brain.

PubMed

Fernandes, Alda; Li, Yu-Wen

2017-09-01

Ketamine produces rapid and long-lasting antidepressant effects in depressive patients. Preclinical studies demonstrate that ketamine stimulates AMPA receptor transmission and activates BDNF/TrkB-Akt/ERK-mTOR signaling cascades, leading to a sustained increase in synaptic protein synthesis and strengthening of synaptic plasticity, a potential mechanism underlying the antidepressant effects. The purpose of this study was to develop an immunohistochemistry (IHC) assay to map the distribution of extracellular signal-regulated kinase (ERK) phosphorylation in the mouse brain in response to systemic ketamine treatment. We established a focused microwave irradiation-assisted IHC assay to detect phosphorylated (phospho) proteins including phospho-ERK, phospho- cAMP-response- element-binding protein (CREB), phospho- glutamate receptor 1 (GluR1) and phospho- calcium/calmodulin-dependent protein kinase II (CaMKII) with greater sensitivity and reproducibility in comparison to conventional IHC methods. A single dose of ketamine produced a robust, dose- and time-dependent increase in phospho-ERK immunoreactive (phospho-ERK-ir) neurons in the medial prefrontal cortex (mPFC) and the central nucleus of the amygdala. Phospho-ERK-ir neurons in the mPFC were primarily located in the prelimbic and anterior cingulate subregions with the morphology resembling pyramidal neurons. An increase in phospho-ERK-ir was also observed in the brainstem dorsal raphe nucleus and locus coeruleus. The NMDA GluN2B subtype receptor antagonist Ro 25-6981 increased phospho-ERK expression in the brain in a similar pattern as ketamine. In summary, we have established a sensitive and reliable focused microwave irradiation-assisted IHC assay, and defined the activation pattern of ERK, in response to systemic ketamine and Ro 25-6981 treatment, in brain regions that are potentially responsible for mediating the antidepressant effects. Copyright © 2017 Elsevier B.V. All rights reserved.

DARPP-32 interaction with adducin may mediate rapid environmental effects on striatal neurons.

PubMed

Engmann, Olivia; Giralt, Albert; Gervasi, Nicolas; Marion-Poll, Lucile; Gasmi, Laila; Filhol, Odile; Picciotto, Marina R; Gilligan, Diana; Greengard, Paul; Nairn, Angus C; Hervé, Denis; Girault, Jean-Antoine

2015-12-07

Environmental enrichment has multiple effects on behaviour, including modification of responses to psychostimulant drugs mediated by striatal neurons. However, the underlying molecular and cellular mechanisms are not known. Here we show that DARPP-32, a hub signalling protein in striatal neurons, interacts with adducins, which are cytoskeletal proteins that cap actin filaments' fast-growing ends and regulate synaptic stability. DARPP-32 binds to adducin MARCKS domain and this interaction is modulated by DARPP-32 Ser97 phosphorylation. Phospho-Thr75-DARPP-32 facilitates β-adducin Ser713 phosphorylation through inhibition of a cAMP-dependent protein kinase/phosphatase-2A cascade. Caffeine or 24-h exposure to a novel enriched environment increases adducin phosphorylation in WT, but not T75A mutant mice. This cascade is implicated in the effects of brief exposure to novel enriched environment on dendritic spines in nucleus accumbens and cocaine locomotor response. Our results suggest a molecular pathway by which environmental changes may rapidly alter responsiveness of striatal neurons involved in the reward system.

Structural basis for glucose-6-phosphate activation of glycogen synthase

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

Baskaran, Sulochanadevi; Roach, Peter J.; DePaoli-Roach, Anna A.

2010-11-22

Regulation of the storage of glycogen, one of the major energy reserves, is of utmost metabolic importance. In eukaryotes, this regulation is accomplished through glucose-6-phosphate levels and protein phosphorylation. Glycogen synthase homologs in bacteria and archaea lack regulation, while the eukaryotic enzymes are inhibited by protein kinase mediated phosphorylation and activated by protein phosphatases and glucose-6-phosphate binding. We determined the crystal structures corresponding to the basal activity state and glucose-6-phosphate activated state of yeast glycogen synthase-2. The enzyme is assembled into an unusual tetramer by an insertion unique to the eukaryotic enzymes, and this subunit interface is rearranged by themore » binding of glucose-6-phosphate, which frees the active site cleft and facilitates catalysis. Using both mutagenesis and intein-mediated phospho-peptide ligation experiments, we demonstrate that the enzyme's response to glucose-6-phosphate is controlled by Arg583 and Arg587, while four additional arginine residues present within the same regulatory helix regulate the response to phosphorylation.« less

Ribosomal Protein S6 Phosphorylation Is Involved in Novelty-Induced Locomotion, Synaptic Plasticity and mRNA Translation

PubMed Central

Puighermanal, Emma; Biever, Anne; Pascoli, Vincent; Melser, Su; Pratlong, Marine; Cutando, Laura; Rialle, Stephanie; Severac, Dany; Boubaker-Vitre, Jihane; Meyuhas, Oded; Marsicano, Giovanni; Lüscher, Christian; Valjent, Emmanuel

2017-01-01

The phosphorylation of the ribosomal protein S6 (rpS6) is widely used to track neuronal activity. Although it is generally assumed that rpS6 phosphorylation has a stimulatory effect on global protein synthesis in neurons, its exact biological function remains unknown. By using a phospho-deficient rpS6 knockin mouse model, we directly tested the role of phospho-rpS6 in mRNA translation, plasticity and behavior. The analysis of multiple brain areas shows for the first time that, in neurons, phospho-rpS6 is dispensable for overall protein synthesis. Instead, we found that phospho-rpS6 controls the translation of a subset of mRNAs in a specific brain region, the nucleus accumbens (Acb), but not in the dorsal striatum. We further show that rpS6 phospho-mutant mice display altered long-term potentiation (LTP) in the Acb and enhanced novelty-induced locomotion. Collectively, our findings suggest a previously unappreciated role of phospho-rpS6 in the physiology of the Acb, through the translation of a selective subclass of mRNAs, rather than the regulation of general protein synthesis. PMID:29311811

The tumour suppressor OPCML promotes AXL inactivation by the phosphatase PTPRG in ovarian cancer.

PubMed

Antony, Jane; Zanini, Elisa; Kelly, Zoe; Tan, Tuan Zea; Karali, Evdoxia; Alomary, Mohammad; Jung, Youngrock; Nixon, Katherine; Cunnea, Paula; Fotopoulou, Christina; Paterson, Andrew; Roy-Nawathe, Sushmita; Mills, Gordon B; Huang, Ruby Yun-Ju; Thiery, Jean Paul; Gabra, Hani; Recchi, Chiara

2018-06-15

In ovarian cancer, the prometastatic RTK AXL promotes motility, invasion and poor prognosis. Here, we show that reduced survival caused by AXL overexpression can be mitigated by the expression of the GPI-anchored tumour suppressor OPCML Further, we demonstrate that AXL directly interacts with OPCML, preferentially so when AXL is activated by its ligand Gas6. As a consequence, AXL accumulates in cholesterol-rich lipid domains, where OPCML resides. Here, phospho-AXL is brought in proximity to the lipid domain-restricted phosphatase PTPRG, which de-phosphorylates the RTK/ligand complex. This prevents AXL-mediated transactivation of other RTKs (cMET and EGFR), thereby inhibiting sustained phospho-ERK signalling, induction of the EMT transcription factor Slug, cell migration and invasion. From a translational perspective, we show that OPCML enhances the effect of the phase II AXL inhibitor R428 in vitro and in vivo We therefore identify a novel mechanism by which two spatially restricted tumour suppressors, OPCML and PTPRG, coordinate to repress AXL-dependent oncogenic signalling. © 2018 The Authors.

Phospho-eNOS Ser-1176 is associated with the nucleoli and the Golgi complex in C6 rat glioma cells.

PubMed

Klinz, Franz-Josef; Herberg, Natalie; Arnhold, Stefan; Addicks, Klaus; Bloch, Wilhelm

2007-06-29

Enzymatic activity of endothelial nitric oxide synthase (eNOS) is controlled by posttranslational modifications, protein-protein interactions, and subcellular localization. For example, N-terminal fatty acid modifications target eNOS to the Golgi complex where it becomes phosphorylated. We show here by immunofluorescence analysis that phospho-eNOS Ser-1176 is enriched in the perinuclear region of interphase C6 rat glioma cells. Confocal double immunofluorescence microscopy with the Golgi marker protein 58K revealed that phospho-eNOS Ser-1176 is associated with the Golgi complex. Surprisingly, we observed several spots in the nucleus of C6 cells that were positive for phospho-eNOS Ser-1176. Confocal double immunofluorescence analysis with the nucleolus marker protein fibrillarin revealed that within the nucleus phospho-eNOS Ser-1176 is exclusively associated with the nucleoli. It is known that in mitotic cells nucleoli are lost during prophase and rebuild during telophase. In agreement with this, we find no nucleoli-like distribution of phospho-eNOS Ser-1176 in metaphase and anaphase C6 glioma cells. Our finding that phospho-eNOS Ser-1176 is selectively associated with the nucleoli points to a so far unknown role for eNOS in interphase glioma cells.

Nuclear localization of CPI-17, a protein phosphatase-1 inhibitor protein, affects histone H3 phosphorylation and corresponds to proliferation of cancer and smooth muscle cells

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

Eto, Masumi, E-mail: masumi.eto@jefferson.edu; Kirkbride, Jason A.; Chugh, Rishika

2013-04-26

Highlights: •Non-canonical roles of the myosin phosphatase inhibitor (CPI-17) were studied. •CPI-17 is localized in the nucleus of hyperplastic cancer and smooth muscle cells. •CPI-17 Ser12 phosphorylation may regulate the nuclear import. •CPI-17 regulates histone H3 phosphorylation and cell proliferation. •The nuclear CPI-17-PP1 axis plays a proliferative role in cells. -- Abstract: CPI-17 (C-kinase-activated protein phosphatase-1 (PP1) inhibitor, 17 kDa) is a cytoplasmic protein predominantly expressed in mature smooth muscle (SM) that regulates the myosin-associated PP1 holoenzyme (MLCP). Here, we show CPI-17 expression in proliferating cells, such as pancreatic cancer and hyperplastic SM cells. Immunofluorescence showed that CPI-17 was concentratedmore » in nuclei of human pancreatic cancer (Panc1) cells. Nuclear accumulation of CPI-17 was also detected in the proliferating vascular SM cell culture and cells at neointima of rat vascular injury model. The N-terminal 21-residue tail domain of CPI-17 was necessary for the nuclear localization. Phospho-mimetic Asp-substitution of CPI-17 at Ser12 attenuated the nuclear import. CPI-17 phosphorylated at Ser12 was not localized at nuclei, suggesting a suppressive role of Ser12 phosphorylation in the nuclear import. Activated CPI-17 bound to all three isoforms of PP1 catalytic subunit in Panc1 nuclear extracts. CPI-17 knockdown in Panc1 resulted in dephosphorylation of histone H3 at Thr3, Ser10 and Thr11, whereas it had no effects on the phosphorylation of myosin light chain and merlin, the known targets of MLCP. In parallel, CPI-17 knockdown suppressed Panc1 proliferation. We propose that CPI-17 accumulated in the nucleus through the N-terminal tail targets multiple PP1 signaling pathways regulating cell proliferation.« less

Synthetic physical interactions map kinetochore regulators and regions sensitive to constitutive Cdc14 localization.

PubMed

Ólafsson, Guðjón; Thorpe, Peter H

2015-08-18

The location of proteins within eukaryotic cells is often critical for their function and relocation of proteins forms the mainstay of regulatory pathways. To assess the importance of protein location to cellular homeostasis, we have developed a methodology to systematically create binary physical interactions between a query protein and most other members of the proteome. This method allows us to rapidly assess which of the thousands of possible protein interactions modify a phenotype. As proof of principle we studied the kinetochore, a multiprotein assembly that links centromeres to the microtubules of the spindle during cell division. In budding yeast, the kinetochores from the 16 chromosomes cluster together to a single location within the nucleus. The many proteins that make up the kinetochore are regulated through ubiquitylation and phosphorylation. By systematically associating members of the proteome to the kinetochore, we determine which fusions affect its normal function. We identify a number of candidate kinetochore regulators, including the phosphatase Cdc14. We examine where within the kinetochore Cdc14 can act and show that the effect is limited to regions that correlate with known phosphorylation sites, demonstrating the importance of serine phospho-regulation for normal kinetochore homeostasis.

Pathway-based identification of biomarkers for targeted therapeutics: personalized oncology with PI3K pathway inhibitors.

PubMed

Andersen, Jannik N; Sathyanarayanan, Sriram; Di Bacco, Alessandra; Chi, An; Zhang, Theresa; Chen, Albert H; Dolinski, Brian; Kraus, Manfred; Roberts, Brian; Arthur, William; Klinghoffer, Rich A; Gargano, Diana; Li, Lixia; Feldman, Igor; Lynch, Bethany; Rush, John; Hendrickson, Ronald C; Blume-Jensen, Peter; Paweletz, Cloud P

2010-08-04

Although we have made great progress in understanding the complex genetic alterations that underlie human cancer, it has proven difficult to identify which molecularly targeted therapeutics will benefit which patients. Drug-specific modulation of oncogenic signaling pathways in specific patient subpopulations can predict responsiveness to targeted therapy. Here, we report a pathway-based phosphoprofiling approach to identify and quantify clinically relevant, drug-specific biomarkers for phosphatidylinositol 3-kinase (PI3K) pathway inhibitors that target AKT, phosphoinositide-dependent kinase 1 (PDK1), and PI3K-mammalian target of rapamycin (mTOR). We quantified 375 nonredundant PI3K pathway-relevant phosphopeptides, all containing AKT, PDK1, or mitogen-activated protein kinase substrate recognition motifs. Of these phosphopeptides, 71 were drug-regulated, 11 of them by all three inhibitors. Drug-modulated phosphoproteins were enriched for involvement in cytoskeletal reorganization (filamin, stathmin, dynamin, PAK4, and PTPN14), vesicle transport (LARP1, VPS13D, and SLC20A1), and protein translation (S6RP and PRAS40). We then generated phosphospecific antibodies against selected, drug-regulated phosphorylation sites that would be suitable as biomarker tools for PI3K pathway inhibitors. As proof of concept, we show clinical translation feasibility for an antibody against phospho-PRAS40(Thr246). Evaluation of binding of this antibody in human cancer cell lines, a PTEN (phosphatase and tensin homolog deleted from chromosome 10)-deficient mouse prostate tumor model, and triple-negative breast tumor tissues showed that phospho-PRAS40(Thr246) positively correlates with PI3K pathway activation and predicts AKT inhibitor sensitivity. In contrast to phosphorylation of AKT(Thr308), the phospho-PRAS40(Thr246) epitope is highly stable in tissue samples and thus is ideal for immunohistochemistry. In summary, our study illustrates a rational approach for discovery of drug-specific biomarkers toward development of patient-tailored treatments.

Increased phospho-adducin immunoreactivity in a murine model of amyotrophic lateral sclerosis.

PubMed

Shan, X; Hu, J H; Cayabyab, F S; Krieger, C

2005-01-01

Adducins alpha, beta and gamma are proteins that link spectrin and actin in the regulation of cytoskeletal architecture and are substrates for protein kinase C and other signaling molecules. Previous studies have shown that expressions of phosphorylated adducin (phospho-adducin) and protein kinase C are increased in spinal cord tissue from patients who died with amyotrophic lateral sclerosis, a neurodegenerative disorder of motoneurons and other cells. However, the distribution of phospho-adducin immunoreactivity has not been described in the mammalian spinal cord. We have evaluated the distribution of immunoreactivity to serine/threonine-dependent phospho-adducin at a region corresponding to the myristoylated alanine-rich C kinase substrate-related domain of adducin in spinal cords of mice over-expressing mutant human superoxide dismutase, an animal model of amyotrophic lateral sclerosis, and in control littermates. We find phospho-adducin immunoreactivity in control spinal cord in ependymal cells surrounding the central canal, neurons and astrocytes. Phospho-adducin immunoreactivity is localized to the cell bodies, dendrites and axons of some motoneurons, as well as to astrocytes in the gray and white matter. Spinal cords of mutant human superoxide dismutase mice having motoneuron loss exhibit significantly increased phospho-adducin immunoreactivity in ventral and dorsal horn spinal cord regions, but not in ependyma surrounding the central canal, compared with control animals. Increased phospho-adducin immunoreactivity localizes predominantly to astrocytes and likely increases as a consequence of the astrogliosis that occurs in the mutant human superoxide dismutase mouse with disease progression. These findings demonstrate increased immunoreactivity against phosphorylated adducin at the myristoylated alanine-rich C kinase substrate domain in a murine model of amyotrophic lateral sclerosis. As adducin is a substrate for protein kinase C at the myristoylated alanine-rich C kinase substrate domain, the increased phospho-adducin immunoreactivity is likely a consequence of protein kinase C activation in neurons and astrocytes of the spinal cord and evidence for aberrant phosphorylation events in mutant human superoxide dismutase mice that may affect neuron survival.

XLID CUL4B mutants are defective in promoting TSC2 degradation and positively regulating mTOR signaling in neocortical neurons.

PubMed

Wang, Hung-Li; Chang, Ning-Chun; Weng, Yi-Hsin; Yeh, Tu-Hsueh

2013-04-01

Truncating or missense mutation of cullin 4B (CUL4B) is one of the most prevalent causes underlying X-linked intellectual disability (XLID). CUL4B-RING E3 ubiquitin ligase promotes ubiquitination and degradation of various proteins. Consistent with previous studies, overexpression of wild-type CUL4B in 293 cells enhanced ubiquitylation and degradation of TSC2 or cyclin E. The present study shows that XLID mutant (R388X), (R572C) or (V745A) CULB failed to promote ubiquitination and degradation of TSC2 or cyclin E. Adenoviruses-mediated expression of wild-type CUL4B decreased protein level of TSC2 or cyclin E in cultured neocortical neurons of frontal lobe. Furthermore, shRNA-mediated CUL4B knockdown caused an upregulation of TSC2 or cyclin E. XLID mutant (R388X), (R572C) or (V745A) CUL4B did not downregulate protein expression of TSC2 or cyclin E in neocortical neurons. By promoting TSC2 degradation, CUL4B could positively regulate mTOR activity in neocortical neurons of frontal cortex. Consistent with this hypothesis, CUL4B knockdown-induced upregulation of TSC2 in neocortical neurons resulted in a decreased protein level of active phospho-mTOR(Ser2448) and a reduced expression of active phospho-p70S6K(Thr389) and phospho-4E-BP1(Thr37/46), two main substrates of mTOR-mediated phosphorylation. Wild-type CUL4B also increased protein level of active phospho-mTOR(Ser2448), phospho-p70S6K(Thr389) or phospho-4E-BP1(Thr37/46). XLID CUL4B mutants did not affect protein level of active phospho-mTOR(Ser2448), phospho-p70S6K(Thr389) or phospho-4E-BP1(Thr37/46). Our results suggest that XLID CUL4B mutants are defective in promoting TSC2 degradation and positively regulating mTOR signaling in neocortical neurons. Copyright © 2013 Elsevier B.V. All rights reserved.

Loss of the Greatwall Kinase Weakens the Spindle Assembly Checkpoint.

PubMed

Diril, M Kasim; Bisteau, Xavier; Kitagawa, Mayumi; Caldez, Matias J; Wee, Sheena; Gunaratne, Jayantha; Lee, Sang Hyun; Kaldis, Philipp

2016-09-01

The Greatwall kinase/Mastl is an essential gene that indirectly inhibits the phosphatase activity toward mitotic Cdk1 substrates. Here we show that although Mastl knockout (MastlNULL) MEFs enter mitosis, they progress through mitosis without completing cytokinesis despite the presence of misaligned chromosomes, which causes chromosome segregation defects. Furthermore, we uncover the requirement of Mastl for robust spindle assembly checkpoint (SAC) maintenance since the duration of mitotic arrest caused by microtubule poisons in MastlNULL MEFs is shortened, which correlates with premature disappearance of the essential SAC protein Mad1 at the kinetochores. Notably, MastlNULL MEFs display reduced phosphorylation of a number of proteins in mitosis, which include the essential SAC kinase MPS1. We further demonstrate that Mastl is required for multi-site phosphorylation of MPS1 as well as robust MPS1 kinase activity in mitosis. In contrast, treatment of MastlNULL cells with the phosphatase inhibitor okadaic acid (OKA) rescues the defects in MPS1 kinase activity, mislocalization of phospho-MPS1 as well as Mad1 at the kinetochore, and premature SAC silencing. Moreover, using in vitro dephosphorylation assays, we demonstrate that Mastl promotes persistent MPS1 phosphorylation by inhibiting PP2A/B55-mediated MPS1 dephosphorylation rather than affecting Cdk1 kinase activity. Our findings establish a key regulatory function of the Greatwall kinase/Mastl->PP2A/B55 pathway in preventing premature SAC silencing.

Loss of the Greatwall Kinase Weakens the Spindle Assembly Checkpoint

PubMed Central

Kitagawa, Mayumi; Caldez, Matias J.; Gunaratne, Jayantha; Lee, Sang Hyun

2016-01-01

The Greatwall kinase/Mastl is an essential gene that indirectly inhibits the phosphatase activity toward mitotic Cdk1 substrates. Here we show that although Mastl knockout (MastlNULL) MEFs enter mitosis, they progress through mitosis without completing cytokinesis despite the presence of misaligned chromosomes, which causes chromosome segregation defects. Furthermore, we uncover the requirement of Mastl for robust spindle assembly checkpoint (SAC) maintenance since the duration of mitotic arrest caused by microtubule poisons in MastlNULL MEFs is shortened, which correlates with premature disappearance of the essential SAC protein Mad1 at the kinetochores. Notably, MastlNULL MEFs display reduced phosphorylation of a number of proteins in mitosis, which include the essential SAC kinase MPS1. We further demonstrate that Mastl is required for multi-site phosphorylation of MPS1 as well as robust MPS1 kinase activity in mitosis. In contrast, treatment of MastlNULL cells with the phosphatase inhibitor okadaic acid (OKA) rescues the defects in MPS1 kinase activity, mislocalization of phospho-MPS1 as well as Mad1 at the kinetochore, and premature SAC silencing. Moreover, using in vitro dephosphorylation assays, we demonstrate that Mastl promotes persistent MPS1 phosphorylation by inhibiting PP2A/B55-mediated MPS1 dephosphorylation rather than affecting Cdk1 kinase activity. Our findings establish a key regulatory function of the Greatwall kinase/Mastl->PP2A/B55 pathway in preventing premature SAC silencing. PMID:27631493

Regulation of tyrosine hydroxylase activity and phosphorylation at Ser(19) and Ser(40) via activation of glutamate NMDA receptors in rat striatum.

PubMed

Lindgren, N; Xu, Z Q; Lindskog, M; Herrera-Marschitz, M; Goiny, M; Haycock, J; Goldstein, M; Hökfelt, T; Fisone, G

2000-06-01

The activity of tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by phosphorylation. In this study, we examined the effects of activation of NMDA receptors on the state of phosphorylation and activity of tyrosine hydroxylase in rat striatal slices. NMDA produced a time-and concentration-dependent increase in the levels of phospho-Ser(19)-tyrosine hydroxylase in nigrostriatal nerve terminals. This increase was not associated with any changes in the basal activity of tyrosine hydroxylase, measured as DOPA accumulation. Forskolin, an activator of adenylyl cyclase, stimulated tyrosine hydroxylase phosphorylation at Ser(40) and caused a significant increase in DOPA accumulation. NMDA reduced forskolin-mediated increases in both Ser(40) phosphorylation and DOPA accumulation. In addition, NMDA reduced the increase in phospho-Ser(40)-tyrosine hydroxylase produced by okadaic acid, an inhibitor of protein phosphatase 1 and 2A, but not by a cyclic AMP analogue, 8-bromo-cyclic AMP. These results indicate that, in the striatum, glutamate decreases tyrosine hydroxylase phosphorylation at Ser(40) via activation of NMDA receptors by reducing cyclic AMP production. They also provide a mechanism for the demonstrated ability of NMDA to decrease tyrosine hydroxylase activity and dopamine synthesis.

PhosphoBase: a database of phosphorylation sites.

PubMed Central

Blom, N; Kreegipuu, A; Brunak, S

1998-01-01

PhosphoBase is a database of experimentally verified phosphorylation sites. Version 1.0 contains 156 entries and 398 experimentally determined phosphorylation sites. Entries are compiled and revised from the literature and from major protein sequence databases such as SwissProt and PIR. The entries provide information about the phosphoprotein and the exact position of its phosphorylation sites. Furthermore, part of the entries contain information about kinetic data obtained from enzyme assays on specific peptides. To illustrate the use of data extracted from PhosphoBase we present a sequence logo displaying the overall conservation of positions around serines phosphorylated by protein kinase A (PKA). PhosphoBase is available on the WWW at http://www.cbs.dtu.dk/databases/PhosphoBase/ PMID:9399879

Recent findings and technological advances in phosphoproteomics for cells and tissues.

PubMed

von Stechow, Louise; Francavilla, Chiara; Olsen, Jesper V

2015-01-01

Site-specific phosphorylation is a fast and reversible covalent post-translational modification that is tightly regulated in cells. The cellular machinery of enzymes that write, erase and read these modifications (kinases, phosphatases and phospho-binding proteins) is frequently deregulated in different diseases, including cancer. Large-scale studies of phosphoproteins - termed phosphoproteomics - strongly rely on the use of high-performance mass spectrometric instrumentation. This powerful technology has been applied to study a great number of phosphorylation-based phenotypes. Nevertheless, many technical and biological challenges have to be overcome to identify biologically relevant phosphorylation sites in cells and tissues. This review describes different technological strategies to identify and quantify phosphorylation sites with high accuracy, without significant loss of analysis speed and reproducibility in tissues and cells. Moreover, computational tools for analysis, integration and biological interpretation of phosphorylation events are discussed.

STC1 promotes cell apoptosis via NF-κB phospho-P65 Ser536 in cervical cancer cells

PubMed Central

Pan, Xi; Jiang, Binyuan; Liu, Jianhao; Ding, Juan; Li, Yuehui; Sun, Ruili; Peng, Li; Qin, Changfei; Fang, Shujuan; Li, Guancheng

2017-01-01

Stanniocalin-1 (STC1) is a secreted glycoprotein hormone and involved in various types of human malignancies. Our previous studies revealed that STC1 inhibited cell proliferation and invasion of cervical cancer cells through NF-κB P65 activation, but the mechanism is poorly understood. In our studies, we found overexpression of STC1 promoted cell apoptosis while silencing of STC1 promoted cell growth of cervical cancer. Phospho-protein profiling and Western blotting results showed the expression of NF-κB related phosphorylation sites including NF-κB P65 (Ser536), IκBα, IKKβ, PI3K, and AKT was altered in STC1-overexpressed cervical cancer cells. Moreover, PI3K inhibitor LY294002, AKT-shRNA and IκBα-shRNA could decrease the protein content of phospho-P65 (Ser536), phospho-IκBα, phospho-AKT and phospho-IKKβ while increasing the level of P65 compared to STC1 overexpression groups in cervical cancer cells. Also, PI3K inhibitor LY294002, AKT-shRNA and IκBα-shRNA elevated the percentage of apoptosis and suppressed the G1/S transition in those cells. Additionally, STC1 level was decreased in cervical cancer, especial in stage II and III. The results of immunohistochemistry for the cervical cancer microarray showed that a lower level of STC1, phospho-PI3K and P65 protein expression in tumor tissues than that in normal tissues, and a higher level of phospho-P65 protein expression in tumor tissues, which is consistent with the results of the Western blotting. These data demonstrated that STC1 can promote cell apoptosis via NF-κB phospho-P65 (Ser536) by PI3K/AKT, IκBα and IKK signaling in cervical cancer cells. Our results offer the first mechanism that explains the link between STC1 and cell apoptosis in cervical cancer. PMID:28545028

STC1 promotes cell apoptosis via NF-κB phospho-P65 Ser536 in cervical cancer cells.

PubMed

Pan, Xi; Jiang, Binyuan; Liu, Jianhao; Ding, Juan; Li, Yuehui; Sun, Ruili; Peng, Li; Qin, Changfei; Fang, Shujuan; Li, Guancheng

2017-07-11

Stanniocalin-1 (STC1) is a secreted glycoprotein hormone and involved in various types of human malignancies. Our previous studies revealed that STC1 inhibited cell proliferation and invasion of cervical cancer cells through NF-κB P65 activation, but the mechanism is poorly understood. In our studies, we found overexpression of STC1 promoted cell apoptosis while silencing of STC1 promoted cell growth of cervical cancer. Phospho-protein profiling and Western blotting results showed the expression of NF-κB related phosphorylation sites including NF-κB P65 (Ser536), IκBα, IKKβ, PI3K, and AKT was altered in STC1-overexpressed cervical cancer cells. Moreover, PI3K inhibitor LY294002, AKT-shRNA and IκBα-shRNA could decrease the protein content of phospho-P65 (Ser536), phospho-IκBα, phospho-AKT and phospho-IKKβ while increasing the level of P65 compared to STC1 overexpression groups in cervical cancer cells. Also, PI3K inhibitor LY294002, AKT-shRNA and IκBα-shRNA elevated the percentage of apoptosis and suppressed the G1/S transition in those cells. Additionally, STC1 level was decreased in cervical cancer, especial in stage II and III. The results of immunohistochemistry for the cervical cancer microarray showed that a lower level of STC1, phospho-PI3K and P65 protein expression in tumor tissues than that in normal tissues, and a higher level of phospho-P65 protein expression in tumor tissues, which is consistent with the results of the Western blotting. These data demonstrated that STC1 can promote cell apoptosis via NF-κB phospho-P65 (Ser536) by PI3K/AKT, IκBα and IKK signaling in cervical cancer cells. Our results offer the first mechanism that explains the link between STC1 and cell apoptosis in cervical cancer.

Anaplastic ganglioglioma arising from a Lhermitte-Duclos-like lesion. Case report.

PubMed

Takei, Hidehiro; Dauser, Robert; Su, Jack; Chintagumpala, Murali; Bhattacharjee, Meenakshi B; Jones, Jeremy; Adesina, Adekunle M

2007-08-01

The authors report the case of a 7-year-old boy with a history of developmental delay who presented with aggressive behavior. A magnetic resonance (MR) image showed a mass lesion originating from the cerebellar vermis with an atypical folial pattern and contrast enhancement. Histologically, the subtotally resected specimen consisted mostly of neuropil with nodular foci of ganglion cells. Lhermitte-Duclos disease (LDD) was diagnosed in the patient. A retrospective review of the tissue sections showed a nidus of associated astrocytic proliferation, suggesting a diagnosis of ganglioglioma. Five years later, the patient experienced an altered mental state and a facial droop. An MR image revealed a cerebellar mass with cystic areas and an enhancing nodule. The resected tissue specimen consisted primarily of a mixed proliferation of glial and ganglion cells consistent with a ganglioglioma. Two years later, a third craniectomy was performed in the patient for worsening headache and ataxia. Histologically, the tumor showed progressive anaplasia and was most accurately classified as an anaplastic ganglioglioma. Immunohistochemically, most of the tumor cells were immunoreactive for anti-phospho-mammalian target of rapamycin (mTOR) and phospho-S6 ribosomal protein antibodies. In contrast, the subpopulation of neoplastic ganglion cells in the tissue, particularly from the first surgery, did not express phosphatase and tensin homolog deleted from chromosome 10 (PTEN). This immunohistochemical pattern suggests that the large dysplastic ganglion cells (the gangliocytomatous component) forming the greater part of the lesion were associated with activation of the phosphatidylinositol 3-kinase-PTEN/Akt/mTOR signaling pathway, a feature previously reported in LDD. This case represents the first report of an anaplastic ganglioglioma arising in an LDD-like lesion.

Inhibitor of the Tyrosine Phosphatase STEP Reverses Cognitive Deficits in a Mouse Model of Alzheimer's Disease

PubMed Central

Xu, Jian; Chatterjee, Manavi; Baguley, Tyler D.; Brouillette, Jonathan; Kurup, Pradeep; Ghosh, Debolina; Kanyo, Jean; Zhang, Yang; Seyb, Kathleen; Ononenyi, Chimezie; Foscue, Ethan; Anderson, George M.; Gresack, Jodi; Cuny, Gregory D.; Glicksman, Marcie A.; Greengard, Paul; Lam, TuKiet T.; Tautz, Lutz; Nairn, Angus C.; Ellman, Jonathan A.; Lombroso, Paul J.

2014-01-01

STEP (STriatal-Enriched protein tyrosine Phosphatase) is a neuron-specific phosphatase that regulates N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking, as well as ERK1/2, p38, Fyn, and Pyk2 activity. STEP is overactive in several neuropsychiatric and neurodegenerative disorders, including Alzheimer's disease (AD). The increase in STEP activity likely disrupts synaptic function and contributes to the cognitive deficits in AD. AD mice lacking STEP have restored levels of glutamate receptors on synaptosomal membranes and improved cognitive function, results that suggest STEP as a novel therapeutic target for AD. Here we describe the first large-scale effort to identify and characterize small-molecule STEP inhibitors. We identified the benzopentathiepin 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (known as TC-2153) as an inhibitor of STEP with an IC50 of 24.6 nM. TC-2153 represents a novel class of PTP inhibitors based upon a cyclic polysulfide pharmacophore that forms a reversible covalent bond with the catalytic cysteine in STEP. In cell-based secondary assays, TC-2153 increased tyrosine phosphorylation of STEP substrates ERK1/2, Pyk2, and GluN2B, and exhibited no toxicity in cortical cultures. Validation and specificity experiments performed in wild-type (WT) and STEP knockout (KO) cortical cells and in vivo in WT and STEP KO mice suggest specificity of inhibitors towards STEP compared to highly homologous tyrosine phosphatases. Furthermore, TC-2153 improved cognitive function in several cognitive tasks in 6- and 12-mo-old triple transgenic AD (3xTg-AD) mice, with no change in beta amyloid and phospho-tau levels. PMID:25093460

Streptozotocin-Induced Autophagy Reduces Intracellular Insulin in Insulinoma INS-1E Cells.

PubMed

Yoo, Yeong-Min; Park, Yung Chul

2018-03-01

Streptozotocin (STZ), a glucose analog, induces diabetes in experimental animals by inducing preferential cytotoxicity in pancreatic beta cells. We investigated whether STZ reduced the production of intracellular insulin through autophagy in insulinoma INS-1E cells. Typically, 2 mM STZ treatment for 24 h significantly decreased cell survival. STZ treatment led to significant decrease in phospho-AMP-activated protein kinase (p-AMPK) level; reduction in levels of phospho-protein kinase R-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme 1α (IRE1α); significant reduction in levels of p85α, p110, phospho-serine and threonine kinase/protein kinase B (p-Akt/PKB) (Ser473), phospho-extracellular-regulated kinase (p-ERK), and phospho-mammalian target of rapamycin (p-mTOR); increase in levels of Cu/Zn-superoxide dismutase (SOD), Mn-SOD, and catalase; decrease in B-cell lymphoma 2 (Bcl-2) expression; increase in Bcl-2-associated X protein (Bax) expression; increase in levels of microtubule-associated protein 1 light chain 3 (LC3) and Beclin 1; and reduction in production of intracellular insulin. These results suggest that insulin synthesis during STZ treatment involves autophagy in INS-1E cells and, subsequently, results in a decrease in intracellular production of insulin.

Structure of the Arabidopsis Glucan Phosphatase LIKE SEX FOUR2 Reveals a Unique Mechanism for Starch Dephosphorylation[W

PubMed Central

Meekins, David A.; Guo, Hou-Fu; Husodo, Satrio; Paasch, Bradley C.; Bridges, Travis M.; Santelia, Diana; Kötting, Oliver; Vander Kooi, Craig W.; Gentry, Matthew S.

2013-01-01

Starch is a water-insoluble, Glc-based biopolymer that is used for energy storage and is synthesized and degraded in a diurnal manner in plant leaves. Reversible phosphorylation is the only known natural starch modification and is required for starch degradation in planta. Critical to starch energy release is the activity of glucan phosphatases; however, the structural basis of dephosphorylation by glucan phosphatases is unknown. Here, we describe the structure of the Arabidopsis thaliana starch glucan phosphatase LIKE SEX FOUR2 (LSF2) both with and without phospho-glucan product bound at 2.3Å and 1.65Å, respectively. LSF2 binds maltohexaose-phosphate using an aromatic channel within an extended phosphatase active site and positions maltohexaose in a C3-specific orientation, which we show is critical for the specific glucan phosphatase activity of LSF2 toward native Arabidopsis starch. However, unlike other starch binding enzymes, LSF2 does not possess a carbohydrate binding module domain. Instead we identify two additional glucan binding sites located within the core LSF2 phosphatase domain. This structure is the first of a glucan-bound glucan phosphatase and provides new insights into the molecular basis of this agriculturally and industrially relevant enzyme family as well as the unique mechanism of LSF2 catalysis, substrate specificity, and interaction with starch granules. PMID:23832589

Dehydration-responsive nuclear proteome landscape of chickpea (Cicer arietinum L.) reveals phosphorylation-mediated regulation of stress response.

PubMed

Barua, Pragya; Lande, Nilesh Vikram; Subba, Pratigya; Gayen, Dipak; Pinto, Sneha; Prasad, T S Keshav; Chakraborty, Subhra; Chakraborty, Niranjan

2018-05-10

Non-availability of water or dehydration remains recurring climatic disorder affecting yield of major food crops, legumes in particular. Nuclear proteins (NP) and phosphoproteins (NPPs) execute crucial cellular functions that form the regulatory hub for coordinated stress response. Phosphoproteins hold enormous influence over cellular signalling. Four-week-old seedlings of a grain legume, chickpea, were subjected to gradual dehydration and nuclear proteins were extracted from unstressed control as well as from 72 and 144 h stressed tissues. We identified 4832 NPs and 478 phosphosites, corresponding to 299 unique NPPs involved in multivariate cellular processes including protein modification and gene expression regulation, among others. The identified proteins included several novel kinases, phosphatases and transcription factors, besides 660 uncharacterised proteins. Spliceosome complex and splicing related proteins were dominant among differentially regulated NPPs, indicating their dehydration modulated regulation. Phospho-motif analysis revealed stress-induced enrichment of proline-directed serine phosphorylation. Association mapping of NPPs revealed predominance of differential phosphorylation of spliceosome and splicing associated proteins. Also, regulatory proteins of key processes viz., protein degradation, regulation of flowering time and circadian clock were observed to undergo dehydration-induced dephosphorylation. The characterization of novel regulatory proteins would provide new insights into stress adaptation and enable directed genetic manipulations for developing climate-resilient crops. This article is protected by copyright. All rights reserved.

A method for direct assessment of tissue-nonspecific alkaline phosphatase (TNAP) inhibitors in blood samples.

PubMed

Sergienko, Eduard A; Sun, Qing; Ma, Chen-Ting

2013-01-01

Tissue nonspecific alkaline phosphatase (TNAP) is one of four human alkaline phosphatases (AP), a family of exocytic enzymes that catalyze hydrolysis of phospho-monoesters in bone, liver, kidney, and various other tissues. Overexpression of TNAP gives rise to excessive bone and soft tissue mineralization, including blood vessel calcification. Our prior screening campaigns have found several leads against this attractive therapeutic target using in vitro assay with a recombinant enzyme; these compounds were further optimized using medicinal chemistry approaches. To prioritize compounds for their use in animal models, we have designed and developed a biomarker assay for in situ detection of TNAP activity within human and mouse blood samples at physiological pH. This assay is suitable for screening compounds in 1,536-well plates using blood plasma from different mammalian species. The user may choose from two different substrates based on the need for greater assay simplicity or sensitivity.

A Phosphorylation Switch Regulates the Transcriptional Activation of Cell Cycle Regulator p21 by Histone Deacetylase Inhibitors*

PubMed Central

Simboeck, Elisabeth; Sawicka, Anna; Zupkovitz, Gordin; Senese, Silvia; Winter, Stefan; Dequiedt, Franck; Ogris, Egon; Di Croce, Luciano; Chiocca, Susanna; Seiser, Christian

2010-01-01

Histone deacetylase inhibitors induce cell cycle arrest and apoptosis in tumor cells and are, therefore, promising anti-cancer drugs. The cyclin-dependent kinase inhibitor p21 is activated in histone deacetylase (HDAC) inhibitor-treated tumor cells, and its growth-inhibitory function contributes to the anti-tumorigenic effect of HDAC inhibitors. We show here that induction of p21 by trichostatin A involves MAP kinase signaling. Activation of the MAP kinase signaling pathway by growth factors or stress signals results in histone H3 serine 10 phosphorylation at the p21 promoter and is crucial for acetylation of the neighboring lysine 14 and recruitment of activated RNA polymerase II in response to trichostatin A treatment. In non-induced cells, the protein phosphatase PP2A is associated with the p21 gene and counteracts its activation. Induction of p21 is linked to simultaneous acetylation and phosphorylation of histone H3. The dual modification mark H3S10phK14ac at the activated p21 promoter is recognized by the phospho-binding protein 14-3-3ζ, which protects the phosphoacetylation mark from being processed by PP2A. Taken together we have revealed a cross-talk of reversible phosphorylation and acetylation signals that controls the activation of p21 by HDAC inhibitors and identify the phosphatase PP2A as chromatin-associated transcriptional repressor in mammalian cells. PMID:20952396

TC-PTP directly interacts with connexin43 to regulate gap junction intercellular communication

PubMed Central

Li, Hanjun; Spagnol, Gaelle; Naslavsky, Naava; Caplan, Steve; Sorgen, Paul L.

2014-01-01

ABSTRACT Protein kinases have long been reported to regulate connexins; however, little is known about the involvement of phosphatases in the modulation of intercellular communication through gap junctions and the subsequent downstream effects on cellular processes. Here, we identify an interaction between the T-cell protein tyrosine phosphatase (TC-PTP, officially known as PTPN2) and the carboxyl terminus of connexin43 (Cx43, officially known as GJA1). Two cell lines, normal rat kidney (NRK) cells endogenously expressing Cx43 and an NRK-derived cell line expressing v-Src with temperature-sensitive activity, were used to demonstrate that EGF and v-Src stimulation, respectively, induced TC-PTP to colocalize with Cx43 at the plasma membrane. Cell biology experiments using phospho-specific antibodies and biophysical assays demonstrated that the interaction is direct and that TC-PTP dephosphorylates Cx43 residues Y247 and Y265, but does not affect v-Src. Transfection of TC-PTP also indirectly led to the dephosphorylation of Cx43 S368, by inactivating PKCα and PKCδ, with no effect on the phosphorylation of S279 and S282 (MAPK-dependent phosphorylation sites). Dephosphorylation maintained Cx43 gap junctions at the plaque and partially reversed the channel closure caused by v-Src-mediated phosphorylation of Cx43. Understanding dephosphorylation, along with the well-documented roles of Cx43 phosphorylation, might eventually lead to methods to modulate the regulation of gap junction channels, with potential benefits for human health. PMID:24849651

Estimation of low-dose radiation-responsive proteins in the absence of genomic instability in normal human fibroblast cells.

PubMed

Yim, Ji-Hye; Yun, Jung Mi; Kim, Ji Young; Nam, Seon Young; Kim, Cha Soon

2017-11-01

Low-dose radiation has various biological effects such as adaptive responses, low-dose hypersensitivity, as well as beneficial effects. However, little is known about the particular proteins involved in these effects. Here, we sought to identify low-dose radiation-responsive phosphoproteins in normal fibroblast cells. We assessed genomic instability and proliferation of fibroblast cells after γ-irradiation by γ-H2AX foci and micronucleus formation analyses and BrdU incorporation assay, respectively. We screened fibroblast cells 8 h after low-dose (0.05 Gy) γ-irradiation using Phospho Explorer Antibody Microarray and validated two differentially expressed phosphoproteins using Western blotting. Cell proliferation proceeded normally in the absence of genomic instability after low-dose γ-irradiation. Phospho antibody microarray analysis and Western blotting revealed increased expression of two phosphoproteins, phospho-NFκB (Ser536) and phospho-P70S6K (Ser418), 8 h after low-dose radiation. Our findings suggest that low-dose radiation of normal fibroblast cells activates the expression of phospho-NFκB (Ser536) and phospho-P70S6K (Ser418) in the absence of genomic instability. Therefore, these proteins may be involved in DNA damage repair processes.

Preanalytical variables and phosphoepitope expression in FFPE tissue: quantitative epitope assessment after variable cold ischemic time.

PubMed

Vassilakopoulou, Maria; Parisi, Fabio; Siddiqui, Summar; England, Allison M; Zarella, Elizabeth R; Anagnostou, Valsamo; Kluger, Yuval; Hicks, David G; Rimm, David L; Neumeister, Veronique M

2015-03-01

Individualized targeted therapies for cancer patients require accurate and reproducible assessment of biomarkers to be able to plan treatment accordingly. Recent studies have shown highly variable effects of preanalytical variables on gene expression profiling and protein levels of different tissue types. Several publications have described protein degradation of tissue samples as a direct result of delay of formalin fixation of the tissue. Phosphorylated proteins are more labile and epitope degradation can happen within 30 min of cold ischemic time. To address this issue, we evaluated the change in antigenicity of a series of phosphoproteins in paraffin-embedded samples from breast tumors as a function of time to formalin fixation. A tissue microarray consisting of 93 breast cancer specimens with documented time-to-fixation was used to evaluate changes in antigenicity of 12 phosphoepitopes frequently used in research settings as a function of cold ischemic time. Analysis was performed in a quantitative manner using the AQUA technology for quantitative immunofluorescence. For each marker, least squares univariate linear regression was performed and confidence intervals were computed using bootstrapping. The majority of the epitopes tested revealed changes in expression levels with increasing time to formalin fixation. Some phosphorylated proteins, such as phospho-HSP27 and phospho-S6 RP, involved in post-translational modification and stress response pathways increased in expression or phosphorylation levels. Others (like phospho-AKT, phosphor-ERK1/2, phospho-Tyrosine, phospho-MET, and others) are quite labile and loss of antigenicity can be reported within 1-2 h of cold ischemic time. Therefore specimen collection should be closely monitored and subjected to quality control measures to ensure accurate measurement of these epitopes. However, a few phosphoepitopes (like phospho-JAK2 and phospho-ER) are sufficiently robust for routine usage in companion diagnostic testing.

Low-dose occupational exposure to benzene and signal transduction pathways involved in the regulation of cellular response to oxidative stress.

PubMed

Fenga, Concettina; Gangemi, Silvia; Giambò, Federica; Tsitsimpikou, Christina; Golokhvast, Kirill; Tsatsakis, Aristidis; Costa, Chiara

2016-02-15

Benzene metabolism seems to modulate NF-κB, p38-MAPK (mitogen-activated protein kinase) and signal transducer and activator of transcription 3 (STAT3) signalling pathways via the production of reactive oxygen species. This study aims to evaluate the effects of low-dose, long-term exposure on NF-κB, STAT3, p38-MAPK and stress-activated protein kinase/Jun amino-terminal kinase (SAPK/JNK) signal transduction pathways in peripheral blood mononuclear cells in gasoline station attendants. The influence of consumption of vegetables and fruits on these pathways has also been evaluated. A total of 91 men, employed in gasoline stations located in eastern Sicily, were enrolled for this study and compared with a control group of 63 male office workers with no history of exposure to benzene. The exposure was assessed by measuring urinary trans,trans-muconic acid (t,t-MA) concentration. Quantitative analyses were performed for proteins NF-κB p65, phospho-NF-κB p65, phospho-IκB-α, phospho-SAPK/JNK, phospho-p38 MAPK and phospho-STAT3 using an immunoenzymatic assay. The results of this study indicate significantly higher t,t-MA levels in gasoline station attendants. With regard to NF-κB, phospho-IκB-α and phospho-STAT3 proteins, statistically significant differences were observed in workers exposed to benzene. However, no differences were observed in SAPK/JNK and p38-MAPK activation. These changes were positively correlated with t,t-MA levels, but only phospho-NF-κB p65 was associated with the intake of food rich in antioxidant active principles. Chronic exposure to low-dose benzene can modulate signal transduction pathways activated by oxidative stress and involved in cell proliferation and apoptosis. This could represent a possible mechanism of carcinogenic action of chronic benzene exposure. Copyright © 2016 Elsevier Inc. All rights reserved.

Autophagy Signaling in Prostate Cancer: Identification of a Novel Phosphatase

DTIC Science & Technology

2011-08-01

the transmembrane and cytosolic residues). We measured PTPRS activity using a phospho-tyrosine (pTyr) peptide with malachite green free phosphate...vitro using a 100 uM phosphotyrosine peptide substrate and malachite green detection of released free phosphates. Activity is expressed as picomoles...Upstate) at 37°C for 15 minutes. Released phosphates were detected with malachite green (Upstate) and absorbance measured at 650 nm. Background levels

PKC Epsilon: A Novel Oncogenic Player in Prostate Cancer

DTIC Science & Technology

2015-11-01

control laboratory diet . Group 2 mice were fed with rofecoxib diet . Protein expression of (A) Akt, phospho-Akt, mTOR, phospho-mTOR, (B) Stat3 and...DISTRIBUTION / AVAILABILITY STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Protein kinase C epsilon (PKC...mechanisms orchestrating prostate cancer development and progression. Studies have recognized protein kinase C (PKC) isozymes as eminent players of cancer

Okadaic acid and microcystin insensitive PPP-family phosphatases may represent novel biotechnology targets.

PubMed

Uhrig, R Glen; Moorhead, Greg B

2011-12-01

Reversible protein phosphorylation is of central importance to the proper cellular functioning of all living organisms. Catalyzed by the opposing reactions of protein kinases and phosphatases, dysfunction in reversible protein phosphorylation can result in a wide variety of cellular aberrations. In eukaryotic organisms there exists four classes of protein phosphatases, of which the PPP-family protein phosphatases have documented susceptibility to a range of protein and small molecule inhibitors. These inhibitors have been of great importance to the biochemical characterization of PPP-family protein phosphatases since their discovery, but also maintain in natura biological significance with their endogenous regulatory properties (protein inhibitors) and toxicity (small molecule inhibitors). Recently, two unique PPP-family protein phosphatases, named the Shewanella-like protein phosphatases (SLP phosphatases), from Arabidopsis thaliana were characterized and found to be phylogenetically similar to the PPP-family protein phosphatases protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), while completely lacking sensitivity to the classic PPP-family phosphatase small molecule inhibitors okadaic acid and microcystin-LR. SLP phosphatases were also found to be absent in metazoans, but present in a wide range of bacteria, fungi and protozoa responsible for human disease. The unique biochemical properties and evolutionary heritage of SLP phosphatases suggests they could not only be potential biotechnology targets for agriculture, but may also prove to be of interest for future therapeutic drug development. © 2011 Landes Bioscience

PP2B and PP1α cooperatively disrupt 7SK snRNP to release P-TEFb for transcription in response to Ca2+ signaling

PubMed Central

Chen, Ruichuan; Liu, Min; Li, Huan; Xue, Yuhua; Ramey, Wanichaya N.; He, Nanhai; Ai, Nanping; Luo, Haohong; Zhu, Ying; Zhou, Nan; Zhou, Qiang

2008-01-01

The positive transcription elongation factor b (P-TEFb), consisting of Cdk9 and cyclin T, stimulates RNA polymerase II elongation and cotranscriptional pre-mRNA processing. To accommodate different growth conditions and transcriptional demands, a reservoir of P-TEFb is kept in an inactive state in the multisubunit 7SK snRNP. Under certain stress or disease conditions, P-TEFb is released to activate transcription, although the signaling pathway(s) that controls this is largely unknown. Here, through analyzing the UV- or hexamethylene bisacetamide (HMBA)-induced release of P-TEFb from 7SK snRNP, an essential role for the calcium ion (Ca2+)–calmodulin–protein phosphatase 2B (PP2B) signaling pathway is revealed. However, Ca2+ signaling alone is insufficient, and PP2B must act sequentially and cooperatively with protein phosphatase 1α (PP1α) to disrupt 7SK snRNP. Activated by UV/HMBA and facilitated by a PP2B-induced conformational change in 7SK snRNP, PP1α releases P-TEFb through dephosphorylating phospho-Thr186 in the Cdk9 T-loop. This event is also necessary for the subsequent recruitment of P-TEFb by the bromodomain protein Brd4 to the preinitiation complex, where Cdk9 remains unphosphorylated and inactive until after the synthesis of a short RNA. Thus, through cooperatively dephosphorylating Cdk9 in response to Ca2+ signaling, PP2B and PP1α alter the P-TEFb functional equilibrium through releasing P-TEFb from 7SK snRNP for transcription. PMID:18483222

Phosphorylation-mediated PTEN conformational closure and deactivation revealed with protein semisynthesis

PubMed Central

Bolduc, David; Rahdar, Meghdad; Tu-Sekine, Becky; Sivakumaren, Sindhu Carmen; Raben, Daniel; Amzel, L Mario; Devreotes, Peter; Gabelli, Sandra B; Cole, Philip

2013-01-01

The tumor suppressor PIP3 phosphatase PTEN is phosphorylated on four clustered Ser/Thr on its C-terminal tail (aa 380–385) and these phosphorylations are proposed to induce a reduction in PTEN’s plasma membrane recruitment. How these phosphorylations affect the structure and enzymatic function of PTEN is poorly understood. To gain insight into the mechanistic basis of PTEN regulation by phosphorylation, we generated semisynthetic site-specifically tetra-phosphorylated PTEN using expressed protein ligation. By employing a combination of biophysical and enzymatic approaches, we have found that purified tail-phosphorylated PTEN relative to its unphosphorylated counterpart shows reduced catalytic activity and membrane affinity and undergoes conformational compaction likely involving an intramolecular interaction between its C-tail and the C2 domain. Our results suggest that there is a competition between membrane phospholipids and PTEN phospho-tail for binding to the C2 domain. These findings reveal a key aspect of PTEN’s regulation and suggest pharmacologic approaches for direct PTEN activation. DOI: http://dx.doi.org/10.7554/eLife.00691.001 PMID:23853711

Redox-Regulated Pathway of Tyrosine Phosphorylation Underlies NF-κB Induction by an Atypical Pathway Independent of the 26S Proteasome

PubMed Central

Cullen, Sarah; Ponnappan, Subramaniam; Ponnappan, Usha

2015-01-01

Alternative redox stimuli such as pervanadate or hypoxia/reoxygenation, induce transcription factor NF-κB by phospho-tyrosine-dependent and proteasome-independent mechanisms. While considerable attention has been paid to the absence of proteasomal regulation of tyrosine phosphorylated IκBα, there is a paucity of information regarding proteasomal regulation of signaling events distinct from tyrosine phosphorylation of IκBα. To delineate roles for the ubiquitin-proteasome pathway in the phospho-tyrosine dependent mechanism of NF-κB induction, we employed the proteasome inhibitor, Aclacinomycin, and the phosphotyrosine phosphatase inhibitor, pervanadate (PV). Results from these studies demonstrate that phospho-IκBα (Tyr-42) is not subject to proteasomal degradation in a murine stromal epithelial cell line, confirming results previously reported. Correspondingly, proteasome inhibition had no discernable effect on the key signaling intermediaries, Src and ERK1/2, involved in the phospho-tyrosine mechanisms regulating PV-mediated activation of NF-κB. Consistent with previous reports, a significant redox imbalance leading to the activation of tyrosine kinases, as occurs with pervanadate, is required for the induction of NF-κB. Strikingly, our studies demonstrate that proteasome inhibition can potentiate oxidative stress associated with PV-stimulation without impacting kinase activation, however, other cellular implications for this increase in intracellular oxidation remain to be fully delineated. PMID:25671697

The mechanism of signal transduction by two-component systems.

PubMed

Casino, Patricia; Rubio, Vicente; Marina, Alberto

2010-12-01

Two-component systems, composed of a homodimeric histidine kinase (HK) and a response regulator (RR), are major signal transduction devices in bacteria. Typically the signal triggers HK autophosphorylation at one His residue, followed by phosphoryl transfer from the phospho-His to an Asp residue in the RR. Signal extinction frequently involves phospho-RR dephosphorylation by a phosphatase activity of the HK. Our understanding of these reactions and of the determinants of partner specificity among HK-RR couples has been greatly increased by recent crystal structures and biochemical experiments on HK-RR complexes. Cis-autophosphorylation (one subunit phosphorylates itself) occurs in some HKs while trans-autophosphorylation takes place in others. We review and integrate this new information, discuss the mechanism of the three reactions and propose a model for transmembrane signaling by these systems. Copyright © 2010 Elsevier Ltd. All rights reserved.

Advanced glycation end product Nε-carboxymethyllysine induces endothelial cell injury: the involvement of SHP-1-regulated VEGFR-2 dephosphorylation.

PubMed

Liu, Shing Hwa; Sheu, Wayne Huey Herng; Lee, Maw Rong; Lee, Wen Jane; Yi, Yu Chiao; Yang, Tzung Jie; Jen, Jen Fon; Pan, Hung Chuan; Shen, Chin Chang; Chen, Wen Bao; Tien, Hsing Ru; Sheu, Meei Ling

2013-06-01

N(ε)-carboxymethyllysine (CML), a major advanced glycation end product, plays a crucial role in diabetes-induced vascular injury. The roles of protein tyrosine phosphatases and vascular endothelial growth factor (VEGF) receptors in CML-related endothelial cell injury are still unclear. Human umbilical vein endothelial cells (HUVECs) are a commonly used human EC type. Here, we tested the hypothesis that NADPH oxidase/reactive oxygen species (ROS)-mediated SH2 domain-containing tyrosine phosphatase-1 (SHP-1) activation by CML inhibits the VEGF receptor-2 (VEGFR-2, KDR/Flk-1) activation, resulting in HUVEC injury. CML significantly inhibited cell proliferation and induced apoptosis and reduced VEGFR-2 activation in parallel with the increased SHP-1 protein expression and activity in HUVECs. Adding recombinant VEGF increased forward biological effects, which were attenuated by CML. The effects of CML on HUVECs were abolished by SHP-1 siRNA transfection. Exposure of HUVECs to CML also remarkably escalated the integration of SHP-1 with VEGFR-2. Consistently, SHP-1 siRNA transfection and pharmacological inhibitors could block this interaction and elevating [(3)H]thymidine incorporation. CML also markedly activated the NADPH oxidase and ROS production. The CML-increased SHP-1 activity in HUVECs was effectively attenuated by antioxidants. Moreover, the immunohistochemical staining of SHP-1 and CML was increased, but phospho-VEGFR-2 staining was decreased in the aortic endothelium of streptozotocin-induced and high-fat diet-induced diabetic mice. We conclude that a pathway of tyrosine phosphatase SHP-1-regulated VEGFR-2 dephosphorylation through NADPH oxidase-derived ROS is involved in the CML-triggered endothelial cell dysfunction/injury. These findings suggest new insights into the development of therapeutic approaches to reduce diabetic vascular complications. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The mechanism by which MEK/ERK regulates JNK and p38 activity in polyamine depleted IEC-6 cells during apoptosis

PubMed Central

Bavaria, Mitul N.; Jin, Shi; Ray, Ramesh M.; Johnson, Leonard R.

2014-01-01

Polyamine-depletion inhibited apoptosis by activating ERK1/2, while, preventing JNK1/2 activation. MKP-1 knockdown by SiRNA increased ERK1/2, JNK1/2, and p38 phosphorylation and apoptosis. Therefore, we predicted that polyamines might regulate MKP1 via MEK/ERK and thereby apoptosis. We examined the role of MEK/ERK in the regulation of MKP1 and JNK, and p38 activities and apoptosis. Inhibition of MKP-1 activity with a pharmacological inhibitor, sanguinarine (SA), increased JNK1/2, p38, and ERK1/2 activities without causing apoptosis. However, pre-activation of these kinases by SA significantly increased camptothecin (CPT)-induced apoptosis suggesting different roles for MAPKs during survival and apoptosis. Inhibition of MEK1 activity prevented the expression of MKP-1 protein and augmented CPT-induced apoptosis, which correlated with increased activities of JNK1/2, caspases, and DNA fragmentation. Polyamine depleted cells had higher levels of MKP-1 protein and decreased JNK1/2 activity and apoptosis. Inhibition of MEK1 prevented MKP-1 expression and increased JNK1/2 and apoptosis. Phospho-JNK1/2, phospho-ERK2, MKP-1, and the catalytic subunit of protein phosphatase 2A (PP2Ac) formed a complex in response to TNF/CPT. Inactivation of PP2Ac had no effect on the association of MKP-1 and JNK1. However, inhibition of MKP-1 activity decreased the formation of the MKP-1, PP2Ac and JNK complex. Following inhibition by SA, MKP-1 localized in the cytoplasm, while basal and CPT-induced MKP-1 remained in the nuclear fraction. These results suggest that nuclear MKP-1 translocates to the cytoplasm, binds phosphorylated JNK and p38 resulting in dephosphorylation and decreased activity. Thus, MEK/ERK activity controls the levels of MKP-1 and, thereby, regulates JNK activity in polyamine-depleted cells. PMID:24253595

Autophagy Signaling in Prostate Cancer: Identification of a Novel Phosphatase

DTIC Science & Technology

2013-01-01

be attributed to, we measured the activity of wildtype or mutant PTPsigma using a phospho-tyrosine (pTyr) peptide and malachite green free... malachite green (Upstate) and absorbance measured at 650 nm. Background levels from enzyme-only and substrate-only (T yr-P or PtdIns3P) reactions were...peptide at the indicated concentrations for 15 minutes at 37°C and released phosphates measured by malachite green quenching and 650 nm absorbance. (B

Temperature sensitivity of phospho-Ser{sup 473}-PKB/AKT

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

Oehler-Jaenne, Christoph; Bueren, Andre O. von; Vuong, Van

2008-10-24

The phospho-PKB/Akt status is often used as surrogate marker to measure activation of the PI3K/Akt/mTOR signal transduction pathway. Though, inconsistencies of the p-Ser{sup 473}-PKB/Akt status have raised doubts in the validity of p-Ser{sup 473}-PKB/Akt phosphorylation as endpoint. Here, we determined that p-Ser{sup 473}-PKB/Akt but not p-Thr{sup 308}-PKB/Akt phosphorylation is highly temperature sensitive. p-Ser{sup 473}-PKB/Akt phosphorylation was rapidly reduced to levels below 50% on exposure to 20-25 deg. C in murine and human cell lines including cells expressing constitutively active PI3K or lacking PTEN. Down-regulation of p-Ser{sup 473}-PKB/Akt was reversible and re-exposure to physiological temperature resulted in increased p-Ser{sup 473}-PKB/Akt phosphorylationmore » levels. Phosphatase activity at low temperature was sustained at 75% baseline level and phosphatase inhibition prevented p-Ser{sup 473}-PKB/Akt dephosphorylation induced by the low temperature shift. Interestingly temperature-dependent deregulation of the p-Ser{sup 473}-PKB/Akt status was also observed in response to irradiation. Thus our data demonstrate that minimal additional stress factors deregulate the PI3K/Akt-survival pathway and the p-Ser{sup 473}-PKB/Akt status as experimental endpoint.« less

Phospho-control of TGF-β superfamily signaling

PubMed Central

Wrighton, Katharine H; Lin, Xia; Feng, Xin-Hua

2010-01-01

Members of the transforming growth factor-β (TGF-β) family control a broad range of cellular responses in metazoan organisms via autocrine, paracrine, and endocrine modes. Thus, aberrant TGF-β signaling can play a key role in the pathogenesis of several diseases, including cancer. TGF-β signaling pathways are activated by a short phospho-cascade, from receptor phosphorylation to the subsequent phosphorylation and activation of downstream signal transducers called R-Smads. R-Smad phosphorylation state determines Smad complex assembly/disassembly, nuclear import/export, transcriptional activity and stability, and is thus the most critical event in TGF-β signaling. Dephosphorylation of R-Smads by specific phosphatases prevents or terminates TGF-β signaling, highlighting the need to consider Smad (de)phosphorylation as a tightly controlled and dynamic event. This article illustrates the essential roles of reversible phosphorylation in controlling the strength and duration of TGF-β signaling and the ensuing physiological responses. PMID:19114991

Characterization and validation of new tools for measuring site-specific cardiac troponin I phosphorylation.

PubMed

Thoemmes, Stephen F; Stutzke, Crystal A; Du, Yanmei; Browning, Michael D; Buttrick, Peter M; Walker, Lori A

2014-01-31

Phosphorylation of cardiac troponin I is a well established mechanism by which cardiac contractility is modulated. However, there are a number of phosphorylation sites on TnI which contribute singly or in combination to influence cardiac function. Accordingly, methods for accurately measuring site-specific TnI phosphorylation are needed. Currently, two strategies are employed: mass spectrometry, which is costly, difficult and has a low throughput; and Western blotting using phospho-specific antibodies, which is limited by the availability of reagents. In this report, we describe a cohort of new site-specific TnI phosphoantibodies, generated against physiologically relevant phosphorylation sites, that are superior to the current commercially available antibodies: to phospho-serine 22/23 which shows a >5-fold phospho-specificity for phosphorylated TnI; to phospho-serine 43, which has >3-fold phospho-specificity for phosphorylated TnI; and phospho-serine 150 which has >2-fold phospho-specificity for phosphorylated TnI. These new antibodies demonstrated greater sensitivity and specificity for the phosphorylated TnI than the most widely used commercially available reagents. For example, at a protein load of 20 μg of total cardiac extract, a commercially available antibody recognized both phosphorylated and dephosphorylated TnI to the same degree. At the same protein load our phospho-serine 22/23 antibody exhibited no cross-reactivity with dephosphorylated TnI. These new tools should allow a more accurate assessment and a better understanding of the role of TnI phosphorylation in the response of the heart to pathologic stress. Copyright © 2013 Elsevier B.V. All rights reserved.

Blood-Based Detection of Radiation Exposure in Humans Based on Novel Phospho-Smc1 ELISA

PubMed Central

Ivey, Richard G.; Moore, Heather D.; Voytovich, Uliana J.; Thienes, Cortlandt P.; Lorentzen, Travis D.; Pogosova-Agadjanyan, Era L.; Frayo, Shani; Izaguirre, Venissa K.; Lundberg, Sally J.; Hedin, Lacey; Badiozamani, Kas Ray; Hoofnagle, Andrew N.; Stirewalt, Derek L.; Wang, Pei; Georges, George E.; Gopal, Ajay K.; Paulovich, Amanda G.

2011-01-01

The structural maintenance of chromosome 1 (Smc1) protein is a member of the highly conserved cohesin complex and is involved in sister chromatid cohesion. In response to ionizing radiation, Smc1 is phosphorylated at two sites, Ser-957 and Ser-966, and these phosphorylation events are dependent on the ATM protein kinase. In this study, we describe the generation of two novel ELISAs for quantifying phospho-Smc1Ser-957 and phospho-Smc1Ser-966. Using these novel assays, we quantify the kinetic and biodosimetric responses of human cells of hematological origin, including immortalized cells, as well as both quiescent and cycling primary human PBMC. Additionally, we demonstrate a robust in vivo response for phospho-Smc1Ser-957 and phospho-Smc1Ser-966 in lymphocytes of human patients after therapeutic exposure to ionizing radiation, including total-body irradiation, partial-body irradiation, and internal exposure to 131I. These assays are useful for quantifying the DNA damage response in experimental systems and potentially for the identification of individuals exposed to radiation after a radiological incident. PMID:21388270

Phosphorylation-dependent autoinhibition of myosin light chain phosphatase accounts for Ca2+ sensitization force of smooth muscle contraction.

PubMed

Khromov, Alexander; Choudhury, Nandini; Stevenson, Andra S; Somlyo, Avril V; Eto, Masumi

2009-08-07

The reversible regulation of myosin light chain phosphatase (MLCP) in response to agonist stimulation and cAMP/cGMP signals plays an important role in the regulation of smooth muscle (SM) tone. Here, we investigated the mechanism underlying the inhibition of MLCP induced by the phosphorylation of myosin phosphatase targeting subunit (MYPT1), a regulatory subunit of MLCP, at Thr-696 and Thr-853 using glutathione S-transferase (GST)-MYPT1 fragments having the inhibitory phosphorylation sites. GST-MYPT1 fragments, including only Thr-696 and only Thr-853, inhibited purified MLCP (IC(50) = 1.6 and 60 nm, respectively) when they were phosphorylated with RhoA-dependent kinase (ROCK). The activities of isolated catalytic subunits of type 1 and type 2A phosphatases (PP1 and PP2A) were insensitive to either fragment. Phospho-GST-MYPT1 fragments docked directly at the active site of MLCP, and this was blocked by a PP1/PP2A inhibitor microcystin (MC)-LR or by mutation of the active sites in PP1. GST-MYPT1 fragments induced a contraction of beta-escin-permeabilized ileum SM at constant pCa 6.3 (EC(50) = 2 microm), which was eliminated by Ala substitution of the fragment at Thr-696 or by ROCK inhibitors or 8Br-cGMP. GST-MYPT1-(697-880) was 5-times less potent than fragments including Thr-696. Relaxation induced by 8Br-cGMP was not affected by Ala substitution at Ser-695, a known phosphorylation site for protein kinase A/G. Thus, GST-MYPT1 fragments are phosphorylated by ROCK in permeabilized SM and mimic agonist-induced inhibition and cGMP-induced activation of MLCP. We propose a model in which MYPT1 phosphorylation at Thr-696 and Thr-853 causes an autoinhibition of MLCP that accounts for Ca(2+) sensitization of smooth muscle force.

Smad phospho-isoforms direct context-dependent TGF-β signaling.

PubMed

Matsuzaki, Koichi

2013-08-01

Better understanding of TGF-β signaling has deepened our appreciation of normal epithelial cell homeostasis and its dysfunction in such human disorders as cancer and fibrosis. Smad proteins, which convey signals from TGF-β receptors to the nucleus, possess intermediate linker regions connecting Mad homology domains. Membrane-bound, cytoplasmic, and nuclear protein kinases differentially phosphorylate Smad2 and Smad3 to create C-tail (C), the linker (L), or dually (L/C) phosphorylated (p, phospho-) isoforms. According to domain-specific phosphorylation, distinct transcriptional responses, and selective metabolism, Smad phospho-isoform pathways can be grouped into 4 types: cytostatic pSmad3C signaling, mitogenic pSmad3L (Ser-213) signaling, invasive/fibrogenic pSmad2L (Ser-245/250/255)/C or pSmad3L (Ser-204)/C signaling, and mitogenic/migratory pSmad2/3L (Thr-220/179)/C signaling. We outline how responses to TGF-β change through the multiple Smad phospho-isoforms as normal epithelial cells mature from stem cells through progenitors to differentiated cells, and further reflect upon how constitutive Ras-activating mutants favor the Smad phospho-isoform pathway promoting tumor progression. Finally, clinical analyses of reversible Smad phospho-isoform signaling during human carcinogenesis could assess effectiveness of interventions aimed at reducing human cancer risk. Spatiotemporally separate, functionally different Smad phospho-isoforms have been identified in specific cells and tissues, answering long-standing questions about context-dependent TGF-β signaling. Copyright © 2013 Elsevier Ltd. All rights reserved.

Functional human sperm capacitation requires both bicarbonate-dependent PKA activation and down-regulation of Ser/Thr phosphatases by Src family kinases.

PubMed

Battistone, M A; Da Ros, V G; Salicioni, A M; Navarrete, F A; Krapf, D; Visconti, P E; Cuasnicú, P S

2013-09-01

In all mammalian species studied so far, sperm capacitation correlates with an increase in protein tyrosine (Tyr) phosphorylation mediated by a bicarbonate-dependent cAMP/protein kinase A (PKA) pathway. Recent studies in mice revealed, however, that a Src family kinase (SFK)-induced inactivation of serine/threonine (Ser/Thr) phosphatases is also involved in the signaling pathways leading to Tyr phosphorylation. In view of these observations and with the aim of getting a better understanding of the signaling pathways involved in human sperm capacitation, in the present work we investigated the involvement of both the cAMP/PKA and SFK/phosphatase pathways in relation to the capacitation state of the cells. For this purpose, different signaling events and sperm functional parameters were analyzed as a function of capacitation time. Results revealed a very early bicarbonate-dependent activation of PKA indicated by the rapid (1 min) increase in both phospho-PKA substrates and cAMP levels (P < 0.05). However, a complete pattern of Tyr phosphorylation was detected only after 6-h incubation at which time sperm exhibited the ability to undergo the acrosome reaction (AR) and to penetrate zona-free hamster oocytes. Sperm capacitated in the presence of the SFK inhibitor SKI606 showed a decrease in both PKA substrate and Tyr phosphorylation levels, which was overcome by exposure of sperm to the Ser/Thr phosphatase inhibitor okadaic acid (OA). However, OA was unable to induce phosphorylation when sperm were incubated under PKA-inhibitory conditions (i.e. in the absence of bicarbonate or in the presence of PKA inhibitor). Moreover, the increase in PKA activity by exposure to a cAMP analog and a phosphodiesterase inhibitor did not overcome the inhibition produced by SKI606. Whereas the presence of SKI606 during capacitation produced a negative effect (P < 0.05) on sperm motility, progesterone-induced AR and fertilizing ability, none of these inhibitions were observed when sperm were exposed to SKI606 and OA. Interestingly, different concentrations of inhibitors were required to modulate human and mouse capacitation revealing the species specificity of the molecular mechanisms underlying this process. In conclusion, our results describe for the first time the involvement of both PKA activation and Ser/Thr phosphatase down-regulation in functional human sperm capacitation and provide convincing evidence that early PKA-dependent phosphorylation is the convergent regulatory point between these two signaling pathways.

14-3-3 zeta is a molecular target in guggulsterone induced apoptosis in head and neck cancer cells.

PubMed

Macha, Muzafar A; Matta, Ajay; Chauhan, Ss; Siu, Kw Michael; Ralhan, Ranju

2010-11-30

The five-year survival rates for head and neck squamous cell carcinoma (HNSCC) patients are less than 50%, and the prognosis has not improved, despite advancements in standard multi-modality therapies. Hence major emphasis is being laid on identification of novel molecular targets and development of multi-targeted therapies. 14-3-3 zeta, a multifunctional phospho-serine/phospho-threonine binding protein, is emerging as an effector of pro-survival signaling by binding to several proteins involved in apoptosis (Bad, FKHRL1 and ASK1) and may serve as an appropriate target for head and neck cancer therapy. Herein, we determined effect of guggulsterone (GS), a farnesoid X receptor antagonist, on 14-3-3 zeta associated molecular pathways for abrogation of apoptosis in head and neck cancer cells. Head and neck cancer cells were treated with guggulsterone (GS). Effect of GS-treatment was evaluated using cell viability (MTT) assay and apoptosis was verified by annexin V, DNA fragmentation and M30 CytoDeath antibody assay. Mechanism of GS-induced apoptosis was determined by western blotting and co-IP assays using specific antibodies. Using in vitro models of head and neck cancer, we showed 14-3-3 zeta as a key player regulating apoptosis in GS treated SCC4 cells. Treatment with GS releases BAD from the inhibitory action of 14-3-3 zeta in proliferating HNSCC cells by activating protein phosphatase 2A (PP2A). These events initiate the intrinsic mitochondrial pathway of apoptosis, as revealed by increased levels of cytochrome c in cytoplasmic extracts of GS-treated SCC4 cells. In addition, GS treatment significantly reduced the expression of anti-apoptotic proteins, Bcl-2, xIAP, Mcl1, survivin, cyclin D1 and c-myc, thus committing cells to apoptosis. These events were followed by activation of caspase 9, caspase 8 and caspase 3 leading to cleavage of its downstream target, poly-ADP-ribose phosphate (PARP). GS targets 14-3-3 zeta associated cellular pathways for reducing proliferation and inducing apoptosis in head and neck cancer cells, warranting its investigation for use in treatment of head and neck cancer.

Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family.

PubMed

Yu, J S; Chen, W J; Ni, M H; Chan, W H; Yang, S D

1998-08-15

Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both autophosphorylation/activation and protein phosphatase-mediated dephosphorylation/inactivation processes. Taken together, our results identify Thr402 as the regulatory autophosphorylation site of auto-kinase, which is a C-terminal catalytic fragment of PAK2.

Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family.

PubMed Central

Yu, J S; Chen, W J; Ni, M H; Chan, W H; Yang, S D

1998-01-01

Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both autophosphorylation/activation and protein phosphatase-mediated dephosphorylation/inactivation processes. Taken together, our results identify Thr402 as the regulatory autophosphorylation site of auto-kinase, which is a C-terminal catalytic fragment of PAK2. PMID:9693111

T-kininogen inhibits kinin-mediated activation of ERK in endothelial cells.

PubMed

Leiva-Salcedo, Elias; Perez, Viviana; Acuña-Castillo, Claudio; Walter, Robin; Sierra, Felipe

2002-01-01

Serum levels of T-kininogen increase dramatically as rats approach the end of their lifespan. Stable expression of the protein in Balb/c 3T3 fibroblasts leads to a dramatic inhibition of cell proliferation, as well as inhibition of the ERK signaling pathway. T-kininogen is a potent inhibitor of cysteine proteinases, and we have described that the inhibition of ERK activity occurs, at least in part, via stabilization of the MAP kinase phosphatase, MKP-1. Since fibroblasts are not a physiological target of T-kininogen, we have now purified the protein from rat serum, and used it to assess the effect of T-kininogen on endothelial cells. Adding purified T-kininogen to EAhy 926 hybridoma cells resulted in inhibition of basal ERK activity levels, as estimated using appropriate anti-phospho ERK antibodies. Furthermore, exogenously added T-kininogen inhibited the activation of the ERK pathway induced by either bradykinin or T-kinin. We conclude that the age-related increase in hepatic T-kininogen gene expression and serum levels of the protein could have dramatic consequences on endothelial cell physiology, both under steady state conditions, and after activation by cell-specific stimuli. Our results are consistent with T-kininogen being an important modulator of the senescent phenotype in vivo.

AKAP79/150 impacts intrinsic excitability of hippocampal neurons through phospho-regulation of A-type K+ channel trafficking.

PubMed

Lin, Lin; Sun, Wei; Kung, Faith; Dell'Acqua, Mark L; Hoffman, Dax A

2011-01-26

Kv4.2, as the primary α-subunit of rapidly inactivating, A-type voltage-gated K(+) (Kv) channels expressed in hippocampal CA1 pyramidal dendrites, plays a critical role in regulating their excitability. Activity-dependent trafficking of Kv4.2 relies on C-terminal protein kinase A (PKA) phosphorylation. A-kinase-anchoring proteins (AKAPs) target PKA to glutamate receptor and ion channel complexes to allow for discrete, local signaling. As part of a previous study, we showed that AKAP79/150 interacts with Kv4.2 complexes and that the two proteins colocalize in hippocampal neurons. However, the nature and functional consequence of their interaction has not been previously explored. Here, we report that the C-terminal domain of Kv4.2 interacts with an internal region of AKAP79/150 that overlaps with its MAGUK (membrane-associated guanylate kinase)-binding domain. We show that AKAP79/150-anchored PKA activity controls Kv4.2 surface expression in heterologous cells and hippocampal neurons. Consistent with these findings, disrupting PKA anchoring led to a decrease in neuronal excitability, while preventing dephosphorylation by the phosphatase calcineurin resulted in increased excitability. These results demonstrate that AKAP79/150 provides a platform for dynamic PKA regulation of Kv4.2 expression, fundamentally impacting CA1 excitability.

Pathway connectivity and signaling coordination in the yeast stress-activated signaling network

PubMed Central

Chasman, Deborah; Ho, Yi-Hsuan; Berry, David B; Nemec, Corey M; MacGilvray, Matthew E; Hose, James; Merrill, Anna E; Lee, M Violet; Will, Jessica L; Coon, Joshua J; Ansari, Aseem Z; Craven, Mark; Gasch, Audrey P

2014-01-01

Stressed cells coordinate a multi-faceted response spanning many levels of physiology. Yet knowledge of the complete stress-activated regulatory network as well as design principles for signal integration remains incomplete. We developed an experimental and computational approach to integrate available protein interaction data with gene fitness contributions, mutant transcriptome profiles, and phospho-proteome changes in cells responding to salt stress, to infer the salt-responsive signaling network in yeast. The inferred subnetwork presented many novel predictions by implicating new regulators, uncovering unrecognized crosstalk between known pathways, and pointing to previously unknown ‘hubs’ of signal integration. We exploited these predictions to show that Cdc14 phosphatase is a central hub in the network and that modification of RNA polymerase II coordinates induction of stress-defense genes with reduction of growth-related transcripts. We find that the orthologous human network is enriched for cancer-causing genes, underscoring the importance of the subnetwork's predictions in understanding stress biology. PMID:25411400

Enhancement of P53-Mutant Human Colorectal Cancer Cells Radiosensitivity by Flavonoid Fisetin

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

Chen Wenshu; Lee Yijang; Yu Yichu

Purpose: The aim of this study was to investigate whether fisetin is a potential radiosensitizer for human colorectal cancer cells, which are relatively resistant to radiotherapy. Methods and Materials: Cell survival was examined by clonogenic survival assay, and DNA fragmentation was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The effects of treatments on cell cycle distribution and apoptosis were examined by flow cytometry. Western blot analysis was performed to ascertain the protein levels of {gamma}-H2AX, phospho-Chk2, active caspase-3, PARP cleavage, phospho-p38, phospho-AKT, and phospho-ERK1/2. Results: Fisetin pretreatment enhanced the radiosensitivity of p53-mutant HT-29 human colorectal cancer cellsmore » but not human keratocyte HaCaT cells; it also prolonged radiation-induced G{sub 2}/M arrest, enhanced radiation-induced cell growth arrest in HT-29 cells, and suppressed radiation-induced phospho-H2AX (Ser-139) and phospho-Chk2 (Thr-68) in p53-mutant HT-29 cells. Pretreatment with fisetin enhanced radiation-induced caspase-dependent apoptosis in HT-29 cells. Fisetin pretreatment augmented radiation-induced phosphorylation of p38 mitogen-activated protein kinase, which is involved in caspase-mediated apoptosis, and SB202190 significantly reduced apoptosis and radiosensitivity in fisetin-pretreated HT-29 cells. By contrast, both phospho-AKT and phospho-ERK1/2, which are involved in cell proliferation and antiapoptotic pathways, were suppressed after irradiation combined with fisetin pretreatment. Conclusions: To our knowledge, this study is the first to provide evidence that fisetin exerts a radiosensitizing effect in p53-mutant HT-29 cells. Fisetin could potentially be developed as a novel radiosensitizer against radioresistant human cancer cells.« less

Two ancient bacterial-like PPP family phosphatases from Arabidopsis are highly conserved plant proteins that possess unique properties.

PubMed

Uhrig, R Glen; Moorhead, Greg B

2011-12-01

Protein phosphorylation, catalyzed by the opposing actions of protein kinases and phosphatases, is a cornerstone of cellular signaling and regulation. Since their discovery, protein phosphatases have emerged as highly regulated enzymes with specificity that rivals their counteracting kinase partners. However, despite years of focused characterization in mammalian and yeast systems, many protein phosphatases in plants remain poorly or incompletely characterized. Here, we describe a bioinformatic, biochemical, and cellular examination of an ancient, Bacterial-like subclass of the phosphoprotein phosphatase (PPP) family designated the Shewanella-like protein phosphatases (SLP phosphatases). The SLP phosphatase subcluster is highly conserved in all plants, mosses, and green algae, with members also found in select fungi, protists, and bacteria. As in other plant species, the nucleus-encoded Arabidopsis (Arabidopsis thaliana) SLP phosphatases (AtSLP1 and AtSLP2) lack genetic redundancy and phylogenetically cluster into two distinct groups that maintain different subcellular localizations, with SLP1 being chloroplastic and SLP2 being cytosolic. Using heterologously expressed and purified protein, the enzymatic properties of both AtSLP1 and AtSLP2 were examined, revealing unique metal cation preferences in addition to a complete insensitivity to the classic serine/threonine PPP protein phosphatase inhibitors okadaic acid and microcystin. The unique properties and high conservation of the plant SLP phosphatases, coupled to their exclusion from animals, red algae, cyanobacteria, archaea, and most bacteria, render understanding the function(s) of this new subclass of PPP family protein phosphatases of particular interest.

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

PubMed Central

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

2013-01-01

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

Anti-Proliferative Effects of Rutin on OLETF Rat Vascular Smooth Muscle Cells Stimulated by Glucose Variability

PubMed Central

Yu, Sung Hoon; Yu, Jae Myung; Lee, Seong Jin; Kang, Dong Hyun; Cho, Young Jung; Kim, Doo Man

2016-01-01

Purpose Proliferation of vascular smooth muscle cells (VSMCs) plays a crucial role in atherosclerosis. Rutin is a major representative of the flavonol subclass of flavonoids and has various pharmacological activities. Currently, data are lacking regarding its effects on VSMC proliferation induced by intermittent hyperglycemia. Here, we demonstrate the effects of rutin on VSMC proliferation and migration according to fluctuating glucose levels. Materials and Methods Primary cultures of male Otsuka Long-Evans Tokushima Fatty (OLETF) rat VSMCs were obtained from enzymatically dissociated rat thoracic aortas. VSMCs were incubated for 72 h with alternating normal (5.5 mmol/L) and high (25.0 mmol/L) glucose media every 12 h. Proliferation and migration of VSMCs, the proliferative molecular pathway [including p44/42 mitogen-activated protein kinases (MAPK), mitogen-activated protein kinase kinase 1/2 (MEK1/2), p38 MAPK, phosphoinositide 3-kinase (PI3K), c-Jun N-terminal protein kinase (JNK), nuclear factor kappa B (NF-κB), and Akt], the migratory pathway (big MAPK 1, BMK1), reactive oxygen species (ROS), and apoptotic pathway were analyzed. Results We found enhanced proliferation and migration of VSMCs when cells were incubated in intermittent high glucose conditions, compared to normal glucose. These effects were lowered upon rutin treatment. Intermittent treatment with high glucose for 72 h increased the expression of phospho-p44/42 MAPK (extracellular signal regulated kinase 1/2, ERK1/2), phospho-MEK1/2, phospho-PI3K, phospho-NF-κB, phospho-BMK1, and ROS, compared to treatment with normal glucose. These effects were suppressed by rutin. Phospho-p38 MAPK, phospho-Akt, JNK, and apoptotic pathways [B-cell lymphoma (Bcl)-xL, Bcl-2, phospho-Bad, and caspase-3] were not affected by fluctuations in glucose levels. Conclusion Fluctuating glucose levels increased proliferation and migration of OLETF rat VSMCs via MAPK (ERK1/2), BMK1, PI3K, and NF-κB pathways. These effects were inhibited by the antioxidant rutin. PMID:26847289

Chemical Visualization of Phosphoproteomes on Membrane*

PubMed Central

Iliuk, Anton; Liu, X. Shawn; Xue, Liang; Liu, Xiaoqi; Tao, W. Andy

2012-01-01

With new discoveries of important roles of phosphorylation on a daily basis, phospho-specific antibodies, as the primary tool for on-membrane detection of phosphoproteins, face enormous challenges. To address an urgent need for convenient and reliable analysis of phosphorylation events, we report a novel strategy for sensitive phosphorylation analysis in the Western blotting format. The chemical reagent, which we termed pIMAGO, is based on a multifunctionalized soluble nanopolymer and is capable of selectively binding to phosphorylated residues independent of amino acid microenvironment, thus offering great promise as a universal tool in biological analyses where the site of phosphorylation is not known or its specific antibody is not available. The specificity and sensitivity of the approach was first examined using a mixture of standard proteins. The method was then applied to monitor phosphorylation changes in in vitro kinase and phosphatase assays. Finally, to demonstrate the unique ability of pIMAGO to measure endogenous phosphorylation, we used it to visualize and determine the differences in phosphorylated proteins that interact with wild-type and kinase dead mutant of Polo-like kinase 1 during mitosis, the results of which were further confirmed by a quantitative phosphoproteomics experiment. PMID:22593177

[Ischemic postconditioning attenuates ischemia/reperfusion injury in isolated hypertrophied rat heart].

PubMed

Peng, Long-yun; Ma, Hong; He, Jian-gui; Gao, Xiu-ren; Zhang, Yan; He, Xiao-hong; Zhai, Yuan-sheng; Zhang, Xue-jiao

2006-08-01

To explore the effects of ischemic postconditioning on ischemia/reperfusion injury in isolated hypertrophied rat heart and investigate the signal transduction pathway changes induced by ischemia postconditioning. Cardiac hypertrophy was induced in rats by abdominal aortic banding, and isolated hypertrophied rat heart ischemia/reperfusion model was made by Langendorff technique to evaluate the effects of ischemia postconditioning on left ventricular systole pressure, coronary artery flow, creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) release, myocardial infarction size, and the level of myocardial phospho-protein kinase B/Akt (Ser473), phospho-glycogen synthase kinase-3beta (Ser9). Following groups were studied (n = 12 each group): IR, 30 min ischemia (I)/60 min Reperfusion (R); Post: 30 min ischemia, 6 circles of 10 s I/10 s R followed by 60 min R; Post Wort: 30 min ischemia, 6 circles of 10 s I/10 s R, wortmannin (10(-7) mol/L) followed by 60 min R; Wort: 30 min ischemia, wortmannin (10(-7) mol/L) followed by 60 min R. Left ventricular systolic pressure and coronary artery flow were significantly increased, myocardial infarction size and the release of CPK, LDH significantly reduced in Post group compared to that in IR group. Phospho-protein kinase B/Akt (Ser473) and phospho-glycogen synthase kinase-3beta (Ser9) levels were also significantly higher in Post group than that in IR group. Phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin prevented the increase of phospho-protein kinase B/Akt (Ser473) and phospho-glycogen synthase kinase-3beta (Ser9) induced by ischemic postconditioning, but only partly abolished the cardioprotection of ischemic postconditioning. Ischemic postconditioning attenuates ischemia/reperfusion injury in isolated hypertrophied rat heart. The cardioprotective effects of ischemic postconditioning were partly mediated through PI3K/Akt/GSK-3beta signaling pathway.

Glutamate Counteracts Dopamine/PKA Signaling via Dephosphorylation of DARPP-32 Ser-97 and Alteration of Its Cytonuclear Distribution.

PubMed

Nishi, Akinori; Matamales, Miriam; Musante, Veronica; Valjent, Emmanuel; Kuroiwa, Mahomi; Kitahara, Yosuke; Rebholz, Heike; Greengard, Paul; Girault, Jean-Antoine; Nairn, Angus C

2017-01-27

The interaction of glutamate and dopamine in the striatum is heavily dependent on signaling pathways that converge on the regulatory protein DARPP-32. The efficacy of dopamine/D1 receptor/PKA signaling is regulated by DARPP-32 phosphorylated at Thr-34 (the PKA site), a process that inhibits protein phosphatase 1 (PP1) and potentiates PKA action. Activation of dopamine/D1 receptor/PKA signaling also leads to dephosphorylation of DARPP-32 at Ser-97 (the CK2 site), leading to localization of phospho-Thr-34 DARPP-32 in the nucleus where it also inhibits PP1. In this study the role of glutamate in the regulation of DARPP-32 phosphorylation at four major sites was further investigated. Experiments using striatal slices revealed that glutamate decreased the phosphorylation states of DARPP-32 at Ser-97 as well as Thr-34, Thr-75, and Ser-130 by activating NMDA or AMPA receptors in both direct and indirect pathway striatal neurons. The effect of glutamate in decreasing Ser-97 phosphorylation was mediated by activation of PP2A. In vitro phosphatase assays indicated that the PP2A/PR72 heterotrimer complex was likely responsible for glutamate/Ca 2+ -regulated dephosphorylation of DARPP-32 at Ser-97. As a consequence of Ser-97 dephosphorylation, glutamate induced the nuclear localization in cultured striatal neurons of dephospho-Thr-34/dephospho-Ser-97 DARPP-32. It also reduced PKA-dependent DARPP-32 signaling in slices and in vivo Taken together, the results suggest that by inducing dephosphorylation of DARPP-32 at Ser-97 and altering its cytonuclear distribution, glutamate may counteract dopamine/D1 receptor/PKA signaling at multiple cellular levels. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Glutamate Counteracts Dopamine/PKA Signaling via Dephosphorylation of DARPP-32 Ser-97 and Alteration of Its Cytonuclear Distribution*

PubMed Central

Nishi, Akinori; Matamales, Miriam; Musante, Veronica; Valjent, Emmanuel; Kuroiwa, Mahomi; Kitahara, Yosuke; Rebholz, Heike; Greengard, Paul; Girault, Jean-Antoine; Nairn, Angus C.

2017-01-01

The interaction of glutamate and dopamine in the striatum is heavily dependent on signaling pathways that converge on the regulatory protein DARPP-32. The efficacy of dopamine/D1 receptor/PKA signaling is regulated by DARPP-32 phosphorylated at Thr-34 (the PKA site), a process that inhibits protein phosphatase 1 (PP1) and potentiates PKA action. Activation of dopamine/D1 receptor/PKA signaling also leads to dephosphorylation of DARPP-32 at Ser-97 (the CK2 site), leading to localization of phospho-Thr-34 DARPP-32 in the nucleus where it also inhibits PP1. In this study the role of glutamate in the regulation of DARPP-32 phosphorylation at four major sites was further investigated. Experiments using striatal slices revealed that glutamate decreased the phosphorylation states of DARPP-32 at Ser-97 as well as Thr-34, Thr-75, and Ser-130 by activating NMDA or AMPA receptors in both direct and indirect pathway striatal neurons. The effect of glutamate in decreasing Ser-97 phosphorylation was mediated by activation of PP2A. In vitro phosphatase assays indicated that the PP2A/PR72 heterotrimer complex was likely responsible for glutamate/Ca2+-regulated dephosphorylation of DARPP-32 at Ser-97. As a consequence of Ser-97 dephosphorylation, glutamate induced the nuclear localization in cultured striatal neurons of dephospho-Thr-34/dephospho-Ser-97 DARPP-32. It also reduced PKA-dependent DARPP-32 signaling in slices and in vivo. Taken together, the results suggest that by inducing dephosphorylation of DARPP-32 at Ser-97 and altering its cytonuclear distribution, glutamate may counteract dopamine/D1 receptor/PKA signaling at multiple cellular levels. PMID:27998980

Protein tyrosine phosphatase 1B inhibitory activity of lavandulyl flavonoids from roots of Sophora flavescens.

PubMed

Sasaki, Tatsunori; Li, Wei; Higai, Koji; Quang, Tran Hong; Kim, Young Ho; Koike, Kazuo

2014-05-01

Protein tyrosine phosphatase 1B is a non-transmembrane protein tyrosine phosphatase and major negative regulator in insulin signaling cascades, and much attention has been paid to protein tyrosine phosphatase 1B inhibitors as potential therapies for diabetes. The screening of a natural compound library led to the discovery of five lavandulyl flavonoids, which were isolated from the roots of Sophora flavescens, as novel PTP1B inhibitors: kuraridin (1), norkurarinone (2), kurarinone (3), 2'-methoxykurarinone (4), and kushenol T (5). The three most potent compounds, 1, 2, and 4 (IC50 < 30 µM), were demonstrated to be noncompetitive inhibitors of protein tyrosine phosphatase 1B based on a kinetic analysis, and they exhibited different inhibitory selectivities against four homologous protein tyrosine phosphatases (T cell protein tyrosine phosphatase, vaccinia H1-related phosphatase, and Src homology domain 2-containing protein tyrosine phosphatases 1 and 2). Compounds 1, 2, and 4 also exhibited cellular activity in the insulin signaling pathway by increasing the insulin-stimulated Akt phosphorylation level in human hepatocellular liver carcinoma HepG2 cells, suggesting their potential for new anti-insulin-resistant drug developments. Georg Thieme Verlag KG Stuttgart · New York.

Teaching resources. Protein phosphatases.

PubMed

Salton, Stephen R

2005-03-01

This Teaching Resource provides lecture notes and slides for a class covering the structure and function of protein phosphatases and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with a discussion of the importance of phosphatases in physiology, recognized by the award of a Nobel Prize in 1992, and then proceeds to describe the two types of protein phosphatases: serine/threonine and tyrosine phosphatases. The information covered includes the structure, regulation, and substrate specificity of protein phosphatases, with an emphasis on their importance in disease and clinical settings.

Live Cell Imaging Reveals Differential Modifications to Cytoplasmic Dynein Properties by Phospho- and Dephospho-mimic Mutations of the Intermediate Chain 2C S84

PubMed Central

Blasier, Kiev R.; Humsi, Michael K.; Ha, Junghoon; Ross, Mitchell W.; Smiley, W. Russell; Inamdar, Nirja A.; Mitchell, David J.; Lo, Kevin W.-H.; Pfister, K. Kevin

2014-01-01

Cytoplasmic dynein is a multi-subunit motor protein responsible for intracellular cargo transport toward microtubule minus ends. There are multiple isoforms of the dynein intermediate chain (DYNC1I, IC) which is encoded by two genes. One way to regulate cytoplasmic dynein is by IC phosphorylation. The IC-2C isoform is expressed in all cells and the functional significance of phosphorylation on IC-2C serine 84 was investigated using live cell imaging of fluorescent protein-tagged wild type IC-2C (WT) and phospho- and dephospho-mimic mutant isoforms in axonal transport model systems. Both mutations modulated dynein functional properties. The dephospho-mimic mutant IC-2C S84A had greater co-localization with mitochondria than IC-2C wild-type (WT) or the phospho-mimic mutant IC-2C S84D. The dephospho-mimic mutant IC-2C S84A was also more likely to be motile than the phospho-mimic mutant IC-2C S84D or IC-2C WT. In contrast, the phospho-mimic mutant IC-2C S84D mutant was more likely to move in the retrograde direction than was the IC-2C S84A mutant. The phospho-mimic IC-2C S84D was also as likely as IC-2C WT to co-localize with mitochondria. Both the S84D phospho- and S84A, dephospho-mimic mutants were found to be capable of microtubule minus end directed (retrograde) movement in axons. They were also observed to be passively transported in the anterograde direction. These data suggest that the IC-2C S84 has a role in modulating dynein properties. PMID:24798412

Reduction of aggregated Tau in neuronal processes but not in the cell bodies after Abeta42 immunisation in Alzheimer's disease.

PubMed

Boche, Delphine; Donald, Jane; Love, Seth; Harris, Scott; Neal, James W; Holmes, Clive; Nicoll, James A R

2010-07-01

Alzheimer's disease (AD) pathology is characterised by aggregation in the brain of amyloid-beta (Abeta) peptide and hyperphosphorylated tau (phospho-tau), although how these proteins interact in disease pathogenesis is unclear. Abeta immunisation results in removal of Abeta from the brain but cognitive decline continues to progress, possibly due to persistent phospho-tau. We quantified phospho-tau and Abeta42 in the brains of 10 AD patients (iAD) who were actively immunised with Abeta42 (AN1792, Elan Pharmaceuticals) compared with 28 unimmunised AD cases (cAD). The phospho-tau load was lower in the iAD than the cAD group in the cerebral cortex (cAD 1.08% vs. iAD 0.72%, P = 0.048), CA1 hippocampus (cAD 2.26% vs. iAD 1.05%; P = 0.001), subiculum (cAD 1.60% vs. iAD 0.31%; P = 0.001) and entorhinal cortex (cAD 1.10% vs. iAD 0.18%; P < 0.001). Assessment of the localisation within neurons of phospho-tau indicated that the Abeta immunotherapy-associated reduction was confined to neuronal processes, i.e. neuropil threads and dystrophic neurites. However, the phospho-tau accumulation in the neuronal cell bodies, contributing to neurofibrillary tangles, appeared not to be affected. In showing that Abeta immunisation can influence phospho-tau pathology, we confirm the position of Abeta as a target for modifying tau accumulation in AD and demonstrate a link between these proteins. However, the continuing progression of cognitive decline in AD patients after Abeta immunisation may be explained by its lack of apparent effect on tangles.

Disruption of Smad-dependent signaling for growth of GST-P-positive lesions from the early stage in a rat two-stage hepatocarcinogenesis model

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

Ichimura, Ryohei, E-mail: red0828@hotmail.co.j; Mizukami, Sayaka, E-mail: non_sugar_life@hotmail.co.j; Takahashi, Miwa, E-mail: mtakahashi@nihs.go.j

2010-08-01

To clarify the involvement of signaling of transforming growth factor (TGF)-{beta} during the hepatocarcinogenesis, the immunohistochemical distribution of related molecules was analyzed in relation with liver cell lesions expressing glutathione S-transferase placental form (GST-P) during liver tumor promotion by fenbendazole, phenobarbital, piperonyl butoxide, or thioacetamide, using rats. Our study focused on early-stage promotion (6 weeks after starting promotion) and late-stage promotion (57 weeks after starting promotion). With regard to Smad-dependent signaling, cytoplasmic accumulation of phosphorylated Smad (phospho-Smad)-2/3 - identified as Smad3 by later immunoblot analysis - increased in the subpopulation of GST-P{sup +} foci, while Smad4, a nuclear transporter ofmore » Smad2/3, decreased during early-stage promotion. By late-stage promotion, GST-P{sup +} lesions lacking phospho-Smad2/3 had increased in accordance with lesion development from foci to carcinomas, while Smad4 largely disappeared in most proliferative lesions. With regard to Smad-independent mitogen-activated protein kinases, GST-P{sup +} foci that co-expressed phospho-p38 mitogen-activated protein kinase increased during early-stage promotion; however, p38-downstream phospho-activating transcriptional factor (ATF)-2, ATF3, and phospho-c-Myc, were inversely downregulated without relation to promotion. By late-stage promotion, proliferative lesions downregulated phospho-ATF2 and phospho-c-Myc along with lesion development, as with downregulation of phospho-p38 in all lesions. These results suggest that from the early stages, carcinogenic processes were facilitated by disruption of tumor suppressor functions of Smad-dependent signaling, while Smad-independent activation of p38 was an early-stage phenomenon. GST-P{sup -} foci induced by promotion with agonists of peroxisome proliferator-activated receptor-{alpha} did not change Smad expression, suggesting an aberration in the Smad-dependent signaling prerequisites for induction of GST-P{sup +} proliferative lesions.« less

Mutational Analysis of Cell Types in Tuberous Sclerosis Complex (TSC)

DTIC Science & Technology

2009-01-01

from mutations in the TSC1 or TSC2 genes that is associated with epilepsy, cognitive disability, and autism . TSC1/TSC2 gene mutations lead to...gene inactivation and leads to activation of the mTOR cascade as evidenced by phosphorylation of ribosomal S6 protein (P-S6). We demonstrate that...phosphorylation of the ribosomal S6 protein (phospho-S6 or P-S6), a marker for enhanced mTOR signaling. We find P-S6 expression in cortex as well as

Crosstalk between mTORC1 and cAMP Signaling

DTIC Science & Technology

2014-07-01

based genome editing to endogenously tag the V1 subunit and introduce point mutations (T175A; phospho-defective and T175D; phospho-mimetic). By...analysis ap- proach, and the other screened small GTPases using RNAi in Drosophila cells [41,48]. There are four Rag proteins in mammals: RagA and RagB (!98...at the lysosome The Rag proteins lack membrane-targeting sequences , unlike other typical small GTPases such as Rheb. Thus, the Rag–mTORC1 complex is

Exposure to rosiglitazone, a PPAR-γ agonist, in late gestation reduces the abundance of factors regulating cardiac metabolism and cardiomyocyte size in the sheep fetus

PubMed Central

Lie, Shervi; Hui, Melisa; McMillen, I. Caroline; Muhlhausler, Beverly S.; Posterino, Giuseppe S.; Dunn, Stacey L.; Wang, Kimberley C.; Botting, Kimberley J.

2014-01-01

It is unknown whether cardiomyocyte hypertrophy and the transition to fatty acid oxidation as the main source of energy after birth is dependent on the maturation of the cardiomyocytes' metabolic system, or on the limitation of substrate availability before birth. This study aimed to investigate whether intrafetal administration of a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, rosiglitazone, during late gestation can stimulate the expression of factors regulating cardiac growth and metabolism in preparation for birth, and the consequences of cardiac contractility in the fetal sheep at ∼140 days gestation. The mRNA expression and protein abundance of key factors regulating growth and metabolism were quantified using quantitative RT-PCR and Western blot analysis, respectively. Cardiac contractility was determined by measuring the Ca2+ sensitivity and maximum Ca2+-activated force of skinned cardiomyocyte bundles. Rosiglitazone-treated fetuses had a lower cardiac abundance of insulin-signaling molecules, including insulin receptor-β, insulin receptor substrate-1 (IRS-1), phospho-IRS-1 (Tyr-895), phosphatidylinositol 3-kinase (PI3K) regulatory subunit p85, PI3K catalytic subunit p110α, phospho-3-phosphoinositide-dependent protein kinase 1 (Ser-241), protein kinase B (Akt-1), phospho-Akt (Ser-273), PKCζ, phospho-PKCζ(Thr-410), Akt substrate 160 kDa (AS160), phospho-AS160 (Thr-642), and glucose transporter type-4. Additionally, cardiac abundance of regulators of fatty acid β-oxidation, including adiponectin receptor 1, AMPKα, phospho-AMPKα (Thr-172), phospho-acetyl CoA carboxylase (Ser-79), carnitine palmitoyltransferase-1, and PGC-1α was lower in the rosiglitazone-treated group. Rosiglitazone administration also resulted in a decrease in cardiomyocyte size. Rosiglitazone administration in the late-gestation sheep fetus resulted in a decreased abundance of factors regulating cardiac glucose uptake, fatty acid β-oxidation, and cardiomyocyte size. These findings suggest that activation of PPAR-γ using rosiglitazone does not promote the maturation of cardiomyocytes; rather, it may decrease cardiac metabolism and compromise cardiac health later in life. PMID:24477540

Exposure to rosiglitazone, a PPAR-γ agonist, in late gestation reduces the abundance of factors regulating cardiac metabolism and cardiomyocyte size in the sheep fetus.

PubMed

Lie, Shervi; Hui, Melisa; McMillen, I Caroline; Muhlhausler, Beverly S; Posterino, Giuseppe S; Dunn, Stacey L; Wang, Kimberley C; Botting, Kimberley J; Morrison, Janna L

2014-03-15

It is unknown whether cardiomyocyte hypertrophy and the transition to fatty acid oxidation as the main source of energy after birth is dependent on the maturation of the cardiomyocytes' metabolic system, or on the limitation of substrate availability before birth. This study aimed to investigate whether intrafetal administration of a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, rosiglitazone, during late gestation can stimulate the expression of factors regulating cardiac growth and metabolism in preparation for birth, and the consequences of cardiac contractility in the fetal sheep at ∼140 days gestation. The mRNA expression and protein abundance of key factors regulating growth and metabolism were quantified using quantitative RT-PCR and Western blot analysis, respectively. Cardiac contractility was determined by measuring the Ca(2+) sensitivity and maximum Ca(2+)-activated force of skinned cardiomyocyte bundles. Rosiglitazone-treated fetuses had a lower cardiac abundance of insulin-signaling molecules, including insulin receptor-β, insulin receptor substrate-1 (IRS-1), phospho-IRS-1 (Tyr-895), phosphatidylinositol 3-kinase (PI3K) regulatory subunit p85, PI3K catalytic subunit p110α, phospho-3-phosphoinositide-dependent protein kinase 1 (Ser-241), protein kinase B (Akt-1), phospho-Akt (Ser-273), PKCζ, phospho-PKCζ(Thr-410), Akt substrate 160 kDa (AS160), phospho-AS160 (Thr-642), and glucose transporter type-4. Additionally, cardiac abundance of regulators of fatty acid β-oxidation, including adiponectin receptor 1, AMPKα, phospho-AMPKα (Thr-172), phospho-acetyl CoA carboxylase (Ser-79), carnitine palmitoyltransferase-1, and PGC-1α was lower in the rosiglitazone-treated group. Rosiglitazone administration also resulted in a decrease in cardiomyocyte size. Rosiglitazone administration in the late-gestation sheep fetus resulted in a decreased abundance of factors regulating cardiac glucose uptake, fatty acid β-oxidation, and cardiomyocyte size. These findings suggest that activation of PPAR-γ using rosiglitazone does not promote the maturation of cardiomyocytes; rather, it may decrease cardiac metabolism and compromise cardiac health later in life.

Molecular basis for TPR domain-mediated regulation of protein phosphatase 5.

PubMed

Yang, Jing; Roe, S Mark; Cliff, Matthew J; Williams, Mark A; Ladbury, John E; Cohen, Patricia T W; Barford, David

2005-01-12

Protein phosphatase 5 (Ppp5) is a serine/threonine protein phosphatase comprising a regulatory tetratricopeptide repeat (TPR) domain N-terminal to its phosphatase domain. Ppp5 functions in signalling pathways that control cellular responses to stress, glucocorticoids and DNA damage. Its phosphatase activity is suppressed by an autoinhibited conformation maintained by the TPR domain and a C-terminal subdomain. By interacting with the TPR domain, heat shock protein 90 (Hsp90) and fatty acids including arachidonic acid stimulate phosphatase activity. Here, we describe the structure of the autoinhibited state of Ppp5, revealing mechanisms of TPR-mediated phosphatase inhibition and Hsp90- and arachidonic acid-induced stimulation of phosphatase activity. The TPR domain engages with the catalytic channel of the phosphatase domain, restricting access to the catalytic site. This autoinhibited conformation of Ppp5 is stabilised by the C-terminal alphaJ helix that contacts a region of the Hsp90-binding groove on the TPR domain. Hsp90 activates Ppp5 by disrupting TPR-phosphatase domain interactions, permitting substrate access to the constitutively active phosphatase domain, whereas arachidonic acid prompts an alternate conformation of the TPR domain, destabilising the TPR-phosphatase domain interface.

Expression and prognostic relevance of MET and phospho-BAD in non-small cell lung cancer.

PubMed

Sun, Wenze; Ai, Ting; Gao, Ying; Zhang, Yingbing; Cui, Jie; Song, Liping

2013-01-01

MET is involved in the progression of several types of human cancers, while phospho-BAD(Ser-136) is a key molecule in apoptosis and might be regulated by MET. The aim of this study was to investigate the correlation between altered expression of MET and phospho-BAD in non-small cell lung cancer (NSCLC) and their association with clinicopathologic parameters and overall survival. MET and phospho-BAD(Ser-136) proteins were evaluated by immunohistochemical analysis in 183 paraffin-embedded specimens and were also assessed by Western blotting analysis in 12 frozen tumor tissue samples, which were representative examples of immunohistochemical staining. Positive expression of MET and phospho-BAD(Ser-136) occurred in 67.2% and 49.2% of the 183 cases of NSCLC, respectively. However, neither MET expression nor phospho-BAD(Ser-136) expression was associated with any clinicopathologic parameter. A significant correlation was found between MET and phospho-BAD(Ser-136) expression levels evaluated by immunohistochemistry (r = 0.268, P < 0.001). Overexpression of MET was significantly associated with shortened overall survival in univariate analysis (P < 0.001). Moreover, patients with a MET+/phospho-BAD(Ser-136)+ phenotype had a poorer prognosis than others (P < 0.001). Multivariate Cox proportional hazard analysis confirmed that MET expression is a prognostic factor for NSCLC. MET expression might be correlated with phospho-BAD(Ser-136) expression, and may be an adverse predictor for NSCLC. Activation of the MET/phospho-BAD(Ser-136) signaling pathway might play a role in the development and progression of NSCLC.

Discovery of a protein phosphatase activity encoded in the genome of bacteriophage lambda. Probable identity with open reading frame 221.

PubMed

Cohen, P T; Cohen, P

1989-06-15

Infection of Escherichia coli with phage lambda gt10 resulted in the appearance of a protein phosphatase with activity towards 32P-labelled casein. Activity reached a maximum near the point of cell lysis and declined thereafter. The phosphatase was stimulated 30-fold by Mn2+, while Mg2+ and Ca2+ were much less effective. Activity was unaffected by inhibitors 1 and 2, okadaic acid, calmodulin and trifluoperazine, distinguishing it from the major serine/threonine-specific protein phosphatases of eukaryotic cells. The lambda phosphatase was also capable of dephosphorylating other substrates in the presence of Mn2+, although activity towards 32P-labelled phosphorylase was 10-fold lower, and activity towards phosphorylase kinase and glycogen synthase 25 50-fold lower than with casein. No casein phosphatase activity was present in either uninfected cells, or in E. coli infected with phage lambda gt11. Since lambda gt11 lacks part of the open reading frame (orf) 221, previously shown to encode a protein with sequence similarity to protein phosphatase-1 and protein phosphatase-2A of mammalian cells [Cohen, Collins, Coulson, Berndt & da Cruz e Silva (1988) Gene 69, 131-134], the results indicate that ORF221 is the protein phosphatase detected in cells infected with lambda gt10. Comparison of the sequence of ORF221 with other mammalian protein phosphatases defines three highly conserved regions which are likely to be essential for function. The first of these is deleted in lambda gt11.

Phospho-selective mechanisms of arrestin conformations and functions revealed by unnatural amino acid incorporation and 19F-NMR

PubMed Central

Yang, Fan; Yu, Xiao; Liu, Chuan; Qu, Chang-Xiu; Gong, Zheng; Liu, Hong-Da; Li, Fa-Hui; Wang, Hong-Mei; He, Dong-Fang; Yi, Fan; Song, Chen; Tian, Chang-Lin; Xiao, Kun-Hong; Wang, Jiang-Yun; Sun, Jin-Peng

2015-01-01

Specific arrestin conformations are coupled to distinct downstream effectors, which underlie the functions of many G-protein-coupled receptors (GPCRs). Here, using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance (19F-NMR) spectroscopy, we demonstrate that distinct receptor phospho-barcodes are translated to specific β-arrestin-1 conformations and direct selective signalling. With its phosphate-binding concave surface, β-arrestin-1 ‘reads' the message in the receptor phospho-C-tails and distinct phospho-interaction patterns are revealed by 19F-NMR. Whereas all functional phosphopeptides interact with a common phosphate binding site and induce the movements of finger and middle loops, different phospho-interaction patterns induce distinct structural states of β-arrestin-1 that are coupled to distinct arrestin functions. Only clathrin recognizes and stabilizes GRK2-specific β-arrestin-1 conformations. The identified receptor-phospho-selective mechanism for arrestin conformation and the spacing of the multiple phosphate-binding sites in the arrestin enable arrestin to recognize plethora phosphorylation states of numerous GPCRs, contributing to the functional diversity of receptors. PMID:26347956

Network inference reveals novel connections in pathways regulating growth and defense in the yeast salt response.

PubMed

MacGilvray, Matthew E; Shishkova, Evgenia; Chasman, Deborah; Place, Michael; Gitter, Anthony; Coon, Joshua J; Gasch, Audrey P

2018-05-01

Cells respond to stressful conditions by coordinating a complex, multi-faceted response that spans many levels of physiology. Much of the response is coordinated by changes in protein phosphorylation. Although the regulators of transcriptome changes during stress are well characterized in Saccharomyces cerevisiae, the upstream regulatory network controlling protein phosphorylation is less well dissected. Here, we developed a computational approach to infer the signaling network that regulates phosphorylation changes in response to salt stress. We developed an approach to link predicted regulators to groups of likely co-regulated phospho-peptides responding to stress, thereby creating new edges in a background protein interaction network. We then use integer linear programming (ILP) to integrate wild type and mutant phospho-proteomic data and predict the network controlling stress-activated phospho-proteomic changes. The network we inferred predicted new regulatory connections between stress-activated and growth-regulating pathways and suggested mechanisms coordinating metabolism, cell-cycle progression, and growth during stress. We confirmed several network predictions with co-immunoprecipitations coupled with mass-spectrometry protein identification and mutant phospho-proteomic analysis. Results show that the cAMP-phosphodiesterase Pde2 physically interacts with many stress-regulated transcription factors targeted by PKA, and that reduced phosphorylation of those factors during stress requires the Rck2 kinase that we show physically interacts with Pde2. Together, our work shows how a high-quality computational network model can facilitate discovery of new pathway interactions during osmotic stress.

Pleiotropic effects of mutations involved in the regulation of Escherichia coli K-12 alkaline phosphatase.

PubMed

Morris, H; Schlesinger, M J; Bracha, M; Yagil, E

1974-08-01

Induction of alkaline phosphatase in wild-type Escherichia coli K-12 leads to the appearance of three new proteins in addition to alkaline phosphatase in the periplasmic space of the bacteria. These proteins are detected in autoradiograms of sodium dodecyl sulfate-acrylamide gel electropherograms of extracts from cells labeled with [(35)S]methionine. Studies with constitutive mutants defective in the three genes phoS, phoT, and phoR that have been shown to regulate alkaline phosphatase synthesis indicate that the three periplasmic proteins are coregulated with alkaline phosphatase. A mutant that has a deletion in the alkaline phosphatase structural gene phoA produces the three proteins, but a newly discovered mutant phoB that has a defect in the expression of alkaline phosphatase fails to produce the three proteins. phoB mutants are shown here to be unable to make detectable amounts of alkaline phosphatase polypeptides, as measured by immunoprecipitins or acrylamide gel electropherograms. On the basis of these results we suggest a new model for the regulation of alkaline phosphatase biosynthesis. In this model, a ternary complex composed of phoB(+) and phoR(+) gene products and an internal metabolite functions as a positive control element to regulate the transcription of several cistrons coding for periplasmic proteins.

Identification of a dual-specificity protein phosphatase that inactivates a MAP kinase from Arabidopsis

NASA Technical Reports Server (NTRS)

Gupta, R.; Huang, Y.; Kieber, J.; Luan, S.; Evans, M. L. (Principal Investigator)

1998-01-01

Mitogen-activated protein kinases (MAPKs) play a key role in plant responses to stress and pathogens. Activation and inactivation of MAPKs involve phosphorylation and dephosphorylation on both threonine and tyrosine residues in the kinase domain. Here we report the identification of an Arabidopsis gene encoding a dual-specificity protein phosphatase capable of hydrolysing both phosphoserine/threonine and phosphotyrosine in protein substrates. This enzyme, designated AtDsPTP1 (Arabidopsis thaliana dual-specificity protein tyrosine phosphatase), dephosphorylated and inactivated AtMPK4, a MAPK member from the same plant. Replacement of a highly conserved cysteine by serine abolished phosphatase activity of AtDsPTP1, indicating a conserved catalytic mechanism of dual-specificity protein phosphatases from all eukaryotes.

Stereochemical course of the reactions catalyzed by the bacterial phosphoenolpyruvate: Mannitol phosphotransferase system

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

Mueller, E.G.; Knowles, J.R.; Khandekar, S.S.

1990-07-24

The authors have determined the overall stereochemical course of the reactions leading to the phosphorylation of D-mannitol by mannitol-specific enzyme II (EII{sup Mtl}) of the Escherichia coli phosphoenolpyruvate- (PEP) dependent phosphotransferase system (PTS). In the presence of enzyme I and HPr of the PTS, and of membranes containing EII{sup Mtl}, the phospho group from ((R)-{sup 16}O, {sup 17}O, {sup 18}O)PEP was transferred to D-mannitol to form mannitol 1-phosphate with overall inversion of the configuration at phosphorus with respect to that of PEP. Since in the course of these reactions enzyme I and HPr are each covalently phosphorylated at a singlemore » site and inversion of the chiral phospho group from PEP indicates an odd number of transfer steps overall, transfer from phospho-HPr to mannitol via EII{sup Mtl} must also occur in an odd number of steps. Taken together with the fact that catalytically important phospho-EII{sup Mtl} intermediates have been demonstrated biochemically, the results imply that EII{sup Mtl} is sequentially phosphorylated at two different sites during phospho transfer from phospho-HPr to mannitol. This conclusion is consistent with the available evidence on phospho-EII{sup Mtl} intermediates and in particular with the recent report that two different phospho peptides can be isolated from the fully phosphorylated protein.« less

Evidence for a latent form of protein phosphatase 1 associated with cardiac myofibrils.

PubMed

Schlender, K K; Wang, W; Wilson, S E

1989-02-28

Detergent-purified myofibrils from bovine heart contained very little spontaneously active protein phosphatase 1 activity. Phosphatase 1, extracted from the myofibrils by freeze-thawing in the presence of 500 mM KCl, was markedly activated by cobalt/trypsin treatment. Myofibril phosphatase 1 was separated from phosphatase 2A by chromatography on heparin-Sepharose. The phosphatase 1 was isolated in a latent form. Pretreatment with trypsin released free catalytic subunit and increased activity about 25-fold. Addition of cobalt with the trypsin increased activity another 2-fold. The latent myofibril phosphatase 1 did not appear to be the same as previously characterized forms of protein phosphatase 1. We suggest that cardiac myofibril phosphatase 1 contains a unique inhibitory subunit which directs the enzyme to the myofibril and regulates the dephosphorylation of myofibril phosphoproteins.

Gender-Specific Potential Inhibitory Role of Ca2+/Calmodulin Dependent Protein Kinase Phosphatase (CaMKP) in Pressure-Overloaded Mouse Heart

PubMed Central

Prévilon, Miresta; Pezet, Mylène; Vinet, Laurent; Mercadier, Jean-Jacques; Rouet-Benzineb, Patricia

2014-01-01

Background Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP) has been proposed as a potent regulator of multifunctional Ca2+/calmodulin-dependent protein kinases (i.e., CaMKII). The CaMKII-dependent activation of myocyte enhancer factor 2 (MEF2) disrupts interactions between MEF2-histone deacetylases (HDACs), thereby de-repressing downstream gene transcription. Whether CaMKP modulates the CaMKII- MEF2 pathway in the heart is unknown. Here, we investigated the molecular and functional consequences of left ventricular (LV) pressure overload in the mouse of both genders, and in particular we evaluated the expression levels and localization of CaMKP and its association with CaMKII-MEF2 signaling. Methodology and Principal Findings Five week-old B6D1/F1 mice of both genders underwent a sham-operation or thoracic aortic constriction (TAC). Thirty days later, TAC was associated with pathological LV hypertrophy characterized by systolic and diastolic dysfunction. Gene expression was assessed by real-time PCR. Fetal gene program re-expression comprised increased RNA levels of brain natriuretic peptide and alpha-skeletal actin. Mouse hearts of both genders expressed both CaMKP transcript and protein. Activation of signalling pathways was studied by Western blot in LV lysates or subcellular fractions (nuclear and cytoplasmic). TAC was associated with increased CaMKP expression in male LVs whereas it tended to be decreased in females. The DNA binding activity of MEF2 was determined by spectrophotometry. CaMKP compartmentalization differed according to gender. In male TAC mice, nuclear CaMKP was associated with inactive CaMKII resulting in less MEF2 activation. In female TAC mice, active CaMKII (phospho-CaMKII) detected in the nuclear fraction, was associated with a strong MEF2 transcription factor-binding activity. Conclusions/Significance Gender-specific CaMKP compartmentalization is associated with CaMKII-mediated MEF2 activation in pressure-overloaded hearts. Therefore, CaMKP could be considered as an important novel cellular target for the development of new therapeutic strategies for heart diseases. PMID:24608696

Gender-specific potential inhibitory role of Ca2+/calmodulin dependent protein kinase phosphatase (CaMKP) in pressure-overloaded mouse heart.

PubMed

Prévilon, Miresta; Pezet, Mylène; Vinet, Laurent; Mercadier, Jean-Jacques; Rouet-Benzineb, Patricia

2014-01-01

Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP) has been proposed as a potent regulator of multifunctional Ca2+/calmodulin-dependent protein kinases (i.e., CaMKII). The CaMKII-dependent activation of myocyte enhancer factor 2 (MEF2) disrupts interactions between MEF2-histone deacetylases (HDACs), thereby de-repressing downstream gene transcription. Whether CaMKP modulates the CaMKII- MEF2 pathway in the heart is unknown. Here, we investigated the molecular and functional consequences of left ventricular (LV) pressure overload in the mouse of both genders, and in particular we evaluated the expression levels and localization of CaMKP and its association with CaMKII-MEF2 signaling. Five week-old B6D1/F1 mice of both genders underwent a sham-operation or thoracic aortic constriction (TAC). Thirty days later, TAC was associated with pathological LV hypertrophy characterized by systolic and diastolic dysfunction. Gene expression was assessed by real-time PCR. Fetal gene program re-expression comprised increased RNA levels of brain natriuretic peptide and alpha-skeletal actin. Mouse hearts of both genders expressed both CaMKP transcript and protein. Activation of signalling pathways was studied by Western blot in LV lysates or subcellular fractions (nuclear and cytoplasmic). TAC was associated with increased CaMKP expression in male LVs whereas it tended to be decreased in females. The DNA binding activity of MEF2 was determined by spectrophotometry. CaMKP compartmentalization differed according to gender. In male TAC mice, nuclear CaMKP was associated with inactive CaMKII resulting in less MEF2 activation. In female TAC mice, active CaMKII (phospho-CaMKII) detected in the nuclear fraction, was associated with a strong MEF2 transcription factor-binding activity. Gender-specific CaMKP compartmentalization is associated with CaMKII-mediated MEF2 activation in pressure-overloaded hearts. Therefore, CaMKP could be considered as an important novel cellular target for the development of new therapeutic strategies for heart diseases.

Phospho1 deficiency transiently modifies bone architecture yet produces consistent modification in osteocyte differentiation and vascular porosity with ageing

PubMed Central

Javaheri, B.; Carriero, A.; Staines, K.A.; Chang, Y.-M.; Houston, D.A.; Oldknow, K.J.; Millan, J.L.; Kazeruni, Bassir N.; Salmon, P.; Shefelbine, S.; Farquharson, C.; Pitsillides, A.A.

2015-01-01

PHOSPHO1 is one of principal proteins involved in initiating bone matrix mineralisation. Recent studies have found that Phospho1 KO mice (Phospho1-R74X) display multiple skeletal abnormalities with spontaneous fractures, bowed long bones, osteomalacia and scoliosis. These analyses have however been limited to young mice and it remains unclear whether the role of PHOSPHO1 is conserved in the mature murine skeleton where bone turnover is limited. In this study, we have used ex-vivo computerised tomography to examine the effect of Phospho1 deletion on tibial bone architecture in mice at a range of ages (5, 7, 16 and 34 weeks of age) to establish whether its role is conserved during skeletal growth and maturation. Matrix mineralisation has also been reported to influence terminal osteoblast differentiation into osteocytes and we have also explored whether hypomineralised bones in Phospho1 KO mice exhibit modified osteocyte lacunar and vascular porosity. Our data reveal that Phospho1 deficiency generates age-related defects in trabecular architecture and compromised cortical microarchitecture with greater porosity accompanied by marked alterations in osteocyte shape, significant increases in osteocytic lacuna and vessel number. Our in vitro studies examining the behaviour of osteoblast derived from Phospho1 KO and wild-type mice reveal reduced levels of matrix mineralisation and modified osteocytogenic programming in cells deficient in PHOSPHO1. Together our data suggest that deficiency in PHOSPHO1 exerts modifications in bone architecture that are transient and depend upon age, yet produces consistent modification in lacunar and vascular porosity. It is possible that the inhibitory role of PHOSPHO1 on osteocyte differentiation leads to these age-related changes in bone architecture. It is also intriguing to note that this apparent acceleration in osteocyte differentiation evident in the hypomineralised bones of Phospho1 KO mice suggests an uncoupling of the interplay between osteocytogenesis and biomineralisation. Further studies are required to dissect the molecular processes underlying the regulatory influences exerted by PHOSPHO1 on the skeleton with ageing. PMID:26232374

Spermine oxidase promotes bile canalicular lumen formation through acrolein production.

PubMed

Uemura, Takeshi; Takasaka, Tomokazu; Igarashi, Kazuei; Ikegaya, Hiroshi

2017-11-01

Spermine oxidase (SMOX) catalyzes oxidation of spermine to generate spermidine, hydrogen peroxide (H 2 O 2 ) and 3-aminopropanal, which is spontaneously converted to acrolein. SMOX is induced by a variety of stimuli including bacterial infection, polyamine analogues and acetaldehyde exposure. However, the physiological functions of SMOX are not yet fully understood. We investigated the physiological role of SMOX in liver cells using human hepatocellular carcinoma cell line HepG2. SMOX localized to the bile canalicular lumen, as determined by F-actin staining. Knockdown of SMOX reduced the formation of bile canalicular lumen. We also found that phospho-Akt (phosphorylated protein kinase B) was localized to canalicular lumen. Treatment with Akt inhibitor significantly reduced the formation of bile canalicular lumen. Acrolein scavenger also inhibited the formation of bile canalicular lumen. PTEN, phosphatase and tensin homolog and an inhibitor of Akt, was alkylated in a SMOX-dependent manner. Our results suggest that SMOX plays a central role in the formation of bile canalicular lumen in liver cells by activating Akt pathway through acrolein production.

Protein phosphatase 2A in stretch-induced endothelial cell proliferation

NASA Technical Reports Server (NTRS)

Murata, K.; Mills, I.; Sumpio, B. E.

1996-01-01

We previously proposed that activation of protein kinase C is a key mechanism for control of cell growth enhanced by cyclic strain [Rosales and Sumpio (1992): Surgery 112:459-466]. Here we examined protein phosphatase 1 and 2A activity in bovine aortic endothelial cells exposed to cyclic stain. Protein phosphatase 2A activity in the cytosol was decreased by 36.1% in response to cyclic strain for 60 min, whereas the activity in the membrane did not change. Treatment with low concentration (0.1 nM) of okadaic acid enhanced proliferation of both static and stretched endothelial cells in 10% fetal bovine serum. These data suggest that protein phosphatase 2A acts as a growth suppressor and cyclic strain may enhance cellular proliferation by inhibiting protein phosphatase 2A as well as stimulating protein kinase C.

Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho-protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma.

PubMed

Li, Congfen; Takahashi, Chikara; Zhang, Liangxuan; Huseni, Mahrukh; Stankovich, Basha; Mashhedi, Haider; Lee, Joanna; French, Dorothy; Anderson, Jeff Eastham; Kim, Doris; Howell, Kathy; Brauer, Matthew J; Kowanetz, Marcin; Yan, Yibing; Humke, Eric; Ebens, Allen; Hampton, Garret; Lackner, Mark R; Hegde, Priti; Jia, Shidong

2013-03-23

The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients. We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application. The phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells. We developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials.

Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho-protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma

PubMed Central

2013-01-01

Background The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients. Methods We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application. Results The phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells. Conclusions We developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials. PMID:23522020

Mangiferin, a Natural Xanthone, Protects Murine Liver in Pb(II) Induced Hepatic Damage and Cell Death via MAP Kinase, NF-κB and Mitochondria Dependent Pathways

PubMed Central

Pal, Pabitra Bikash; Sinha, Krishnendu; Sil, Parames C.

2013-01-01

One of the most well-known naturally occurring environmental heavy metals, lead (Pb) has been reported to cause liver injury and cellular apoptosis by disturbing the prooxidant-antioxidant balance via oxidative stress. Several studies, on the other hand, reported that mangiferin, a naturally occurring xanthone, has been used for a broad range of therapeutic purposes. In the present study, we, therefore, investigated the molecular mechanisms of the protective action of mangiferin against lead-induced hepatic pathophysiology. Lead [Pb(II)] in the form of Pb(NO3)2 (at a dose of 5 mg/kg body weight, 6 days, orally) induced oxidative stress, hepatic dysfunction and cell death in murine liver. Post treatment of mangiferin at a dose of 100 mg/kg body weight (6 days, orally), on the other hand, diminished the formation of reactive oxygen species (ROS) and reduced the levels of serum marker enzymes [alanine aminotranferase (ALT) and alkaline phosphatase (ALP)]. Mangiferin also reduced Pb(II) induced alterations in antioxidant machineries, restored the mitochondrial membrane potential as well as mutual regulation of Bcl-2/Bax. Furthermore, mangiferin inhibited Pb(II)-induced activation of mitogen-activated protein kinases (MAPKs) (phospho-ERK 1/2, phosphor-JNK phospho- p38), nuclear translocation of NF-κB and apoptotic cell death as was evidenced by DNA fragmentation, FACS analysis and histological assessment. In vitro studies using hepatocytes as the working model also showed the protective effect of mangiferin in Pb(II) induced cytotoxicity. All these beneficial effects of mangiferin contributes to the considerable reduction of apoptotic hepatic cell death induced by Pb(II). Overall results demonstrate that mangiferin exhibit both antioxidative and antiapoptotic properties and protects the organ in Pb(II) induced hepatic dysfunction. PMID:23451106

Purification and characterization of a phosphotyrosyl-protein phosphatase from wheat seedlings.

PubMed

Cheng, H F; Tao, M

1989-10-19

A neutral phosphatase which catalyzes the hydrolysis of p-nitrophenylphosphate has been purified to homogeneity from wheat seedlings. The enzyme is a monomeric glycoprotein exhibiting a molecular weight of 35,000, frictional ratio of 1.22, Stokes' radius of 260 nm, and sedimentation coefficient of 3.2 S. That the enzyme is a glycoprotein is surmised from its chromatographic property on Concanavalin A-Sepharose column. An examination of the substrate specificity indicates that the enzyme exhibits a preference for phosphotyrosine over a number of phosphocompounds, including p-nitrophenylphosphate and several glycolytic intermediates. Both phosphoserine and phosphothreonine are not hydrolyzed by the enzyme. The phosphatase activity is not affected by high concentrations of chelating agents and does not require metal ions. Molybdate, orthovanadate, Zn2+, and Hg2+ are all potent inhibitors of the phosphatase activity. The ability of the phosphatase to dephosphorylate protein phosphotyrosine has been investigated. [32P-Tyr]poly(Glu,Tyr)n, [32P-Tyr]alkylated bovine serum albumin, [32P-Tyr]angiotensin-I, and [32P-Tyr]band 3 (from human erythrocyte) are all substrates of the phosphatase. On the other hand, the enzyme has no activity toward protein phosphoserine and phosphothreonine. Our result further indicates that the neutral phosphatase is distinct from the wheat germ acid phosphatase. The latter enzyme is found to dephosphorylate phosphotyrosyl as well as phosphoseryl and phosphothreonyl groups in proteins. In light of the many similarities in properties to phosphotyrosyl protein phosphatases isolated from several sources, it is suggested that the wheat seedling phosphatase may participate in cellular regulation involving protein tyrosine phosphorylation.

Moraxella catarrhalis synthesizes an autotransporter that is an acid phosphatase.

PubMed

Hoopman, Todd C; Wang, Wei; Brautigam, Chad A; Sedillo, Jennifer L; Reilly, Thomas J; Hansen, Eric J

2008-02-01

Moraxella catarrhalis O35E was shown to synthesize a 105-kDa protein that has similarity to both acid phosphatases and autotransporters. The N-terminal portion of the M. catarrhalis acid phosphatase A (MapA) was most similar (the BLAST probability score was 10(-10)) to bacterial class A nonspecific acid phosphatases. The central region of the MapA protein had similarity to passenger domains of other autotransporter proteins, whereas the C-terminal portion of MapA resembled the translocation domain of conventional autotransporters. Cloning and expression of the M. catarrhalis mapA gene in Escherichia coli confirmed the presence of acid phosphatase activity in the MapA protein. The MapA protein was shown to be localized to the outer membrane of M. catarrhalis and was not detected either in the soluble cytoplasmic fraction from disrupted M. catarrhalis cells or in the spent culture supernatant fluid from M. catarrhalis. Use of the predicted MapA translocation domain in a fusion construct with the passenger domain from another predicted M. catarrhalis autotransporter confirmed the translocation ability of this MapA domain. Inactivation of the mapA gene in M. catarrhalis strain O35E reduced the acid phosphatase activity expressed by this organism, and this mutation could be complemented in trans with the wild-type mapA gene. Nucleotide sequence analysis of the mapA gene from six M. catarrhalis strains showed that this protein was highly conserved among strains of this pathogen. Site-directed mutagenesis of a critical histidine residue (H233A) in the predicted active site of the acid phosphatase domain in MapA eliminated acid phosphatase activity in the recombinant MapA protein. This is the first description of an autotransporter protein that expresses acid phosphatase activity.

Processing of alkaline phosphatase precursor to the mature enzyme by an Escherichia coli inner membrane preparation.

PubMed Central

Chang, C N; Inouye, H; Model, P; Beckwith, J

1980-01-01

An inner membrane preparation co-translationally cleaved both the alkaline phosphatase and bacteriophage f1 coat protein precursors to the mature proteins. Post-translational outer membrane proteolysis of pre-alkaline phosphatase generated a protein smaller than the authentic monomer. Images PMID:6991486

Arabidopsis PPP family of serine/threonine protein phosphatases: many targets but few engines.

PubMed

Uhrig, R Glen; Labandera, Anne-Marie; Moorhead, Greg B

2013-09-01

The major plant serine/threonine protein phosphatases belong to the phosphoprotein phosphatase (PPP) family. Over the past few years the complement of Arabidopsis thaliana PPP family of catalytic subunits has been cataloged and many regulatory subunits identified. Specific roles for PPPs have been characterized, including roles in auxin and brassinosteroid signaling, in phototropism, in regulating the target of rapamycin pathway, and in cell stress responses. In this review, we provide a framework for understanding the PPP family by exploring the fundamental role of the phosphatase regulatory subunits that drive catalytic engine specificity. Although there are fewer plant protein phosphatases compared with their protein kinase partners, their function is now recognized to be as dynamic and as regulated as that of protein kinases. Copyright © 2013 Elsevier Ltd. All rights reserved.

A Kinome RNAi Screen in Drosophila Identifies Novel Genes Interacting with Lgl, aPKC, and Crb Cell Polarity Genes in Epithelial Tissues.

PubMed

Parsons, Linda M; Grzeschik, Nicola A; Amaratunga, Kasun; Burke, Peter; Quinn, Leonie M; Richardson, Helena E

2017-08-07

In both Drosophila melanogaster and mammalian systems, epithelial structure and underlying cell polarity are essential for proper tissue morphogenesis and organ growth. Cell polarity interfaces with multiple cellular processes that are regulated by the phosphorylation status of large protein networks. To gain insight into the molecular mechanisms that coordinate cell polarity with tissue growth, we screened a boutique collection of RNAi stocks targeting the kinome for their capacity to modify Drosophila "cell polarity" eye and wing phenotypes. Initially, we identified kinase or phosphatase genes whose depletion modified adult eye phenotypes associated with the manipulation of cell polarity complexes (via overexpression of Crb or aPKC). We next conducted a secondary screen to test whether these cell polarity modifiers altered tissue overgrowth associated with depletion of Lgl in the wing. These screens identified Hippo, Jun kinase (JNK), and Notch signaling pathways, previously linked to cell polarity regulation of tissue growth. Furthermore, novel pathways not previously connected to cell polarity regulation of tissue growth were identified, including Wingless (Wg/Wnt), Ras, and lipid/Phospho-inositol-3-kinase (PI3K) signaling pathways. Additionally, we demonstrated that the "nutrient sensing" kinases Salt Inducible Kinase 2 and 3 ( SIK2 and 3 ) are potent modifiers of cell polarity phenotypes and regulators of tissue growth. Overall, our screen has revealed novel cell polarity-interacting kinases and phosphatases that affect tissue growth, providing a platform for investigating molecular mechanisms coordinating cell polarity and tissue growth during development. Copyright © 2017 Parsons et al.

Alkaline phosphatase protein increases in response to prednisolone in HeLa cells.

PubMed Central

Hanford, W C; Kottel, R H; Fishman, W H

1981-01-01

Quantification of term-placental alkaline phosphatase isoenzyme protein in HeLa TCRC-1 cells grown in the presence and absence of prednisolone indicates that there is a net increase in amount of enzyme-specific protein in prednisolone-stimulated cells. In a similar analysis of HeLa D98AH2 cells, prednisolone treatment causes the appearance of term-placental alkaline phosphatase protein and the loss of the intestinal isoenzyme protein. These results support the interpretation that the response of these cells to corticosteroids is the net accumulation of alkaline phosphatase protein rather than the modification of pre-existing enzyme to a more active state. Images Fig. 1. Fig. 2. PMID:7340849

Legionella pneumophila effector WipA, a bacterial PPP protein phosphatase with PTP activity.

PubMed

Jia, Qian; Lin, Yun; Gou, Xuejing; He, Lei; Shen, Dong; Chen, Dongni; Xie, Wei; Lu, Yongjun

2018-04-26

The gram-negative bacterium Legionella pneumophila invades human's lung and causes Legionnaires' disease. To benefit its survival and replication in cellular milieu, L. pneumophila secrets at least 330 effector proteins into host cells. We found that the effector WipA has the protein tyrosine phosphatase (PTP) activity but does not depend on the classical CX5R motif for activity, suggesting that WipA is an unconventional PTP. Meanwhile, the presence of three other highly conserved motifs typically seen in protein serine/threonine phosphatases and the poor inhibition of WipA activity by okadaic acid led us to propose that WipA is a bacterial protein phosphatase. In addition, the determination of the 2.55-Å crystal structure of WipA revealed that WipA resembles cold-active protein tyrosine phosphatase (CAPTPase), and therefore very likely shares the same catalytic mechanism.

Structural Genomics of Protein Phosphatases

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

Almo,S.; Bonanno, J.; Sauder, J.

The New York SGX Research Center for Structural Genomics (NYSGXRC) of the NIGMS Protein Structure Initiative (PSI) has applied its high-throughput X-ray crystallographic structure determination platform to systematic studies of all human protein phosphatases and protein phosphatases from biomedically-relevant pathogens. To date, the NYSGXRC has determined structures of 21 distinct protein phosphatases: 14 from human, 2 from mouse, 2 from the pathogen Toxoplasma gondii, 1 from Trypanosoma brucei, the parasite responsible for African sleeping sickness, and 2 from the principal mosquito vector of malaria in Africa, Anopheles gambiae. These structures provide insights into both normal and pathophysiologic processes, including transcriptionalmore » regulation, regulation of major signaling pathways, neural development, and type 1 diabetes. In conjunction with the contributions of other international structural genomics consortia, these efforts promise to provide an unprecedented database and materials repository for structure-guided experimental and computational discovery of inhibitors for all classes of protein phosphatases.« less

Protein phosphatase and kinase activities possibly involved in exocytosis regulation in Paramecium tetraurelia.

PubMed Central

Kissmehl, R; Treptau, T; Hofer, H W; Plattner, H

1996-01-01

In Paramecium tetraurelia cells synchronous exocytosis induced by aminoethyldextran (AED) is accompanied by an equally rapid dephosphorylation of a 63 kDa phosphoprotein (PP63) within 80 ms. In vivo, rephosphorylation occurs within a few seconds after AED triggering. In homogenates (P)P63 can be solubilized in all three phosphorylation states (phosphorylated, dephosphorylated and rephosphorylated) and thus tested in vitro. By using chelators of different divalent cations, de- and rephosphorylation of PP63 and P63 respectively can be achieved by an endogenous protein phosphatase/kinase system. Dephosphorylation occurs in the presence of EDTA, whereas in the presence of EGTA this was concealed by phosphorylation by endogenous kinase(s), thus indicating that phosphorylation of P63 is calcium-independent. Results obtained with protein phosphatase inhibitors (okadaic acid, calyculin A) allowed us to exclude a protein serine/threonine phosphatase of type I (with selective sensitivity in Paramecium). Protein phosphatase 2C is also less likely to be a candidate because of its requirement for high Mg2+ concentrations. According to previous evidence a protein serine/threonine phosphatase of type 2B (calcineurin; CaN) is possibly involved. We have now found that bovine brain CaN dephosphorylates PP63 in vitro. Taking into account the specific requirements of this phosphatase in vitro, with p-nitrophenyl phosphate as a substrate, we have isolated a cytosolic phosphatase of similar characteristics by combined preparative gel electrophoresis and affinity-column chromatography. In Paramecium this phosphatase also dephosphorylates PP63 in vitro (after 32P labelling in vivo). Using various combinations of ion exchange, affinity and hydrophobic interaction chromatography we have also isolated three different protein kinases from the soluble fraction, i.e. a cAMP-dependent protein kinase (PKA), a cGMP-dependent protein kinase (PKG) and a casein kinase. Among the kinases tested, PKA cannot phosphorylate P63, whereas either PKG or the casein kinase phosphorylate P63 in vitro. On the basis of these findings we propose that a protein phosphatase/kinase system is involved in the regulation of exocytosis in P. tetraurelia cells. PMID:8694788

Protein tyrosine phosphatases as potential therapeutic targets

PubMed Central

He, Rong-jun; Yu, Zhi-hong; Zhang, Ruo-yu; Zhang, Zhong-yin

2014-01-01

Protein tyrosine phosphorylation is a key regulatory process in virtually all aspects of cellular functions. Dysregulation of protein tyrosine phosphorylation is a major cause of human diseases, such as cancers, diabetes, autoimmune disorders, and neurological diseases. Indeed, protein tyrosine phosphorylation-mediated signaling events offer ample therapeutic targets, and drug discovery efforts to date have brought over two dozen kinase inhibitors to the clinic. Accordingly, protein tyrosine phosphatases (PTPs) are considered next-generation drug targets. For instance, PTP1B is a well-known targets of type 2 diabetes and obesity, and recent studies indicate that it is also a promising target for breast cancer. SHP2 is a bona-fide oncoprotein, mutations of which cause juvenile myelomonocytic leukemia, acute myeloid leukemia, and solid tumors. In addition, LYP is strongly associated with type 1 diabetes and many other autoimmune diseases. This review summarizes recent findings on several highly recognized PTP family drug targets, including PTP1B, Src homology phosphotyrosyl phosphatase 2(SHP2), lymphoid-specific tyrosine phosphatase (LYP), CD45, Fas associated phosphatase-1 (FAP-1), striatal enriched tyrosine phosphatases (STEP), mitogen-activated protein kinase/dual-specificity phosphatase 1 (MKP-1), phosphatases of regenerating liver-1 (PRL), low molecular weight PTPs (LMWPTP), and CDC25. Given that there are over 100 family members, we hope this review will serve as a road map for innovative drug discovery targeting PTPs. PMID:25220640

Tumor suppression effects of bilberry extracts and enzymatically modified isoquercitrin in early preneoplastic liver cell lesions induced by piperonyl butoxide promotion in a two-stage rat hepatocarcinogenesis model.

PubMed

Hara, Shintaro; Morita, Reiko; Ogawa, Takashi; Segawa, Risa; Takimoto, Norifumi; Suzuki, Kazuhiko; Hamadate, Naobumi; Hayashi, Shim-Mo; Odachi, Ayano; Ogiwara, Isao; Shibusawa, Sakae; Yoshida, Toshinori; Shibutani, Makoto

2014-08-01

To investigate the protective effect of bilberry extracts (BBE) and enzymatically modified isoquercitrin (EMIQ) on the hepatocarcinogenic process involving oxidative stress responses, we used a two-stage hepatocarcinogenesis model in N-diethylnitrosamine-initiated and piperonyl butoxide (PBO)-promoted rats. We examined the modifying effect of co-administration with BBE or EMIQ on the liver tissue environment including oxidative stress responses, cell proliferation and apoptosis, and phosphatase and tensin homolog (PTEN)/Akt and transforming growth factor (TGF)-β/Smad signalings on the induction mechanism of preneoplastic lesions during early stages of hepatocellular tumor promotion. PBO increased the numbers and area of glutathione S-transferase placental form (GST-P)(+) liver cell foci and the numbers of Ki-67(+) proliferating cells within GST-P(+) foci. Co-administration of BBE or EMIQ suppressed these effects with the reductions of GST-P(+) foci (area) to 48.9-49.4% and Ki-67(+) cells to 55.5-61.4% of the PBO-promoted cases. Neither BBE nor EMIQ decreased microsomal reactive oxygen species induced by PBO. However, only EMIQ suppressed the level of thiobarbituric acid-reactive substances to 78.4% of the PBO-promoted cases. PBO increased the incidences of phospho-PTEN(-) foci, phospho-Akt substrate(+) foci, phospho-Smad3(-) foci and Smad4(-) foci in GST-P(+) foci. Both BBE and EMIQ decreased the incidences of phospho-PTEN(-) foci in GST-P(+) foci to 59.8-72.2% and Smad4(-) foci to 62.4-71.5% of the PBO-promoted cases, and BBE also suppressed the incidence of phospho-Akt substrate(+) foci in GST-P(+) foci to 75.2-75.7% of the PBO-promoted cases. These results suggest that PBO-induced tumor promotion involves facilitation of PTEN/Akt and disruptive TGF-β/Smad signalings without relation to oxidative stress responses, but this promotion was suppressed by co-treatment with BBE or EMIQ through suppression of cell proliferation activity of preneoplastic liver cells. Copyright © 2014 Elsevier GmbH. All rights reserved.

Understanding the Function of EZH2 as a Determinant of Breast Cancer Invasion and Metastasis

DTIC Science & Technology

2013-04-01

and heat shock protein 27 ( HSP27 ) in breast cancer cell lines when compared to controls (Fig. 3a & Supplementary Fig. 2a). No significant effect on...inhibit the ability of activated p-p38 to phosphorylate downstream targets, such as HSP27 , had no effect on EZH2, SUZ12, EED, or H3K27me3 protein levels...3042), Snail1 (#3879); rabbit polyclonals p38 (#9212), p38a (#9218), p38c (#2307), phospho- HSP27 (Ser82, #2401), phospho-MAPKAPK-2 (#3007); mouse

Both Intrinsic Substrate Preference and Network Context Contribute to Substrate Selection of Classical Tyrosine Phosphatases*

PubMed Central

Tinti, Michele; Paoluzi, Serena; Santonico, Elena; Masch, Antonia; Schutkowski, Mike

2017-01-01

Reversible tyrosine phosphorylation is a widespread post-translational modification mechanism underlying cell physiology. Thus, understanding the mechanisms responsible for substrate selection by kinases and phosphatases is central to our ability to model signal transduction at a system level. Classical protein-tyrosine phosphatases can exhibit substrate specificity in vivo by combining intrinsic enzymatic specificity with the network of protein-protein interactions, which positions the enzymes in close proximity to their substrates. Here we use a high throughput approach, based on high density phosphopeptide chips, to determine the in vitro substrate preference of 16 members of the protein-tyrosine phosphatase family. This approach helped identify one residue in the substrate binding pocket of the phosphatase domain that confers specificity for phosphopeptides in a specific sequence context. We also present a Bayesian model that combines intrinsic enzymatic specificity and interaction information in the context of the human protein interaction network to infer new phosphatase substrates at the proteome level. PMID:28159843

Interrogating Protein Phosphatases with Chemical Activity Probes.

PubMed

Casey, Garrett R; Stains, Cliff I

2018-06-04

Protein phosphatases, while long overlooked, have recently become appreciated as drivers of both normal- and disease-associated signaling events. As a result, the spotlight is now turning torwards this enzyme family and efforts geared towards the development of modern chemical tools for studying these enzymes are well underway. This Minireview focuses on the evolution of chemical activity probes, both optical and covalent, for the study of protein phosphatases. Small-molecule probes, global monitoring of phosphatase activity through the use of covalent modifiers, and targeted fluorescence-based activity probes are discussed. We conclude with an overview of open questions in the field and highlight the potential impact of chemical tools for studying protein phosphatases. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Global phosphorylation analysis of beta-arrestin-mediated signaling downstream of a seven transmembrane receptor (7TMR).

PubMed

Xiao, Kunhong; Sun, Jinpeng; Kim, Jihee; Rajagopal, Sudarshan; Zhai, Bo; Villén, Judit; Haas, Wilhelm; Kovacs, Jeffrey J; Shukla, Arun K; Hara, Makoto R; Hernandez, Marylens; Lachmann, Alexander; Zhao, Shan; Lin, Yuan; Cheng, Yishan; Mizuno, Kensaku; Ma'ayan, Avi; Gygi, Steven P; Lefkowitz, Robert J

2010-08-24

beta-Arrestin-mediated signaling downstream of seven transmembrane receptors (7TMRs) is a relatively new paradigm for signaling by these receptors. We examined changes in protein phosphorylation occurring when HEK293 cells expressing the angiotensin II type 1A receptor (AT1aR) were stimulated with the beta-arrestin-biased ligand Sar(1), Ile(4), Ile(8)-angiotensin (SII), a ligand previously found to signal through beta-arrestin-dependent, G protein-independent mechanisms. Using a phospho-antibody array containing 46 antibodies against signaling molecules, we found that phosphorylation of 35 proteins increased upon SII stimulation. These SII-mediated phosphorylation events were abrogated after depletion of beta-arrestin 2 through siRNA-mediated knockdown. We also performed an MS-based quantitative phosphoproteome analysis after SII stimulation using a strategy of stable isotope labeling of amino acids in cell culture (SILAC). We identified 1,555 phosphoproteins (4,552 unique phosphopeptides), of which 171 proteins (222 phosphopeptides) showed increased phosphorylation, and 53 (66 phosphopeptides) showed decreased phosphorylation upon SII stimulation of the AT1aR. This study identified 38 protein kinases and three phosphatases whose phosphorylation status changed upon SII treatment. Using computational approaches, we performed system-based analyses examining the beta-arrestin-mediated phosphoproteome including construction of a kinase-substrate network for beta-arrestin-mediated AT1aR signaling. Our analysis demonstrates that beta-arrestin-dependent signaling processes are more diverse than previously appreciated. Notably, our analysis identifies an AT1aR-mediated cytoskeletal reorganization network whereby beta-arrestin regulates phosphorylation of several key proteins, including cofilin and slingshot. This study provides a system-based view of beta-arrestin-mediated phosphorylation events downstream of a 7TMR and opens avenues for research in a rapidly evolving area of 7TMR signaling.

Analysis of Substrates of Protein Kinase C Isoforms in Human Breast Cells By The Traceable Kinase Method

PubMed Central

Chen, Xiangyu; Zhao, Xin; Abeyweera, Thushara P.; Rotenberg, Susan A.

2012-01-01

A previous report (Biochemistry 46: 2364–2370, 2007) described the application of The Traceable Kinase Method to identify substrates of PKCα in non-transformed human breast MCF-10A cells. Here, a non-radioactive variation of this method compared the phospho-protein profiles of three traceable PKC isoforms (α, δ and ζ) for the purpose of identifying novel, isoform-selective substrates. Each FLAG-tagged traceable kinase was expressed and co-immunoprecipitated along with high affinity substrates. The isolated kinase and its associated substrates were subjected to an in vitro phosphorylation reaction with traceable kinase-specific N6-phenyl-ATP, and the resulting phospho-proteins were analyzed by Western blot with an antibody that recognizes the phosphorylated PKC consensus site. Phospho-protein profiles generated by PKC-α and -δ were similar and differed markedly from that of PKC-ζ. Mass spectrometry of selected bands revealed known PKC substrates and several potential substrates that included the small GTPase-associated effector protein Cdc42 effector protein-4 (CEP4). Of those potential substrates tested, only CEP4 was phosphorylated by pure PKC-α, –δ, and −ζ isoforms in vitro, and by endogenous PKC isoforms in MCF-10A cells treated with DAG-lactone, a membrane permeable PKC activator. Under these conditions, the stoichiometry of CEP4 phosphorylation was 3.2 ± 0.5 (mol phospho-CEP4/mol CEP4). Following knock-down with isoform-specific shRNA-encoding plasmids, phosphorylation of CEP4 was substantially decreased in response to silencing of each of the three isoforms (PKC–α, –δ, or –ζ), whereas testing of kinase-dead mutants supported a role for only PKC-α and –δ in CEP4 phosphorylation. These findings identify CEP4 as a novel intracellular PKC substrate that is phosphorylated by multiple PKC isoforms. PMID:22897107

[Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells].

PubMed

Sheremet, Ia A; Emets, A I; Azmi, A; Vissenberg, K; Verbelen, J-P; Blium, Ia B

2012-01-01

In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.

Molecular characterization of a tyrosine-specific protein phosphatase encoded by a stress-responsive gene in Arabidopsis.

PubMed Central

Xu, Q; Fu, H H; Gupta, R; Luan, S

1998-01-01

Protein tyrosine kinases and phosphatases play a vital role in the regulation of cell growth and differentiation in animal systems. However, none of these enzymes has been characterized from higher plants. In this study, we isolated a cDNA encoding a putative protein tyrosine phosphatase (PTPase) from Arabidopsis (referred to as AtPTP1). The expression level of AtPTP1 is highly sensitive to environmental stresses. High-salt conditions increased AtPTP1 mRNA levels, whereas cold treatment rapidly eliminated the AtPTP1 transcript. The recombinant AtPTP1 protein specifically hydrolyzed phosphotyrosine, but not phosphoserine/threonine, in protein substrates. Site-directed mutagenesis defined two highly conserved amino acids, cysteine-265 and aspartate-234, as being essential for the phosphatase activity of the AtPTP1 protein, suggesting a common catalytic mechanism for PTPases from all eukaryotic systems. In summary, we have identified AtPTP1 as a tyrosine-specific protein phosphatase that may function in stress responses of higher plants. PMID:9596642

Purification and characterization of two wheat-embryo protein phosphatases.

PubMed

Polya, G M; Haritou, M

1988-04-15

Two protein phosphatases (enzymes I and II) were extensively purified from wheat embryo by a procedure involving chromatography on DEAE-cellulose, phenyl-Sepharose CL-4B, DEAE-Sephacel and Ultrogel AcA 44. Preparations of enzyme I (Mr 197,000) are heterogeneous. Preparations of enzyme II (Mr 35,000) contain only one major polypeptide (Mr 17,500), which exactly co-purifies with protein phosphatase II on gel filtration and is not present in preparations of enzyme I. However, this major polypeptide has been identified as calmodulin. Calmodulin and protein phosphatase II can be separated by further chromatography on phenyl-Sepharose CL-4B. Protein phosphatases I and II do not require Mg2+ or Ca2+ for activity. Both enzymes catalyse the dephosphorylation of phosphohistone H1 (phosphorylated by wheat-germ Ca2+-dependent protein kinase) and of phosphocasein (phosphorylated by wheat-germ Ca2+-independent casein kinase), but neither enzyme dephosphorylates a range of non-protein phosphomonoesters tested. Both enzymes are inhibited by Zn2+, Hg2+, vanadate, molybdate, F-, pyrophosphate and ATP.

Inhibitors of the Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase family (CaMKP and CaMKP-N)

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

Sueyoshi, Noriyuki; Takao, Toshihiko; Nimura, Takaki

2007-11-23

Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear isoform CaMKP-N are unique Ser/Thr protein phosphatases that negatively regulate the Ca{sup 2+}/calmodulin-dependent protein kinase (CaMK) cascade by dephosphorylating multifunctional CaMKI, II, and IV. However, the lack of specific inhibitors of these phosphatases has hampered studies on these enzymes in vivo. In an attempt to obtain specific inhibitors, we searched inhibitory compounds and found that Evans Blue and Chicago Sky Blue 6B served as effective inhibitors for CaMKP. These compounds also inhibited CaMKP-N, but inhibited neither protein phosphatase 2C, another member of PPM family phosphatase, nor calcineurin, a typical PPP familymore » phosphatase. The minimum structure required for the inhibition was 1-amino-8-naphthol-4-sulfonic acid. When Neuro2a cells cotransfected with CaMKIV and CaMKP-N were treated with these compounds, the dephosphorylation of CaMKIV was strongly suppressed, suggesting that these compounds could be used as potent inhibitors of CaMKP and CaMKP-N in vivo as well as in vitro.« less

Dephosphorylation of microtubule-binding sites at the neurofilament-H tail domain by alkaline, acid, and protein phosphatases.

PubMed

Hisanaga, S; Yasugawa, S; Yamakawa, T; Miyamoto, E; Ikebe, M; Uchiyama, M; Kishimoto, T

1993-06-01

The dephosphorylation-induced interaction of neurofilaments (NFs) with microtubules (MTs) was investigated by using several phosphatases. Escherichia coli alkaline and wheat germ acid phosphatases increased the electrophoretic mobility of NF-H and NF-M by dephosphorylation, and induced the binding of NF-H to MTs. The binding of NFs to MTs was observed only after the electrophoretic mobility of NF-H approached the exhaustively dephosphorylated level when alkaline phosphatase was used. The number of phosphate remaining when NF-H began to bind to MTs was estimated by measuring phosphate bound to NF-H. NF-H did not bind to MTs even when about 40 phosphates from the total of 51 had been removed by alkaline phosphatase. The removal of 6 further phosphates finally resulted in the association of NF-H with MTs. A similar finding, that the restricted phosphorylation sites in the NF-H tail domain, but not the total amount of phosphates, were important for binding to MTs, was also obtained with acid phosphatases. In contrast to alkaline and acid phosphatases, four classes of protein phosphatases (protein phosphatases 1, 2A, 2B, and 2C) were ineffective for shifting the electrophoretic mobility of NF proteins and for inducing the association of NFs to MTs.

Transcriptional responses to cantharidin a protein phosphatase inhibitor in Arabidopsis thaliana reveal the involvement of multiple signal transduction pathways

USDA-ARS?s Scientific Manuscript database

Cantharidin is a natural compound isolated from the blister beetle (Epicauta spp.). It is a very potent inhibitor of serine/threonine protein phosphatases PPP, especially PP2A and PP4. Protein phosphatases and kinases maintain a sensitive balance between phosphorylated and dephosphorylated forms of ...

Moraxella catarrhalis Synthesizes an Autotransporter That Is an Acid Phosphatase▿

PubMed Central

Hoopman, Todd C.; Wang, Wei; Brautigam, Chad A.; Sedillo, Jennifer L.; Reilly, Thomas J.; Hansen, Eric J.

2008-01-01

Moraxella catarrhalis O35E was shown to synthesize a 105-kDa protein that has similarity to both acid phosphatases and autotransporters. The N-terminal portion of the M. catarrhalis acid phosphatase A (MapA) was most similar (the BLAST probability score was 10−10) to bacterial class A nonspecific acid phosphatases. The central region of the MapA protein had similarity to passenger domains of other autotransporter proteins, whereas the C-terminal portion of MapA resembled the translocation domain of conventional autotransporters. Cloning and expression of the M. catarrhalis mapA gene in Escherichia coli confirmed the presence of acid phosphatase activity in the MapA protein. The MapA protein was shown to be localized to the outer membrane of M. catarrhalis and was not detected either in the soluble cytoplasmic fraction from disrupted M. catarrhalis cells or in the spent culture supernatant fluid from M. catarrhalis. Use of the predicted MapA translocation domain in a fusion construct with the passenger domain from another predicted M. catarrhalis autotransporter confirmed the translocation ability of this MapA domain. Inactivation of the mapA gene in M. catarrhalis strain O35E reduced the acid phosphatase activity expressed by this organism, and this mutation could be complemented in trans with the wild-type mapA gene. Nucleotide sequence analysis of the mapA gene from six M. catarrhalis strains showed that this protein was highly conserved among strains of this pathogen. Site-directed mutagenesis of a critical histidine residue (H233A) in the predicted active site of the acid phosphatase domain in MapA eliminated acid phosphatase activity in the recombinant MapA protein. This is the first description of an autotransporter protein that expresses acid phosphatase activity. PMID:18065547

The effect of PKA-mediated phosphorylation of ryanodine receptor on SR Ca2+ leak in ventricular myocytes.

PubMed

Bovo, Elisa; Huke, Sabine; Blatter, Lothar A; Zima, Aleksey V

2017-03-01

Functional impact of cardiac ryanodine receptor (type 2 RyR or RyR2) phosphorylation by protein kinase A (PKA) remains highly controversial. In this study, we characterized a functional link between PKA-mediated RyR2 phosphorylation level and sarcoplasmic reticulum (SR) Ca 2+ release and leak in permeabilized rabbit ventricular myocytes. Changes in cytosolic [Ca 2+ ] and intra-SR [Ca 2+ ] SR were measured with Fluo-4 and Fluo-5N, respectively. Changes in RyR2 phosphorylation at two PKA sites, serine-2031 and -2809, were measured with phospho-specific antibodies. cAMP (10μM) increased Ca 2+ spark frequency approximately two-fold. This effect was associated with an increase in SR Ca 2+ load from 0.84 to 1.24mM. PKA inhibitory peptide (PKI; 10μM) abolished the cAMP-dependent increase of SR Ca 2+ load and spark frequency. When SERCA was completely blocked by thapsigargin, cAMP did not affect RyR2-mediated Ca 2+ leak. The lack of a cAMP effect on RyR2 function can be explained by almost maximal phosphorylation of RyR2 at serine-2809 after sarcolemma permeabilization. This high RyR2 phosphorylation level is likely the consequence of a balance shift between protein kinase and phosphatase activity after permeabilization. When RyR2 phosphorylation at serine-2809 was reduced to its "basal" level (i.e. RyR2 phosphorylation level in intact myocytes) using kinase inhibitor staurosporine, SR Ca 2+ leak was significantly reduced. Surprisingly, further dephosphorylation of RyR2 with protein phosphatase 1 (PP1) markedly increased SR Ca 2+ leak. At the same time, phosphorylation of RyR2 at serine 2031 did not significantly change under identical experimental conditions. These results suggest that RyR2 phosphorylation by PKA has a complex effect on SR Ca 2+ leak in ventricular myocytes. At an intermediate level of RyR2 phosphorylation SR Ca 2+ leak is minimal. However, complete dephosphorylation and maximal phosphorylation of RyR2 increases SR Ca 2+ leak. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed Central

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

2007-01-01

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

Role of IKK-alpha in the EGFR Signaling Regulation

DTIC Science & Technology

2014-09-01

Rho family GTPase (VAV2), and Phospholipase C (PLC) (4). These signaling activities regulate cell proliferation, mobility , and differentiation in...correlation coefficient r=0.63, pɘ.05) was found, suggesting that high IKK promotes EGFR phosphorylation in breast cancer cells (Fig. 3G ...phosphorylation (Figure 2E). Since protein phosphorylation sometimes results in a mobility shift in SDS-PAGE, we used a phospho-tag to capture phospho

ERK Oscillation-Dependent Gene Expression Patterns and Deregulation by Stress-Response

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

Waters, Katrina M.; Cummings, Brian S.; Shankaran, Harish

2014-09-15

Studies were undertaken to determine whether ERK oscillations regulate a unique subset of genes in human keratinocytes and subsequently, whether the p38 stress response inhibits ERK oscillations. A DNA microarray identified many genes that were unique to ERK oscillations, and network reconstruction predicted an important role for the mediator complex subunit 1 (MED1) node in mediating ERK oscillation-dependent gene expression. Increased ERK-dependent phosphorylation of MED1 was observed in oscillating cells compared to non-oscillating counterparts as validation. Treatment of keratinocytes with a p38 inhibitor (SB203580) increased ERK oscillation amplitudes and MED1 and phospho-MED1 protein levels. Bromate is a probable human carcinogenmore » that activates p38. Bromate inhibited ERK oscillations in human keratinocytes and JB6 cells and induced an increase in phospho-p38 and decrease in phospho-MED1 protein levels. Treatment of normal rat kidney cells and primary salivary gland epithelial cells with bromate decreased phospho-MED1 levels in a reversible fashion upon treatment with p38 inhibitors (SB202190; SB203580). Our results indicate that oscillatory behavior in the ERK pathway alters homeostatic gene regulation patterns and that the cellular response to perturbation may manifest differently in oscillating vs non-oscillating cells.« less

An SH2 domain model of STAT5 in complex with phospho-peptides define ``STAT5 Binding Signatures''

NASA Astrophysics Data System (ADS)

Gianti, Eleonora; Zauhar, Randy J.

2015-05-01

The signal transducer and activator of transcription 5 (STAT5) is a member of the STAT family of proteins, implicated in cell growth and differentiation. STAT activation is regulated by phosphorylation of protein monomers at conserved tyrosine residues, followed by binding to phospho-peptide pockets and subsequent dimerization. STAT5 is implicated in the development of severe pathological conditions, including many cancer forms. However, nowadays a few STAT5 inhibitors are known, and only one crystal structure of the inactive STAT5 dimer is publicly available. With a view to enabling structure-based drug design, we have: (1) analyzed phospho-peptide binding pockets on SH2 domains of STAT5, STAT1 and STAT3; (2) generated a model of STAT5 bound to phospho-peptides; (3) assessed our model by docking against a class of known STAT5 inhibitors (Müller et al. in ChemBioChem 9:723-727, 2008); (4) used molecular dynamics simulations to optimize the molecular determinants responsible for binding and (5) proposed unique "Binding Signatures" of STAT5. Our results put in place the foundations to address STAT5 as a target for rational drug design, from sequence, structural and functional perspectives.

Phospho-ubiquitin-PARK2 complex as a marker for mitophagy defects.

PubMed

Callegari, Sylvie; Oeljeklaus, Silke; Warscheid, Bettina; Dennerlein, Sven; Thumm, Michael; Rehling, Peter; Dudek, Jan

2017-01-02

The E3 ubiquitin ligase PARK2 and the mitochondrial protein kinase PINK1 are required for the initiation of mitochondrial damage-induced mitophagy. Together, PARK2 and PINK1 generate a phospho-ubiquitin signal on outer mitochondrial membrane proteins that triggers recruitment of the autophagy machinery. This paper describes the detection of a defined 500-kDa phospho-ubiquitin-rich PARK2 complex that accumulates on mitochondria upon treatment with the membrane uncoupler CCCP. Formation of this complex is dependent on the presence of PINK1 and is absent in mutant forms of PARK2, whereby mitophagy is also arrested. These results signify a functional signaling complex that is essential for the progression of mitophagy. The visualization of the PARK2 signaling complex represents a novel marker for this critical step in mitophagy and can be used to monitor mitophagy progression in PARK2 mutants and to uncover additional upstream factors required for PARK2-mediated mitophagy signaling.

Leishmania mexicana: promastigotes and amastigotes secrete protein phosphatases and this correlates with the production of inflammatory cytokines in macrophages.

PubMed

Escalona-Montaño, A R; Ortiz-Lozano, D M; Rojas-Bernabé, A; Wilkins-Rodriguez, A A; Torres-Guerrero, H; Mondragón-Flores, R; Mondragón-Gonzalez, R; Becker, I; Gutiérrez-Kobeh, L; Aguirre-Garcia, M M

2016-09-01

Phosphatase activity of Leishmania spp. has been shown to deregulate the signalling pathways of the host cell. We here show that Leishmania mexicana promastigotes and amastigotes secrete proteins with phosphatase activity to the culture medium, which was higher in the Promastigote Secretion Medium (PSM) as compared with the Amastigote Secretion Medium (ASM) and was not due to cell lysis, since parasite viability was not affected by the secretion process. The biochemical characterization showed that the phosphatase activity present in PSM was higher in dephosphorylating the peptide END (pY) INASL as compared with the peptide RRA (pT)VA. In contrast, the phosphatase activity in ASM showed little dephosphorylating capacity for both peptides. Inhibition assays demonstrated that the phosphatase activity of both PSM and ASM was sensible only to protein tyrosine phosphatases inhibitors. An antibody against a protein phosphatase 2C (PP2C) of Leishmania major cross-reacted with a 44·9 kDa molecule in different cellular fractions of L. mexicana promastigotes and amastigotes, however, in PSM and ASM, the antibody recognized a protein about 70 kDa. By electron microscopy, the PP2C was localized in the flagellar pocket of amastigotes. PSM and ASM induced the production of tumor necrosis factor alpha, IL-1β, IL-12p70 and IL-10 in human macrophages.

Striatal cell signaling in chronically food-restricted rats under basal conditions and in response to brief handling.

PubMed

Pan, Yan; Siregar, Ermanda; Carr, Kenneth D

2006-01-30

Chronic food restriction increases exploratory behavior, cognitive function, and the rewarding effects of abused drugs. Recently, striatal neuroadaptations that may be involved in these effects were observed. Specifically, D-1 dopamine (DA) receptor agonist challenge produced stronger activation of extracellular signal-regulated kinase (ERK), calcium-calmodulin-dependent kinase II (CaMKII), and the nuclear transcription factor cAMP response element binding protein (CREB) in nucleus accumbens (NAc) of food-restricted (FR) relative to ad libitum fed (AL) rats. Further, when FR rats were injected intracerebroventricularly (i.c.v.) with vehicle (saline) they displayed stronger activation of c-Jun N-terminal protein kinase (JNK), ERK and CaMKII than did AL rats. It is not known to what extent the latter effects represent the basal state of FR rats or an amplified response to the brief handling involved in the i.c.v. injection procedure. Using Western blotting it was found that basal phospho-JNK is higher in caudate-putamen (CPu) and NAc of FR relative to AL rats. Interestingly, brief handling decreased phospho-JNK levels in FR subjects. Basal phospho-ERK1/2 also tended to be elevated in CPu and NAc of FR rats but the elevation was not significant. However, phospho-MEK--the activated kinase upstream of ERK1/2--was significantly elevated in NAc of FR rats. Neither ERK1/2 nor MEK were activated by brief handling. CaMKII was selectively activated by handling in NAc of FR rats, suggesting a state-dependent response to a salient event. Given the established involvement of mitogen-activated protein kinase (MAPK) and CaMKII in synaptic plasticity, learning and memory, the increase in basal phospho-MEK and hyperresponsiveness of CaMKII in NAc may represent adaptive cellular responses to persistent negative energy balance that facilitate associative learning in connection with food-seeking.

Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses1[OPEN

PubMed Central

Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; Munemasa, Shintaro; Booker, Matthew A.; Brandt, Benjamin; Waadt, Christian; Nusinow, Dmitri A.; Kay, Steve A.; Kunz, Hans-Henning; Schumacher, Karin; DeLong, Alison; Yates, John R.; Schroeder, Julian I.

2015-01-01

The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases. PMID:26175513

Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses

DOE PAGES

Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; ...

2015-09-04

The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. Furthemore, these analyses, which were confirmed usingmore » bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. Our analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases.« less

Bacterial-like PPP protein phosphatases: novel sequence alterations in pathogenic eukaryotes and peculiar features of bacterial sequence similarity.

PubMed

Kerk, David; Uhrig, R Glen; Moorhead, Greg B

2013-01-01

Reversible phosphorylation is a widespread modification affecting the great majority of eukaryotic cellular proteins, and whose effects influence nearly every cellular function. Protein phosphatases are increasingly recognized as exquisitely regulated contributors to these changes. The PPP (phosphoprotein phosphatase) family comprises enzymes, which catalyze dephosphorylation at serine and threonine residues. Nearly a decade ago, "bacterial-like" enzymes were recognized with similarity to proteins from various bacterial sources: SLPs (Shewanella-like phosphatases), RLPHs (Rhizobiales-like phosphatases), and ALPHs (ApaH-like phosphatases). A recent article from our laboratory appearing in Plant Physiology characterizes their extensive organismal distribution, abundance in plant species, predicted subcellular localization, motif organization, and sequence evolution. One salient observation is the distinct evolutionary trajectory followed by SLP genes and proteins in photosynthetic eukaryotes vs. animal and plant pathogens derived from photosynthetic ancestors. We present here a closer look at sequence data that emphasizes the distinctiveness of pathogen SLP proteins and that suggests that they might represent novel drug targets. A second observation in our original report was the high degree of similarity between the bacterial-like PPPs of eukaryotes and closely related proteins of the "eukaryotic-like" phyla Myxococcales and Planctomycetes. We here reflect on the possible implications of these observations and their importance for future research.

Phosphorylation of G Protein-Coupled Receptors: From the Barcode Hypothesis to the Flute Model.

PubMed

Yang, Zhao; Yang, Fan; Zhang, Daolai; Liu, Zhixin; Lin, Amy; Liu, Chuan; Xiao, Peng; Yu, Xiao; Sun, Jin-Peng

2017-09-01

Seven transmembrane G protein-coupled receptors (GPCRs) are often phosphorylated at the C terminus and on intracellular loops in response to various extracellular stimuli. Phosphorylation of GPCRs by GPCR kinases and certain other kinases can promote the recruitment of arrestin molecules. The arrestins critically regulate GPCR functions not only by mediating receptor desensitization and internalization, but also by redirecting signaling to G protein-independent pathways via interactions with numerous downstream effector molecules. Accumulating evidence over the past decade has given rise to the phospho-barcode hypothesis, which states that ligand-specific phosphorylation patterns of a receptor direct its distinct functional outcomes. Our recent work using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance ( 19 F-NMR) spectroscopy led to the flute model, which provides preliminary insight into the receptor phospho-coding mechanism, by which receptor phosphorylation patterns are recognized by an array of phosphate-binding pockets on arrestin and are translated into distinct conformations. These selective conformations are recognized by various effector molecules downstream of arrestin. The phospho-barcoding mechanism enables arrestin to recognize a wide range of phosphorylation patterns of GPCRs, contributing to their diverse functions. Copyright © 2017 by The Author(s).

Developmental and light regulation of tumor suppressor protein PP2A in the retina

PubMed Central

Rajala, Ammaji; Wang, Yuhong; Abcouwer, Steven F.; Gardner, Thomas W.; Rajala, Raju V.S.

2018-01-01

Protein phosphatases are a group of universal enzymes that are responsible for the dephosphorylation of various proteins and enzymes in cells. Cellular signal transduction events are largely governed by the phosphorylation of key proteins. The length of cellular response depends on the activation of protein phosphatase that dephosphorylates the phosphate groups to halt a biological response, and fine-tune the defined cellular outcome. Dysregulation of these phosphatase(s) results in various disease phenotypes. The retina is a post-mitotic tissue, and oncogenic tyrosine and serine/ threonine kinase activities are important for retinal neuron survival. Aberrant activation of protein phosphatase(s) may have a negative effect on retinal neurons. In the current study, we characterized tumor suppressor protein phosphatase 2 (PP2A), a major serine/ threonine kinase with a broad substrate specificity. Our data suggest that PP2A is developmentally regulated in the retina, localized predominantly in the inner retina, and expressed in photoreceptor inner segments. Our findings indicate that PKCα and mTOR may serve as PP2A substrates. We found that light regulates PP2A activity. Our studies also suggest that rhodopsin regulates PP2A and its substrate(s) dephosphorylation. PP2A substrate phosphorylation is increased in mice lacking the A-subunit of PP2A. However, there is no accompanying effect on retina structure and function. Together, our findings suggest that controlling the activity of PP2A in the retina may be neuroprotective. PMID:29416710

Purification and characterization of the glycogen-bound protein phosphatase from rat liver.

PubMed

Wera, S; Bollen, M; Stalmans, W

1991-01-05

Glycogen-bound protein phosphatase G from rat liver was transferred from glycogen to beta-cyclodextrin (cycloheptaamylose) linked to Sepharose 6B. After removal of the catalytic subunit and of contaminating proteins with 2 M NaCl, elution with beta-cyclodextrin yielded a single protein on native polyacrylamide gel electrophoresis and two polypeptides (161 and 54 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Several lines of evidence indicate that the latter polypeptides are subunits of the protein phosphatase G holoenzyme. First, these polypeptides were also present, together with the catalytic subunit, in the extensively purified holoenzyme. Also, polyclonal antibodies against these polypeptides were able to bind the holoenzyme. Further, while bound to cyclodextrin-Sepharose, the polypeptides were able to recombine with separately purified type-1 (AMD) catalytic subunit, but not with type-2A (PCS) catalytic subunit. The characteristics of the reconstituted enzyme resembled those of the nonpurified protein phosphatase G. At low dilutions, the spontaneous phosphorylase phosphatase activity of the reconstituted enzyme was about 10 times lower than that of the catalytic subunit, but it was about 1000-fold more resistant to inhibition by the modulator protein (inhibitor-2). In contrast with the free catalytic subunit, the reconstituted enzyme co-sedimented with glycogen, and it was able to activate purified liver glycogen synthase b. Also, the synthase phosphatase activity was synergistically increased by a cytosolic phosphatase and inhibited by physiological concentrations of phosphorylase alpha and of Ca2+.

Combining affinity proteomics and network context to identify new phosphatase substrates and adapters in growth pathways

PubMed Central

Sacco, Francesca; Boldt, Karsten; Calderone, Alberto; Panni, Simona; Paoluzi, Serena; Castagnoli, Luisa; Ueffing, Marius; Cesareni, Gianni

2014-01-01

Protein phosphorylation homoeostasis is tightly controlled and pathological conditions are caused by subtle alterations of the cell phosphorylation profile. Altered levels of kinase activities have already been associated to specific diseases. Less is known about the impact of phosphatases, the enzymes that down-regulate phosphorylation by removing the phosphate groups. This is partly due to our poor understanding of the phosphatase-substrate network. Much of phosphatase substrate specificity is not based on intrinsic enzyme specificity with the catalytic pocket recognizing the sequence/structure context of the phosphorylated residue. In addition many phosphatase catalytic subunits do not form a stable complex with their substrates. This makes the inference and validation of phosphatase substrates a non-trivial task. Here, we present a novel approach that builds on the observation that much of phosphatase substrate selection is based on the network of physical interactions linking the phosphatase to the substrate. We first used affinity proteomics coupled to quantitative mass spectrometry to saturate the interactome of eight phosphatases whose down regulations was shown to affect the activation of the RAS-PI3K pathway. By integrating information from functional siRNA with protein interaction information, we develop a strategy that aims at inferring phosphatase physiological substrates. Graph analysis is used to identify protein scaffolds that may link the catalytic subunits to their substrates. By this approach we rediscover several previously described phosphatase substrate interactions and characterize two new protein scaffolds that promote the dephosphorylation of PTPN11 and ERK by DUSP18 and DUSP26, respectively. PMID:24847354

Negative regulation of multifunctional Ca2+/calmodulin-dependent protein kinases: physiological and pharmacological significance of protein phosphatases

PubMed Central

Ishida, A; Sueyoshi, N; Shigeri, Y; Kameshita, I

2008-01-01

Multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) play pivotal roles in intracellular Ca2+ signaling pathways. There is growing evidence that CaMKs are involved in the pathogenic mechanisms underlying various human diseases. In this review, we begin by briefly summarizing our knowledge of the involvement of CaMKs in the pathogenesis of various diseases suggested to be caused by the dysfunction/dysregulation or aberrant expression of CaMKs. It is widely known that the activities of CaMKs are strictly regulated by protein phosphorylation/dephosphorylation of specific phosphorylation sites. Since phosphorylation status is balanced by protein kinases and protein phosphatases, the mechanism of dephosphorylation/deactivation of CaMKs, corresponding to their ‘switching off', is extremely important, as is the mechanism of phosphorylation/activation corresponding to their ‘switching on'. Therefore, we focus on the regulation of multifunctional CaMKs by protein phosphatases. We summarize the current understanding of negative regulation of CaMKs by protein phosphatases. We also discuss the biochemical properties and physiological significance of a protein phosphatase that we designated as Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP), and those of its homologue CaMKP-N. Pharmacological applications of CaMKP inhibitors are also discussed. These compounds may be useful not only for exploring the physiological functions of CaMKP/CaMKP-N, but also as novel chemotherapies for various diseases. PMID:18454172

AMP-activated protein kinase is involved in neural stem cell growth suppression and cell cycle arrest by 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside and glucose deprivation by down-regulating phospho-retinoblastoma protein and cyclin D.

PubMed

Zang, Yi; Yu, Li-Fang; Nan, Fa-Jun; Feng, Lin-Yin; Li, Jia

2009-03-06

The fate of neural stem cells (NSCs), including their proliferation, differentiation, survival, and death, is regulated by multiple intrinsic signals and the extrinsic environment. We had previously reported that 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) directly induces astroglial differentiation of NSCs by activation of the Janus kinase (JAK)/Signal transducer and activator of transcription 3 (STAT3) pathway independently of AMP-activated protein kinase (AMPK). Here, we reported the observation that AICAR inhibited NSC proliferation and its underlying mechanism. Analysis of caspase activity and cell cycle showed that AICAR induced G1/G0 cell cycle arrest in NSCs, associated with decreased levels of poly(ADP-ribose) polymerase, phospho-retinoblastoma protein (Rb), and cyclin D but did not cause apoptosis. Iodotubericidin and Compound C, inhibitors of adenosine kinase and AMPK, respectively, or overexpression of a dominant-negative mutant of AMPK, but not JAK inhibitor, were able to reverse the anti-proliferative effect of AICAR. Glucose deprivation also activated the AMPK pathway, induced G0/G1 arrest, and suppressed the proliferation of NSCs, an effect associated with decreased levels of phospho-Rb and cyclin D protein. Furthermore, Compound C and overexpression of dominant-negative AMPK in C17.2 NSCs could block the glucose deprivation-mediated down-regulation of cyclin D and partially reverse the suppression of proliferation. These results suggest that AICAR and glucose deprivation might induce G1/G0 cell cycle arrest and suppress proliferation of NSCs via phospho-Rb and cyclin D down-regulation. AMPK, but not JAK/STAT3, activation is key for this inhibitory effect and may play an important role in the responses of NSCs to metabolic stresses such as glucose deprivation.

A low molecular weight protein tyrosine phosphatase from Synechocystis sp. strain PCC 6803: enzymatic characterization and identification of its potential substrates

PubMed Central

Mukhopadhyay, Archana; Kennelly, Peter J.

2011-01-01

The predicted protein product of open reading frame slr0328 from Synechocystis sp. PCC 6803, SynPTP, possesses significant amino acid sequence similarity with known low molecular weight protein tyrosine phosphatases (PTPs). To determine the functional properties of this hypothetical protein, open reading frame slr0328 was expressed in Escherichia coli. The purified recombinant protein, SynPTP, displayed its catalytic phosphatase activity towards several tyrosine, but not serine, phosphorylated exogenous protein substrates. The protein phosphatase activity of SynPTP was inhibited by sodium orthovanadate, a known inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor for many serine/threonine phosphatases. Kinetic analysis indicated that the Km and Vmax values for SynPTP towards p-nitrophenyl phosphate are similar to those of other known bacterial low molecular weight PTPs. Mutagenic alteration of the predicted catalytic cysteine of PTP, Cys7, to serine abolished enzyme activity. Using a combination of immunodetection, mass spectrometric analysis and mutagenically altered Cys7SerAsp125Ala-SynPTP, we identified PsaD (photosystem I subunit II), CpcD (phycocyanin rod linker protein) and phycocyanin-α and -β subunits as possible endogenous substrates of SynPTP in this cyanobacterium. These results indicate that SynPTP might be involved in the regulation of photosynthesis in Synechocystis sp. PCC 6803. PMID:21288886

Outer membrane protein e of Escherichia coli K-12 is co-regulated with alkaline phosphatase.

PubMed

Tommassen, J; Lugtenberg, B

1980-07-01

Outer membrane protein e is induced in wild-type cells, just like alkaline phosphatase and some other periplasmic proteins, by growth under phosphatase limitation. nmpA and nmpB mutants, which synthesize protein e constitutively, are shown also to produce the periplasmic enzyme alkaline phosphatase constitutively. Alternatively, individual phoS, phoT, and phoR mutants as well as pit pst double mutants, all of which are known to produce alkaline phosphatase constitutively, were found to be constitutive for protein e. Also, the periplasmic space of most nmpA mutants and of all nmpB mutants grown in excess phosphate was found to contain, in addition to alkaline phosphatase, at least two new proteins, a phenomenon known for individual phoT and phoR mutants as well as for pit pst double mutants. The other nmpA mutants as well as phoS mutants lacked one of these extra periplasmic proteins, namely the phosphate-binding protein. From these data and from the known positions of the mentioned genes on the chromosomal map, it is concluded that nmpB mutants are identical to phoR mutants. Moreover, some nmpA mutants were shown to be identical to phoS mutants, whereas other nmpA mutants are likely to contain mutations in one of the genes phoS, phoT, or pst.

Ppm1E is an in cellulo AMP-activated protein kinase phosphatase.

PubMed

Voss, Martin; Paterson, James; Kelsall, Ian R; Martín-Granados, Cristina; Hastie, C James; Peggie, Mark W; Cohen, Patricia T W

2011-01-01

Activation of 5'-AMP-activated protein kinase (AMPK) is believed to be the mechanism by which the pharmaceuticals, metformin and phenformin, exert their beneficial effects for treatment of type 2 diabetes. These biguanide drugs elevate 5'-AMP, which allosterically activates AMPK and promotes phosphorylation on Thr172 of AMPK catalytic α subunits. Although kinases phosphorylating this site have been identified, phosphatases that dephosphorylate it are unknown. The aim of this study is to identify protein phosphatase(s) that dephosphorylate AMPKα-Thr172 within cells. Our initial data indicated that members of the protein phosphatase Mg/Mn(2+)-dependent [corrected] (PPM) family and not those of the PPP family of protein serine/threonine phosphatases may be directly or indirectly inhibited by phenformin. Using antibodies raised to individual Ppm phosphatases that facilitated the assessment of their activities, phenformin stimulation of cells was found to decrease the Mg(2+)/Mn(2+)-dependent [corrected] protein serine/threonine phosphatase activity of Ppm1E and Ppm1F, but not that attributable to other PPM family members, including Ppm1A/PP2Cα. Depletion of Ppm1E, but not Ppm1A, using lentiviral-mediated stable gene silencing, increased AMPKα-Thr172 phosphorylation approximately three fold in HEK293 cells. In addition, incubation of cells with low concentrations of phenformin and depletion of Ppm1E increased AMPK phosphorylation synergistically. Ppm1E and the closely related Ppm1F interact weakly with AMPK and assays with lysates of cells stably depleted of Ppm1F suggest [corrected] that this phosphatase contributes to dephosphorylation of AMPK. The data indicate that Ppm1E and probably PpM1F are in cellulo AMPK phosphatases and that Ppm1E is a potential anti-diabetic drug target. Copyright © 2010 Elsevier Inc. All rights reserved.

Identification of an additional member of the protein-tyrosine-phosphatase family: evidence for alternative splicing in the tyrosine phosphatase domain.

PubMed Central

Matthews, R J; Cahir, E D; Thomas, M L

1990-01-01

Protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.13.48) have been implicated in the regulation of cell growth; however, to date few tyrosine phosphatases have been characterized. To identify additional family members, the cDNA for the human tyrosine phosphatase leukocyte common antigen (LCA; CD45) was used to screen, under low stringency, a mouse pre-B-cell cDNA library. Two cDNA clones were isolated and sequence analysis predicts a protein sequence of 793 amino acids. We have named the molecule LRP (LCA-related phosphatase). RNA transfer analysis indicates that the cDNAs were derived from a 3.2-kilobase mRNA. The LRP mRNA is transcribed in a wide variety of tissues. The predicted protein structure can be divided into the following structural features: a short 19-amino acid leader sequence, an exterior domain of 123 amino acids that is predicted to be highly glycosylated, a 24-amino acid membrane-spanning region, and a 627-amino acid cytoplasmic region. The cytoplasmic region contains two approximately 260-amino acid domains, each with homology to the tyrosine phosphatase family. One of the cDNA clones differed in that it had a 108-base-pair insertion that, while preserving the reading frame, would disrupt the first protein-tyrosine-phosphatase domain. Analysis of genomic DNA indicates that the insertion is due to an alternatively spliced exon. LRP appears to be evolutionarily conserved as a putative homologue has been identified in the invertebrate Styela plicata. Images PMID:2162042

Aerobic exercise training improves oxidative stress and ubiquitin proteasome system activity in heart of spontaneously hypertensive rats.

PubMed

de Andrade, Luiz Henrique Soares; de Moraes, Wilson Max Almeida Monteiro; Matsuo Junior, Eduardo Hiroshi; de Orleans Carvalho de Moura, Elizabeth; Antunes, Hanna Karen Moreira; Montemor, Jairo; Antonio, Ednei Luiz; Bocalini, Danilo Sales; Serra, Andrey Jorge; Tucci, Paulo José Ferreira; Brum, Patricia Chakur; Medeiros, Alessandra

2015-04-01

The activity of the ubiquitin proteasome system (UPS) and the level of oxidative stress contribute to the transition from compensated cardiac hypertrophy to heart failure in hypertension. Moreover, aerobic exercise training (AET) is an important therapy for the treatment of hypertension, but its effects on the UPS are not completely known. The aim of this study was to evaluate the effect of AET on UPS's activity and oxidative stress level in heart of spontaneously hypertensive rats (SHR). A total of 53 Wistar and SHR rats were randomly divided into sedentary and trained groups. The AET protocol was 5×/week in treadmill for 13 weeks. Exercise tolerance test, non-invasive blood pressure measurement, echocardiographic analyses, and left ventricle hemodynamics were performed during experimental period. The expression of ubiquitinated proteins, 4-hydroxynonenal (4-HNE), Akt, phospho-Akt(ser473), GSK3β, and phospho-GSK3β(ser9) were analyzed by western blotting. The evaluation of lipid hydroperoxide concentration was performed using the xylenol orange method, and the proteasomal chymotrypsin-like activity was measured by fluorimetric assay. Sedentary hypertensive group presented cardiac hypertrophy, unaltered expression of total Akt, phospho-Akt, total GSK3β and phospho-GSK3β, UPS hyperactivity, increased lipid hydroperoxidation as well as elevated expression of 4-HNE but normal cardiac function. In contrast, AET significantly increased exercise tolerance, decreased resting systolic blood pressure and heart rate in hypertensive animals. In addition, the AET increased phospho-Akt expression, decreased phospho-GSK3β, and did not alter the expression of total Akt, total GSK3β, and ubiquitinated proteins, however, significantly attenuated 4-HNE levels, lipid hydroperoxidation, and UPS's activity toward normotensive group levels. Our results provide evidence for the main effect of AET on attenuating cardiac ubiquitin proteasome hyperactivity and oxidative stress in SHR rats.

Phosphorylation-Dependent Regulation of Ryanodine Receptors

PubMed Central

Marx, Steven O.; Reiken, Steven; Hisamatsu, Yuji; Gaburjakova, Marta; Gaburjakova, Jana; Yang, Yi-Ming; Rosemblit, Nora; Marks, Andrew R.

2001-01-01

Ryanodine receptors (RyRs), intracellular calcium release channels required for cardiac and skeletal muscle contraction, are macromolecular complexes that include kinases and phosphatases. Phosphorylation/dephosphorylation plays a key role in regulating the function of many ion channels, including RyRs. However, the mechanism by which kinases and phosphatases are targeted to ion channels is not well understood. We have identified a novel mechanism involved in the formation of ion channel macromolecular complexes: kinase and phosphatase targeting proteins binding to ion channels via leucine/isoleucine zipper (LZ) motifs. Activation of kinases and phosphatases bound to RyR2 via LZs regulates phosphorylation of the channel, and disruption of kinase binding via LZ motifs prevents phosphorylation of RyR2. Elucidation of this new role for LZs in ion channel macromolecular complexes now permits: (a) rapid mapping of kinase and phosphatase targeting protein binding sites on ion channels; (b) predicting which kinases and phosphatases are likely to regulate a given ion channel; (c) rapid identification of novel kinase and phosphatase targeting proteins; and (d) tools for dissecting the role of kinases and phosphatases as modulators of ion channel function. PMID:11352932

Protein tyrosine phosphatase receptor R and Z1 expression as independent prognostic indicators in oral squamous cell carcinoma.

PubMed

Duś-Szachniewicz, Kamila; Woźniak, Marta; Nelke, Kamil; Gamian, Elżbieta; Gerber, Hanna; Ziółkowski, Piotr

2015-12-01

The actions of tyrosine phosphorylation and dephosphorylation are controlled by tyrosine kinases and phosphatases. Although substantial previous data have revealed the role of several protein tyrosine phosphatases (PTPs) in various cancers, the function of protein tyrosine phosphatase receptor R (PTPRR) and protein tyrosine phosphatase, receptor-type, Z polypeptide 1 (PTPRZ1) proteins in oral cavity squamous cell carcinoma (SCC) has not been studied to date. The PTPRR and PTPRZ1 immunoreactivity in 67 formalin-fixed and paraffin-embedded oral cancer tissues at different stages were analyzed with the technique of immunohistochemistry (IHC). The presence of PTPRR in cancerous tissue was confirmed by Western blotting. The occurrence of PTPRR and PTPRZ1 proteins in the cancer specimens was more frequent in lower grade tumors. In addition, the association between the immunoreactivity of both examined proteins and improved patients survival was detected. Moreover, the PTPRR expression was found to be related to the absence of synchronous lymph node involvement. The above results indicate that the PTPRR and PTPRZ1 protein expression should be monitored in oral cancer for patients' prognostic stratification. © 2015 Wiley Periodicals, Inc.

The leukocyte common antigen (CD45): a putative receptor-linked protein tyrosine phosphatase.

PubMed Central

Charbonneau, H; Tonks, N K; Walsh, K A; Fischer, E H

1988-01-01

A major protein tyrosine phosphatase (PTPase 1B) has been isolated in essentially homogeneous form from the soluble and particulate fractions of human placenta. Unexpectedly, partial amino acid sequences displayed no homology with the primary structures of the protein Ser/Thr phosphatases deduced from cDNA clones. However, the sequence is strikingly similar to the tandem C-terminal homologous domains of the leukocyte common antigen (CD45). A 157-residue segment of PTPase 1B displayed 40% and 33% sequence identity with corresponding regions from cytoplasmic domains I and II of human CD45. Similar degrees of identity have been observed among the catalytic domains of families of regulatory proteins such as protein kinases and cyclic nucleotide phosphodiesterases. On this basis, it is proposed that the CD45 family has protein tyrosine phosphatase activity and may represent a set of cell-surface receptors involved in signal transduction. This suggests that the repertoire of signal transduction mechanisms may include the direct control of an intracellular protein tyrosine phosphatase, offering the possibility of a regulatory balance with those protein tyrosine kinases that act at the internal surface of the membrane. Images PMID:2845400

Requirement of phosphorylatable endothelial nitric oxide synthase at Ser-1177 for vasoinhibin-mediated inhibition of endothelial cell migration and proliferation in vitro.

PubMed

García, Celina; Nuñez-Anita, Rosa Elvira; Thebault, Stéphanie; Arredondo Zamarripa, David; Jeziorsky, Michael C; Martínez de la Escalera, Gonzalo; Clapp, Carmen

2014-03-01

Endothelial nitric oxide synthase (eNOS)-derived nitric oxide is a major vasorelaxing factor and a mediator of vasopermeability and angiogenesis. Vasoinhibins, a family of antiangiogenic prolactin fragments that include 16 K prolactin, block most eNOS-mediated vascular effects. Vasoinhibins activate protein phosphatase 2A, causing eNOS inactivation through dephosphorylation of eNOS at serine residue 1179 in bovine endothelial cells and thereby blocking vascular permeability. In this study, we examined whether human eNOS phosphorylation at S1177 (analogous to bovine S1179) influences other actions of vasoinhibins. Bovine umbilical vein endothelial cells were stably transfected with human wild-type eNOS (WT) or with phospho-mimetic (S1177D) or non-phosphorylatable (S1177A) eNOS mutants. Vasoinhibins inhibited the increases in eNOS activity, migration, and proliferation following the overexpression of WT eNOS but did not affect these responses in cells expressing S1177D and S1177A eNOS mutants. We conclude that eNOS inhibition by dephosphorylation of S1177 is fundamental for the inhibition of endothelial cell migration and proliferation by vasoinhibins.

Widespread presence of "bacterial-like" PPP phosphatases in eukaryotes.

PubMed

Andreeva, Alexandra V; Kutuzov, Mikhail A

2004-11-19

In eukaryotes, PPP (protein phosphatase P) family is one of the two known protein phosphatase families specific for Ser and Thr. The role of PPP phosphatases in multiple signaling pathways in eukaryotic cell has been extensively studied. Unlike eukaryotic PPP phosphatases, bacterial members of the family have broad substrate specificity or may even be Tyr-specific. Moreover, one group of bacterial PPPs are diadenosine tetraphosphatases, indicating that bacterial PPP phosphatases may not necessarily function as protein phosphatases. We describe the presence in eukaryotes of three groups of expressed genes encoding "non-conventional" phosphatases of the PPP family. These enzymes are more closely related to bacterial PPP phosphatases than to the known eukaryotic members of the family. One group, found exclusively in land plants, is most closely related to PPP phosphatases from some alpha-Proteobacteria, including Rhizobiales, Rhodobacterales and Rhodospirillaceae. This group is therefore termed Rhizobiales / Rhodobacterales / Rhodospirillaceae-like phosphatases, or Rhilphs. Phosphatases of the other group are found in Viridiplantae, Rhodophyta, Trypanosomatidae, Plasmodium and some fungi. They are structurally related to phosphatases from psychrophilic bacteria Shewanella and Colwellia, and are termed Shewanella-like phosphatases, or Shelphs. Phosphatases of the third group are distantly related to ApaH, bacterial diadenosine tetraphosphatases, and are termed ApaH-like phosphatases, or Alphs. Patchy distribution of Alphs in animals, plants, fungi, diatoms and kinetoplasts suggests that these phosphatases were present in the common ancestor of eukaryotes but were independently lost in many lineages. Rhilphs, Shelphs and Alphs form PPP clades, as divergent from "conventional" eukaryotic PPP phosphatases as they are from each other and from major bacterial clades. In addition, comparison of primary structures revealed a previously unrecognised (I/L/V)D(S/T)G motif, conserved in all bacterial and "bacterial-like" eukaryotic PPPs, but not in "conventional" eukaryotic and archaeal PPPs. Our findings demonstrate that many eukaryotes possess diverse "bacterial-like" PPP phosphatases, the enzymatic characteristics, physiological roles and precise evolutionary history of which have yet to be determined.

Interaction of Myosin Phosphatase Target Subunit (MYPT1) with Myosin Phosphatase-RhoA Interacting Protein (MRIP): A Role of Glutamic Acids in the Interaction.

PubMed

Lee, Eunhee; Stafford, Walter F

2015-01-01

Scaffold proteins bind to and functionally link protein members of signaling pathways. Interaction of the scaffold proteins, myosin phosphatase target subunit (MYPT1) and myosin phosphatase-RhoA interacting protein (MRIP), causes co-localization of myosin phosphatase and RhoA to actomyosin. To examine biophysical properties of interaction of MYPT1 with MRIP, we employed analytical ultracentrifugation and surface plasmon resonance. In regard to MRIP, its residues 724-837 are sufficient for the MYPT1/MRIP interaction. Moreover, MRIP binds to MYPT1 as either a monomer or a dimer. With respect to MYPT1, its leucine repeat region, LR (residues 991-1030) is sufficient to account for the MYPT1/MRIP interaction. Furthermore, point mutations that replace glutamic acids 998-1000 within LR reduced the binding affinity toward MRIP. This suggests that the glutamic acids of MYPT1 play an important role in the interaction.

Molecular dynamics simulations of human E3 ubiquitin ligase Parkin

PubMed Central

Qiu, Shi; Zhu, Shun; Xu, Shan; Han, Yanyan; Liu, Wen; Zuo, Ji

2017-01-01

Human E3 ubiquitin protein ligase parkin (Parkin) mediates mitophagy to maintain mitochondrial homeostasis. Parkin mutations are common genetic causes of early onset familial Parkinson's disease. The molecular mechanism of Parkin activation has been widely studied with emerging evidence suggesting an essential role of the phosphorylated (phospho)-ubiquitin interaction. However, the underlying mechanism of the phospho-ubiquitin interaction remains elusive. In the present study, replica exchange molecular dynamics simulations were performed to examine the conformational dynamics of Parkin in monomer and phospho-ubiquitin-bound states. In the Parkin monomer state, high structural flexibilities were observed in the majority of regions of Parkin particularly in the loop domain between the ubiquitin-like (UBL) and really interesting new gene (RING)0 domain. Binding of phospho-ubiquitin stabilizes the RING1/RING in between RING interface but destabilizes the RING1-UBL interface. Furthermore, using steered molecular dynamics simulations of Parkin mutations, it was demonstrated that salt bridge interactions contribute significantly to the interdomain interactions between the RING1 and UBL domain. Taken together, the results of the present study revealed the conformational dynamics of human full-length Parkin in monomer and phospho-ubiquitin-bound states, providing insights into designing potential therapeutics against Parkinson's disease. PMID:28765939

An extract of Perilla stem inhibits Src homology phosphatase-1 (SHP)-1 and influences insulin signaling.

PubMed

Peng, Liu; Lei, Zhang; Xiao-na, Xie; Deli, Wang; Jing, Sun; Yong-sen, Wang; Zhi, Wang; Shu, Xing; Jun-feng, Ma; Wan-nan, Li; Xue-qi, Fu

2015-03-01

Protein tyrosine phosphatases (PTPs) are enzymes that catalyze protein tyrosine dephosphorylation of which Src homology phosphatase-1 (SHP-1) is one of the best-validated, a widely distributed intracellular tyrosine phosphatase that contains two SH2 domains. Down regulation of SHP-1 tyrosine phosphatases was significantly increased sensitivity to insulin in insulin signaling pathway. Through in vitro enzymatic reaction kinetics experiment, we found that the extract of Perilla stem was a potential inhibitor to δSHP-1, the catalytic domain of SHP-1 protein tyrosine phosphatase, and its IC(50) was 4ug/ml, and was more sensitive towards SHP-1than other PTPs, which indicated that SHP-1 might be a target of the extract of Perilla stem. It can strengthened the level of tyrosine phosphorylation of insulin receptor (IR) and extracellular signal-regulated protein kinase (ERK) in HepG2 cells, and then activated the insulin signaling pathway through inhibiting the protein phosphorylation of SHP-1. These results demonstrated that the extract of Perilla stem could play an important role for diabetes treatment through inhibiting the level of SHP-1 in insulin signaling pathway.

The possible involvement of protein phosphatase 1 in thrombin-induced Ca2+ influx of human platelets.

PubMed

Murata, K; Sakon, M; Kambayashi, J; Yukawa, M; Yano, Y; Fujitani, K; Kawasaki, T; Shiba, E; Mori, T

1993-04-01

Protein phosphatase 1 is considered to be involved in thrombin-induced platelet activation (Murata et al., Biochem Int 26:327-334, 1992). To clarify the mechanism, we examined the effects of protein phosphatase 1 and 2A inhibitors (calyculin A, tautomycin, okadaic acid) on Ca2+ influx. In the presence of 1 mM Ca2+, thrombin- (0.1 U/ml) induced platelet aggregation and ATP release were inhibited by calyculin A, while this inhibitory effect was abolished in the absence of Ca2+ (EGTA 1 mM). Furthermore, thrombin-induced Mn2+ influx but not intracellular Ca2+ mobilization was inhibited by calyculin A in a dose-related manner. Calyculin A also blocked the ongoing Ca2+ influx when added 3 min after thrombin stimulation. Similar inhibitory effects were observed with okadaic acid and tautomycin in the same potency sequence as the reported one for protein phosphatase 1 (calyculin A > tautomycin > okadaic acid). These results suggest that the anti-platelet effects of phosphatase inhibitors are due to the inhibition of Ca2+ influx and that protein phosphatase 1 plays a key role in the regulation of receptor operated Ca2+ channel of human platelets.

Leukocyte common antigen-related phosphatase (LRP) gene structure: Conservation of the genomic organization of transmembrane protein tyrosine phosphatases

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

Wong, E.C.C.; Mullersman, J.E.; Thomas, M.L.

1993-07-01

The leukocyte common antigen-related protein tyrosine phosphatase (LRP) is a widely expressed transmembrane glycoprotein thought to be involved in cell growth and differentiation. Similar to most other transmembrane protein tyrosine phosphatases, LRP contains two tandem cytoplasmic phosphatase domains. To understand further the regulation and evolution of LRP, the authors have isolated and characterized mouse [lambda] genomic clones. Thirteen genomic clones could be divided into two non-overlapping clusters. The first cluster contained the transcription initiation site and the exon encoding most of the 5[prime] untranslated region. The second cluster contained the remaining exons encoding the protein and the 3[prime] untranslated region.more » The gene consists of 22 exons spanning over 75 kb. The distance between exon 1 and exon 2 is at least 25 kb. Characterization of the 5[prime] ends of LRP mRNA by S1 nuclease protection identifies putative initiation start sites within a G/C-rich region. The upstream region does not contain a TATA box. Comparison of the LRP gene structure to the mammalian protein tyrosine phosphatase gene, CD45, shows striking similarities in size and genomic organization. 29 refs., 5 figs., 1 tab.« less

Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation

PubMed Central

Kazlauskaite, Agne; Martínez-Torres, R Julio; Wilkie, Scott; Kumar, Atul; Peltier, Julien; Gonzalez, Alba; Johnson, Clare; Zhang, Jinwei; Hope, Anthony G; Peggie, Mark; Trost, Matthias; van Aalten, Daan MF; Alessi, Dario R; Prescott, Alan R; Knebel, Axel; Walden, Helen; Muqit, Miratul MK

2015-01-01

Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser65)—which lies within its ubiquitin-like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser65. How Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitinPhospho-Ser65 binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser65 by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitinPhospho-Ser65, thereby promoting Parkin Ser65 phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser65 phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitinPhospho-Ser65 to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser65. Finally, purified Parkin maximally phosphorylated at Ser65 in vitro cannot be further activated by the addition of ubiquitinPhospho-Ser65. Our results thus suggest that a major role of ubiquitinPhospho-Ser65 is to promote PINK1-mediated phosphorylation of Parkin at Ser65, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser65-binding pocket on the surface of Parkin that is critical for the ubiquitinPhospho-Ser65 interaction. This study provides new mechanistic insights into Parkin activation by ubiquitinPhospho-Ser65, which could aid in the development of Parkin activators that mimic the effect of ubiquitinPhospho-Ser65. PMID:26116755

Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation.

PubMed

Kazlauskaite, Agne; Martínez-Torres, R Julio; Wilkie, Scott; Kumar, Atul; Peltier, Julien; Gonzalez, Alba; Johnson, Clare; Zhang, Jinwei; Hope, Anthony G; Peggie, Mark; Trost, Matthias; van Aalten, Daan M F; Alessi, Dario R; Prescott, Alan R; Knebel, Axel; Walden, Helen; Muqit, Miratul M K

2015-08-01

Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser(65))--which lies within its ubiquitin-like domain (Ubl)--and indirectly through phosphorylation of ubiquitin at Ser(65). How Ser(65)-phosphorylated ubiquitin (ubiquitin(Phospho-Ser65)) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitin(Phospho-Ser65) binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser(65) by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitin(Phospho-Ser65), thereby promoting Parkin Ser(65) phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser(65) phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitin(Phospho-Ser65) to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser(65). Finally, purified Parkin maximally phosphorylated at Ser(65) in vitro cannot be further activated by the addition of ubiquitin(Phospho-Ser65). Our results thus suggest that a major role of ubiquitin(Phospho-Ser65) is to promote PINK1-mediated phosphorylation of Parkin at Ser(65), leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser(65)-binding pocket on the surface of Parkin that is critical for the ubiquitin(Phospho-Ser65) interaction. This study provides new mechanistic insights into Parkin activation by ubiquitin(Phospho-Ser65), which could aid in the development of Parkin activators that mimic the effect of ubiquitin(Phospho-Ser65). © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

Structural basis of O-GlcNAc recognition by mammalian 14-3-3 proteins.

PubMed

Toleman, Clifford A; Schumacher, Maria A; Yu, Seok-Ho; Zeng, Wenjie; Cox, Nathan J; Smith, Timothy J; Soderblom, Erik J; Wands, Amberlyn M; Kohler, Jennifer J; Boyce, Michael

2018-06-05

O-GlcNAc is an intracellular posttranslational modification that governs myriad cell biological processes and is dysregulated in human diseases. Despite this broad pathophysiological significance, the biochemical effects of most O-GlcNAcylation events remain uncharacterized. One prevalent hypothesis is that O-GlcNAc moieties may be recognized by "reader" proteins to effect downstream signaling. However, no general O-GlcNAc readers have been identified, leaving a considerable gap in the field. To elucidate O-GlcNAc signaling mechanisms, we devised a biochemical screen for candidate O-GlcNAc reader proteins. We identified several human proteins, including 14-3-3 isoforms, that bind O-GlcNAc directly and selectively. We demonstrate that 14-3-3 proteins bind O-GlcNAc moieties in human cells, and we present the structures of 14-3-3β/α and γ bound to glycopeptides, providing biophysical insights into O-GlcNAc-mediated protein-protein interactions. Because 14-3-3 proteins also bind to phospho-serine and phospho-threonine, they may integrate information from O-GlcNAc and O-phosphate signaling pathways to regulate numerous physiological functions.

Kappa Opioid Receptor Activation of p38 MAPK Is GRK3- and Arrestin-dependent in Neurons and Astrocytes*

PubMed Central

Bruchas, Michael R.; Macey, Tara A.; Lowe, Janet D.; Chavkin, Charles

2007-01-01

AtT-20 cells expressing the wild-type kappa opioid receptor (KOR) increased phospho-p38 MAPK following treatment with the kappa agonist U50,488. The increase was blocked by the kappa antagonist norbinaltorphimine and not evident in untransfected cells. In contrast, U50,488 treatment of AtT-20 cells expressing KOR having alanine substituted for serine-369 (KSA) did not increase phospho-p38. Phosphorylation of serine 369 in the KOR carboxyl terminus by G-protein receptor kinase 3 (GRK3) was previously shown to be required for receptor desensitization, and the results suggest that p38 MAPK activation by KOR may require arrestin recruitment. This hypothesis was tested by transfecting arrestin3-(R170E), a dominant positive form of arrestin that does not require receptor phosphorylation for activation. AtT-20 cells expressing both KSA and arrestin3-(R170E) responded to U50,488 treatment with an increase in phospho-p38 consistent with the hypothesis. Primary cultured astrocytes (glial fibrillary acidic protein-positive) and neurons (γ-aminobutyric acid-positive) isolated from mouse striata also responded to U50,488 by increasing phospho-p38 immunolabeling. p38 activation was not evident in either striatal astrocytes or neurons isolated from KOR knock-out mice or GRK3 knock-out mice. Astrocytes pretreated with small interfering RNA for arrestin3 were also unable to activate p38 in response to U50,488 treatment. Furthermore, in striatal neurons, the kappa-mediated phospho-p38 labeling was colocalized with arrestin3. These findings suggest that KOR may activate p38 MAPK in brain by a GRK3 and arrestin-dependent mechanism. PMID:16648139

Parathyroid hormone regulation of the human bone sialoprotein gene transcription is mediated through two cAMP response elements.

PubMed

Araki, Shouta; Mezawa, Masaru; Sasaki, Yoko; Yang, Li; Li, Zhengyang; Takai, Hideki; Nakayama, Youhei; Ogata, Yorimasa

2009-03-01

Parathyroid hormone (PTH) regulates serum calcium and inorganic phosphate levels through its actions on kidney and bone. Bone sialoprotein (BSP) is an early marker of osteoblast differentiation and bone metabolism. We here report that two cAMP response elements (CRE) in the human BSP gene promoter are target of PTH. In human osteoblast-like Saos2 cells, PTH (human 1-34 PTH, 10 nM) increased BSP mRNA and protein levels at 3 h. From transient transfection assays, 2- to 2.5-fold increase in transcription by PTH was observed at 3 and 6 h in -184, -211, -428, -868, and -927 luciferase constructs that included the human BSP gene promoter. Effect of PTH was abrogated by 2 bp mutations in either the CRE1 (-79 to -72) or CRE2 (-674 to -667). Luciferase activities induced by PTH were blocked by protein kinase A inhibitor H89 and tyrosine kinase inhibitor herbimycin A. Gel shift analyses showed that PTH increased binding of nuclear proteins to the CRE1 and CRE2 elements. The CRE1-protein and CRE2-protein complexes were disrupted by CRE binding protein 1 (CREB1) antibodies and supershifted by phospho-CREB1 antibody. ChIP assays detected binding of CREB1 and phospho-CREB1 to a chromatin fragment containing CRE1 and CRE2, and increased binding of phospho-CREB1 to the both sites. These studies demonstrate that PTH stimulates human BSP gene transcription by targeting the two CREs in the promoter of the human BSP gene.

A Global Protein Kinase and Phosphatase Interaction Network in Yeast

PubMed Central

Breitkreutz, Ashton; Choi, Hyungwon; Sharom, Jeffrey R.; Boucher, Lorrie; Neduva, Victor; Larsen, Brett; Lin, Zhen-Yuan; Breitkreutz, Bobby-Joe; Stark, Chris; Liu, Guomin; Ahn, Jessica; Dewar-Darch, Danielle; Reguly, Teresa; Tang, Xiaojing; Almeida, Ricardo; Qin, Zhaohui Steve; Pawson, Tony; Gingras, Anne-Claude; Nesvizhskii, Alexey I.; Tyers, Mike

2011-01-01

The interactions of protein kinases and phosphatases with their regulatory subunits and substrates underpin cellular regulation. We identified a kinase and phosphatase interaction (KPI) network of 1844 interactions in budding yeast by mass spectrometric analysis of protein complexes. The KPI network contained many dense local regions of interactions that suggested new functions. Notably, the cell cycle phosphatase Cdc14 associated with multiple kinases that revealed roles for Cdc14 in mitogen-activated protein kinase signaling, the DNA damage response, and metabolism, whereas interactions of the target of rapamycin complex 1 (TORC1) uncovered new effector kinases in nitrogen and carbon metabolism. An extensive backbone of kinase-kinase interactions cross-connects the proteome and may serve to coordinate diverse cellular responses. PMID:20489023

The Protein Phosphatases of Synechocystis sp. Strain PCC 6803: Open Reading Frames sll1033 and sll1387 Encode Enzymes That Exhibit both Protein-Serine and Protein-Tyrosine Phosphatase Activity In Vitro.

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

Li, Ruiliang; Potters, M B.; Shi, Liang

2005-09-01

The open reading frames (ORFs) encoding two potential protein-serine/threonine phosphatases from the cyanobacterium Synechocystis sp. strain PCC 6803 were cloned and their protein products expressed in Escherichia coli cells. The product of ORF sll1033, SynPPM3, is a homologue of the PPM family of protein-serine/threonine phosphatases found in all eukaryotes as well as many members of the Bacteria. Surprisingly, the recombinant protein phosphatase dephosphorylated phosphotyrosine- as well as phosphoserine-containing proteins in vitro. While kinetic analyses indicate that the enzyme was more efficient at dephosphorylating the latter, replacement of Asp(608) by asparagine enhanced activity toward a phosphotyrosine-containing protein fourfold. The product ofmore » ORF sll1387, SynPPP1, is the sole homolog of the PPP family of protein phosphatases encoded by the genome of Synechocystis sp. strain PCC 6803. Like many other bacterial PPPs, the enzyme dephosphorylated phosphoserine- and phosphotyrosine-containing proteins with comparable efficiencies. However, while previously described PPPs from prokaryotic organisms required the addition of exogenous metal ion cofactors, such as Mg(2+) or Mn(2+), for activity, recombinantly produced SynPPP1 displayed near-maximal activity in the absence of added metals. Inductively coupled plasma mass spectrometry indicated that recombinant SynPPP1 contained significant quantities, 0.32 to 0.44 mol/mole total, of Mg and Mn. In this respect, the cyanobacterial enzyme resembled eukaryotic members of the PPP family, which are metalloproteins. mRNA encoding SynPPP1 or SynPPM3 could be detected in cells grown under many, but not all, environmental conditions.« less

The Role of Bacterial Protein Tyrosine Phosphatases in the Regulation of the Biosynthesis of Secreted Polysaccharides

PubMed Central

Morona, Renato

2014-01-01

Abstract Significance: Tyrosine phosphorylation and associated protein tyrosine phosphatases are gaining prominence as critical mechanisms in the regulation of fundamental processes in a wide variety of bacteria. In particular, these phosphatases have been associated with the control of the biosynthesis of capsular polysaccharides and extracellular polysaccharides, critically important virulence factors for bacteria. Recent Advances: Deletion and overexpression of the phosphatases result in altered polysaccharide biosynthesis in a range of bacteria. The recent structures of associated auto-phosphorylating tyrosine kinases have suggested that the phosphatases may be critical for the cycling of the kinases between monomers and higher order oligomers. Critical Issues: Additional substrates of the phosphatases apart from cognate kinases are currently being identified. These are likely to be critical to our understanding of the mechanism by which polysaccharide biosynthesis is regulated. Future Directions: Ultimately, these protein tyrosine phosphatases are an attractive target for the development of novel antimicrobials. This is particularly the case for the polymerase and histidinol phosphatase family, which is predominantly found in bacteria. Furthermore, the determination of bacterial tyrosine phosphoproteomes will likely help to uncover the fundamental roles, mechanism, and critical importance of these phosphatases in a wide range of bacteria. Antioxid. Redox Signal. 20, 2274–2289. PMID:24295407

A novel transmembrane Ser/Thr kinase complexes with protein phosphatase-1 and inhibitor-2.

PubMed

Wang, Hong; Brautigan, David L

2002-12-20

Protein kinases and protein phosphatases exert coordinated control over many essential cellular processes. Here, we describe the cloning and characterization of a novel human transmembrane protein KPI-2 (Kinase/Phosphatase/Inhibitor-2) that was identified by yeast two-hybrid using protein phosphatase inhibitor-2 (Inh2) as bait. KPI-2 mRNA was predominantly expressed in skeletal muscle. KPI-2 is a 1503-residue protein with two predicted transmembrane helices at the N terminus, a kinase domain, followed by a C-terminal domain. The transmembrane helices were sufficient for targeting proteins to the membrane. KPI-2 kinase domain has about 60% identity with its closest relative, a tyrosine kinase. However, it only exhibited serine/threonine kinase activity in autophosphorylation reactions or with added substrates. KPI-2 kinase domain phosphorylated protein phosphatase-1 (PP1C) at Thr(320), which attenuated PP1C activity. KPI-2 C-terminal domain directly associated with PP1C, and this required a VTF motif. Inh2 associated with KPI-2 C-terminal domain with and without PP1C. Thus, KPI-2 is a kinase with sites to associate with PP1C and Inh2 to form a regulatory complex that is localized to membranes.

Fluorescence turn-on detection of alkaline phosphatase activity based on controlled release of PEI-capped Cu nanoclusters from MnO2 nanosheets.

PubMed

Zhang, Yunyi; Li, Yongxin; Zhang, Cuiyun; Zhang, Qingfeng; Huang, Xinan; Yang, Meiding; Shahzad, Sohail Anjum; Lo, Kenneth Kam-Wing; Yu, Cong; Jiang, Shichun

2017-08-01

A fluorescence turn-on assay for alkaline phosphatase (ALP) activity is developed through the controlled release of polyethyleneimine-capped copper nanoclusters (PEI-capped CuNCs) from the MnO 2 nanosheets. In an aqueous solution, the positively charged PEI-capped CuNCs could be adsorbed onto the surface of the negatively charged MnO 2 nanosheets. Such adsorption through favorable electrostatic interactions could efficiently quench the nanocluster fluorescence emission via resonance energy transfer from the PEI-capped CuNCs to the MnO 2 nanosheets. 2-Phospho-L-ascorbic acid (AAP) could be hydrolyzed to L-ascorbic acid (AA) in the presence of ALP. AA could reduce MnO 2 into Mn 2+ and trigger the disintegration of the MnO 2 nanosheets. As a result, the CuNCs were released and the quenched fluorescence was recovered efficiently. The detection strategy is simple, inexpensive, sensitive, selective, with low toxicity, and has better biocompatibility. The newly fabricated biosensor for ALP activity will potentially make it a robust candidate for numerous biological and biomedical applications.

Conditional Deletion of Pten Causes Bronchiolar Hyperplasia

PubMed Central

Davé, Vrushank; Wert, Susan E.; Tanner, Tiffany; Thitoff, Angela R.; Loudy, Dave E.; Whitsett, Jeffrey A.

2008-01-01

Tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase that regulates multiple cellular processes including cell polarity, migration, proliferation, and carcinogenesis. In this work, we demonstrate that conditional deletion of Pten (PtenΔ/Δ) in the respiratory epithelial cells of the developing mouse lung caused epithelial cell proliferation and hyperplasia as early as 4 to 6 weeks of age. While bronchiolar cell differentiation was normal, as indicated by β-tubulin and FOXJ1 expression in ciliated cells and by CCSP expression in nonciliated cells, cell proliferation (detected by expression of Ki-67, phospho-histone-H3, and cyclin D1) was increased and associated with activation of the AKT/mTOR survival pathway. Deletion of Pten caused papillary epithelial hyperplasia characterized by a hypercellular epithelium lining papillae with fibrovascular cores that protruded into the airway lumens. Cell polarity, as assessed by subcellular localization of cadherin, β-catenin, and zonula occludens-1, was unaltered. PTEN is required for regulation of epithelial cell proliferation in the lung and for the maintenance of the normal simple columnar epithelium characteristics of bronchi and bronchioles. PMID:17921358

Conditional deletion of Pten causes bronchiolar hyperplasia.

PubMed

Davé, Vrushank; Wert, Susan E; Tanner, Tiffany; Thitoff, Angela R; Loudy, Dave E; Whitsett, Jeffrey A

2008-03-01

Tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase that regulates multiple cellular processes including cell polarity, migration, proliferation, and carcinogenesis. In this work, we demonstrate that conditional deletion of Pten (Pten(Delta/Delta)) in the respiratory epithelial cells of the developing mouse lung caused epithelial cell proliferation and hyperplasia as early as 4 to 6 weeks of age. While bronchiolar cell differentiation was normal, as indicated by beta-tubulin and FOXJ1 expression in ciliated cells and by CCSP expression in nonciliated cells, cell proliferation (detected by expression of Ki-67, phospho-histone-H3, and cyclin D1) was increased and associated with activation of the AKT/mTOR survival pathway. Deletion of Pten caused papillary epithelial hyperplasia characterized by a hypercellular epithelium lining papillae with fibrovascular cores that protruded into the airway lumens. Cell polarity, as assessed by subcellular localization of cadherin, beta-catenin, and zonula occludens-1, was unaltered. PTEN is required for regulation of epithelial cell proliferation in the lung and for the maintenance of the normal simple columnar epithelium characteristics of bronchi and bronchioles.

PhosphoSitePlus, 2014: mutations, PTMs and recalibrations

PubMed Central

Hornbeck, Peter V.; Zhang, Bin; Murray, Beth; Kornhauser, Jon M.; Latham, Vaughan; Skrzypek, Elzbieta

2015-01-01

PhosphoSitePlus® (PSP, http://www.phosphosite.org/), a knowledgebase dedicated to mammalian post-translational modifications (PTMs), contains over 330 000 non-redundant PTMs, including phospho, acetyl, ubiquityl and methyl groups. Over 95% of the sites are from mass spectrometry (MS) experiments. In order to improve data reliability, early MS data have been reanalyzed, applying a common standard of analysis across over 1 000 000 spectra. Site assignments with P > 0.05 were filtered out. Two new downloads are available from PSP. The ‘Regulatory sites’ dataset includes curated information about modification sites that regulate downstream cellular processes, molecular functions and protein-protein interactions. The ‘PTMVar’ dataset, an intersect of missense mutations and PTMs from PSP, identifies over 25 000 PTMVars (PTMs Impacted by Variants) that can rewire signaling pathways. The PTMVar data include missense mutations from UniPROTKB, TCGA and other sources that cause over 2000 diseases or syndromes (MIM) and polymorphisms, or are associated with hundreds of cancers. PTMVars include 18 548 phosphorlyation sites, 3412 ubiquitylation sites, 2316 acetylation sites, 685 methylation sites and 245 succinylation sites. PMID:25514926

Phylogenetic and genetic linkage between novel atypical dual-specificity phosphatases from non-metazoan organisms.

PubMed

Romá-Mateo, Carlos; Sacristán-Reviriego, Almudena; Beresford, Nicola J; Caparrós-Martín, José Antonio; Culiáñez-Macià, Francisco A; Martín, Humberto; Molina, María; Tabernero, Lydia; Pulido, Rafael

2011-04-01

Dual-specificity phosphatases (DSPs) constitute a large protein tyrosine phosphatase (PTP) family, with examples in distant evolutive phyla. PFA-DSPs (Plant and Fungi Atypical DSPs) are a group of atypical DSPs present in plants, fungi, kinetoplastids, and slime molds, the members of which share structural similarity with atypical- and lipid phosphatase DSPs from mammals. The analysis of the PFA-DSPs from the plant Arabidopsis thaliana (AtPFA-DSPs) showed differential tissue mRNA expression, substrate specificity, and catalytic activity for these proteins, suggesting different functional roles among plant PFA-DSPs. Bioinformatic analysis revealed the existence of novel PFA-DSP-related proteins in fungi (Oca1, Oca2, Oca4 and Oca6 in Saccharomyces cerevisiae) and protozoa, which were segregated from plant PFA-DSPs. The closest yeast homolog for these proteins was the PFA-DSP from S. cerevisiae ScPFA-DSP1/Siw14/Oca3. Oca1, Oca2, Siw14/Oca3, Oca4, and Oca6 were involved in the yeast response to caffeine and rapamycin stresses. Siw14/Oca3 was an active phosphatase in vitro, whereas no phosphatase activity could be detected for Oca1. Remarkably, overexpression of Siw14/Oca3 suppressed the caffeine sensitivity of oca1, oca2, oca4, and oca6 deleted strains, indicating a genetic linkage and suggesting a functional relationship for these proteins. Functional studies on mutations targeting putative catalytic residues from the A. thaliana AtPFA-DSP1/At1g05000 protein indicated the absence of canonical amino acids acting as the general acid/base in the phosphor-ester hydrolysis, which suggests a specific mechanism of reaction for PFA-DSPs and related enzymes. Our studies demonstrate the existence of novel phosphatase protein families in fungi and protozoa, with active and inactive enzymes linked in common signaling pathways. This illustrates the catalytic and functional complexity of the expanding family of atypical dual-specificity phosphatases in non-metazoans, including parasite organisms responsible for infectious human diseases.

PLD2 has both enzymatic and cell proliferation-inducing capabilities, that are differentially regulated by phosphorylation and dephosphorylation

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

Henkels, Karen M.; Short, Stephen; Peng, Hong-Juan

2009-11-13

Phospholipase D2 (PLD2) overexpression in mammalian cells results in cell transformation. We have hypothesized that this is due to an increase of de novo DNA synthesis. We show here that overexpression of PLD2-WT leads to an increased DNA synthesis, as measured by the expression levels of the proliferation markers PCNA, p27{sup KIP1} and phospho-histone-3. The enhancing effect was even higher with phosphorylation-deficient PLD2-Y179F and PLD2-Y511F mutants. The mechanism for this did not involve the enzymatic activity of the lipase, but, rather, the presence of the protein tyrosine phosphatase CD45, as silencing with siRNA for CD45 abrogated the effect. The twomore » Y{yields}F mutants had in common a YxN consensus site that, in the phosphorylated counterparts, could be recognized by SH2-bearing proteins, such as Grb2. Even though Y179F and Y511F cannot bind Grb2, they could still find other protein partners, one of which, we have reasoned, could be CD45 itself. Affinity purified PLD2 is indeed activated by Grb2 and deactivated by CD45 in vitro. We concluded that phosphorylated PLD2, aided by Grb2, mediates lipase activity, whereas dephosphorylated PLD2 mediates an induction of cell proliferation, and the specific residues involved in this newly discovered regulation of PLD2 are Y{sup 179} and Y{sup 511}.« less

Testicular Lumicrine Factors Regulate ERK, STAT, and NFKB Pathways in the Initial Segment of the Rat Epididymis to Prevent Apoptosis1

PubMed Central

Xu, Bingfang; Abdel-Fattah, Rana; Yang, Ling; Crenshaw, Sallie A.; Black, Michael B.; Hinton, Barry T.

2011-01-01

The initial segment of the epididymis is vital for male fertility; therefore, it is important to understand the mechanisms that regulate this important region. Deprival of testicular luminal fluid factors/lumicrine factors from the epididymis results in a wave of apoptosis in the initial segment. In this study, a combination of protein array and microarray analyses was used to examine the early changes in downstream signal transduction pathways following loss of lumicrine factors. We discovered the following cascade of events leading to the loss of protection and eventual apoptosis: in the first 6 h after loss of lumicrine factors, down-regulation of the ERK pathway components was observed at the mRNA expression and protein activity levels. Microarray analysis revealed that mRNA levels of several key components of the ERK pathway, Dusp6, Dusp5, and Etv5, decreased sharply, while the analysis from the protein array revealed a decline in the activities of MAP2K1/2 and MAPK1. Immunostaining of phospho-MAPK3/1 indicated that down-regulation of the ERK pathway was specific to the epithelial cells of the initial segment. Subsequently, after 12 h of loss of lumicrine factors, levels of mRNA expression of STAT and NFKB pathway components increased, mRNA levels of several genes encoding cell cycle inhibitors increased, and levels of protein expression of several proapoptotic phosphatases increased. Finally, after 18 h of loss of protection from lumicrine factors, apoptosis was observed. In conclusion, testicular lumicrine factors protect the cells of the initial segment by activating the ERK pathway, repressing STAT and NFKB pathways, and thereby preventing apoptosis. PMID:21311037

Natural products possessing protein tyrosine phosphatase 1B (PTP1B) inhibitory activity found in the last decades

PubMed Central

Jiang, Cheng-shi; Liang, Lin-fu; Guo, Yue-wei

2012-01-01

This article provides an overview of approximately 300 secondary metabolites with inhibitory activity against protein tyrosine phosphatase 1B (PTP1B), which were isolated from various natural sources or derived from synthetic process in the last decades. The structure-activity relationship and the selectivity of some compounds against other protein phosphatases were also discussed. Potential pharmaceutical applications of several PTP1B inhibitors were presented. PMID:22941286

Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of East Asian-type Helicobacter pylori strains.

PubMed

Lind, Judith; Backert, Steffen; Hoffmann, Rebecca; Eichler, Jutta; Yamaoka, Yoshio; Perez-Perez, Guillermo I; Torres, Javier; Sticht, Heinrich; Tegtmeyer, Nicole

2016-09-02

Highly virulent strains of the gastric pathogen Helicobacter pylori encode a type IV secretion system (T4SS) that delivers the effector protein CagA into gastric epithelial cells. Translocated CagA undergoes tyrosine phosphorylation by members of the oncogenic c-Src and c-Abl host kinases at EPIYA-sequence motifs A, B and D in East Asian-type strains. These phosphorylated EPIYA-motifs serve as recognition sites for various SH2-domains containing human proteins, mediating interactions of CagA with host signaling factors to manipulate signal transduction pathways. Recognition of phospho-CagA is mainly based on the use of commercial pan-phosphotyrosine antibodies that were originally designed to detect phosphotyrosines in mammalian proteins. Specific anti-phospho-EPIYA antibodies for each of the three sites in CagA are not forthcoming. This study was designed to systematically analyze the detection preferences of each phosphorylated East Asian CagA EPIYA-motif by pan-phosphotyrosine antibodies and to determine a minimal recognition sequence. We synthesized phospho- and non-phosphopeptides derived from each predominant EPIYA-site, and determined the recognition patterns by seven different pan-phosphotyrosine antibodies using Western blotting, and also investigated representative East Asian H. pylori isolates during infection. The results indicate that a total of only 9-11 amino acids containing the phosphorylated East Asian EPIYA-types are required and sufficient to detect the phosphopeptides with high specificity. However, the sequence recognition by the different antibodies was found to bear high variability. From the seven antibodies used, only four recognized all three phosphorylated EPIYA-motifs A, B and D similarly well. Two of the phosphotyrosine antibodies preferentially bound primarily to the phosphorylated motif A and D, while the seventh antibody failed to react with any of the phosphorylated EPIYA-motifs. Control experiments confirmed that none of the antibodies reacted with non-phospho-CagA peptides and in accordance were able to recognize phosphotyrosine proteins in human cells. The results of this study disclose the various binding preferences of commercial anti-phosphotyrosine antibodies for phospho-EPIYA-motifs, and are valuable in the application for further characterization of CagA phosphorylation events during infection with H. pylori and risk prediction for gastric disease development.

Inhibitory Effect of Dried Pomegranate Concentration Powder on Melanogenesis in B16F10 Melanoma Cells; Involvement of p38 and PKA Signaling Pathways

PubMed Central

Kang, Su Jin; Choi, Beom Rak; Lee, Eun Kyoung; Kim, Seung Hee; Yi, Hae Yeon; Park, Hye Rim; Song, Chang Hyun; Lee, Young Joon; Ku, Sae Kwang

2015-01-01

Plants rich in antioxidant substances may be useful for preventing skin aging. Pomegranates, containing flavonoids and other polyphenolic compounds, are widely consumed due to their beneficial properties. We examined the underlying mechanisms of dried pomegranate concentrate powder (PCP) on melanin synthesis in B16F10 melanoma cells. The antioxidant effects of PCP were determined by measuring free radical scavenging capacity and transcript levels of antioxidant enzymes. To explore the inhibitory effects of PCP on melanin synthesis, we measured tyrosinase activity and melanin content in α-melanocyte stimulating hormone (α-MSH)-stimulated B16F10 cells. In addition, the levels of tyrosinase-related protein-1 (TRP-1), TRP-2, tyrosinase, and microphthalmia-associated transcription factor (MITF) expression were determined by Western blotting. Changes in the phosphorylation status of protein kinase A (PKA), cAMP response element-binding protein (CREB), mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K), serine/threonine kinase Akt, and glycogen kinase 3β (GSK3β) were also examined. The free radical scavenging activity of PCP increased in a dose-dependent manner. In PCP-treated B16F10 cells, transcript levels of glutathione peroxidase-1 (GPx-1) were increased compared with α-MSH-stimulated cells. In addition, PCP led to the down-regulation of phospho-p38, phospho-PKA, phospho-CREB, phospho-GSK3β, MITF, and TRP-1 compared with α-MSH-stimulated B16F10 cells. We believe this effect may be associated with PCP activity, which leads to the inhibition of melanin production and tyrosinase activity. These results suggest that PCP decreases tyrosinase activity and melanin production via inactivation of the p38 and PKA signaling pathways, and subsequently decreases phosphorylation of CREB, MITF, and melanogenic enzymes. These observations provided new insights on the molecular mechanisms of the skin-whitening property of PCP. PMID:26473849

Selective activators of protein phosphatase 5 target the auto-inhibitory mechanism.

PubMed

Haslbeck, Veronika; Drazic, Adrian; Eckl, Julia M; Alte, Ferdinand; Helmuth, Martin; Popowicz, Grzegorz; Schmidt, Werner; Braun, Frank; Weiwad, Matthias; Fischer, Gunter; Gemmecker, Gerd; Sattler, Michael; Striggow, Frank; Groll, Michael; Richter, Klaus

2015-04-20

Protein phosphatase 5 (PP5) is an evolutionary conserved serine/threonine phosphatase. Its dephosphorylation activity modulates a diverse set of cellular factors including protein kinases and the microtubule-associated tau protein involved in neurodegenerative disorders. It is auto-regulated by its heat-shock protein (Hsp90)-interacting tetratricopeptide repeat (TPR) domain and its C-terminal α-helix. In the present study, we report the identification of five specific PP5 activators [PP5 small-molecule activators (P5SAs)] that enhance the phosphatase activity up to 8-fold. The compounds are allosteric modulators accelerating efficiently the turnover rate of PP5, but do barely affect substrate binding or the interaction between PP5 and the chaperone Hsp90. Enzymatic studies imply that the compounds bind to the phosphatase domain of PP5. For the most promising compound crystallographic comparisons of the apo PP5 and the PP5-P5SA-2 complex indicate a relaxation of the auto-inhibited state of PP5. Residual electron density and mutation analyses in PP5 suggest activator binding to a pocket in the phosphatase/TPR domain interface, which may exert regulatory functions. These compounds thus may expose regulatory mechanisms in the PP5 enzyme and serve to develop optimized activators based on these scaffolds. © 2015 Authors.

DELAY OF GERMINATION1 requires PP2C phosphatases of the ABA signalling pathway to control seed dormancy.

PubMed

Née, Guillaume; Kramer, Katharina; Nakabayashi, Kazumi; Yuan, Bingjian; Xiang, Yong; Miatton, Emma; Finkemeier, Iris; Soppe, Wim J J

2017-07-13

The time of seed germination is a major decision point in the life of plants determining future growth and development. This timing is controlled by seed dormancy, which prevents germination under favourable conditions. The plant hormone abscisic acid (ABA) and the protein DELAY OF GERMINATION 1 (DOG1) are essential regulators of dormancy. The function of ABA in dormancy is rather well understood, but the role of DOG1 is still unknown. Here, we describe four phosphatases that interact with DOG1 in seeds. Two of them belong to clade A of type 2C protein phosphatases: ABA-HYPERSENSITIVE GERMINATION 1 (AHG1) and AHG3. These phosphatases have redundant but essential roles in the release of seed dormancy epistatic to DOG1. We propose that the ABA and DOG1 dormancy pathways converge at clade A of type 2C protein phosphatases.The DOG1 protein is a major regulator of seed dormancy in Arabidopsis. Here, Née et al. provide evidence that DOG1 can interact with the type 2C protein phosphatases AHG1 and AHG3 and that this represents the convergence point of the DOG1-regulated dormancy pathway and signalling by the plant hormone abscisic acid.

Post-translational generation of constitutively active cores from larger phosphatases in the malaria parasite, Plasmodium falciparum: implications for proteomics

PubMed Central

Kumar, Rajinder; Musiyenko, Alla; Oldenburg, Anja; Adams, Brian; Barik, Sailen

2004-01-01

Background Although the complete genome sequences of a large number of organisms have been determined, the exact proteomes need to be characterized. More specifically, the extent to which post-translational processes such as proteolysis affect the synthesized proteins has remained unappreciated. We examined this issue in selected protein phosphatases of the protease-rich malaria parasite, Plasmodium falciparum. Results P. falciparum encodes a number of Ser/Thr protein phosphatases (PP) whose catalytic subunits are composed of a catalytic core and accessory domains essential for regulation of the catalytic activity. Two examples of such regulatory domains are found in the Ca+2-regulated phosphatases, PP7 and PP2B (calcineurin). The EF-hand domains of PP7 and the calmodulin-binding domain of PP2B are essential for stimulation of the phosphatase activity by Ca+2. We present biochemical evidence that P. falciparum generates these full-length phosphatases as well as their catalytic cores, most likely as intermediates of a proteolytic degradation pathway. While the full-length phosphatases are activated by Ca+2, the processed cores are constitutively active and either less responsive or unresponsive to Ca+2. The processing is extremely rapid, specific, and occurs in vivo. Conclusions Post-translational cleavage efficiently degrades complex full-length phosphatases in P. falciparum. In the course of such degradation, enzymatically active catalytic cores are produced as relatively stable intermediates. The universality of such proteolysis in other phosphatases or other multi-domain proteins and its potential impact on the overall proteome of a cell merits further investigation. PMID:15230980

Crystallization and preliminary crystallographic analysis of two Streptococcus agalactiae proteins: the family II inorganic pyrophosphatase and the serine/threonine phosphatase

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

Rantanen, Mika K.; Lehtiö, Lari; Rajagopal, Lakshmi

Two S. agalactiae proteins, the inorganic pyrophosphatase and the serine/threonine phosphatase, were crystallized and diffraction data were collected and processed from these crystals. The data from the two protein crystals extended to 2.80 and 2.65 Å, respectively. Streptococcus agalactiae, which infects human neonates and causes sepsis and meningitis, has recently been shown to possess a eukaryotic-like serine/threonine protein phosphorylation signalling cascade. Through their target proteins, the S. agalactiae Ser/Thr kinase and Ser/Thr phosphatase together control the growth as well as the morphology and virulence of this organism. One of the targets is the S. agalactiae family II inorganic pyrophosphatase. Themore » inorganic pyrophosphatase and the serine/threonine phosphatase have therefore been purified and crystallized and diffraction data have been collected from their crystals. The data were processed using XDS. The inorganic pyrosphosphatase crystals diffracted to 2.80 Å and the Ser/Thr phosphatase crystals to 2.65 Å. Initial structure-solution experiments indicate that structure solution will be successful in both cases. Solving the structure of the proteins involved in this cascade is the first step towards understanding this phenomenon in atomic detail.« less

A peptide export-import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay.

PubMed

Perego, M

1997-08-05

The phosphorelay signal transduction system activates developmental transcription in sporulation of Bacillus subtilis by phosphorylation of aspartyl residues of the Spo0F and Spo0A response regulators. The phosphorylation level of these response regulators is determined by the opposing activities of protein kinases and protein aspartate phosphatases that interpret positive and negative signals for development in a signal integration circuit. The RapA protein aspartate phosphatase of the phosphorelay is regulated by a peptide that directly inhibits its activity. This peptide is proteolytically processed from an inactive pre-inhibitor protein encoded in the phrA gene. The pre-inhibitor is cleaved by the protein export apparatus to a putative pro-inhibitor that is further processed to the active inhibitor peptide and internalized by the oligopeptide permease. This export-import circuit is postulated to be a mechanism for timing phosphatase activity where the processing enzymes regulate the rate of formation of the active inhibitor. The processing events may, in turn, be controlled by a regulatory hierarchy. Chromosome sequencing has revealed several other phosphatase-prepeptide gene pairs in B. subtilis, suggesting that the use of this mechanism may be widespread in signal transduction.

Anchored phosphatases modulate glucose homeostasis

PubMed Central

Hinke, Simon A; Navedo, Manuel F; Ulman, Allison; Whiting, Jennifer L; Nygren, Patrick J; Tian, Geng; Jimenez-Caliani, Antonio J; Langeberg, Lorene K; Cirulli, Vincenzo; Tengholm, Anders; Dell'Acqua, Mark L; Santana, L Fernando; Scott, John D

2012-01-01

Endocrine release of insulin principally controls glucose homeostasis. Nutrient-induced exocytosis of insulin granules from pancreatic β-cells involves ion channels and mobilization of Ca2+ and cyclic AMP (cAMP) signalling pathways. Whole-animal physiology, islet studies and live-β-cell imaging approaches reveal that ablation of the kinase/phosphatase anchoring protein AKAP150 impairs insulin secretion in mice. Loss of AKAP150 impacts L-type Ca2+ currents, and attenuates cytoplasmic accumulation of Ca2+ and cAMP in β-cells. Yet surprisingly AKAP150 null animals display improved glucose handling and heightened insulin sensitivity in skeletal muscle. More refined analyses of AKAP150 knock-in mice unable to anchor protein kinase A or protein phosphatase 2B uncover an unexpected observation that tethering of phosphatases to a seven-residue sequence of the anchoring protein is the predominant molecular event underlying these metabolic phenotypes. Thus anchored signalling events that facilitate insulin secretion and glucose homeostasis may be set by AKAP150 associated phosphatase activity. PMID:22940692

Mechanism of phospho-ubiquitin induced PARKIN activation

PubMed Central

Wauer, Tobias; Simicek, Michal; Schubert, Alexander; Komander, David

2016-01-01

Summary The E3 ubiquitin ligase PARKIN (encoded by PARK2) and the protein kinase PINK1 (encoded by PARK6) are mutated in autosomal recessive juvenile Parkinsonism (AR-JP) and work together in the disposal of damaged mitochondria by mitophagy1–3. PINK1 is stabilised on the outside of depolarised mitochondria, and phosphorylates poly-ubiquitin (polyUb)4–8 as well as the PARKIN Ub-like (Ubl) domain9,10. These phosphorylation events lead to PARKIN recruitment to mitochondria, and activation by an unknown allosteric mechanism4–12. Here we present the crystal structure of Pediculus humanus PARKIN in complex with Ser65-phosphorylated ubiquitin (phosphoUb), revealing the molecular basis for PARKIN recruitment and activation. The phosphoUb binding site on PARKIN comprises a conserved phosphate pocket and harbours residues mutated in AR-JP patients. PhosphoUb binding leads to straightening of a helix in the RING1 domain, and the resulting conformational changes release the Ubl domain from the PARKIN core; this activates PARKIN. Moreover, phosphoUb-mediated Ubl release enhances Ubl phosphorylation by PINK1, leading to conformational changes within the Ubl domain and stabilisation of an open, active conformation of PARKIN. We redefine the role of the Ubl domain not only as an inhibitory13 but also as an activating element that is restrained in inactive PARKIN and released by phosphoUb. Our work opens new avenues to identify small molecule PARKIN activators. PMID:26161729

Protein tyrosine kinase regulation by ubiquitination: Critical roles of Cbl-family ubiquitin ligases

PubMed Central

Mohapatra, Bhopal; Ahmad, Gulzar; Nadeau, Scott; Zutshi, Neha; An, Wei; Scheffe, Sarah; Dong, Lin; Feng, Dan; Goetz, Benjamin; Arya, Priyanka; Bailey, Tameka A.; Palermo, Nicholas; Borgstahl, Gloria E.O.; Natarajan, Amarnath; Raja, Srikumar M.; Naramura, Mayumi; Band, Vimla; Band, Hamid

2012-01-01

Protein tyrosine kinases (PTKs) coordinate a broad spectrum of cellular responses to extracellular stimuli and cell–cell interactions during development, tissue homeostasis, and responses to environmental challenges. Thus, an understanding of the regulatory mechanisms that ensure physiological PTK function and potential aberrations of these regulatory processes during diseases such as cancer are of broad interest in biology and medicine. Aside from the expected role of phospho-tyrosine phosphatases, recent studies have revealed a critical role of covalent modification of activated PTKs with ubiquitin as a critical mechanism of their negative regulation. Members of the Cbl protein family (Cbl, Cbl-b and Cbl-c in mammals) have emerged as dominant “activated PTK-selective” ubiquitin ligases. Structural, biochemical and cell biological studies have established that Cbl protein-dependent ubiquitination targets activated PTKs for degradation either by facilitating their endocytic sorting into lysosomes or by promoting their proteasomal degradation. This mechanism also targets PTK signaling intermediates that become associated with Cbl proteins in a PTK activation-dependent manner. Cellular and animal studies have established that the relatively broadly expressed mammalian Cbl family members Cbl and Cbl-b play key physiological roles, including their critical functions to prevent the transition of normal immune responses into autoimmune disease and as tumor suppressors; the latter function has received validation from human studies linking mutations in Cbl to human leukemia. These newer insights together with embryonic lethality seen in mice with a combined deletion of Cbl and Cbl-b genes suggest an unappreciated role of the Cbl family proteins, and by implication the ubiquitin-dependent control of activated PTKs, in stem/progenitor cell maintenance. Future studies of existing and emerging animal models and their various cell lineages should help test the broader implications of the evolutionarily-conserved Cbl family protein-mediated, ubiquitin-dependent, negative regulation of activated PTKs in physiology and disease. PMID:23085373

IGF-I Stimulates Cooperative Interaction between the IGF-I Receptor and CSK Homologous Kinase that Regulates SHPS-1 Phosphorylation in Vascular Smooth Muscle Cells

PubMed Central

Radhakrishnan, Yashwanth; Shen, Xinchun; Maile, Laura A.; Xi, Gang

2011-01-01

IGF-I plays an important role in smooth muscle cell proliferation and migration. In vascular smooth muscle cells cultured in 25 mm glucose, IGF-I stimulated a significant increase in Src homology 2 domain containing protein tyrosine phosphatase substrate-1 (SHPS-1) phosphorylation compared with 5 mm glucose and this increase was required for smooth muscle cell proliferation. A proteome-wide screen revealed that carboxyl-terminal SRC kinase homologous kinase (CTK) bound directly to phosphotyrosines in the SHPS-1 cytoplasmic domain. Because the kinase(s) that phosphorylates these tyrosines in response to IGF-I is unknown, we determined the roles of IGF-I receptor (IGF-IR) and CTK in mediating SHPS-1 phosphorylation. After IGF-I stimulation, CTK was recruited to IGF-IR and subsequently to phospho-SHPS-1. Expression of an IGF-IR mutant that eliminated CTK binding reduced CTK transfer to SHPS-1, SHPS-1 phosphorylation, and cell proliferation. IGF-IR phosphorylated SHPS-1, which provided a binding site for CTK. CTK recruitment to SHPS-1 resulted in a further enhancement of SHPS-1 phosphorylation. CTK knockdown also impaired IGF-I-stimulated SHPS-1 phosphorylation and downstream signaling. Analysis of specific tyrosines showed that mutation of tyrosines 428/452 in SHPS-1 to phenylalanine reduced SHPS-1 phosphorylation but allowed CTK binding. In contrast, the mutation of tyrosines 469/495 inhibited IGF-IR-mediated the phosphorylation of SHPS-1 and CTK binding, suggesting that IGF-IR phosphorylated Y469/495, allowing CTK binding, and that CTK subsequently phosphorylated Y428/452. Based on the above findings, we conclude that after IGF-I stimulation, CTK is recruited to IGF-IR and its recruitment facilitates CTK's subsequent association with phospho-SHPS-1. This results in the enhanced CTK transfer to SHPS-1, and the two kinases then fully phosphorylate SHPS-1, which is necessary for IGF-I stimulated cellular proliferation. PMID:21799000

Angiotensin II receptor one (AT1) mediates dextrose induced endoplasmic reticulum stress and superoxide production in human coronary artery endothelial cells.

PubMed

Haas, Michael J; Onstead-Haas, Luisa; Lee, Tracey; Torfah, Maisoon; Mooradian, Arshag D

2016-10-01

Renin-angiotensin-aldosterone system (RAAS) has been implicated in diabetes-related vascular complications partly through oxidative stress. To determine the role of angiotensin II receptor subtype one (AT1) in dextrose induced endoplasmic reticulum (ER) stress, another cellular stress implicated in vascular disease. Human coronary artery endothelial cells with or without AT1 receptor knock down were treated with 27.5mM dextrose for 24h in the presence of various pharmacologic blockers of RAAS and ER stress and superoxide (SO) production were measured. Transfection of cells with AT1 antisense RNA knocked down cellular AT1 by approximately 80%. The ER stress was measured using the placental alkaline phosphatase (ES-TRAP) assay and western blot analysis of glucose regulated protein 78 (GRP78), c-jun-N-terminal kinase 1 (JNK1), phospho-JNK1, eukaryotic translation initiation factor 2α (eIF2α) and phospho-eIF2α measurements. Superoxide (SO) generation was measured using the superoxide-reactive probe 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride (MCLA) chemiluminescence. In cells with AT1 knock down, dextrose induced ER stress was significantly blunted and treatment with 27.5mM dextrose resulted in significantly smaller increase in SO production compared to 27.5mM dextrose treated and sham transfected cells. Dextrose induced ER stress was reduced with pharmacologic blockers of AT1 (losartan and candesartan) and mineralocorticoid receptor blocker (spironolactone) but not with angiotensin converting enzyme inhibitors (captopril and lisinopril). The dextrose induced SO generation was inhibited by all pharmacologic blockers of RAAS tested. The results indicate that dextrose induced ER stress and SO production in endothelial cells are mediated at least partly through AT1 receptor activation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Investigation of Dracocephalum kostchyi plant extract on the effective inflammatory transcription factors and mediators in activated macrophages.

PubMed

Kalantar, Kurosh; Gholijani, Nasser; Mousaei, Nashmin; Yazdani, Malihe; Amirghofran, Zahra

2018-06-07

Dracocephalum kotschyi is traditionally used for its anti-inflammatory effects. We aimed to investigate the effects of ethyl acetate extract of D. kotschyi on the expression of key inflammatory mediators and main signaling molecules involved in regulation of inflammation. Lipopolysaccharide (LPS)-stimulated J774.1 mouse macrophages were cultured in the presence of the plant extract and examined by the real time-PCR for gene expressions of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. Cytokine levels and phosphorylated forms of stress-activated protein kinases/c-Jun N-terminal kinase (SAPK/JNK), signal transducer and activator of transcription (STAT)-3, p38, IκB-α and nuclear factor (NF)-κB p65 were determined using ELISA. The extract significantly reduced the expression of key mediators of inflammation. iNOS expression level decreased from 138±8.5 fold in LPS-only treated cells to 6.5±2.6 fold after treatment with 25 ug/ml of the extract (p<0.001). Similarly, COX-2 expression decreased from 632 ±98.8 fold in control to 124 ±24.6 fold (p<0.01). Treatment of cells with the extract significantly reduced IL-1β and TNF-α cytokines at both gene and protein expression levels. The extract at 25 µg/ml caused significant decreases in phospho-SAPK/JNK and phospho-STAT3 levels in macrophages (p<0.01). Proteins of phospho-p38, NFκB-p65 and phospho-NF-κB p65 had a reduced levels in treated cells (p<0.05). No significant change in phospho-IĸB level was observed. These findings suggested that D. kotschyi with inhibition of NF-κB, SAPK/JNK, STAT-3 and p-38 might have reduced the expression levels of key inflammatory mediators and thus possibly have potential beneficial impact on inflammatory diseases. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Palmitate-induced ER stress and inhibition of protein synthesis in cultured myotubes does not require Toll-like receptor 4.

PubMed

Perry, Ben D; Rahnert, Jill A; Xie, Yang; Zheng, Bin; Woodworth-Hobbs, Myra E; Price, S Russ

2018-01-01

Saturated fatty acids, such as palmitate, are elevated in metabolically dysfunctional conditions like type 2 diabetes mellitus. Palmitate has been shown to impair insulin sensitivity and suppress protein synthesis while upregulating proteolytic systems in skeletal muscle. Increased sarco/endoplasmic reticulum (ER) stress and subsequent activation of the unfolded protein response may contribute to the palmitate-induced impairment of muscle protein synthesis. In some cell types, ER stress occurs through activation of the Toll-like receptor 4 (TLR4). Given the link between ER stress and suppression of protein synthesis, we investigated whether palmitate induces markers of ER stress and protein synthesis by activating TLR4 in cultured mouse C2C12 myotubes. Myotubes were treated with vehicle, a TLR4-specific ligand (lipopolysaccharides), palmitate, or a combination of palmitate plus a TLR4-specific inhibitor (TAK-242). Inflammatory indicators of TLR4 activation (IL-6 and TNFα) and markers of ER stress were measured, and protein synthesis was assessed using puromycin incorporation. Palmitate substantially increased the levels of IL-6, TNF-α, CHOP, XBP1s, and ATF 4 mRNAs and augmented the levels of CHOP, XBP1s, phospho-PERK and phospho-eIF2α proteins. The TLR4 antagonist attenuated both acute palmitate and LPS-induced increases in IL-6 and TNFα, but did not reduce ER stress signaling with either 6 h or 24 h palmitate treatment. Similarly, treating myotubes with palmitate for 6 h caused a 43% decline in protein synthesis consistent with an increase in phospho-eIF2α, and the TLR4 antagonist did not alter these responses. These results suggest that palmitate does not induce ER stress through TLR4 in muscle, and that palmitate impairs protein synthesis in skeletal muscle in part by induction of ER stress.

Phospho-TCTP as a therapeutic target of dihydroartemisinin for aggressive breast cancer cells

PubMed Central

Lucibello, Maria; Adanti, Sara; Antelmi, Ester; Dezi, Dario; Ciafrè, Stefania; Carcangiu, Maria Luisa; Zonfrillo, Manuela; Nicotera, Giuseppe; Sica, Lorenzo; De Braud, Filippo; Pierimarchi, Pasquale

2015-01-01

Upregulation of Translationally Controlled Tumor Protein (TCTP) is associated with poorly differentiated aggressive tumors, including breast cancer, but the underlying mechanism(s) are still debated. Here, we show that in breast cancer cell lines TCTP is primarily localized in the nucleus, mostly in the phosphorylated form. The effects of Dihydroartemisinin (DHA), an anti-malaria agent that binds TCTP, were tested on breast cancer cells. DHA decreases cell proliferation and induces apoptotic cell death by targeting the phosphorylated form of TCTP. Remarkably, DHA enhances the anti-tumor effects of Doxorubicin in triple negative breast cancer cells resulting in an increased level of apoptosis. DHA also synergizes with Trastuzumab, used to treat HER2/neu positive breast cancers, to induce apoptosis of tumor cells. Finally, we present new clinical data that nuclear phospho-TCTP overexpression in primary breast cancer tissue is associated with high histological grade, increase expression of Ki-67 and with ER-negative breast cancer subtypes. Notably, phospho-TCTP expression levels increase in trastuzumab-resistant breast tumors, suggesting a possible role of phospho-TCTP as a new prognostic marker. In conclusion, the anti-tumor effect of DHA in vitro with conventional chemotherapeutics suggests a novel therapeutic strategy and identifies phospho-TCTP as a new promising target for advanced breast cancer. PMID:25779659

Association of protein tyrosine phosphatase, non-receptor type 22 +1858C→T polymorphism and susceptibility to vitiligo: Systematic review and meta-analysis.

PubMed

Agarwal, Silky; Changotra, Harish

2017-01-01

Protein tyrosine phosphatase, non-receptor type 22 gene, which translates to lymphoid tyrosine phosphatase, is considered to be a susceptibility gene marker associated with several autoimmune diseases. Several studies have demonstrated the association of protein tyrosine phosphatase, non-receptor type 22 +1858C→T polymorphism with vitiligo. However, these studies showed conflicting results. Meta-analysis of the same was conducted earlier that included fewer number of publications in their study. We performed a meta-analysis of a total of seven studies consisting of 2094 cases and 3613 controls to evaluate the possible association of protein tyrosine phosphatase, non-receptor type 22 +1858C>T polymorphism with vitiligo susceptibility. We conducted a literature search in PubMed, Google Scholar and Dogpile for all published paper on protein tyrosine phosphatase, non-receptor type 22 +1858C→T polymorphism and vitiligo risk till June 2016. Data analysis was performed by RevMan 5.3 and comprehensive meta-analysis v3.0 software. Meta-analysis showed an overall significant association of protein tyrosine phosphatase, non- receptor type 22 +1858C→T polymorphism with vitiligo in all models (allelic model [T vs. C]: odds ratio = 1.50, 95% confidence interval [1.32-1.71], P< 0.001; dominant model [TT + CT vs. CC]: odds ratio = 1.61, 95% confidence interval [1.16-2.24], P = 0.004; recessive model [TT vs. CT + CC]: odds ratio = 4.82, 95% confidence interval [1.11-20.92], P = 0.04; homozygous model [TT vs. CC]: odds ratio = 5.34, 95% confidence interval [1.23-23.24], P = 0.03; co-dominant model [CT vs. CC]: odds ratio = 1.52, 95% confidence interval [1.09-2.13], P = 0.01). No publication bias was detected in the funnel plot study. Limited ethnic-based studies, unable to satisfy data by gender or vitiligo-type are some limitations of the present meta-analysis. Stratifying data by ethnicity showed an association of protein tyrosine phosphatase, non-receptor type 22 +1858C→T with vitiligo in European population (odds ratio = 1.53, 95% confidence interval [1.34-1.75], P< 0.001) but not in Asian population (odds ratio = 0.59, 95% confidence interval [0.26-1.32], P = 0.2). In conclusion, protein tyrosine phosphatase, non-receptor type 22 +1858 T allele predisposes European individuals to vitiligo.

Applying a Targeted Label-free Approach using LC-MS AMT Tags to Evaluate Changes in Protein Phosphorylation Following Phosphatase Inhibition

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

Yang, Feng; Jaitly, Navdeep; Jayachandran, Hemalatha

2007-10-12

To identify phosphoproteins regulated by the phosphoprotein phosphatase (PPP) family of S/T phosphatases, we performed a large-scale characterization of changes in protein phosphorylation on extracts from HeLa cells treated with or without calyculin A, a potent PPP enzyme inhibitor. A label-free comparative Phosphoproteomics approach using immobilized metal ion affinity chromatography and targeted tandem mass spectrometry was employed to discover and identify signatures based upon distinctive changes in abundance. Overall, 232 proteins were identified as either direct or indirect targets for PPP enzyme regulation. Most of the present identifications represent novel PPP enzyme targets at the level of both phosphorylation sitemore » and protein. These include phosphorylation sites within signaling proteins such as p120 Catenin, A Kinase Anchoring Protein 8, JunB, and Type II Phosphatidyl Inositol 4 Kinase. These data can be used to define underlying signaling pathways and events regulated by the PPP family of S/T phosphatases.« less

Entire mitogen activated protein kinase (MAPK) pathway is present in preimplantation mouse embryos.

PubMed

Wang, Yingchun; Wang, Fangfei; Sun, Tong; Trostinskaia, Anna; Wygle, Dana; Puscheck, Elizabeth; Rappolee, Daniel A

2004-09-01

To understand how mitogenic signals are transduced into the trophoblasts in preimplantation embryos, the expression of mitogen-activated protein kinase (MAPK) pathway molecules was tested. We used immunocytochemical means and reverse transcriptase-polymerase chain reaction to test whether MAPK pathway molecule gene products exist at the protein and phosphoprotein level in the zygote and the RNA level in the egg and zygote. In addition, all antibodies detected the correct-sized major band in Westerns of placental cell lines representing the most prevalent cell type in preimplantation embryos. A majority of mRNA transcripts of MAPK pathway genes were detected in unfertilized eggs, and all were expressed in the zygote. We found that the MAPK pathway protein set consisting of the following gene products was present: FRS2 alpha, GRB2, GAB1, SOS1, Ha-ras, Raf1/RafB, MEK1,2,5, MAPK/ERK1,2, MAPK/ERK5, and RSK1,2,3 (see abbreviations). These proteins were detected in trophoblasts in embryonic day (E) 3.5 embryos when they could mediate mitogenic fibroblast growth factor signals from the embryo or colony stimulating factor-1 signals from the uterus. The phosphorylation state and position of the phosphoproteins in the cells suggested that they might function in mediating mitogenic signals. Interestingly, a subtle transition from maternal MAPK function to zygotic function was suggested by the localization for three MAPK pathway enzymes between E2.5 and E3.5, Raf1 phospho is largely cell membrane-localized at E2.5 and E3.5, and MEK1,2 phospho accumulates in the nucleus on E2.5 and E3.5. However, MAPK phospho shifts from nuclear accumulation at E2.5 to cytoplasmic accumulation at E3.5. This finding is similar to the cytoplasmic MAPK phospho localization reported in fibroblast growth factor signaling fields in postimplantation embryos (Corson et al. [2003] Development 130:4527-4537). This spatial and temporal expression study lays a foundation to plan and analyze perturbation studies aimed at understanding the role of the major mitogenic pathway in preimplantation mouse embryos.

Deep Insight into the Phosphatomes of Parasitic Protozoa and a Web Resource ProtozPhosDB.

PubMed

Anwar, Tamanna; Gourinath, Samudrala

2016-01-01

Phosphorylation dynamically regulates the function of proteins by maintaining a balance between protein kinase and phosphatase activity. A comprehensive understanding of the role phosphatases in cellular signaling is lacking in case of protozoans of medical and veterinary importance worldwide. The drugs used to treat protozoal diseases have many undesired effects and the development of resistance, highlights the need for new effective and safer antiprotozoal agents. In the present study we have analyzed phosphatomes of 15 protozoans of medical significance. We identified ~2000 phosphatases, out of which 21% are uncharacterized proteins. A significant positive correlation between phosphatome and proteome size was observed except for E. histolytica, having highest density of phosphatases irrespective of its proteome size. A difference in the number of phosphatases among different genera shows the variation in the signaling pathways they are involved in. The phosphatome of parasites is dominated by ser/thr phosphatases contrary to the vertebrate host dominated by tyrosine phosphatases. Phosphatases were widely distributed throughout the cell suggesting physiological adaptation of the parasite to regulate its host. 20% to 45% phosphatome of different protozoa consists of ectophosphatases, i.e. crucial for the survival of parasites. A database and a webserver "ProtozPhosDB" can be used to explore the phosphatomes of protozoans of medical significance.

Pro-oncogenic function of HIP-55/Drebrin-like (DBNL) through Ser269/Thr291-phospho-sensor motifs

PubMed Central

Park, Hae Ryon; Shi, Zhi; Li, Zenggang; Pham, Cau Dinh; Du, Yuhong; Khuri, Fadlo R.; Zhang, Youyi; Han, Qide

2014-01-01

HIP-55 (HPK1-interacting protein of 55 kDa, also named DBNL, SH3P7, and mAbp1) is a multidomain adaptor protein that is critical for organ development and the immune response. Here, we report the coupling of HIP-55 to cell growth control through its 14-3-3-binding phospho-Ser/Thr-sensor sites. Using affinity chromatography, we found HIP-55 formed a complex with 14-3-3 proteins, revealing a new node in phospho-Ser/Thr-mediated signaling networks. In addition, we demonstrated that HIP-55 is required for proper cell growth control. Enforced HIP-55 expression promoted proliferation, colony formation, migration, and invasion of lung cancer cells while silencing of HIP-55 reversed these effects. Importantly, HIP-55 was found to be upregulated in lung cancer cell lines and in tumor tissues of lung cancer patients. Upregulated HIP-55 was required to promote the growth of tumors in a xenograft animal model. However, tumors with S269A/T291A-mutated HIP-55, which ablates 14-3-3 binding, exhibited significantly reduced sizes, supporting a vital role of the HIP-55/14-3-3 protein interaction node in transmitting oncogenic signals. Mechanistically, HIP-55-mediated tumorigenesis activity appears to be in part mediated by antagonizing the tumor suppressor function of HPK1. Thus, the HIP-55–mediated oncogenic pathway, through S269/T291, may be exploited for the development of new therapeutic strategies. PMID:24912570

Perturbing NR2B-PSD-95 interaction relieves neuropathic pain by inactivating CaMKII-CREB signaling.

PubMed

Xu, Fangxia; Zhao, Xin; Liu, Lin; Song, Jia; Zhu, Yingjun; Chu, Shuaishuai; Shao, Xueming; Li, Xiuxiu; Ma, Zhengliang; Gu, Xiaoping

2017-09-06

Neuropathic pain is characterized by central sensitization. The interaction between N-methyl-D-aspartate receptors (NMDARs) and postsynaptic density protein-95 (PSD-95) plays a major role in central sensitization. Here, we aimed to investigate the analgesic effect of disruption of the interaction between NMDAR and PSD-95. Chronic dorsal root ganglia compression model rats were used to mimic sciatica. Thermal hyperalgesia and mechanical allodynia were evaluated. The expression of spinal phospho-NR2B, PSD-95, calcium/calmodulin-dependent protein kinase II (CaMKII), and cAMP response element binding protein (CREB) was measured using western blotting. A mimetic peptide Myr-NR2B9c was injected intrathecally to disrupt the interaction between PSD-95 and NR2B and detected by coimmunoprecipitation. Chronic dorsal root ganglia compression surgery induced thermal hyperalgesia and mechanical allodynia, and upregulated pain-related proteins such as phospho-NR2B, PSD-95, CaMKII, and CREB expressions in the spinal cord. Myr-NR2B9c disrupted the interaction between NR2B-containing NMDARs and PSD-95 in the spinal cord. Intrathecal administration of Myr-NR2B9c attenuated neuropathic pain behaviors and downregulated the expressions of phospho-NR2B, PSD-95, CaMKII, and CREB in the spinal cord. The present study indicates that dissociation of NR2B-containing NMDARs from PSD-95 inactivates CaMKII and CREB signaling and relieves pain.

Proteomic analysis of protein phosphatase Z1 from Candida albicans

PubMed Central

Pfliegler, Walter P.; Petrényi, Katalin; Boros, Enikő; Pócsi, István; Tőzsér, József; Dombrádi, Viktor

2017-01-01

Protein phosphatase Z is a “novel type” fungus specific serine/threonine protein phosphatase. Previously our research group identified the CaPPZ1 gene in the opportunistic pathogen Candida albicans and reported that the gene deletion had several important physiological consequences. In order to reveal the protein targets and the associated mechanisms behind the functions of the phosphatase a proteomic method was adopted for the comparison of the cappz1 deletion mutant and the genetically matching QMY23 control strain. Proteins extracted from the control and deletion mutant strains were separated by two-dimensional gel electrophoresis and the protein spots were stained with RuBPS and Pro-Q Diamond in order to visualize the total proteome and the phosphoproteome, respectively. The alterations in spot intensities were determined by densitometry and were analysed with the Delta2D (Decodon) software. Spots showing significantly different intensities between the mutant and control strains were excised from the gels and were digested with trypsin. The resulting peptides were identified by LC-MS/MS mass spectrometry. As many as 15 protein spots were found that exhibited significant changes in their intensity upon the deletion of the phosphatase and 20 phosphoproteins were identified in which the level of phosphorylation was modified significantly in the mutant. In agreement with previous findings we found that the affected proteins function in protein synthesis, oxidative stress response, regulation of morphology and metabolism. Among these proteins we identified two potential CaPpz1 substrates (Eft2 and Rpp0) that may regulate the elongation step of translation. RT-qPCR experiments revealed that the expression of the genes coding for the affected proteins was not altered significantly. Thus, the absence of CaPpz1 exerted its effects via protein synthesis/degradation and phosphorylation/dephosphorylation. In addition, our proteomics data strongly suggested a role for CaPpz1 in biofilm formation, was confirmed experimentally. Thus our unbiased proteomic approach lead to the discovery of a novel function for this phosphatase in C. albicans. PMID:28837603

Noni (Morinda citrifolia L.) fruit juice delays immunosenescence in the lymphocytes in lymph nodes of old F344 rats.

PubMed

Pratap, Uday P; Priyanka, Hannah P; Ramanathan, Karthik R; Raman, Vishak; Hima, Lalgi; Thyagarajan, Srinivasan

2018-05-01

Aging is associated with the development of diseases because of immunosuppression and altered functioning of the neuroendocrine system. The medicinal properties of Morinda citrifolia L. have been widely exploited for the treatment of age-associated diseases. This study aims to investigate the in vitro and in vivo effects of noni (M. citrifolia) fruit juice (NFJ) on neuro-immunomodulation in the lymph node lymphocytes of F344 rats. Lymphocytes isolated from axillary and inguinal lymph nodes of young (3-4 months) and old (18-21 months) rats were treated in vitro with different concentrations (0.0001%, 0.01%, and 1%) of NFJ for a period of 24 h. In the in vivo study, old (16-17 months) male F344 rats were treated with 5 mL/kg body weight of 5%, 10% and 20% of NFJ, twice a day, by oral gavage, and lymph node lymphocytes were isolated after 60 d. Concanavalin A (Con A)-induced lymphocyte proliferation, interleukin-2 (IL-2) and interferon-γ (IFN-γ) production and expression of intracellular markers, such as phospho-extracellular signal-regulated kinase (p-ERK1/2), phospho-cAMP response element-binding protein, phospho-protein kinase B (p-Akt), phospho-tyrosine hydroxylase (p-TH), phospho-nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor-α (p-IκB-α) and phospho-nuclear factor-κB (p-NF-κB p65 and p50) were examined in the lymphocytes of lymph nodes. NFJ increased Con A-induced lymphocyte proliferation, IL-2 and IFN-γ production, and p-ERK1/2 expression both in vitro and in vivo. In in vivo NFJ-treated old rats, lymph node lymphocytes showed increased expression of p-TH and Akt, nitric oxide production and decreased expression of p-NF-κB p65 and p50. These results suggest that the immunostimulatory properties of NFJ are facilitated through intracellular signaling pathways involving ERK1/2, Akt and NF-κB. Copyright © 2018 Shanghai Changhai Hospital. Published by Elsevier B.V. All rights reserved.

Norartocarpetin from a folk medicine Artocarpus communis plays a melanogenesis inhibitor without cytotoxicity in B16F10 cell and skin irritation in mice

PubMed Central

2013-01-01

Background Many natural products used in preventive medicine have also been developed as cosmeceutical ingredients in skin care products, such as Scutellaria baicalensis and Gardenia jasminoides. Norartocarpetin is one of the antioxidant and antityrosinase activity compound in Artocarpus communis; however, the cytotoxicity, skin irritation and antimelanogenesis mechanisms of norartocarpetin have not been investigated yet. Methods In the present study, cell viability in vitro and skin irritation in vivo are used to determine the safety of norartocarpetin. The melanogenesis inhibition of norartocarpetin was determined by cellular melanin content and tyrosinase in B16F10 melanoma cell. Moreover, we examined the related-melanogenesis protein by western blot analysis for elucidating the antimelanogenesis mechanism of norartocarpin. Results The result of the present study demonstrated that norartocarpetin not only present non-cytotoxic in B16F10 and human fibroblast cells but also non-skin irritation in mice. Moreover, our result also first found that norartocarpetin downregulated phospho-cAMP response element-binding (phospho-CREB) and microphthalmia-associated transcription factor (MITF) expression, which in turn decreased both synthesis of tyrosinases (TRP-1 and TRP-2) and cellular melanin content. This process is dependent on norartocarpetin phosphorylation by mitogen-activated protein kinases such as phospho-JNK and phospho-p38, and it results in decreased melanogenesis. Conclusion The present study suggests that norartocarpetin could be used as a whitening agent in medicine and/or cosmetic industry and need further clinical study. PMID:24325567

Vanadate monomers and dimers both inhibit the human prostatic acid phosphatase.

PubMed

Crans, D C; Simone, C M; Saha, A K; Glew, R H

1989-11-30

A combination of enzyme kinetics and 51V NMR spectroscopy was used to identify the species of vanadate that inhibits acid phosphatases. Monomeric vanadate was shown to inhibit wheat germ and potato acid phosphatases. At pH 5.5, the vanadate dimer inhibits the human prostatic acid phosphatase whereas at pH 7.0 it is the vanadate monomer that inhibits this enzyme. The pH-dependent shift in the affinity of the prostatic phosphatase for vanadate is presumably due to deprotonation of an amino acid side chain in or near the binding site resulting in a conformational change in the protein. pH may be a subtle effector of the insulin-like vanadate activity in biological systems and may explain some of the differences in selectivity observed with the protein phosphatases.

Regulation of Src homology 2-containing tyrosine phosphatase 1 during activation of human neutrophils. Role of protein kinase C.

PubMed

Brumell, J H; Chan, C K; Butler, J; Borregaard, N; Siminovitch, K A; Grinstein, S; Downey, G P

1997-01-10

The tyrosine phosphorylation of several proteins induced in neutrophils by soluble and particulate stimuli is thought to be crucial for initiating antimicrobial responses. Although activation of tyrosine kinases is thought to mediate this event, the role of tyrosine phosphatases in the initiation and modulation of neutrophil responses remains largely undefined. We investigated the role of Src homology 2-containing tyrosine phosphatase 1 (SHP-1; also known as protein tyrosine phosphatase 1C (PTP1C), hematopoetic cell phosphatase, PTP-N6, and SHPTP-1), a phosphatase expressed primarily in hemopoietic cells, in the activation of human neutrophils. SHP-1 mRNA and protein were detected in these cells, and the enzyme was found to be predominantly localized to the cytosol in unstimulated cells. Following stimulation with neutrophil agonists such as phorbol ester, chemotactic peptide, or opsonized zymosan, a fraction of the phosphatase redistributed to the cytoskeleton. Agonist treatment also induced significant decreases (30-60%) in SHP-1 activity, which correlated temporally with increases in the cellular phosphotyrosine content. Phosphorylation of SHP-1 on serine residues was associated with the inhibition of its enzymatic activity, suggesting a causal relationship. Accordingly, both the agonist-evoked phosphorylation of SHP-1 and the inhibition of its catalytic activity were blocked by treatment with bisindolylmaleimide I, a potent and specific inhibitor of protein kinase C (PKC) activity. Immunoprecipitated SHP-1 was found to be phosphorylated efficiently by purified PKC in vitro. Such phosphorylation also caused a decrease in the phosphatase activity of SHP-1. Together, these data suggest that inhibition of SHP-1 by PKC-mediated serine phosphorylation plays a role in facilitating the accumulation of tyrosine-phosphorylated proteins following neutrophil stimulation. These findings provide a new link between the PKC and tyrosine phosphorylation branches of the signaling cascade that triggers antimicrobial responses in human neutrophils.

Crystallization and preliminary X-ray diffraction analysis of a high-affinity phosphate-binding protein endowed with phosphatase activity from Pseudomonas aeruginosa PAO1

PubMed Central

Djeghader, Ahmed; Gotthard, Guillaume; Suh, Andrew; Gonzalez, Daniel; Scott, Ken; Chabriere, Eric; Elias, Mikael

2013-01-01

In prokaryotes, phosphate starvation induces the expression of numerous phosphate-responsive genes, such as the pst operon including the high-affinity phosphate-binding protein (PBP or pstS) and alkaline phosphatases such as PhoA. This response increases the cellular inorganic phosphate import efficiency. Notably, some Pseudomonas species secrete, via a type-2 secretion system, a phosphate-binding protein dubbed LapA endowed with phosphatase activity. Here, the expression, purification, crystallization and X-ray data collection at 0.87 Å resolution of LapA are described. Combined with biochemical and enzymatic characterization, the structure of this intriguing phosphate-binding protein will help to elucidate the molecular origin of its phosphatase activity and to decipher its putative role in phosphate uptake. PMID:24100568

SHIP, a novel factor to ameliorate extracellular matrix accumulation via suppressing PI3K/Akt/CTGF signaling in diabetic kidney disease.

PubMed

Li, Fan; Li, Lisha; Cheng, Meijuan; Wang, Xiumin; Hao, Jun; Liu, Shuxia; Duan, Huijun

2017-01-22

Tubular interstitial extracellular matrix accumulation, which plays a key role in the pathogenesis and progression of diabetic kidney disease (DKD), is believed to be mediated by activation of PI3K/Akt signal pathway. However, it is still not clear whether SH2 domain-containing inositol 5'-phosphatase (SHIP), known as a negative regulator of PI3K/Akt pathway is also involved in extracellular matrix metabolism of diabetic kidney. In the present study, decreased SHIP and increased phospho-Akt (Ser 473, Thr 308) were found in renal tubular cells of diabetic mice accompanied by overexpression of connective tissue growth factor (CTGF) and extracellular matrix deposition versus normal mice. Again, high glucose attenuated SHIP expression in a time-dependent manner, concomitant with activation of PI3K/Akt signaling and extracellular matrix production in human renal proximal tubular epithelial cells (HK2) cultured in vitro, which was significantly prevented by transfection of M90-SHIP vector. Furthermore, in vivo delivery of rAd-INPP5D vector (SHIP expression vector) via intraperitoneal injection in diabetic mice increased SHIP expression by 3.36 times followed by 65.26%, 70.38% and 46.71% decreases of phospho-Akt (Ser 473), phospho-Akt (Thr 308) and CTGF expression versus diabetic mice receiving rAd-EGFP vector. Meanwhile, increased renal extracellular matrix accumulation of diabetic mice was also inhibited with intraperitoneal injection of rAd-INPP5D vector. These above data suggested that overexpression of SHIP might be a potent method to lessen renal extracellular matrix accumulation via inactivation of PI3K/Akt pathway and suppression of CTGF expression in DKD. Copyright © 2016 Elsevier Inc. All rights reserved.

Reactivation of oxidized PTP1B and PTEN by Thioredoxin 1

PubMed Central

Schwertassek, Ulla; Haque, Aftabul; Krishnan, Navasona; Greiner, Romy; Weingarten, Lars; Dick, Tobias P.; Tonks, Nicholas K.

2014-01-01

The transient inactivation of protein phosphatases contributes to the efficiency and temporal control of kinase-dependent signal transduction. In particular, members of the protein tyrosine phosphatase family are known to undergo reversible oxidation of their active site cysteine. The thiol oxidation step requires activation of co-localized NADPH oxidases and is mediated by locally produced ROS, in particular H2O2. How oxidized phosphatases are returned to the reduced active state is less well studied. Both major thiol reductive systems, the thioredoxin and the glutathione systems, have been implicated in the reactivation of phosphatases. Here, we show that the protein tyrosine phosphatase PTP1B and the dual-specificity phosphatase PTEN are preferentially reactivated by the thioredoxin system. We show that inducible depletion of TRX1 slows down PTEN re-activation in intact living cells. Finally, using a mechanism-based trapping approach we demonstrate direct thiol disulfide exchange between the active sites of thioredoxin and either phosphatase. The application of thioredoxin trapping mutants represents a complementary approach to direct assays of PTP oxidation in elucidating the significance of redox regulation of PTP function in the control of cell signaling. PMID:24976139

Monitoring UV-induced signalling pathways in an ex vivo skin organ culture model using phospho-antibody array.

PubMed

Lenain, Christelle; Gamboa, Bastien; Perrin, Agnes; Séraïdaris, Alexia; Bertino, Béatrice; Rival, Yves; Bernardi, Mathieu; Piwnica, David; Méhul, Bruno

2018-05-01

We investigated UV-induced signalling in an ex vivo skin organ culture model using phospho-antibody array. Phosphorylation modulations were analysed in time-course experiments following exposure to solar-simulated UV and validated by Western blot analyses. We found that UV induced P-p38 and its substrates, P-ERK1/2 and P-AKT, which were previously shown to be upregulated by UV in cultured keratinocytes and in vivo human skin. This indicates that phospho-antibody array applied to ex vivo skin organ culture is a relevant experimental system to investigate signalling events following perturbations. As the identified proteins are components of pathways implicated in skin tumorigenesis, UV-exposed skin organ culture model could be used to investigate the effect on these pathways of NMSC cancer drug candidates. In addition, we found that phospho-HCK is induced upon UV exposure, producing a new candidate for future studies investigating its role in the skin response to UV and UV-induced carcinogenesis. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Reduced expression of CD45 Protein-Tyrosine Phosphatase Pr

DTIC Science & Technology

2009-05-08

H S /D T R A on A ugust 19, 2009 w w w .jbc.org D ow nloaded from PTP1B , CD45, TCPTP, LMPTP-A, LMPTP-B, MEG1, MEG2, HePTP, PTP), three belong to...the dual specificity phosphatase VHR or the protein-tyrosine phosphatase PTP1B . Given these FIGURE 5. Mice expressing intermediate CD45 levels survive

Structural Basis for the Catalytic Activity of Human Serine/Threonine Protein Phosphatase type 5 (PP5)

NASA Technical Reports Server (NTRS)

Swingle, Mark R.; Ciszak, Ewa M.; Honkanen, Richard E.

2004-01-01

Serine/threonine protein phosphatase-5 (PP5) is a member of the PPP-gene family of protein phosphatases that is widely expressed in mammalian tissues and is highly conserved among eukaryotes. PP5 associates with several proteins that affect signal transduction networks, including the glucocorticoid receptor (GR)-heat shock protein-90 (Hsp90)-heterocomplex, the CDC16 and CDC27 subunits of the anaphase-promoting complex, elF2alpha kinase, the A subunit of PP2A, the G12-alpha / G13-alpha subunits of heterotrimeric G proteins and DNA-PK. The catalytic domain of PP5 (PP5c) shares 35-45% sequence identity with the catalytic domains of other PPP-phosphatases, including protein phosphatase-1 (PP1), -2A (PP2A), -2B / calcineurin (PP2B), -4 (PP4), -6 (PP6), and -7 (PP7). Like PP1, PP2A and PP4, PP5 is also sensitive to inhibition by okadaic acid, microcystin, cantharidin, tautomycin, and calyculin A. Here we report the crystal structure of the PP5 catalytic domain (PP5c) at a resolution of 1.6 angstroms. From this structure we propose a mechanism for PP5-mediated hydrolysis of phosphoprotein substrates, which requires the precise positioning of two metal ions within a conserved Asp(sup 271)-M(sub 1):M(sub 2)-W(sup 1)-His(sup 304)-Asp(sup 274) catalytic motif. The structure of PP5c provides a possible structural basis for explaining the exceptional catalytic proficiency of protein phosphatases, which are among the most powerful known catalysts. Resolution of the entire C-terminus revealed a novel subdomain, and the structure of the PP5c should also aid development of type-specific inhibitors.

Conservation of the PTEN catalytic motif in the bacterial undecaprenyl pyrophosphate phosphatase, BacA/UppP.

PubMed

Bickford, Justin S; Nick, Harry S

2013-12-01

Isoprenoid lipid carriers are essential in protein glycosylation and bacterial cell envelope biosynthesis. The enzymes involved in their metabolism (synthases, kinases and phosphatases) are therefore critical to cell viability. In this review, we focus on two broad groups of isoprenoid pyrophosphate phosphatases. One group, containing phosphatidic acid phosphatase motifs, includes the eukaryotic dolichyl pyrophosphate phosphatases and proposed recycling bacterial undecaprenol pyrophosphate phosphatases, PgpB, YbjB and YeiU/LpxT. The second group comprises the bacterial undecaprenol pyrophosphate phosphatase, BacA/UppP, responsible for initial formation of undecaprenyl phosphate, which we predict contains a tyrosine phosphate phosphatase motif resembling that of the tumour suppressor, phosphatase and tensin homologue (PTEN). Based on protein sequence alignments across species and 2D structure predictions, we propose catalytic and lipid recognition motifs unique to BacA/UppP enzymes. The verification of our proposed active-site residues would provide new strategies for the development of substrate-specific inhibitors which mimic both the lipid and pyrophosphate moieties, leading to the development of novel antimicrobial agents.

Memantine Attenuates Alzheimer’s Disease-Like Pathology and Cognitive Impairment

PubMed Central

Wang, Xiaochuan; Blanchard, Julie; Iqbal, Khalid

2015-01-01

Deficiency of protein phosphatase-2A is a key event in Alzheimer’s disease. An endogenous inhibitor of protein phosphatase-2A, inhibitor-1, I1 PP2A, which inhibits the phosphatase activity by interacting with its catalytic subunit protein phosphatase-2Ac, is known to be upregulated in Alzheimer’s disease brain. In the present study, we overexpressed I1 PP2A by intracerebroventricular injection with adeno-associated virus vector-1-I1 PP2A in Wistar rats. The I1 PP2A rats showed a decrease in brain protein phosphatase-2A activity, abnormal hyperphosphorylation of tau, neurodegeneration, an increase in the level of activated glycogen synthase kinase-3beta, enhanced expression of intraneuronal amyloid-beta and spatial reference memory deficit; littermates treated identically but with vector only, i.e., adeno-associated virus vector-1-enhanced GFP, served as a control. Treatment with memantine, a noncompetitive NMDA receptor antagonist which is an approved drug for treatment of Alzheimer’s disease, rescued protein phosphatase-2A activity by decreasing its demethylation at Leu309 selectively and attenuated Alzheimer’s disease-like pathology and cognitive impairment in adeno-associated virus vector-1-I1 PP2A rats. These findings provide new clues into the possible mechanism of the beneficial therapeutic effect of memantine in Alzheimer’s disease patients. PMID:26697860

An RNAi Screen To Identify Protein Phosphatases That Function Within the Drosophila Circadian Clock.

PubMed

Agrawal, Parul; Hardin, Paul E

2016-12-07

Circadian clocks in eukaryotes keep time via cell-autonomous transcriptional feedback loops. A well-characterized example of such a transcriptional feedback loop is in Drosophila, where CLOCK-CYCLE (CLK-CYC) complexes activate transcription of period (per) and timeless (tim) genes, rising levels of PER-TIM complexes feed-back to repress CLK-CYC activity, and degradation of PER and TIM permits the next cycle of CLK-CYC transcription. The timing of CLK-CYC activation and PER-TIM repression is regulated posttranslationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Previous behavioral screens identified several kinases that control CLK, PER, and TIM levels, subcellular localization, and/or activity, but two phosphatases that function within the clock were identified through the analysis of candidate genes from other pathways or model systems. To identify phosphatases that play a role in the clock, we screened clock cell-specific RNA interference (RNAi) knockdowns of all annotated protein phosphatases and protein phosphatase regulators in Drosophila for altered activity rhythms. This screen identified 19 protein phosphatases that lengthened or shortened the circadian period by ≥1 hr (p ≤ 0.05 compared to controls) or were arrhythmic. Additional RNAi lines, transposon inserts, overexpression, and loss-of-function mutants were tested to independently confirm these RNAi phenotypes. Based on genetic validation and molecular analysis, 15 viable protein phosphatases remain for future studies. These candidates are expected to reveal novel features of the circadian timekeeping mechanism in Drosophila that are likely to be conserved in all animals including humans. Copyright © 2016 Agrawal and Hardin.

Dysfunction of protein kinase FA/GSK-3 alpha in lymphocytes of patients with schizophrenic disorder.

PubMed

Yang, S D; Yu, J S; Lee, T T; Yang, C C; Ni, M H; Yang, Y Y

1995-09-01

As compared to normal people, the lymphocytes of patients with schizophrenia were found to have an impairment of ATP.Mg-dependent protein phosphatase activation. More importantly, the impaired protein phosphatase activation in the lymphocytes of schizophrenic patients could be consistently and completely restored to normal by exogenous pure protein kinase FA/glycogen synthase kinase-3 alpha (kinase FA/GSK-3 alpha) (the activating factor of ATP.Mg-dependent protein phosphatase), indicating that the molecular mechanism for the impaired protein phosphatase activation in schizophrenic patients may be due to a functional loss of kinase FA/GSK-3 alpha. Immunoblotting and kinase activity analysis in an anti-kinase FA/GSK-3 alpha immunoprecipitate further demonstrate that both cellular activities and protein levels of kinase FA/GSK-3 alpha in the lymphocytes of schizophrenic patients were greatly impared as compared to normal controls. Statistical analysis revealed that the lymphocytes isolated from 37 normal people contain kinase FA/GSK-3 alpha activity in the high levels of 14.8 +/- 2.4 units/mg of cell protein, whereas the lymphocytes of 48 patients with schizophrenic disorder contain kinase FA/GSK-3 alpha activity in the low levels of 2.8 +/- 1.6 units/mg, indicating that the different levels of kinase FA/GSK-3 alpha activity between schizophrenic patients and normal people are statistically significant. Taken together, the results provide initial evidence that patients with schizophrenic disorder may have a common impairment in the protein levels and cellular activities of kinase FA/GSK-3 alpha, a multisubstrate protein kinase and a multisubstrate protein phosphatase activator in their lymphocytes.

Use of double-stranded RNA-mediated interference to determine the substrates of protein tyrosine kinases and phosphatases.

PubMed

Muda, Marco; Worby, Carolyn A; Simonson-Leff, Nancy; Clemens, James C; Dixon, Jack E

2002-08-15

Despite the wealth of information generated by genome-sequencing projects, the identification of in vivo substrates of specific protein kinases and phosphatases is hampered by the large number of candidate enzymes, overlapping enzyme specificity and sequence similarity. In the present study, we demonstrate the power of RNA interference (RNAi) to dissect signal transduction cascades involving specific kinases and phosphatases. RNAi is used to identify the cellular tyrosine kinases upstream of the phosphorylation of Down-Syndrome cell-adhesion molecule (Dscam), a novel cell-surface molecule of the immunoglobulin-fibronectin super family, which has been shown to be important for axonal path-finding in Drosophila. Tyrosine phosphorylation of Dscam recruits the Src homology 2 domain of the adaptor protein Dock to the receptor. Dock, the ortho- logue of mammalian Nck, is also essential for correct axonal path-finding in Drosophila. We further determined that Dock is tyrosine-phosphorylated in vivo and identified DPTP61F as the protein tyrosine phosphatase responsible for maintaining Dock in its non-phosphorylated state. The present study illustrates the versatility of RNAi in the identification of the physiological substrates for protein kinases and phosphatases.

Protein Kinases and Phosphatases in the Control of Cell Fate

PubMed Central

Bononi, Angela; Agnoletto, Chiara; De Marchi, Elena; Marchi, Saverio; Patergnani, Simone; Bonora, Massimo; Giorgi, Carlotta; Missiroli, Sonia; Poletti, Federica; Rimessi, Alessandro; Pinton, Paolo

2011-01-01

Protein phosphorylation controls many aspects of cell fate and is often deregulated in pathological conditions. Several recent findings have provided an intriguing insight into the spatial regulation of protein phosphorylation across different subcellular compartments and how this can be finely orchestrated by specific kinases and phosphatases. In this review, the focus will be placed on (i) the phosphoinositide 3-kinase (PI3K) pathway, specifically on the kinases Akt and mTOR and on the phosphatases PP2a and PTEN, and on (ii) the PKC family of serine/threonine kinases. We will look at general aspects of cell physiology controlled by these kinases and phosphatases, highlighting the signalling pathways that drive cell division, proliferation, and apoptosis. PMID:21904669

Localization of inclusion bodies in Escherichia coli overproducing beta-lactamase or alkaline phosphatase.

PubMed Central

Georgiou, G; Telford, J N; Shuler, M L; Wilson, D B

1986-01-01

High-level synthesis of the periplasmic protein beta-lactamase in Escherichia coli caused the formation of insoluble protein precipitates called inclusion bodies. beta-Lactamase inclusion bodies differed from those reported previously in that they appeared to be localized in the periplasmic space, not in the cytoplasm. The inclusion bodies contained mature beta-lactamase and were solubilized more easily than has been reported for cytoplasmic inclusion bodies. In contrast, overproduction of the periplasmic protein alkaline phosphatase caused the formation of cytoplasmic inclusion bodies containing alkaline phosphatase precursor. Images PMID:3539017

Electromagnetic-pulse-induced activation of p38 MAPK pathway and disruption of blood-retinal barrier.

PubMed

Li, Hai-Juan; Guo, Liang-Mei; Yang, Long-Long; Zhou, Yong-Chun; Zhang, Yan-Jun; Guo, Juan; Xie, Xue-Jun; Guo, Guo-Zhen

2013-06-20

The blood-retinal barrier (BRB) is critical for maintaining retina homeostasis and low permeability. In this study, we evaluated the effects of electromagnetic pulse (EMP) exposure on the permeability of BRB, alterations of tight junction (TJ) proteins of BRB and if any, involvement of mitogen-activated protein kinase (MAPK) pathway. Male Sprague-Dawley (SD) rats and RF/6A cells which were pretreated with or without MAPKs inhibitors were sham exposed or exposed to EMP at 200kV/m for 200 pulses. The alteration of BRB permeability was examined through fluorescence microscope and quantitatively assessed using Evans blue (EB) and endogenous albumin as tracers. The expressions of TJ proteins and some signaling molecules of MAPK pathway were measured by Western blots. The observations were that EMP exposure resulted in increased BRB permeability concurrent with the decreased expressions of occludin and claudin-5, which were correlated with the increased expressions of phospho-p38, phospho-JNK and phospho-ERK and could be blocked when pretreated with p38 MAPK inhibitor. Thus, the results suggested that the alterations of occludin and claudin-5 may play an important role in the disruption of TJs, which may lead to the transient breakdown of BRB after EMP exposure with the involvement of p38 MAPK pathway through phosphorylation of signaling molecules. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Epigenetic modification in neurons of the mollusc Pomacea canaliculata after immune challenge.

PubMed

Ottaviani, Enzo; Accorsi, Alice; Rigillo, Giovanna; Malagoli, Davide; Blom, Joan M C; Tascedda, Fabio

2013-11-06

In human and rodents, the transcriptional response of neurons to stress is related to epigenetic modifications of both DNA and histone proteins. To assess the suitability of simple invertebrate models in studying the basic mechanisms of stress-related epigenetic modifications, we analyzed epigenetic modifications in neurons of the freshwater snail Pomacea canaliculata after the injection of Escherichia coli-derived lipopolysaccharide (LPS). The phospho-acetylation of histone H3, together with the induction of stress-related factors, c-Fos and HSP70, were evaluated in large and small neurons of the pedal ganglia of sham- and LPS-injected snails. Immunocytochemical investigations showed that after LPS injection, the immunopositivity towards phospho (Ser10)-acetyl (Lys14)-histone H3 and c-Fos increases in the nuclei of small gangliar neurons. Western blot analysis confirmed a significant increase of phospho (Ser10)-acetyl (Lys14)-histone H3 in nuclear extracts from 2h LPS-injected animals. c-Fos protein levels were significantly augmented 6h after LPS injection. Immunocytochemistry and western blot indicated that no changes occurred in HSP70 distribution and protein levels. To our knowledge this is the first demonstration of epigenetic changes in molluscan neurons after an immune challenge and indicate the gastropod P. canaliculata as a suitable model for evolutionary and translational studies on stress-related epigenetic modifications. © 2013 Published by Elsevier B.V.

p38 Mitogen-Activated Protein Kinase in Metastasis Associated with Transforming Growth Factor Beta

DTIC Science & Technology

2006-06-01

Functional Analysis of cells • Expression of HA-tagged TβRI • Signaling (p-Smad2, p- HSP27 , p-p38) • Transcription (SBE-Lux, 3TP-Lux) • Proliferation...when they also affected lls expressing DN-p38alpha phosphorylation of HSP27 was effectively blocked (data not shown). However, cells e studies show...231-ALK5-T204D cells treated with kinase inhibitors U0126, SB202190, SP600125 (JNK). (B) Immunoblot of phospho-Smad2/3, phospho- HSP27 , and alpha

Shigeru Tsuiki: a pioneer in the research fields of complex carbohydrates and protein phosphatases.

PubMed

Miyagi, Taeko; Kikuchi, Kunimi; Tamura, Shinri

2011-11-01

Dr Tsuiki made three major contributions during his illustrious career as a biochemist. First, he developed the procedure for mucin isolation from bovine submaxillary glands. His work became the basis for mucin biochemistry. Second, he identified four distinct molecular species of mammalian sialidase. Subsequent studies based on his work led to the discovery that sialidase plays a unique role as an intracellular signalling factor involved in the regulation of a variety of cellular functions. Finally, he established the molecular basis for the diversity of mammalian protein phosphatases through protein purification and molecular cloning. His work prompted the functional studies of protein phosphatases.

Structural Basis of Response Regulator Dephosphorylation by Rap Phosphatases

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

V Parashar; N Mirouze; D Dubnau

2011-12-31

Bacterial Rap family proteins have been most extensively studied in Bacillus subtilis, where they regulate activities including sporulation, genetic competence, antibiotic expression, and the movement of the ICEBs1 transposon. One subset of Rap proteins consists of phosphatases that control B. subtilis and B. anthracis sporulation by dephosphorylating the response regulator Spo0F. The mechanistic basis of Rap phosphatase activity was unknown. Here we present the RapH-Spo0F X-ray crystal structure, which shows that Rap proteins consist of a 3-helix bundle and a tetratricopeptide repeat domain. Extensive biochemical and genetic functional studies reveal the importance of the observed RapH-Spo0F interactions, including the catalyticmore » role of a glutamine in the RapH 3-helix bundle that inserts into the Spo0F active site. We show that in addition to dephosphorylating Spo0F, RapH can antagonize sporulation by sterically blocking phosphoryl transfer to and from Spo0F. Our structure-function analysis of the RapH-Spo0F interaction identified Rap protein residues critical for Spo0F phosphatase activity. This information enabled us to assign Spo0F phosphatase activity to a Rap protein based on sequence alone, which was not previously possible. Finally, as the ultimate test of our newfound understanding of the structural requirements for Rap phosphatase function, a non-phosphatase Rap protein that inhibits the binding of the response regulator ComA to DNA was rationally engineered to dephosphorylate Spo0F. In addition to revealing the mechanistic basis of response regulator dephosphorylation by Rap proteins, our studies support the previously proposed T-loop-Y allostery model of receiver domain regulation that restricts the aromatic 'switch' residue to an internal position when the {beta}4-{alpha}4 loop adopts an active-site proximal conformation.« less

Chondroitin sulfatases differentially regulate Wnt signaling in prostate stem cells through effects on SHP2, phospho-ERK1/2, and Dickkopf Wnt signaling pathway inhibitor (DKK3)

PubMed Central

Bhattacharyya, Sumit; Feferman, Leo; Tobacman, Joanne K.

2017-01-01

The chondroitin sulfatases N-acetylgalactosamine-4-sulfatase (ARSB) and galactosamine-N-acetyl-6-sulfatase (GALNS) remove either the 4-sulfate group at the non-reducing end of chondroitin 4-sulfate (C4S) and dermatan sulfate, or the 6-sulfate group of chondroitin 6-sulfate, chondroitin 4,6-disulfate (chondroitin sulfate E), or keratan sulfate. In human prostate cancer tissues, the ARSB activity was reduced and the GALNS activity was increased, compared to normal prostate tissue. In human prostate stem cells, when ARSB was reduced by silencing or GALNS was increased by overexpression, activity of SHP2, the ubiquitous non-receptor tyrosine phosphatase, declined, attributable to increased binding of SHP2 with C4S. This led to increases in phospho-ERK1/2, Myc/Max nuclear DNA binding, DNA methyltransferase (DNMT) activity and expression, and methylation of the Dickkopf Wnt signaling pathway inhibitor (DKK)3 promoter and to reduced DKK3 expression. Since DKK3 negatively regulates Wnt/β-catenin signaling, silencing of ARSB or overexpression of GALNS disinhibited (increased) Wnt/β-catenin signaling. These findings indicate that the chondroitin sulfatases can exert profound effects on Wnt-mediated processes, due to epigenetic effects that modulate Wnt signaling. PMID:29245974

Pseudotargeted MS Method for the Sensitive Analysis of Protein Phosphorylation in Protein Complexes.

PubMed

Lyu, Jiawen; Wang, Yan; Mao, Jiawei; Yao, Yating; Wang, Shujuan; Zheng, Yong; Ye, Mingliang

2018-05-15

In this study, we presented an enrichment-free approach for the sensitive analysis of protein phosphorylation in minute amounts of samples, such as purified protein complexes. This method takes advantage of the high sensitivity of parallel reaction monitoring (PRM). Specifically, low confident phosphopeptides identified from the data-dependent acquisition (DDA) data set were used to build a pseudotargeted list for PRM analysis to allow the identification of additional phosphopeptides with high confidence. The development of this targeted approach is very easy as the same sample and the same LC-system were used for the discovery and the targeted analysis phases. No sample fractionation or enrichment was required for the discovery phase which allowed this method to analyze minute amount of sample. We applied this pseudotargeted MS method to quantitatively examine phosphopeptides in affinity purified endogenous Shc1 protein complexes at four temporal stages of EGF signaling and identified 82 phospho-sites. To our knowledge, this is the highest number of phospho-sites identified from the protein complexes. This pseudotargeted MS method is highly sensitive in the identification of low abundance phosphopeptides and could be a powerful tool to study phosphorylation-regulated assembly of protein complex.

The protein histidine phosphatase LHPP is a tumour suppressor.

PubMed

Hindupur, Sravanth K; Colombi, Marco; Fuhs, Stephen R; Matter, Matthias S; Guri, Yakir; Adam, Kevin; Cornu, Marion; Piscuoglio, Salvatore; Ng, Charlotte K Y; Betz, Charles; Liko, Dritan; Quagliata, Luca; Moes, Suzette; Jenoe, Paul; Terracciano, Luigi M; Heim, Markus H; Hunter, Tony; Hall, Michael N

2018-03-29

Histidine phosphorylation, the so-called hidden phosphoproteome, is a poorly characterized post-translational modification of proteins. Here we describe a role of histidine phosphorylation in tumorigenesis. Proteomic analysis of 12 tumours from an mTOR-driven hepatocellular carcinoma mouse model revealed that NME1 and NME2, the only known mammalian histidine kinases, were upregulated. Conversely, expression of the putative histidine phosphatase LHPP was downregulated specifically in the tumours. We demonstrate that LHPP is indeed a protein histidine phosphatase. Consistent with these observations, global histidine phosphorylation was significantly upregulated in the liver tumours. Sustained, hepatic expression of LHPP in the hepatocellular carcinoma mouse model reduced tumour burden and prevented the loss of liver function. Finally, in patients with hepatocellular carcinoma, low expression of LHPP correlated with increased tumour severity and reduced overall survival. Thus, LHPP is a protein histidine phosphatase and tumour suppressor, suggesting that deregulated histidine phosphorylation is oncogenic.

An atlas of the human kinome reveals the mutational landscape underlying dysregulated phosphorylation cascades in cancer

PubMed Central

Olow, Aleksandra; Chen, Zhongzhong; Niedner, R. Hannes; Wolf, Denise M.; Yau, Christina; Pankov, Aleksandr; Lee, Evelyn Pei Rong; Brown-Swigart, Lamorna; van’t Veer, Laura J.; Coppé, Jean-Philippe

2016-01-01

Kinase inhibitors are used widely to treat various cancers, but adaptive reprogramming of kinase cascades and activation of feedback loop mechanisms often contribute to therapeutic resistance. Determining comprehensive, accurate maps of kinase circuits may therefore help elucidate mechanisms of response and resistance to kinase inhibitor therapies. In this study, we identified and validated phosphorylatable target sites across human cell and tissue types to generate PhosphoAtlas, a map of 1,733 functionally interconnected proteins comprising the human phospho-reactome. A systematic curation approach was used to distill protein phosphorylation data cross-referenced from 38 public resources. We demonstrated how a catalog of 2,617 stringently verified heptameric peptide regions at the catalytic interface of kinases and substrates could expose mutations that recurrently perturb specific phospho-hubs. In silico mapping of 2,896 nonsynonymous tumor variants identified from thousands of tumor tissues, also revealed that normal and aberrant catalytic interactions co-occur frequently, showing how tumors systematically hijack, as well as spare, particular sub-networks. Overall, our work provides an important new resource for interrogating the human tumor kinome to strategically identify therapeutically actionable kinase networks which drive tumorigenesis. PMID:26921330

Arctigenin, a Potent Ingredient of Arctium lappa L., Induces Endothelial Nitric Oxide Synthase and Attenuates Subarachnoid Hemorrhage-Induced Vasospasm through PI3K/Akt Pathway in a Rat Model

PubMed Central

Chang, Chih-Zen; Wu, Shu-Chuan; Chang, Chia-Mao; Kwan, Aij-Lie

2015-01-01

Upregulation of protein kinase B (PKB, also known as Akt) is observed within the cerebral arteries of subarachnoid hemorrhage (SAH) animals. This study is of interest to examine Arctigenin, a potent antioxidant, on endothelial nitric oxide synthase (eNOS) and Akt pathways in a SAH in vitro study. Basilar arteries (BAs) were obtained to examine phosphatidylinositol-3-kinase (PI3K), phospho-PI3K, Akt, phospho-Akt (Western blot) and morphological examination. Endothelins (ETs) and eNOS evaluation (Western blot and immunostaining) were also determined. Arctigenin treatment significantly alleviates disrupted endothelial cells and tortured internal elastic layer observed in the SAH groups (p < 0.01). The reduced eNOS protein and phospho-Akt expression in the SAH groups were relieved by the treatment of Arctigenin (p < 0.01). This result confirmed that Arctigenin might exert dural effects in preventing SAH-induced vasospasm through upregulating eNOS expression via the PI3K/Akt signaling pathway and attenuate endothelins after SAH. Arctigenin shows therapeutic promise in the treatment of cerebral vasospasm following SAH. PMID:26539501

Arctigenin, a Potent Ingredient of Arctium lappa L., Induces Endothelial Nitric Oxide Synthase and Attenuates Subarachnoid Hemorrhage-Induced Vasospasm through PI3K/Akt Pathway in a Rat Model.

PubMed

Chang, Chih-Zen; Wu, Shu-Chuan; Chang, Chia-Mao; Lin, Chih-Lung; Kwan, Aij-Lie

2015-01-01

Upregulation of protein kinase B (PKB, also known as Akt) is observed within the cerebral arteries of subarachnoid hemorrhage (SAH) animals. This study is of interest to examine Arctigenin, a potent antioxidant, on endothelial nitric oxide synthase (eNOS) and Akt pathways in a SAH in vitro study. Basilar arteries (BAs) were obtained to examine phosphatidylinositol-3-kinase (PI3K), phospho-PI3K, Akt, phospho-Akt (Western blot) and morphological examination. Endothelins (ETs) and eNOS evaluation (Western blot and immunostaining) were also determined. Arctigenin treatment significantly alleviates disrupted endothelial cells and tortured internal elastic layer observed in the SAH groups (p < 0.01). The reduced eNOS protein and phospho-Akt expression in the SAH groups were relieved by the treatment of Arctigenin (p < 0.01). This result confirmed that Arctigenin might exert dural effects in preventing SAH-induced vasospasm through upregulating eNOS expression via the PI3K/Akt signaling pathway and attenuate endothelins after SAH. Arctigenin shows therapeutic promise in the treatment of cerebral vasospasm following SAH.

Suppression of AKT Potentiates Synergistic Cytotoxicity of Apigenin with TRAIL in Anaplastic Thyroid Carcinoma Cells.

PubMed

Kim, Si Hyoung; Kang, Jun Goo; Kim, Chul Sik; Ihm, Sung-Hee; Choi, Moon Gi; Yoo, Hyung Joon; Lee, Seong Jin

2015-12-01

We studied the effect of apigenin in combination with tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on cell survival and the influence of AKT inhibition on the combined effect of apigenin with TRAIL in anaplastic thyroid carcinoma (ATC) cells. The human 8505C and CAL62 ATC cell lines were used. Apigenin in combination with TRAIL, compared to apigenin alone, reduced cell viability and Bcl2 protein levels, elevated the percentage of dead cells, as well as the protein levels of cleaved PARP and phospho-ERK1/2. The protein levels of Bcl-xL, Bax, Bid, total ERK1/2, and total and phospho-AKT were unchanged. Administration of wortmannin further reduced cell viability, and elevated the percentage of dead cells, cytotoxic activity and cleaved PARP protein levels. Apigenin synergizes with TRAIL through regulation of Bcl2 family proteins in inducing cytotoxicity, and suppression of AKT potentiates synergistic cytotoxicity of apigenin with TRAIL in ATC cells. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Centromeric binding and activity of Protein Phosphatase 4

PubMed Central

Lipinszki, Zoltan; Lefevre, Stephane; Savoian, Matthew S.; Singleton, Martin R.; Glover, David M.; Przewloka, Marcin R.

2015-01-01

The cell division cycle requires tight coupling between protein phosphorylation and dephosphorylation. However, understanding the cell cycle roles of multimeric protein phosphatases has been limited by the lack of knowledge of how their diverse regulatory subunits target highly conserved catalytic subunits to their sites of action. Phosphoprotein phosphatase 4 (PP4) has been recently shown to participate in the regulation of cell cycle progression. We now find that the EVH1 domain of the regulatory subunit 3 of Drosophila PP4, Falafel (Flfl), directly interacts with the centromeric protein C (CENP-C). Unlike other EVH1 domains that interact with proline-rich ligands, the crystal structure of the Flfl amino-terminal EVH1 domain bound to a CENP-C peptide reveals a new target-recognition mode for the phosphatase subunit. We also show that binding of Flfl to CENP-C is required to bring PP4 activity to centromeres to maintain CENP-C and attached core kinetochore proteins at chromosomes during mitosis. PMID:25562660

Voltage-sensing phosphatase: its molecular relationship with PTEN.

PubMed

Okamura, Yasushi; Dixon, Jack E

2011-02-01

Voltage-sensing phosphoinositide phosphatase (VSP) contains voltage sensor and cytoplasmic phosphatase domains. A unique feature of this protein is that depolarization-induced motions of the voltage sensor activate PtdIns(3,4,5)P(3) and PtdIns(4,5)P(2) phosphatase activities. VSP exhibits remarkable structural similarities with PTEN, the phosphatase and tensin homolog deleted on chromosome 10. These similarities include the cytoplasmic phosphatase region, the phosphoinositide binding region, and the putative membrane interacting C2 domain.

Human cytomegalovirus carries serine/threonine protein phosphatases PP1 and a host-cell derived PP2A.

PubMed Central

Michelson, S; Turowski, P; Picard, L; Goris, J; Landini, M P; Topilko, A; Hemmings, B; Bessia, C; Garcia, A; Virelizier, J L

1996-01-01

Human cytomegalovirus (CMV), a herpesvirus, is an important cause of morbidity and mortality in immunocompromised patients. When studying hyper-immediate-early events after contact between CMV virions and the cell membrane, we observed a hypophosphorylation of cellular proteins within 10 min. This can be explained in part by our finding that purified CMV contains serine/threonine protein phosphatase activities. Biochemical analyses indicate that this protein phosphatase activity has all characteristics of type 1 and 2A protein phosphatases (PP1 and PP2A). Specifically, PP1 accounts for approximately 30% and PP2A accounts for the remaining 70% of the phosphorylase phosphatase activity found. CMV produced in astrocytoma cells stably expressing an amino-terminally tagged PP2A catalytic subunit contained tagged enzyme, thus demonstrating the cellular origin of CMV-associated PP2A. PP2A is specifically found inside the virus, associated with the nucleocapsid fraction. Western blot (immunoblot) analysis of purified virus revealed the presence of the catalytic subunits of PP2A and PP1. Furthermore, the catalytic subunit of PP2A appears to be complexed to the regulatory subunits PR65 and PR55, which is also the most abundant configuration of this enzyme found in the host cells. Incubation of virus with okadaic acid before contact of CMV with cells prevented hypophosphorylation of cellular proteins, thus demonstrating the role of CMV-associated phosphatases in this phenomenon. CMV can thus transport an active enzyme from one cell to another. PMID:8627658

Membrane-bound 2,3-diphosphoglycerate phosphatase of human erythrocytes.

PubMed

Schröter, W; Neuvians, M

1970-12-01

Gradual osmotic hemolysis of human erythrocytes reduces the cell content of whole protein, hemoglobin, 2,3-diphosphoglycerate and triosephosphate isomerase extensively, but not that of membrane protein and 2,3-diphosphoglycerate phosphatase. After the refilling of the ghosts with 2,3-diphosphoglycerate and reconstitution of the membrane, the 2,3-diphosphoglycerate phosphatase activity equals that of intact red cells. The membrane-bound 2,3-diphosphoglycerate phosphatase can be activated by sodium hyposulfite. The enzyme system of ghosts seems to differ from that of intact red cells with regard to the optima of pH and temperature. It remains to be elucidated if the membrane binding of the 2,3-diphosphoglycerate phosphatase is related to the transfer of inorganic phosphate across the red cell membrane.

Demonstration of separate phosphotyrosyl- and phosphoseryl- histone phosphatase activities in the plasma membranes of a human astrocytoma.

PubMed

Leis, J F; Knowles, A F; Kaplan, N O

1985-06-01

A plasma membrane preparation from a human astrocytoma contained p-nitrophenyl phosphate (pNPP), phosphotyrosyl histone, and phosphoseryl histone hydrolysis activities. The pNPPase and phosphotyrosyl histone phosphatase activities were inhibited by vanadate, whereas the phosphoseryl histone phosphatase activity was not; the latter activity was inhibited by pyrophosphate and nucleoside di- and triphosphates. When the membranes were solubilized by Triton X-100 and the solubilized proteins were subjected to column chromatography on DEAE-Sephadex, Sepharose 6B-C1, and wheat germ agglutinin-Sepharose 4B columns, the pNPPase activity from the phosphoseryl histone phosphatase activity. The results from column chromatography also indicated that there may be multiple phosphotyrosyl and phosphoseryl protein phosphatases in the plasma membranes.

Evaluation of elevated dietary corn fiber from corn germ meal in growing female pigs.

PubMed

Weber, T E; Trabue, S L; Ziemer, C J; Kerr, B J

2010-01-01

To evaluate the effects of dietary hemicellulose from corn on growth and metabolic measures, female pigs (n = 48; initial BW 30.8 kg) were fed diets containing 0 to 38.6% solvent-extracted corn germ meal for 28 d. Increasing the hemicellulose level had no impact on ADG or ADFI, but resulted in a quadratic response (P < 0.03) on G:F. To investigate physiological changes that occur with increased dietary hemicellulose, blood, colon contents, and tissue samples from the liver and intestine were obtained from a subset (n = 16; 8 pigs/treatment) of pigs fed the least and greatest hemicellulose levels. The abundance of phospho-adenosine monophosphate-activated protein kinase (AMPK) and the mitochondrial respiratory protein, cytochrome C oxidase II (COXII) were determined in liver, jejunum, ileum, and colon by Western blotting. The mRNA expression levels of AMPKalpha1, AMPKalpha2, PPAR coactivator 1alpha (PGC1-alpha), PPARgamma2, and sirtuin 1 (Sirt1) were determined in liver and intestinal tissues. When compared with pigs fed the control diet, pigs fed the high hemicellulose diet had increased (P < 0.02) plasma triglycerides, but there was no difference in plasma cholesterol, glucose, or insulin. Absolute and relative liver weights were decreased (P < 0.03) in pigs consuming the high hemicellulose diet. The high-fiber diet led to a tendency (P < 0.12) for decreased liver triglyceride content. In pigs fed the high hemicellulose diet, ileal mucosal alkaline phosphatase activity was increased (P < 0.08) and sucrase activity tended (P < 0.12) to be increased. The high hemicellulose diet had no effect on phospho-AMPK, AMPK mRNA, or colonic VFA, but in pigs consuming the high fiber diet there was a greater (P < 0.05) abundance of COXII in colon tissue. The expression of PGC1-alpha, PPARgamma, or Sirt1 mRNA was not altered by dietary fiber in liver, jejunum, or ileum tissue. In colon tissue from pigs fed the high fiber diet there was an increase (P < 0.09) in Sirt1 mRNA and a trend (P < 0.12) toward increased of PGC1-alpha mRNA. These data suggest that alterations in metabolism involved in adaptation to a diet high in hemicellulose are associated with increased colonic Sirt1 mRNA and COXII expression, indicating an increased propensity for oxidative metabolism by the intestine.

Protein Tyrosine Phosphatases: From Housekeeping Enzymes to Master-Regulators of Signal Transduction

PubMed Central

Tonks, Nicholas K.

2013-01-01

There are many misconceptions surrounding the roles of protein phosphatases in the regulation of signal transduction, perhaps the most damaging of which is the erroneous view that these enzymes exert their effects merely as constitutively active housekeeping enzymes. On the contrary, the phosphatases are critical, specific regulators of signaling in their own right and serve an essential function, in a coordinated manner with the kinases, to determine the response to a physiological stimulus. This review is a personal perspective on the development of our understanding of the protein tyrosine phosphatase (PTP) family of enzymes. I have discussed various aspects of the structure, regulation and function of the PTP family, which I hope will illustrate the fundamental importance of these enzymes to the control of signal transduction. PMID:23176256

Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

NASA Astrophysics Data System (ADS)

Qi, Fei; Guo, Huarong; Wang, Jian

2008-02-01

Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 (PP1) is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase 1(PP1cb), was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPP1cb, by the rapid amplification of cDNA ends (RACE) technique. The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame (ORF), flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B (PP2B). And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates, and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXX ATGG, which is different from mammalian in two positions A-6 and G-3, indicating the possibility of different initiation of translation in turbot, and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals, especially zebrafish. The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.

Phosphoinositide 5-phosphatase activities control cell motility in glioblastoma: Two phosphoinositides PI(4,5)P2 and PI(3,4)P2 are involved.

PubMed

Ramos, Ana Raquel; Elong Edimo, William's; Erneux, Christophe

2018-01-01

Inositol polyphosphate 5-phosphatases or phosphoinositide 5-phosphatases (PI 5-phosphatases) are enzymes that can act on soluble inositol phosphates and/or phosphoinositides (PIs). Several PI 5-phosphatases have been linked to human genetic diseases, in particular the Lowe protein or OCRL which is mutated in the Lowe syndrome. There are 10 different members of this family and 9 of them can use PIs as substrate. One of these substrates, PI(3,4,5)P3 binds to specific PH domains and recruits as effectors specific proteins to signaling complexes. Protein kinase B is one target protein and activation of the kinase will have a major impact on cell proliferation, survival and cell metabolism. Two other PIs, PI(4,5)P2 and PI(3,4)P2, are produced or used as substrates of PI 5-phosphatases (OCRL, INPP5B, SHIP1/2, SYNJ1/2, INPP5K, INPP5J, INPP5E). The inositol lipids may influence many aspects of cytoskeletal organization, lamellipodia formation and F-actin polymerization. PI 5-phosphatases have been reported to control cell migration, adhesion, polarity and cell invasion particularly in cancer cells. In glioblastoma, reducing SHIP2 expression can positively or negatively affect the speed of cell migration depending on the glioblastoma cell type. The two PI 5-phosphatases SHIP2 or SKIP could be localized at the plasma membrane and can reduce either PI(3,4,5)P3 or PI(4,5)P2 abundance. In the glioblastoma 1321 N1 cells, SHIP2 controls plasma membrane PI(4,5)P2 thereby participating in the control of cell migration. Copyright © 2017 Elsevier Ltd. All rights reserved.

Protein tyrosine phosphatases: Ligand interaction analysis and optimisation of virtual screening.

PubMed

Ghattas, Mohammad A; Atatreh, Noor; Bichenkova, Elena V; Bryce, Richard A

2014-07-01

Docking-based virtual screening is an established component of structure-based drug discovery. Nevertheless, scoring and ranking of computationally docked ligand libraries still suffer from many false positives. Identifying optimal docking parameters for a target protein prior to virtual screening can improve experimental hit rates. Here, we examine protocols for virtual screening against the important but challenging class of drug target, protein tyrosine phosphatases. In this study, common interaction features were identified from analysis of protein-ligand binding geometries of more than 50 complexed phosphatase crystal structures. It was found that two interactions were consistently formed across all phosphatase inhibitors: (1) a polar contact with the conserved arginine residue, and (2) at least one interaction with the P-loop backbone amide. In order to investigate the significance of these features on phosphatase-ligand binding, a series of seeded virtual screening experiments were conducted on three phosphatase enzymes, PTP1B, Cdc25b and IF2. It was observed that when the conserved arginine and P-loop amide interactions were used as pharmacophoric constraints during docking, enrichment of the virtual screen significantly increased in the three studied phosphatases, by up to a factor of two in some cases. Additionally, the use of such pharmacophoric constraints considerably improved the ability of docking to predict the inhibitor's bound pose, decreasing RMSD to the crystallographic geometry by 43% on average. Constrained docking improved enrichment of screens against both open and closed conformations of PTP1B. Incorporation of an ordered water molecule in PTP1B screening was also found to generally improve enrichment. The knowledge-based computational strategies explored here can potentially inform structure-based design of new phosphatase inhibitors using docking-based virtual screening. Copyright © 2014 Elsevier Inc. All rights reserved.

Amyloid-β protein precursor regulates phosphorylation and cellular compartmentalization of microtubule associated protein tau.

PubMed

Nizzari, Mario; Barbieri, Federica; Gentile, Maria Teresa; Passarella, Daniela; Caorsi, Calentina; Diaspro, Alberto; Taglialatela, Maurizio; Pagano, Aldo; Colucci-D'Amato, Luca; Florio, Tullio; Russo, Claudio

2012-01-01

Tau is a multifunctional protein detected in different cellular compartments in neuronal and non-neuronal cells. When hyperphosphorylated and aggregated in atrophic neurons, tau is considered the culprit for neuronal death in familial and sporadic tauopathies. With regards to Alzheimer's disease (AD) pathogenesis, it is not yet established whether entangled tau represents a cause or a consequence of neurodegeneration. In fact, it is unquestionably accepted that amyloid-β protein precursor (AβPP) plays a pivotal role in the genesis of the disease, and it is postulated that the formation of toxic amyloid-β peptides from AβPP is the primary event that subsequently induces abnormal tau phosphorylation. In this work, we show that in the brain of AD patients there is an imbalance between the nuclear and the cytoskeletal pools of phospho-tau. We observed that in non-AD subjects, there is a stable pool of phospho-tau which remains strictly confined to neuronal nuclei, while nuclear localization of phospho-tau is significantly underrepresented in neurons of AD patients bearing neurofibrillary tangles. A specific phosphorylation of tau is required during mitosis in vitro and in vivo, likely via a Grb2-ERK1/2 signaling cascade. In differentiated neuronal A1 cells, the overexpression of AβPP modulates tau phosphorylation, altering the ratio between cytoskeletal and nuclear pools, and correlates with cell death. Altogether our data provide evidence that AβPP, in addition to amyloid formation, modulates the phosphorylation of tau and its subcellular compartmentalization, an event that may lead to the formation of neurofibrillary tangles and to neurodegeneration when occurring in postmitotic neurons.

eIF4A RNA Helicase Associates with Cyclin-Dependent Protein Kinase A in Proliferating Cells and Is Modulated by Phosphorylation1[OPEN

PubMed Central

Bush, Maxwell S.; Pierrat, Olivier; Nibau, Candida; Mikitova, Veronika; Zheng, Tao; Corke, Fiona M. K.; Mayberry, Laura K.; Browning, Karen S.

2016-01-01

Eukaryotic initiation factor 4A (eIF4A) is a highly conserved RNA-stimulated ATPase and helicase involved in the initiation of messenger RNA translation. Previously, we found that eIF4A interacts with cyclin-dependent kinase A (CDKA), the plant ortholog of mammalian CDK1. Here, we show that this interaction occurs only in proliferating cells where the two proteins coassociate with 5′-cap-binding protein complexes, eIF4F or the plant-specific eIFiso4F. CDKA phosphorylates eIF4A on a conserved threonine residue (threonine-164) within the RNA-binding motif 1b TPGR. In vivo, a phospho-null (APGR) variant of the Arabidopsis (Arabidopsis thaliana) eIF4A1 protein retains the ability to functionally complement a mutant (eif4a1) plant line lacking eIF4A1, whereas a phosphomimetic (EPGR) variant fails to complement. The phospho-null variant (APGR) rescues the slow growth rate of roots and rosettes, together with the ovule-abortion and late-flowering phenotypes. In vitro, wild-type recombinant eIF4A1 and its phospho-null variant both support translation in cell-free wheat germ extracts dependent upon eIF4A, but the phosphomimetic variant does not support translation and also was deficient in ATP hydrolysis and helicase activity. These observations suggest a mechanism whereby CDK phosphorylation has the potential to down-regulate eIF4A activity and thereby affect translation. PMID:27388680

Crystal structure of SP-PTP, a low molecular weight protein tyrosine phosphatase from Streptococcus pyogenes.

PubMed

Ku, Bonsu; Keum, Chae Won; Lee, Hye Seon; Yun, Hye-Yeoung; Shin, Ho-Chul; Kim, Bo Yeon; Kim, Seung Jun

2016-09-23

Streptococcus pyogenes, or Group A Streptococcus (GAS), is a pathogenic bacterium that causes a variety of infectious diseases. The GAS genome encodes one protein tyrosine phosphatase, SP-PTP, which plays an essential role in the replication and virulence maintenance of GAS. Herein, we present the crystal structure of SP-PTP at 1.9 Å resolution. Although SP-PTP has been reported to have dual phosphatase specificity for both phosphorylated tyrosine and serine/threonine, three-dimensional structural analysis showed that SP-PTP shares high similarity with typical low molecular weight protein tyrosine phosphatases (LMWPTPs), which are specific for phosphotyrosine, but not with dual-specificity phosphatases, in overall folding and active site composition. In the dephosphorylation activity test, SP-PTP consistently acted on phosphotyrosine substrates, but not or only minimally on phosphoserine/phosphothreonine substrates. Collectively, our structural and biochemical analyses verified SP-PTP as a canonical tyrosine-specific LMWPTP. Copyright © 2016 Elsevier Inc. All rights reserved.

EX VIVIO DETECTION OF KINASE AND PHOSPHATASE ACTIVITIES IN HUMAN BRONCHIAL BIOPSIES

EPA Science Inventory

Protein phosphorylation is a posttranslational modification involved in every aspect cellular function. Levels of protein phosphotyrosine, phosphoserine and phosphothreonine are regulated by the opposing activities of kinases and phosphatases, the expression of which can be alt...

Select polyphenolic fractions from dried plum enhance osteoblast activity through BMP-2 signaling.

PubMed

Graef, Jennifer L; Rendina-Ruedy, Elizabeth; Crockett, Erica K; Ouyang, Ping; King, Jarrod B; Cichewicz, Robert H; Lucas, Edralin A; Smith, Brenda J

2018-05-01

Dried plum supplementation has been shown to enhance bone formation while suppressing bone resorption. Evidence from previous studies has demonstrated that these responses can be attributed in part to the fruit's polyphenolic compounds. The purpose of this study was to identify the most bioactive polyphenolic fractions of dried plum with a focus on their osteogenic activity and to investigate their mechanisms of action under normal and inflammatory conditions. Utilizing chromatographic techniques, six fractions of polyphenolic compounds were prepared from a crude extract of dried plum. Initial screening assays revealed that two fractions (DP-FrA and DP-FrB) had the greatest osteogenic potential. Subsequent experiments using primary bone-marrow-derived osteoblast cultures demonstrated these two fractions enhanced extracellular alkaline phosphatase (ALP), an indicator of osteoblast activity, and mineralized nodule formation under normal conditions. Both fractions enhanced bone morphogenetic protein (BMP) signaling, as indicated by increased Bmp2 and Runx2 gene expression and protein levels of phosphorylated Smad1/5. DP-FrB was most effective at up-regulating Tak1 and Smad1, as well as protein levels of phospho-p38. Under inflammatory conditions, TNF-α suppressed ALP and tended to decrease nodule formation (P=.0674). This response coincided with suppressed gene expression of Bmp2 and the up-regulation of Smad6, an inhibitor of BMP signaling. DP-FrA and DP-FrB partially normalized these responses. Our results show that certain fractions of polyphenolic compounds in dried plum up-regulate osteoblast activity by enhancing BMP signaling, and when this pathway is inhibited by TNF-α, the osteogenic response is attenuated. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of ethanol on hydrogen peroxide-induced AMPK phosphorylation.

PubMed

Liangpunsakul, Suthat; Wou, Sung-Eun; Zeng, Yan; Ross, Ruth A; Jayaram, Hiremagalur N; Crabb, David W

2008-12-01

AMP-activated protein kinase (AMPK) responds to oxidative stress. Previous work has shown that ethanol treatment of cultured hepatoma cells and of mice inhibited the activity of AMPK and reduced the amount of AMPK protein. Ethanol generates oxidative stress in the liver. Since AMPK is activated by reactive oxygen species, it seems paradoxical that ethanol would inhibit AMPK in the hepatoma cells. In an attempt to understand the mechanism whereby ethanol inhibits AMPK, we studied the effect of ethanol on AMPK activation by exogenous hydrogen peroxide. The effects of ethanol, hydrogen peroxide, and inhibitors of protein phosphatase 2A (PP2A) [either okadaic acid or PP2A small interference RNA (siRNA)] on AMPK phosphorylation and activity were examined in rat hepatoma cells (H4IIEC3) and HeLa cells. In H4IIEC3 cells, hydrogen peroxide (H(2)O(2), 1 mM) transiently increased the level of phospho-AMPK to 1.5-fold over control (P < 0.05). Similar findings were observed in HeLa cells, which do not express the upstream AMPK kinase, LKB1. H(2)O(2) markedly increased the phosphorylation of LKB1 in H4IIEC3 cells. Ethanol significantly inhibited the phosphorylation of PKC-zeta, LKB1, and AMPK caused by exposure to H(2)O(2). This inhibitory effect of ethanol required its metabolism. More importantly, the inhibitory effects of ethanol on H(2)O(2)-induced AMPK phosphorylation were attenuated by the presence of the PP2A inhibitor, okadaic acid, or PP2A siRNA. The inhibitory effect of ethanol on AMPK phosphorylation is exerted through the inhibition of PKC-zeta and LKB1 phosphorylation and the activation of PP2A.

Lung cancer pathogenesis associated with wood smoke exposure.

PubMed

Delgado, Javier; Martinez, Luis M; Sánchez, Therasa T; Ramirez, Alejandra; Iturria, Cecilia; González-Avila, Georgina

2005-07-01

Tobacco is considered the most important cause of lung cancer, but other factors could also be involved in its pathogenesis. The aim of the present work was to establish an association between wood smoke exposure and lung cancer pathogenesis, and to analyze the effects of wood smoke on p53 and murine double minute 2 (MDM2) protein expression. Blood samples were obtained from 62 lung cancer patients, 9 COPD patients, and 9 control subjects. Of the 62 lung cancer patients, 23 were tobacco smokers (lung cancer associated with tobacco [LCT] group), 24 were exposed to wood smoke (lung cancer associated with wood smoke [LCW] group), and 15 could not be included in these groups. Western blot assays were performed to identify the presence of p53, phospho-p53, and murine double minute 2 (MDM2) isoforms in plasma samples. Densitometric analysis was used to determine the intensity of p53, phospho-p53, and MDM2 bands. Approximately 38.7% of the lung cancer patients examined had an association with wood smoke exposure, most of them women living in rural areas. Adenocarcinoma was present in 46.7% of these patients. The p53 and phospho-p53 proteins were significantly increased in LCW samples (56,536.8 +/- 4,629 densitometry units [DU] and 58,244.8 +/- 7,492 DU, respectively [+/- SD]), in comparison with the other groups. The 57-kD MDM2 isoform plasma concentration was very high in LCW and LCT samples (75,696.4 +/- 11,979 DU and 78,551.7 +/- 11,548 DU, respectively). MDM2-p53 complexes were present in a high concentration in control and COPD subjects. This allows p53 degradation and explains the low concentrations of p53 found in these groups. MDM2-phospho-p53 complexes were observed in COPD but not in the other samples. This correlates with the low concentration of p53 observed in the COPD group (13,657 +/- 2,012 DU), and could explain the different clinic evolution of this smoker population in comparison with the LCT subjects. This study suggests that there is a possible association of lung cancer with wood smoke exposure. Likewise, our findings demonstrate that wood smoke could produce similar effects on p53, phospho-p53, and MDM2 protein expression as tobacco.

Increased level of apoptosis in rat brains and SH-SY5Y cells exposed to excessive fluoride--a mechanism connected with activating JNK phosphorylation.

PubMed

Liu, Yan-Jie; Guan, Zhi-Zhong; Gao, Qin; Pei, Jin-Jing

2011-07-28

In order to reveal the mechanism of the brain injury induced by chronic fluorosis, the levels of apoptosis and c-Jun N-terminal kinases (JNK) in brains of rats and SH-SY5Y cells exposed to different concentrations of sodium fluoride (NaF) were detected. The dental fluorosis and fluoride contents in blood, urine and bones of rats were measured to evaluate the exhibition of fluorosis. The apoptotic death rate was measured by flow cytometry and the expression of JNK at protein level by Western blotting. The results showed that as compared with controls, the apoptotic death rate was obviously increased in brains of the rats exposed to high-fluoride (50ppm) for 6 months with a concentration dependent manner, but no significant change for 3 months. In SH-SY5Y cells treated with high concentration (50ppm) of fluoride, the increased apoptotic death rate was obviously observed as compared to controls. In addition, the expressions of phospho-JNK at protein level were raised by 20.5% and 107.6%, respectively, in brains of the rats exposed to low-fluoride (5ppm) and high-fluoride for 6 months; while no significant changes were found between the rats exposed to fluoride and the controls for 3 months. The protein level of phospho-JNK was also increased in SH-SY5Y cells exposed to high-fluoride. There were no changes of total-JNK both in the rats and in the SH-SY5Y cells exposed to excessive fluoride as compared to controls. When SH-SY5Y cells were singly treated with SP600125, an inhibitor of phospho-JNK, the decreased expression of phospho-JNK, but no apoptosis, was detected. Interestingly, after JNK phosphorylation in the cultured cells was inhibited by SP600125, the treatment with high-fluoride did not induce the increase of apoptosis. In addition, there was a positive correlation between the expression of phospho-JNK and the apoptotic death rate in rat brains or SH-SY5Y cells treated with high-fluoride. The results indicated that exposure to excessive fluoride resulted in the increase of apoptosis in rat brains and SH-SY5Y cells, in which one of the mechanisms might be activating JNK phosphorylation. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

A protein tyrosine phosphatase-like protein from baculovirus has RNA 5′-triphosphatase and diphosphatase activities

PubMed Central

Takagi, Toshimitsu; Taylor, Gregory S.; Kusakabe, Takahiro; Charbonneau, Harry; Buratowski, Stephen

1998-01-01

The superfamily of protein tyrosine phosphatases (PTPs) includes at least one enzyme with an RNA substrate. We recently showed that the RNA triphosphatase domain of the Caenorhabditis elegans mRNA capping enzyme is related to the PTP enzyme family by sequence similarity and mechanism. The PTP most similar in sequence to the capping enzyme triphosphatase is BVP, a dual-specificity PTP encoded by the Autographa californica nuclear polyhedrosis virus. Although BVP previously has been shown to have modest tyrosine and serine/threonine phosphatase activity, we find that it is much more potent as an RNA 5′-phosphatase. BVP sequentially removes γ and β phosphates from the 5′ end of triphosphate-terminated RNA, leaving a 5′-monophosphate end. The activity was specific for polynucleotides; nucleotide triphosphates were not hydrolyzed. A mutant protein in which the active site cysteine was replaced with serine was inactive. Three other dual-specificity PTPs (VH1, VHR, and Cdc14) did not exhibit detectable RNA phosphatase activity. Therefore, capping enzyme and BVP are members of a distinct PTP-like subfamily that can remove phosphates from RNA. PMID:9707557

Membrane topology analysis of Escherichia coli K-12 Mtr permease by alkaline phosphatase and beta-galactosidase fusions.

PubMed

Sarsero, J P; Pittard, A J

1995-01-01

The mtr gene of Escherichia coli K-12 encodes an inner membrane protein which is responsible for the active transport of trypotophan into the cell. It has been proposed that the Mtr permease has a novel structure consisting of 11 hydrophobic transmembrane spans, with a cytoplasmically disposed amino terminus and a carboxyl terminus located in the periplasmic space (J.P. Sarsero, P. J. Wookey, P. Gollnick, C. Yanofsky, and A.J. Pittard, J. Bacteriol. 173:3231-3234, 1991). The validity of this model was examined by the construction of fusion proteins between the Mtr permease and alkaline phosphatase or beta-galactosidase. In addition to the conventional methods, in which the reporter enzyme replaces a carboxyl-terminal portion of the membrane protein, the recently developed alkaline phosphatase sandwich fusion technique was utilized, in which alkaline phosphatase is inserted into an otherwise intact membrane protein. A cluster of alkaline phosphatase fusions to the carboxyl-terminal end of the Mtr permease exhibited high levels of alkaline phosphatase activity, giving support to the proposition of a periplasmically located carboxyl terminus. The majority of fusion proteins produced enzymatic activities which were in agreement with the positions of the fusion sites on the proposed topological model of the permease. The synthesis of a small cluster of hybrid proteins, whose enzymatic activity did not agree with the location of their fusion sites within putative transmembrane span VIII or the preceding periplasmic loop, was not detected by immunological techniques and did not necessitate modification of the proposed model in this region. Slight alterations may need to be made in the positioning of the carboxyl-terminal end of transmembrane span X.

Chimeric proteins combining phosphatase and cellulose-binding activities: proof-of-concept and application in the hydrolysis of paraoxon.

PubMed

Gonçalves, Larissa M; Chaimovich, Hernan; Cuccovia, Iolanda M; Marana, Sandro R

2014-05-01

Phosphatases for organophosphate degradation and carbohydrate-binding domains (CBMs) have potential biotechnological applications. As a proof-of-concept, a soluble chimeric protein that combines acid phosphatase (AppA) from Escherichia coli and a CBM from Xanthomonas axonopodis pv. citri (AppA-CBM) was produced in E.coli. AppACBM adsorbed in microcrystalline cellulose Avicel PH101 catalyzed the hydrolysis of p-nitrophenyl phosphate (PNPP). The binding to microcrystalline cellulose displayed saturation behavior with an apparent binding constant (Kb) of 22 ± 5 mg and a maximum binding (Bmax) of 1.500 ± 0.001 enzyme units. Binding was highest at pH 2.5 and decreased above pH 6.5, as previously observed for family 2 CBMs. The Km values for PNPP of AppA-CBM and native AppA were identical (2.7 mM). To demonstrate that this strategy for protein engineering has practical applications and is largely functional, even for phosphatases exhibiting diverse folds, a chimeric protein combining human paraoxonase 1 (hPON1) and the CBM was produced. Both PON1-CBM and hPON1 had identical K_m values for paraoxon (1.3 mM). Additionally, hPON1 bound to microcrystalline cellulose with a Kb of 27 ± 3 mg, the same as that observed for AppA-CBM. These data show that the phosphatase domains are as functional in both of the chimeric proteins as they are in the native enzymes and that the CBM domain maintains the same cellulose affinity. Therefore, the engineering of chimeric proteins combining domains of phosphatases and CBMs is fully feasible, resulting in chimeric enzymes that exhibit potential for OP detoxification.

Genetic and chemical reductions in protein phosphatase activity alter auxin transport, gravity response, and lateral root growth

NASA Technical Reports Server (NTRS)

Rashotte, A. M.; DeLong, A.; Muday, G. K.; Brown, C. S. (Principal Investigator)

2001-01-01

Auxin transport is required for important growth and developmental processes in plants, including gravity response and lateral root growth. Several lines of evidence suggest that reversible protein phosphorylation regulates auxin transport. Arabidopsis rcn1 mutant seedlings exhibit reduced protein phosphatase 2A activity and defects in differential cell elongation. Here we report that reduced phosphatase activity alters auxin transport and dependent physiological processes in the seedling root. Root basipetal transport was increased in rcn1 or phosphatase inhibitor-treated seedlings but showed normal sensitivity to the auxin transport inhibitor naphthylphthalamic acid (NPA). Phosphatase inhibition reduced root gravity response and delayed the establishment of differential auxin-induced gene expression across a gravity-stimulated root tip. An NPA treatment that reduced basipetal transport in rcn1 and cantharidin-treated wild-type plants also restored a normal gravity response and asymmetric auxin-induced gene expression, indicating that increased basipetal auxin transport impedes gravitropism. Increased auxin transport in rcn1 or phosphatase inhibitor-treated seedlings did not require the AGR1/EIR1/PIN2/WAV6 or AUX1 gene products. In contrast to basipetal transport, root acropetal transport was normal in phosphatase-inhibited seedlings in the absence of NPA, although it showed reduced NPA sensitivity. Lateral root growth also exhibited reduced NPA sensitivity in rcn1 seedlings, consistent with acropetal transport controlling lateral root growth. These results support the role of protein phosphorylation in regulating auxin transport and suggest that the acropetal and basipetal auxin transport streams are differentially regulated.

In Vivo Activity of Insulin-Like Growth Factor Binding Protein-3 in Prevention of Prostate Cancer Progression

DTIC Science & Technology

2008-10-01

cell metastasis and survival through level of expression and differential phosphorylation. Phospho- HSP27 modulates cell motility; blocking...Phospho Hsp27 expression in prostate Compared to wt mice 0 10 20 30 40 50 60 9wk 11wk 13wk 15wk 17wk 19wk LPB-Tag LPB-Tag x PGKBP-3 LPB-Tag x PGKBP...3mut     Figure 7 Relative change in expression of phoshorylated HSP27 by immunoblot analyses using anti-pHSP27 (Santa Cruz; pHSP27(ser78): sc-16568

Cross-talk between oxidative stress and pro-inflammatory cytokines in acute pancreatitis: a key role for protein phosphatases.

PubMed

Escobar, Javier; Pereda, Javier; Arduini, Alessandro; Sandoval, Juan; Sabater, Luis; Aparisi, Luis; López-Rodas, Gerardo; Sastre, Juan

2009-01-01

Acute pancreatitis is an acute inflammatory process localized in the pancreatic gland that frequently involves peripancreatic tissues. It is still under investigation why an episode of acute pancreatitis remains mild affecting only the pancreas or progresses to a severe form leading to multiple organ failure and death. Proinflammatory cytokines and oxidative stress play a pivotal role in the early pathophysiological events of the disease. Cytokines such as interleukin 1beta and tumor necrosis factor alpha initiate and propagate almost all consequences of the systemic inflammatory response syndrome. On the other hand, depletion of pancreatic glutathione is an early hallmark of acute pancreatitis and reactive oxygen species are also associated with the inflammatory process. Changes in thiol homestasis and redox signaling decisively contribute to amplification of the inflammatory cascade through mitogen activated protein kinase (MAP kinase) pathways. This review focuses on the relationship between oxidative stress, pro-inflammatory cytokines and MAP kinase/protein phosphatase pathways as major modulators of the inflammatory response in acute pancreatitis. Redox sensitive signal transduction mediated by inactivation of protein phosphatases, particularly protein tyrosin phosphatases, is highlighted.

Effect of Chronic Administration of Low Dose Rapamycin on Development and Immunity in Young Rats.

PubMed

Lu, Zhenya; Liu, Furong; Chen, Linglin; Zhang, Huadan; Ding, Yuemin; Liu, Jianxiang; Wong, Michael; Zeng, Ling-Hui

2015-01-01

Mammalian target of rapamycin (mTOR) regulates cell growth, cell differentiation and protein synthesis. Rapamycin, an inhibitor of mTOR, has been widely used as an immunosuppressant and anti-cancer drug. Recently, mTOR inhibitors have also been reported to be a potential anti-epileptic drug, which may be effective when used in young patients with genetic epilepsy. Thus, a suitable dose of rapamycin which can maintain the normal function of mTOR and has fewer side effects ideally should be identified. In the present study, we first detected changes in marker proteins of mTOR signaling pathway during development. Then we determined the dose of rapamycin by treating rats of 2 weeks of age with different doses of rapamycin for 3 days and detected its effect on mTOR pathway. Young rats were then treated with a suitable dose of rapamycin for 4 weeks and the effect of rapamycin on mTOR, development and immunity were investigated. We found that the expression of the marker proteins of mTOR pathway was changed during development in brain hippocampus and neocortex. After 3 days of treanent, 0.03 mg/kg rapamycin had no effect on phospho-S6, whereas 0.1, 0.3, 1.0 and 3.0 mg/kg rapamycin inhibited phospho-S6 in a dose-dependent manner. However, only 1.0 mg/kg and 3.0 mg/kg rapamycin inhibited phospho-S6 after 4 weeks treatment of rapamycin. Parallel to this result, rats treated with 0.1 and 0.3 mg/kg rapamycin had no obvious adverse effects, whereas rats treated with 1.0 and 3.0 mg/kg rapamycin showed significant decreases in body, spleen and thymus weight. Additionally, rats treated with 1.0 and 3.0 mg/kg rapamycin exhibited cognitive impairment and anxiety as evident by maze and open field experiments. Furthermore, the content of IL-1β, IL-2, IFN-γ, TNF-α in serum and cerebral cortex were significantly decreased in 1.0 and 3.0 mg/kg rapamycin-treated rats. The expression of DCX was also significantly decreased in 1.0 and 3.0 mg/kg rapamycin-treated rats. However, rats treated with 1.0 mg/kg rapamycin exhibited fewer and milder side effects than those treated with 3.0 mg/kg. In summary, all these data suggest that there is not a rapamycin dose that can inhibit mTOR for epilepsy without causing any side effects, but 1 mg/kg may be the optimal dose for young rats for suppressing mTOR with relatively few side effects.

Effect of Chronic Administration of Low Dose Rapamycin on Development and Immunity in Young Rats

PubMed Central

Lu, Zhenya; Liu, Furong; Chen, Linglin; Zhang, Huadan; Ding, Yuemin; Liu, Jianxiang; Wong, Michael; Zeng, Ling-Hui

2015-01-01

Mammalian target of rapamycin (mTOR) regulates cell growth, cell differentiation and protein synthesis. Rapamycin, an inhibitor of mTOR, has been widely used as an immunosuppressant and anti-cancer drug. Recently, mTOR inhibitors have also been reported to be a potential anti-epileptic drug, which may be effective when used in young patients with genetic epilepsy. Thus, a suitable dose of rapamycin which can maintain the normal function of mTOR and has fewer side effects ideally should be identified. In the present study, we first detected changes in marker proteins of mTOR signaling pathway during development. Then we determined the dose of rapamycin by treating rats of 2 weeks of age with different doses of rapamycin for 3 days and detected its effect on mTOR pathway. Young rats were then treated with a suitable dose of rapamycin for 4 weeks and the effect of rapamycin on mTOR, development and immunity were investigated. We found that the expression of the marker proteins of mTOR pathway was changed during development in brain hippocampus and neocortex. After 3 days of treanent, 0.03 mg/kg rapamycin had no effect on phospho-S6, whereas 0.1, 0.3, 1.0 and 3.0 mg/kg rapamycin inhibited phospho-S6 in a dose-dependent manner. However, only 1.0 mg/kg and 3.0 mg/kg rapamycin inhibited phospho-S6 after 4 weeks treatment of rapamycin. Parallel to this result, rats treated with 0.1 and 0.3 mg/kg rapamycin had no obvious adverse effects, whereas rats treated with 1.0 and 3.0 mg/kg rapamycin showed significant decreases in body, spleen and thymus weight. Additionally, rats treated with 1.0 and 3.0 mg/kg rapamycin exhibited cognitive impairment and anxiety as evident by maze and open field experiments. Furthermore, the content of IL-1β, IL-2, IFN-γ, TNF-α in serum and cerebral cortex were significantly decreased in 1.0 and 3.0 mg/kg rapamycin-treated rats. The expression of DCX was also significantly decreased in 1.0 and 3.0 mg/kg rapamycin-treated rats. However, rats treated with 1.0 mg/ kg rapamycin exhibited fewer and milder side effects than those treated with 3.0 mg/kg. In summary, all these data suggest that there is not a rapamycin dose that can inhibit mTOR for epilepsy without causing any side effects, but 1 mg /kg may be the optimal dose for young rats for suppressing mTOR with relatively few side effects. PMID:26248290

Hematopoietic Colony Formation from Human Growth Factor-Dependent TF1 Cells and Human Cord Blood Myeloid Progenitor Cells Depends on SHP2 Phosphatase Function

PubMed Central

Etienne-Julan, Maryse; Gotoh, Akihiko; Braun, Stephen E.; Lu, Li; Cooper, Scott; Feng, Gen-Sheng; Li, Xing Jun

2013-01-01

The protein tyrosine phosphatase, SHP2, is widely expressed; however, previous studies demonstrated that hematopoietic cell development more stringently requires Shp2 expression compared to other tissues. Furthermore, somatic gain-of-function SHP2 mutants are commonly found in human myeloid leukemias. Given that pharmacologic inhibitors to SHP2 phosphatase activity are currently in development as putative antileukemic agents, we conducted a series of experiments examining the necessity of SHP2 phosphatase activity for human hematopoiesis. Anti-sense oligonucleotides to human SHP2 coding sequences reduced human cord blood- and human cell line, TF1-derived colony formation. Expression of truncated SHP2 bearing its Src homology 2 (SH2) domains, but lacking the phosphatase domain similarly reduced human cord blood- and TF1-derived colony formation. Mechanistically, expression of truncated SHP2 reduced the interaction between endogenous, full-length SHP2 with the adapter protein, Grb2. To verify the role of SHP2 phosphatase function in human hematopoietic cell development, human cord blood CD34+ cells were transduced with a leukemia-associated phosphatase gain-of-function SHP2 mutant or with a phosphatase dead SHP2 mutant, which indicated that increased phosphatase function enhanced, while decreased SHP2 phosphatase function reduced, human cord blood-derived colonies. Collectively, these findings indicate that SHP2 phosphatase function regulates human hematopoietic cell development and imply that the phosphatase component of SHP2 may serve as a pharmacologic target in human leukemias bearing increased SHP2 phosphatase activity. PMID:23082805

Hematopoietic colony formation from human growth factor-dependent TF1 cells and human cord blood myeloid progenitor cells depends on SHP2 phosphatase function.

PubMed

Broxmeyer, Hal E; Etienne-Julan, Maryse; Gotoh, Akihiko; Braun, Stephen E; Lu, Li; Cooper, Scott; Feng, Gen-Sheng; Li, Xing Jun; Chan, Rebecca J

2013-03-15

The protein tyrosine phosphatase, SHP2, is widely expressed; however, previous studies demonstrated that hematopoietic cell development more stringently requires Shp2 expression compared to other tissues. Furthermore, somatic gain-of-function SHP2 mutants are commonly found in human myeloid leukemias. Given that pharmacologic inhibitors to SHP2 phosphatase activity are currently in development as putative antileukemic agents, we conducted a series of experiments examining the necessity of SHP2 phosphatase activity for human hematopoiesis. Anti-sense oligonucleotides to human SHP2 coding sequences reduced human cord blood- and human cell line, TF1-derived colony formation. Expression of truncated SHP2 bearing its Src homology 2 (SH2) domains, but lacking the phosphatase domain similarly reduced human cord blood- and TF1-derived colony formation. Mechanistically, expression of truncated SHP2 reduced the interaction between endogenous, full-length SHP2 with the adapter protein, Grb2. To verify the role of SHP2 phosphatase function in human hematopoietic cell development, human cord blood CD34+ cells were transduced with a leukemia-associated phosphatase gain-of-function SHP2 mutant or with a phosphatase dead SHP2 mutant, which indicated that increased phosphatase function enhanced, while decreased SHP2 phosphatase function reduced, human cord blood-derived colonies. Collectively, these findings indicate that SHP2 phosphatase function regulates human hematopoietic cell development and imply that the phosphatase component of SHP2 may serve as a pharmacologic target in human leukemias bearing increased SHP2 phosphatase activity.

Dimerization and phosphatase activity of calcyclin-binding protein/Siah-1 interacting protein: the influence of oxidative stress

PubMed Central

Topolska-Woś, Agnieszka M.; Shell, Steven M.; Kilańczyk, Ewa; Szczepanowski, Roman H.; Chazin, Walter J.; Filipek, Anna

2015-01-01

CacyBP/SIP [calcyclin-binding protein/Siah-1 [seven in absentia homolog 1 (Siah E3 ubiquitin protein ligase 1)] interacting protein] is a multifunctional protein whose activity includes acting as an ERK1/2 phosphatase. We analyzed dimerization of mouse CacyBP/SIP in vitro and in mouse neuroblastoma cell line (NB2a) cells, as well as the structure of a full-length protein. Moreover, we searched for the CacyBP/SIP domain important for dimerization and dephosphorylation of ERK2, and we analyzed the role of dimerization in ERK1/2 signaling in NB2a cells. Cell-based assays showed that CacyBP/SIP forms a homodimer in NB2a cell lysate, and biophysical methods demonstrated that CacyBP/SIP forms a stable dimer in vitro. Data obtained using small-angle X-ray scattering supported a model in which CacyBP/SIP occupies an anti-parallel orientation mediated by the N-terminal dimerization domain. Site-directed mutagenesis established that the N-terminal domain is indispensable for full phosphatase activity of CacyBP/SIP. We also demonstrated that the oligomerization state of CacyBP/SIP as well as the level of post-translational modifications and subcellular distribution of CacyBP/SIP change after activation of the ERK1/2 pathway in NB2a cells due to oxidative stress. Together, our results suggest that dimerization is important for controlling phosphatase activity of CacyBP/SIP and for regulating the ERK1/2 signaling pathway.—Topolska-Woś, A. M., Shell, S. M., Kilańczyk, E., Szczepanowski, R. H., Chazin, W. J., Filipek, A. Dimerization and phosphatase activity of calcyclin-binding protein/Siah-1 interacting protein: the influence of oxidative stress. PMID:25609429

Differential regulation of the androgen receptor by protein phosphatase regulatory subunits

PubMed Central

Grey, James; Jones, Dominic; Wilson, Laura; Nakjang, Sirintra; Clayton, Jake; Temperley, Richard; Clark, Emma; Gaughan, Luke; Robson, Craig

2018-01-01

The Androgen Receptor (AR) is a key molecule in the development, maintenance and progression of prostate cancer (PC). However, the relationship between the AR and co-regulatory proteins that facilitate AR activity in castrate resistant settings remain understudied. Here we show that protein phosphatase 1 regulatory subunits, identified from a phosphatase RNAi screen, direct PP1 catalytic subunits to a varied yet significant response in AR function. As such, we have characterised the PP1β holoenzyme, myosin phosphatase (MLCP), as a novel ligand independent regulator of the AR. Sustained MLCP activity through down-regulation of the MLCP inhibitory subunit, PPP1R14C, results in impaired AR nuclear translocation, protein stability and transcriptional activity in distinct models of PC progression, culminating in restoration of a non-malignant prostate genotype. Phenotypically, a marked reduction in cell proliferation and migration, characterised by G1 cell cycle arrest is observed, confirming PP1 holoenzyme disruption as a novel treatment approach in PC. PMID:29423094

P-TEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatase

PubMed Central

Myers, Michael P.; Stolarov, Javor P.; Eng, Charis; Li, Jing; Wang, Steven I.; Wigler, Michael H.; Parsons, Ramon; Tonks, Nicholas K.

1997-01-01

Protein tyrosine phosphatases (PTPs) have long been thought to play a role in tumor suppression due to their ability to antagonize the growth promoting protein tyrosine kinases. Recently, a candidate tumor suppressor from 10q23, termed P-TEN, was isolated, and sequence homology was demonstrated with members of the PTP family, as well as the cytoskeletal protein tensin. Here we show that recombinant P-TEN dephosphorylated protein and peptide substrates phosphorylated on serine, threonine, and tyrosine residues, indicating that P-TEN is a dual-specificity phosphatase. In addition, P-TEN exhibited a high degree of substrate specificity, showing selectivity for extremely acidic substrates in vitro. Furthermore, we demonstrate that mutations in P-TEN, identified from primary tumors, tumor cells lines, and a patient with Bannayan–Zonana syndrome, resulted in the ablation of phosphatase activity, demonstrating that enzymatic activity of P-TEN is necessary for its ability to function as a tumor suppressor. PMID:9256433

Evolution of bacterial-like phosphoprotein phosphatases in photosynthetic eukaryotes features ancestral mitochondrial or archaeal origin and possible lateral gene transfer.

PubMed

Uhrig, R Glen; Kerk, David; Moorhead, Greg B

2013-12-01

Protein phosphorylation is a reversible regulatory process catalyzed by the opposing reactions of protein kinases and phosphatases, which are central to the proper functioning of the cell. Dysfunction of members in either the protein kinase or phosphatase family can have wide-ranging deleterious effects in both metazoans and plants alike. Previously, three bacterial-like phosphoprotein phosphatase classes were uncovered in eukaryotes and named according to the bacterial sequences with which they have the greatest similarity: Shewanella-like (SLP), Rhizobiales-like (RLPH), and ApaH-like (ALPH) phosphatases. Utilizing the wealth of data resulting from recently sequenced complete eukaryotic genomes, we conducted database searching by hidden Markov models, multiple sequence alignment, and phylogenetic tree inference with Bayesian and maximum likelihood methods to elucidate the pattern of evolution of eukaryotic bacterial-like phosphoprotein phosphatase sequences, which are predominantly distributed in photosynthetic eukaryotes. We uncovered a pattern of ancestral mitochondrial (SLP and RLPH) or archaeal (ALPH) gene entry into eukaryotes, supplemented by possible instances of lateral gene transfer between bacteria and eukaryotes. In addition to the previously known green algal and plant SLP1 and SLP2 protein forms, a more ancestral third form (SLP3) was found in green algae. Data from in silico subcellular localization predictions revealed class-specific differences in plants likely to result in distinct functions, and for SLP sequences, distinctive and possibly functionally significant differences between plants and nonphotosynthetic eukaryotes. Conserved carboxyl-terminal sequence motifs with class-specific patterns of residue substitutions, most prominent in photosynthetic organisms, raise the possibility of complex interactions with regulatory proteins.

The Membrane and Lipids as Integral Participants in Signal Transduction: Lipid Signal Transduction for the Non-Lipid Biochemist

ERIC Educational Resources Information Center

Eyster, Kathleen M.

2007-01-01

Reviews of signal transduction have often focused on the cascades of protein kinases and protein phosphatases and their cytoplasmic substrates that become activated in response to extracellular signals. Lipids, lipid kinases, and lipid phosphatases have not received the same amount of attention as proteins in studies of signal transduction.…

Integrative Transcriptome Profiling of Cognitive Aging and Its Preservation through Ser/Thr Protein Phosphatase Regulation.

PubMed

Park, C Sehwan; Valomon, Amandine; Welzl, Hans

2015-01-01

Environmental enrichment has been reported to delay or restore age-related cognitive deficits, however, a mechanism to account for the cause and progression of normal cognitive decline and its preservation by environmental enrichment is lacking. Using genome-wide SAGE-Seq, we provide a global assessment of differentially expressed genes altered with age and environmental enrichment in the hippocampus. Qualitative and quantitative proteomics in naïve young and aged mice was used to further identify phosphorylated proteins differentially expressed with age. We found that increased expression of endogenous protein phosphatase-1 inhibitors in aged mice may be characteristic of long-term environmental enrichment and improved cognitive status. As such, hippocampus-dependent performances in spatial, recognition, and associative memories, which are sensitive to aging, were preserved by environmental enrichment and accompanied by decreased protein phosphatase activity. Age-associated phosphorylated proteins were also found to correspond to the functional categories of age-associated genes identified through transcriptome analysis. Together, this study provides a comprehensive map of the transcriptome and proteome in the aging brain, and elucidates endogenous protein phosphatase-1 inhibition as a potential means through which environmental enrichment may ameliorate age-related cognitive deficits.

A peptide export–import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay

PubMed Central

Perego, Marta

1997-01-01

The phosphorelay signal transduction system activates developmental transcription in sporulation of Bacillus subtilis by phosphorylation of aspartyl residues of the Spo0F and Spo0A response regulators. The phosphorylation level of these response regulators is determined by the opposing activities of protein kinases and protein aspartate phosphatases that interpret positive and negative signals for development in a signal integration circuit. The RapA protein aspartate phosphatase of the phosphorelay is regulated by a peptide that directly inhibits its activity. This peptide is proteolytically processed from an inactive pre-inhibitor protein encoded in the phrA gene. The pre-inhibitor is cleaved by the protein export apparatus to a putative pro-inhibitor that is further processed to the active inhibitor peptide and internalized by the oligopeptide permease. This export–import circuit is postulated to be a mechanism for timing phosphatase activity where the processing enzymes regulate the rate of formation of the active inhibitor. The processing events may, in turn, be controlled by a regulatory hierarchy. Chromosome sequencing has revealed several other phosphatase–prepeptide gene pairs in B. subtilis, suggesting that the use of this mechanism may be widespread in signal transduction. PMID:9238025

Increased PTP1B expression and phosphatase activity in colorectal cancer results in a more invasive phenotype and worse patient outcome.

PubMed

Hoekstra, Elmer; Das, Asha M; Swets, Marloes; Cao, Wanlu; van der Woude, C Janneke; Bruno, Marco J; Peppelenbosch, Maikel P; Kuppen, Peter J K; Ten Hagen, Timo L M; Fuhler, Gwenny M

2016-04-19

Cell signaling is dependent on the balance between phosphorylation of proteins by kinases and dephosphorylation by phosphatases. This balance if often disrupted in colorectal cancer (CRC), leading to increased cell proliferation and invasion. For many years research has focused on the role of kinases as potential oncogenes in cancer, while phosphatases were commonly assumed to be tumor suppressive. However, this dogma is currently changing as phosphatases have also been shown to induce cancer growth. One of these phosphatases is protein tyrosine phosphatase 1B (PTP1B). Here we report that the expression of PTP1B is increased in colorectal cancer as compared to normal tissue, and that the intrinsic enzymatic activity of the protein is also enhanced. This suggests a role for PTP1B phosphatase activity in CRC formation and progression. Furthermore, we found that increased PTP1B expression is correlated to a worse patient survival and is an independent prognostic marker for overall survival and disease free survival. Knocking down PTP1B in CRC cell lines results in a less invasive phenotype with lower adhesion, migration and proliferation capabilities. Together, these results suggest that inhibition of PTP1B activity is a promising new target in the treatment of colorectal cancer and the prevention of metastasis.

A screen for over-secretion of proteins by yeast based on a dual component cellular phosphatase and immuno-chromogenic stain for exported bacterial alkaline phosphatase reporter

PubMed Central

2013-01-01

Background To isolate over-secretors, we subjected to saturation mutagenesis, a strain of P.pastoris exporting E. coli alkaline phosphatase (EAP) fused to the secretory domain of the yeast α factor pheromone through cellular PHO1/KEX2 secretory processing signals as the α-sec-EAP reporter protein. Direct chromogenic staining for α-sec-EAP activity is non-specific as its NBT/BCIP substrate cross-reacts with cellular phosphatases which can be inhibited with Levulinic acid. However, the parental E(P) strain only exports detectable levels of α-sec-EAP at 69 hours and not within the 36 hour period post-seeding required for effective screening with the consequent absence of a reference for secretion. We substituted the endogenous cellular phosphatase activity as a comparative reference for secretion rate and levels as well as for colony alignment while elevating specificity and sensitivity of detection of the exported protein with other innovative modifications of the immuno-chromogenic staining application for screening protein export mutants. Results Raising the specificity and utility of staining for α-sec-EAP activity required 5 modifications including some to published methods. These included, exploitation of endogenous phosphatase activity, reduction of the cell/protein burden, establishment of the direct relation between concentrations of transcriptional inducer and exported membrane immobilized protein and concentrations of protein exported into growth media, amplification of immuno-specificity and sensitivity of detection of α-sec-EAP reporter enzyme signal and restriction of staining to optimal concentrations of antisera and time periods. The resultant immuno-chromogenic screen allows for the detection of early secretion and as little as 1.3 fold over-secretion of α-sec-EAP reporter protein by E(M) mutants in the presence of 10 fold -216 fold higher concentrations of HSA. Conclusions The modified immuno-chromogenic screen is sensitive, specific and has led to the isolation of mutants E(M) over-secreting the α-sec-EAP reporter protein by a minimum of 50 fold higher levels than that exported by non-mutagenized E(P) parental strains. Unselected proteins were also over-secreted. PMID:23602005

A macro-enzyme cause of an isolated increase of alkaline phosphatase.

PubMed

Cervinski, Mark A; Lee, Hong Kee; Martin, Isabella W; Gavrilov, Dimitar K

2015-02-02

Macroenzyme complexes of serum enzymes and antibody can increase the circulating enzymatic activity and may lead to unnecessary additional testing and procedures. Laboratory physicians and scientists need to be aware of techniques to identify macroenzyme complexes when suspected. To investigate the possibility of a macro-alkaline phosphatase in the serum of a 74 year old male with persistently increased alkaline phosphatase we coupled a protein A/G agarose affinity chromatography technique with isoenzyme electrophoresis to look for the presence of macro-alkaline phosphatase. The majority of the alkaline phosphatase activity in the patient's serum sample was bound to the column and only a minor fraction (25%) of alkaline phosphatase activity was present in the column flow-through. The alkaline phosphatase activity was also found to co-elute with the immunoglobulins in the patient sample. The alkaline phosphatase activity in a control serum sample concurrently treated in the same manner did not bind to the column and was found in the column flow-through. The use of protein A/G agarose affinity chromatography is a rapid and simple method that can be applied to the investigation of other macro-enzyme complexes. Copyright © 2014 Elsevier B.V. All rights reserved.

Francisella DnaK Inhibits Tissue-nonspecific Alkaline Phosphatase*

PubMed Central

Arulanandam, Bernard P.; Chetty, Senthilnath Lakshmana; Yu, Jieh-Juen; Leonard, Sean; Klose, Karl; Seshu, Janakiram; Cap, Andrew; Valdes, James J.; Chambers, James P.

2012-01-01

Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related Gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella. PMID:22923614

Francisella DnaK inhibits tissue-nonspecific alkaline phosphatase.

PubMed

Arulanandam, Bernard P; Chetty, Senthilnath Lakshmana; Yu, Jieh-Juen; Leonard, Sean; Klose, Karl; Seshu, Janakiram; Cap, Andrew; Valdes, James J; Chambers, James P

2012-10-26

Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella.

Contrasting Pathology of the Stress Granule Proteins TIA-1 and G3BP in Tauopathies

PubMed Central

Vanderweyde, Tara; Yu, Haung; Varnum, Megan; Liu-Yesucevitz, Liqun; Citro, Allison; Ikezu, Tsuneya; Duff, Karen; Wolozin, Benjamin

2012-01-01

Stress induces aggregation of RNA-binding proteins to form inclusions, termed stress granules (SGs). Recent evidence suggests that SG proteins also colocalize with neuropathological structures, but whether this occurs in Alzheimer’s disease is unknown. We examined the relationship between SG proteins and neuropathology in brain tissue from P301L Tau transgenic mice, as well as in cases of Alzheimer’s disease and FTDP-17. The pattern of SG pathology differs dramatically based on the RNA-binding protein examined. SGs positive for T-cell intracellular antigen-1 (TIA-1) or tristetraprolin (TTP) initially do not colocalize with tau pathology, but then merge with tau inclusions as disease severity increases. In contrast, G3BP (ras GAP-binding protein) identifies a novel type of molecular pathology that shows increasing accumulation in neurons with increasing disease severity, but often is not associated with classic markers of tau pathology. TIA-1 and TTP both bind phospho-tau, and TIA-1 overexpression induces formation of inclusions containing phospho-tau. These data suggest that SG formation might stimulate tau pathophysiology. Thus, study of RNA-binding proteins and SG biology highlights novel pathways interacting with the pathophysiology of AD, providing potentially new avenues for identifying diseased neurons and potentially novel mechanisms regulating tau biology. PMID:22699908

Cardioprotective effects of BMS-180448, a prototype mitoK(ATP) channel opener, and the role of salvage kinases, in the rat model of global ischemia and reperfusion heart injury.

PubMed

Lee, Ju-Han; Jung, In-Sang; Lee, Sung-Hun; Yang, Min-Kyu; Hwang, Ji-Hye; Lee, Hak-Dong; Cho, Yu-Sun; Song, Min-Jin; Yi, Kyu-Yang; Yoo, Sung-Eun; Kwon, Suk-Hyung; Kim, Bokyung; Lee, Chang-Soo; Shin, Hwa-Sup

2007-05-01

To investigate the involvement of reperfusion-induced salvage kinases (RISK) as possible signaling molecules for the cardioprotective effects of BMS-180448, a prototype mitochondrial ATP-sensitive K+ (mitoK(ATP)) channel opener, we measured its cardioprotective effects in a rat model of ischemia/reperfusion (I/R) heart injury, together with western blotting analysis of five different signaling proteins. In isolated rat hearts subjected to 30-min global ischemia followed by 30-min reperfusion, BMS-180448 (1, 3 and 10 microM) significantly increased reperfusion left ventricular developed pressure (LVDP) and 30-min reperfusion double product (heart rate x LVDP) in a concentration-dependent manner, while decreasing left ventricular end-diastolic pressure (LVEDP) throughout reperfusion period in a concentration-dependent manner. SDS-PAGE/western blotting analysis of left ventricle reperfused for 30 min revealed that BMS-180448 significantly decreased phospho-GSK3beta at high concentration, whereas it tended to increase slightly phospho-eNOS and phospho-p70S6K with concentration. However, BMS-180448 had no effect on phospho-Akt and phospho-Bad. These results suggest that the cardioprotective effects of BMS-180448 against I/R heart injury may result from direct activation of mitoK(ATP) channel in cardiomyocytes, with the minimal role of RISK pathway in the activation of this channel and the cardioprotective effects of BMS-180448.

Ferulic acid regulates the AKT/GSK-3β/CRMP-2 signaling pathway in a middle cerebral artery occlusion animal model

PubMed Central

Gim, Sang-A; Sung, Jin-Hee; Shah, Fawad-Ali; Kim, Myeong-Ok

2013-01-01

Ferulic acid, a component of the plants Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort, exerts a neuroprotective effect by regulating various signaling pathways. This study showed that ferulic acid treatment prevents the injury-induced increase of collapsin response mediator protein 2 (CRMP-2) in focal cerebral ischemia. Glycogen synthase kinase-3β (GSK-3β) regulates CRMP-2 function through phosphorylation of CRMP-2. Moreover, the pro-apoptotic activity of GSK-3β is inactivated by phosphorylation by Akt. This study investigated whether ferulic acid modulates the expression of CRMP-2 and its upstream targets, Akt and GSK-3β, in focal cerebral ischemia. Male rats were treated immediately with ferulic acid (100 mg/kg, i.v.) or vehicle after middle cerebral artery occlusion (MCAO), and then cerebral cortices were collected 24 hr after MCAO. MCAO resulted in decreased levels of phospho-Akt and phospho-GSK-3β, while ferulic acid treatment prevented the decrease in the levels of these proteins. Moreover, phospho-CRMP-2 and CRMP-2 levels increased during MCAO, whereas ferulic acid attenuated these injury-induced increases. These results demonstrate that ferulic acid regulates the Akt/GSK-3β/CRMP-2 signaling pathway in focal cerebral ischemic injury, thereby protecting against brain injury. PMID:23825478

Essential role for cyclic-AMP responsive element binding protein 1 (CREB) in the survival of acute lymphoblastic leukemia.

PubMed

van der Sligte, Naomi E; Kampen, Kim R; ter Elst, Arja; Scherpen, Frank J G; Meeuwsen-de Boer, Tiny G J; Guryev, Victor; van Leeuwen, Frank N; Kornblau, Steven M; de Bont, Eveline S J M

2015-06-20

Acute lymphoblastic leukemia (ALL) relapse remains a leading cause of cancer related death in children, therefore, new therapeutic options are needed. Recently, we showed that a peptide derived from Cyclic-AMP Responsive Element Binding Protein (CREB) was highly phosphorylated in pediatric leukemias. In this study, we determined CREB phosphorylation and mRNA levels showing that CREB expression was significantly higher in ALL compared to normal bone marrow (phosphorylation: P < 0.0001, mRNA: P = 0.004). High CREB and phospho-CREB expression was correlated with a lower median overall survival in a cohort of 140 adult ALL patients. ShRNA mediated knockdown of CREB in ALL cell lines blocked leukemic cell growth by inducing cell cycle arrest and apoptosis. Gene expression array analysis showed downregulation of CREB target genes regulating cell proliferation and glucose metabolism and upregulation of apoptosis inducing genes. Similar to CREB knockdown, the CREB inhibitor KG-501 decreased leukemic cell viability and induced apoptosis in ALL cell lines, as well as primary T-ALL samples, with cases showing high phospho-CREB levels being more sensitive than those with lower phospho-CREB levels. Together, these in vitro findings support an important role for CREB in the survival of ALL cells and identify this transcription factor as a potential target for treatment.

Expression of the growth factor pleiotrophin and its receptor protein tyrosine phosphatase beta/zeta in the serum, cartilage and subchondral bone of patients with osteoarthritis.

PubMed

Kaspiris, Angelos; Mikelis, Constantinos; Heroult, Melanie; Khaldi, Lubna; Grivas, Theodoros B; Kouvaras, Ioannis; Dangas, Spyridon; Vasiliadis, Elias; Lioté, Frédéric; Courty, José; Papadimitriou, Evangelia

2013-07-01

Pleiotrophin is a heparin-binding growth factor expressed in embryonic but not mature cartilage, suggesting a role in cartilage development. Elucidation of the molecular changes observed during the remodelling process in osteoarthritis is of paramount importance. This study aimed to investigate serum pleiotrophin levels and expression of pleiotrophin and its receptor protein tyrosine phosphatase beta/zeta in the cartilage and subchondral bone of osteoarthritis patients. Serum samples derived from 16 osteoarthritis patients and 18 healthy donors. Pleiotrophin and receptor protein tyrosine phosphatase beta/zeta in the cartilage and subchondral bone were studied in 29 patients who had undergone total knee or hip replacement for primary osteoarthritis and in 10 control patients without macroscopic osteoarthritis changes. Serum pleiotrophin levels and expression of pleiotrophin in chondrocytes and subchondral bone osteocytes significantly increased in osteoarthritis patients graded Ahlback II to III. Receptor protein tyrosine phosphatase beta/zeta was mainly detected in the subchondral bone osteocytes of patients with moderate osteoarthritis and as disease severity increased, in the osteocytes and bone lining cells of the distant trabeculae. These data render pleiotrophin and receptor protein tyrosine phosphatase beta/zeta promising candidates for further studies towards developing targeted therapeutic schemes for osteoarthritis. Copyright © 2012 Société française de rhumatologie. Published by Elsevier SAS. All rights reserved.

Alcohol alters hepatic FoxO1, p53, and mitochondrial SIRT5 deacetylation function

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

Lieber, Charles S.; Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029; Leo, Maria Anna

2008-08-22

Chronic alcohol consumption affects the gene expression of a NAD-dependent deacetylase Sirtuis 1 (SIRT1) and the peroxisome proliferator-activated receptor-{gamma} coactivator1{alpha} (PGC-1{alpha}). Our aim was to verify that it also alters the forkhead (FoxO1) and p53 transcription factor proteins, critical in the hepatic response to oxidative stress and regulated by SIRT1 through its deacetylating capacity. Accordingly, rats were pair-fed the Lieber-DeCarli alcohol-containing liquid diets for 28 days. Alcohol increased hepatic mRNA expression of FoxO1 (p = 0.003) and p53 (p = 0.001) while corresponding protein levels remained unchanged. However phospho-FoxO1 and phospho-Akt (protein kinase) were both decreased by alcohol consumption (pmore » = 0.04 and p = 0.02, respectively) while hepatic p53 was found hyperacetylated (p = 0.017). Furthermore, mitochondrial SIRT5 was reduced (p = 0.0025), and PGC-1{alpha} hyperacetylated (p = 0.027), establishing their role in protein modification. Thus, alcohol consumption disrupts nuclear-mitochondrial interactions by post-translation protein modifications, which contribute to alteration of mitochondrial biogenesis through the newly discovered reduction of SIRT5.« less

Laforin, a dual specificity phosphatase that dephosphorylates complex carbohydrates.

PubMed

Worby, Carolyn A; Gentry, Matthew S; Dixon, Jack E

2006-10-13

Laforin is the only phosphatase in the animal kingdom that contains a carbohydrate-binding module. Mutations in the gene encoding laforin result in Lafora disease, a fatal autosomal recessive neurodegenerative disorder, which is diagnosed by the presence of intracellular deposits of insoluble complex carbohydrates known as Lafora bodies. We demonstrate that laforin interacts with proteins known to be involved in glycogen metabolism and rule out several of these proteins as potential substrates. Surprisingly, we find that laforin displays robust phosphatase activity against a phosphorylated complex carbohydrate. Furthermore, this activity is unique to laforin, since several other phosphatases are unable to dephosphorylate polysaccharides. Finally, fusing the carbohydrate-binding module of laforin to the dual specific phosphatase VHR does not result in the ability of this phosphatase to dephosphorylate polysaccharides. Therefore, we hypothesize that laforin is unique in its ability to utilize a phosphorylated complex carbohydrate as a substrate and that this function may be necessary for the maintenance of normal cellular glycogen.

Characterization of two distinct dual specificity phosphatases encoded in alternative open reading frames of a single gene located on human chromosome 10q22.2.

PubMed

Chen, Hsu-Hsin; Luche, Ralf; Wei, Bo; Tonks, Nicholas K

2004-10-01

Dual specificity phosphatases (DSPs) are members of the protein-tyrosine phosphatase superfamily that dephosphorylate both phosphotyrosine and phosphoserine/threonine residues in vitro. Many DSPs have been found to play important roles in various aspects of cellular function and to be involved in human disease. We have identified a gene located on human chromosome 10q22.2, which utilizes alternative open reading frames (ORFs) to encode the following two distinct DSPs: the previously described testis and skeletal muscle-specific dual specificity phosphatase (TMDP) and a novel DSP, muscle-restricted dual specificity phosphatase (MDSP). Use of alternative ORFs encoding distinct proteins from a single gene is extremely rare in eukaryotes, and in all previously reported cases the two proteins produced from one gene are unrelated. To our knowledge this is the first example of a gene from which two distinct proteins of the same family are expressed using alternative ORFs. Here we provide evidence that both MDSP and TMDP proteins are expressed in vivo and are restricted to specific tissues, skeletal muscle and testis, respectively. Most interestingly, the protein expression profiles of both MDSP and TMDP during mouse postnatal development are strikingly similar. MDSP is expressed at very low levels in myotubes and early postnatal muscle. TMDP is not detectable in testis lysate in the first 3 weeks of life. The expression of both MDSP and TMDP proteins was markedly increased at approximately the 3rd week after birth and continued to increase gradually into adulthood, implying that the physiological functions of both DSPs are specific to the mature/late-developing organs. The conserved gene structure and the similarity in postnatal expression profile of these two proteins suggest biological significance of the unusual gene arrangement.

Clonidine preconditioning improved cerebral ischemia-induced learning and memory deficits in rats via ERK1/2-CREB/ NF-κB-NR2B pathway.

PubMed

Li, Yanli; Yu, Min; Zhao, Bo; Wang, Yan; Zha, Yunhong; Li, Zicheng; Yu, Lingling; Yan, Lingling; Chen, Zhangao; Zhang, Wenjuan; Zeng, Xiaoli; He, Zhi

2018-01-05

Clonidine, a classical α-2 adrenergic agonists, has been shown to antagonize brain damage caused by hypoxia, cerebral ischemia and excitotoxicity and reduce cerebral infarction volume in recent studies. We herein investigate the regulatory effect and possible underlying mechanism of clonidine on learning and memory in rats with cerebral ischemia. The cerebral ischemia rat model was established by right middle cerebral artery occlusion for 2h and reperfusion for 28 days. Drugs were administrated to the rats for consecutive 7 days intraperitoneally and once again on the day of surgery. The learning and memory in rats was assayed by Morris water maze. Moreover, protein expression levels of NMDAR2B (NR2B)/ phosphor - NR2B, ERK1/2/phosphor- ERK1/2, CREB/phosphor-CREB and NF-κB/phosphor-NF-κB in the cortex and hippocampus of the rats were assayed by western blotting. Our results demonstrated that clonidine treatment significantly abrogated the negative effect induced by cerebral ischemia on the learning and memory in the rats. In the Western blotting assay, clonidine treatment led to significant up-regulation of the expression level of NR2B and Phospho-NR2B in the hippocampus of the rats when compared with the cerebral ischemia group. Furthermore, clonidine also significantly decreased the protein expression levels of ERK1/2, Phospho-ERK1/2, CREB, Phospho-CREB and Phospho-NF-κB in the hippocampus of the rats when compared with the cerebral ischemia group. In conclusion, clonidine could improve the learning and memory ability of rats with cerebral ischemia, and NR2B, ERK1/2, CREB, NF-κB were involved in this effect. Copyright © 2017 Elsevier B.V. All rights reserved.

Hyperactive ERK and persistent mTOR signaling characterize vemurafenib resistance in papillary thyroid cancer cells.

PubMed

Hanly, Elyse K; Tuli, Neha Y; Bednarczyk, Robert B; Suriano, Robert; Geliebter, Jan; Moscatello, Augustine L; Darzynkiewicz, Zbigniew; Tiwari, Raj K

2016-02-23

Clinical studies evaluating targeted BRAFV600E inhibitors in advanced thyroid cancer patients are currently underway. Vemurafenib (BRAFV600E inhibitor) monotherapy has shown promising results thus far, although development of resistance is a clinical challenge. The objective of this study was to characterize development of resistance to BRAFV600E inhibition and to identify targets for effective combination therapy. We created a line of BCPAP papillary thyroid cancer cells resistant to vemurafenib by treating with increasing concentrations of the drug. The resistant BCPAP line was characterized and compared to its sensitive counterpart with respect to signaling molecules thought to be directly related to resistance. Expression and phosphorylation of several critical proteins were analyzed by Western blotting and dimerization was evaluated by immunoprecipitation. Resistance to vemurafenib in BCPAP appeared to be mediated by constitutive overexpression of phospho-ERK and by resistance to inhibition of both phospho-mTOR and phospho-S6 ribosomal protein after vemurafenib treatment. Expression of potential alternative signaling molecule, CRAF, was not increased in the resistant line, although formation of CRAF dimers appeared increased. Expression of membrane receptors HER2 and HER3 was greatly amplified in the resistant cancer cells. Papillary thyroid cancer cells were capable of overcoming targeted BRAFV600E inhibition by rewiring of cell signal pathways in response to prolonged vemurafenib therapy. Our study suggests that in vitro culture of cancer cells may be useful in assessing molecular resistance pathways. Potential therapies in advanced thyroid cancer patients may combine vemurafenib with inhibitors of CRAF, HER2/HER3, ERK, and/or mTOR to delay or abort development of resistance.

Phosphorylated Heat Shock Protein 20 (HSPB6) Regulates Transforming Growth Factor-α-Induced Migration and Invasion of Hepatocellular Carcinoma Cells.

PubMed

Matsushima-Nishiwaki, Rie; Toyoda, Hidenori; Nagasawa, Tomoaki; Yasuda, Eisuke; Chiba, Naokazu; Okuda, Seiji; Maeda, Atsuyuki; Kaneoka, Yuji; Kumada, Takashi; Kozawa, Osamu

2016-01-01

Human hepatocellular carcinoma (HCC) is one of the major malignancies in the world. Small heat shock proteins (HSPs) are reported to play an important role in the regulation of a variety of cancer cell functions, and the functions of small HSPs are regulated by post-translational modifications such as phosphorylation. We previously reported that protein levels of a small HSP, HSP20 (HSPB6), decrease in vascular invasion positive HCC compared with those in the negative vascular invasion. Therefore, in the present study, we investigated whether HSP20 is implicated in HCC cell migration and the invasion using human HCC-derived HuH7 cells. The transforming growth factor (TGF)-α-induced migration and invasion were suppressed in the wild-type-HSP20 overexpressed cells in which phosphorylated HSP20 was detected. Phospho-mimic-HSP20 overexpression reduced the migration and invasion compared with unphosphorylated HSP20 overexpression. Dibutyryl cAMP, which enhanced the phosphorylation of wild-type-HSP20, significantly reduced the TGF-α-induced cell migration of wild-type HSP20 overexpressed cells. The TGF-α-induced cell migration was inhibited by SP600125, a c-Jun N-terminal kinases (JNK) inhibitor. In phospho-mimic-HSP20 overexpressed HuH7 cells, TGF-α-stimulated JNK phosphorylation was suppressed compared with the unphosphorylated HSP20 overexpressed cells. Moreover, the level of phospho-HSP20 protein in human HCC tissues was significantly correlated with tumor invasion. Taken together, our findings strongly suggest that phosphorylated HSP20 inhibits TGF-α-induced HCC cell migration and invasion via suppression of the JNK signaling pathway.

2'-Hydroxyflavanone ameliorates mesenteric angiogenesis and portal-systemic collaterals in rats with liver fibrosis.

PubMed

Hsin, I-Fang; Lee, Jing-Yi; Huo, Teh-Ia; Lee, Fa-Yauh; Huang, Hui-Chun; Hsu, Shao-Jung; Wang, Sun-Sang; Ho, Hsin-Ling; Lin, Han-Chieh; Lee, Shou-Dong

2016-05-01

Portal-systemic collaterals lead to dreadful consequences in patients with cirrhosis. Angiogenesis participates in the development of liver fibrosis, hyperdynamic circulation, and portal-systemic collaterals. 2'-Hydroxyflavanone (2'-HF), one of the citrus fruits flavonoids, is known to have antiangiogenesis effect without adverse response. However, the relevant effects in liver fibrosis have not been surveyed. Male Wistar rats received thioacetamide (TAA, 100 mg/kg tiw, i.p.) for 6 weeks to induce liver fibrosis. On the 29th to 42nd day, rats randomly received 2'-HF (100 mg/kg, qod, i.p.) or vehicle (corn oil). On the 43rd day, after hemodynamic measurements, the followings were surveyed: (i) severity of collaterals; (ii) mesenteric angiogenesis; (iii) mesenteric proangiogenic factors protein expressions; (iv) Mesenteric vascular endothelial cells apoptosis; and (v) Mesenteric expressions of proteins regulating apoptosis. Compared with the vehicle group, 2'-HF did not significantly change body weight, mean arterial pressure, heart rate, and portal pressure in TAA rats. 2'-HF significantly alleviated the severity of collaterals, but the mesenteric phospho-ERK, ERK, phospho-Akt, Akt, COX1, COX2, VEGF, and VEGFR-2 protein expressions were not altered. The apoptotic index of 2'-HF group was significantly higher and the mesenteric protein expressions of pro-apoptotic factors, NFkB 50, NFkB 65, Bax, phospho-p53, 17 kD cleaved caspase 3, and 17 kD casepase 3 were up-regulated. 2'-HF does not influence the hemodynamics but alleviated the severity of collaterals in rats with liver fibrosis and early portal hypertension. This is, at least partly, attributed to enhanced apoptosis of mesenteric vascular endothelial cells. © 2015 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

INHIBITION OF PROTEIN TYROSINE PHOSPHATASE ACTIVITY MEDIATES EPIDERMAL GROWTH FACTOR RECEPTOR SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

EPA Science Inventory

Epidemiological studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads t...

Serine/threonine protein phosphatases: multi-purpose enzymes in control of defense mechanisms

USDA-ARS?s Scientific Manuscript database

Serine/threonine protein phosphatases are a group of enzymes involved in the regulation of defense mechanisms in plants. This paper describes the effects of an inhibitor of these enzymes on the expression of all of the genes associated with these defense mechanisms. The results suggest that inhibi...

MECHANISM OF PROTEIN TYROSINE PHOSPHATASE INHIBITION IN HUMAN AIRWAY EPITHELIAL CELLS (HAEC) EXPOSED TO ZN2+

EPA Science Inventory

A number of studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to Zn2+ inhibits protein tyrosine phosphatase (PTP) activity and leads to activation of epidermal growth factor receptor (EGFR) signaling in ...

Timely Degradation of Wip1 Phosphatase by APC/C Activator Protein Cdh1 is Necessary for Normal Mitotic Progression.

PubMed

Jeong, Ho-Chang; Gil, Na-Yeon; Lee, Ho-Soo; Cho, Seung-Ju; Kim, Kyungtae; Chun, Kwang-Hoon; Cho, Hyeseong; Cha, Hyuk-Jin

2015-08-01

Wip1 belongs to the protein phosphatase C (PP2C) family, of which expression is up-regulated by a number of external stresses, and serves as a stress modulator in normal physiological conditions. When overexpressed, premature dephosphorylation of stress-mediators by Wip1 results in abrogation of tumor surveillance, thus Wip1 acts as an oncogene. Previously, the functional regulation of Wip1 in cell-cycle progression by counteracting cellular G1 and G2/M checkpoint activity in response to DNA damage was reported. However, other than in stress conditions, the function and regulatory mechanism of Wip1 has not been fully determined. Herein, we demonstrated that protein regulation of Wip1 occurs in a cell cycle-dependent manner, which is directly governed by APC/C(Cdh1) at the end of mitosis. In particular, we also showed evidence that Wip1 phosphatase activity is closely associated with its own protein stability, suggesting that reduced phosphatase activity of Wip1 during mitosis could trigger its degradation. Furthermore, to verify the physiological role of its phosphatase activity during mitosis, we established doxycycline-inducible cell models, including a Wip1 wild type (WT) and phosphatase dead mutant (Wip1 DA). When ectopically expressing Wip1 WT, we observed a delay in the transition from metaphase to anaphase. In conclusion, these studies show that mitotic degradation of Wip1 by APC/C(Cdh1) is important for normal mitotic progression. © 2015 Wiley Periodicals, Inc.

Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling.

PubMed Central

Janssens, V; Goris, J

2001-01-01

Protein phosphatase 2A (PP2A) comprises a family of serine/threonine phosphatases, minimally containing a well conserved catalytic subunit, the activity of which is highly regulated. Regulation is accomplished mainly by members of a family of regulatory subunits, which determine the substrate specificity, (sub)cellular localization and catalytic activity of the PP2A holoenzymes. Moreover, the catalytic subunit is subject to two types of post-translational modification, phosphorylation and methylation, which are also thought to be important regulatory devices. The regulatory ability of PTPA (PTPase activator), originally identified as a protein stimulating the phosphotyrosine phosphatase activity of PP2A, will also be discussed, alongside the other regulatory inputs. The use of specific PP2A inhibitors and molecular genetics in yeast, Drosophila and mice has revealed roles for PP2A in cell cycle regulation, cell morphology and development. PP2A also plays a prominent role in the regulation of specific signal transduction cascades, as witnessed by its presence in a number of macromolecular signalling modules, where it is often found in association with other phosphatases and kinases. Additionally, PP2A interacts with a substantial number of other cellular and viral proteins, which are PP2A substrates, target PP2A to different subcellular compartments or affect enzyme activity. Finally, the de-regulation of PP2A in some specific pathologies will be touched upon. PMID:11171037

Deletion of Dual Specificity Phosphatase 1 Does Not Predispose Mice to Increased Spontaneous Osteoarthritis

PubMed Central

Pest, Michael Andrew; Pest, Courtney Alice; Bellini, Melina Rodrigues; Feng, Qingping; Beier, Frank

2015-01-01

Background Osteoarthritis (OA) is a degenerative joint disease with poorly understood etiology and pathobiology. Mitogen activated protein kinases (MAPKs) including ERK and p38 play important roles in the mediation of downstream pathways involved in cartilage degenerative processes. Dual specificity phosphatase 1 (DUSP1) dephosphorylates the threonine/serine and tyrosine sites on ERK and p38, causing deactivation of downstream signalling. In this study we examined the role of DUSP1 in spontaneous OA development at 21 months of age using a genetically modified mouse model deficient in Dusp1 (DUSP1 knockout mouse). Results Utilizing histochemical stains of paraffin embedded knee joint sections in DUSP1 knockout and wild type female and male mice, we showed similar structural progression of cartilage degeneration associated with OA at 21 months of age. A semi-quantitative cartilage degeneration scoring system also demonstrated similar scores in the various aspects of the knee joint articular cartilage in DUSP1 knockout and control mice. Examination of overall articular cartilage thickness in the knee joint demonstrated similar results between DUSP1 knockout and wild type mice. Immunostaining for cartilage neoepitopes DIPEN, TEGE and C1,2C was similar in the cartilage lesion sites and chondrocyte pericellular matrix of both experimental groups. Likewise, immunostaining for phosphoERK and MMP13 showed similar intensity and localization between groups. SOX9 immunostaining demonstrated a decreased number of positive cells in DUSP1 knockout mice, with correspondingly decreased staining intensity. Analysis of animal walking patterns (gait) did not show a discernable difference between groups. Conclusion Loss of DUSP1 does not cause changes in cartilage degeneration and gait in a mouse model of spontaneous OA at 21 months of age. Altered staining was observed in SOX9 immunostaining which may prove promising for future studies examining the role of DUSPs in cartilage and OA, as well as models of post-traumatic OA. PMID:26562438

Noonan syndrome-associated SHP2/PTPN11 mutants cause EGF-dependent prolonged GAB1 binding and sustained ERK2/MAPK1 activation.

PubMed

Fragale, Alessandra; Tartaglia, Marco; Wu, Jie; Gelb, Bruce D

2004-03-01

Noonan syndrome is a developmental disorder with dysmorphic facies, short stature, cardiac defects, and skeletal anomalies, which can be caused by missense PTPN11 mutations. PTPN11 encodes Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2 or SHP-2), a protein tyrosine phosphatase that acts in signal transduction downstream to growth factor, hormone, and cytokine receptors. We compared the functional effects of three Noonan syndrome-causative PTPN11 mutations on SHP2's phosphatase activity, interaction with a binding partner, and signal transduction. All SHP2 mutants had significantly increased basal phosphatase activity compared to wild type, but that activity varied significantly between mutants and was further increased after epidermal growth factor stimulation. Cells expressing SHP2 mutants had prolonged extracellular signal-regulated kinase 2 activation, which was ligand-dependent. Binding of SHP2 mutants to Grb2-associated binder-1 was increased and sustained, and tyrosine phosphorylation of both proteins was prolonged. Coexpression of Grb2-associated binder-1-FF, which lacks SHP2 binding motifs, blocked the epidermal growth factor-mediated increase in SHP2's phosphatase activity and resulted in a dramatic reduction of extracellular signal-regulated kinase 2 activation. Taken together, these results document that Noonan syndrome-associated PTPN11 mutations increase SHP2's basal phosphatase activity, with greater activation when residues directly involved in binding at the interface between the N-terminal Src homology 2 and protein tyrosine phosphatase domains are altered. The SHP2 mutants prolonged signal flux through the RAS/mitogen-activated protein kinase (ERK2/MAPK1) pathway in a ligand-dependent manner that required docking through Grb2-associated binder-1 (GAB1), leading to increased cell proliferation. Copyright 2004 Wiley-Liss, Inc.

Streptococcus pneumoniae Phosphotyrosine Phosphatase CpsB and Alterations in Capsule Production Resulting from Changes in Oxygen Availability

PubMed Central

Geno, K. Aaron; Hauser, Jocelyn R.; Gupta, Kanupriya

2014-01-01

Streptococcus pneumoniae produces a protective capsular polysaccharide whose production must be modulated for bacterial survival within various host niches. Capsule production is affected in part by a phosphoregulatory system comprised of CpsB, CpsC, and CpsD. Here, we found that growth of serotype 2 strain D39 under conditions of increased oxygen availability resulted in decreased capsule levels concurrent with an ∼5-fold increase in Cps2B-mediated phosphatase activity. The change in Cps2B phosphatase activity did not result from alterations in the levels of either the cps2B transcript or the Cps2B protein. Recombinant Cps2B expressed in Escherichia coli similarly exhibited increased phosphatase activity under conditions of high-oxygen growth. S. pneumoniae D39 derivatives with defined deletion or point mutations in cps2B demonstrated reduced phosphatase activity with corresponding increases in levels of Cps2D tyrosine phosphorylation. There was, however, no correlation between these phenotypes and the level of capsule production. During growth under reduced-oxygen conditions, the Cps2B protein was essential for parental levels of capsule, but phosphatase activity alone could be eliminated without an effect on capsule. Under increased-oxygen conditions, deletion of cps2B did not affect capsule levels. These results indicate that neither Cps2B phosphatase activity nor Cps2D phosphorylation levels per se are determinants of capsule levels, whereas the Cps2B protein is important for capsule production during growth under conditions of reduced but not enhanced oxygen availability. Roles for factors outside the capsule locus, possible interactions between capsule regulatory proteins, and links to other cellular processes are also suggested by the results described in this study. PMID:24659769

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

PubMed

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

2012-10-19

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

A Novel Inositol Pyrophosphate Phosphatase in Saccharomyces cerevisiae: Siw14 PROTEIN SELECTIVELY CLEAVES THE β-PHOSPHATE FROM 5-DIPHOSPHOINOSITOL PENTAKISPHOSPHATE (5PP-IP5).

PubMed

Steidle, Elizabeth A; Chong, Lucy S; Wu, Mingxuan; Crooke, Elliott; Fiedler, Dorothea; Resnick, Adam C; Rolfes, Ronda J

2016-03-25

Inositol pyrophosphates are high energy signaling molecules involved in cellular processes, such as energetic metabolism, telomere maintenance, stress responses, and vesicle trafficking, and can mediate protein phosphorylation. Although the inositol kinases underlying inositol pyrophosphate biosynthesis are well characterized, the phosphatases that selectively regulate their cellular pools are not fully described. The diphosphoinositol phosphate phosphohydrolase enzymes of the Nudix protein family have been demonstrated to dephosphorylate inositol pyrophosphates; however, theSaccharomyces cerevisiaehomolog Ddp1 prefers inorganic polyphosphate over inositol pyrophosphates. We identified a novel phosphatase of the recently discovered atypical dual specificity phosphatase family as a physiological inositol pyrophosphate phosphatase. Purified recombinant Siw14 hydrolyzes the β-phosphate from 5-diphosphoinositol pentakisphosphate (5PP-IP5or IP7)in vitro. In vivo,siw14Δ yeast mutants possess increased IP7levels, whereas heterologousSIW14overexpression eliminates IP7from cells. IP7levels increased proportionately whensiw14Δ was combined withddp1Δ orvip1Δ, indicating independent activity by the enzymes encoded by these genes. We conclude that Siw14 is a physiological phosphatase that modulates inositol pyrophosphate metabolism by dephosphorylating the IP7isoform 5PP-IP5to IP6. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Integrative proteomics and biochemical analyses define Ptc6p as the Saccharomyces cerevisiae pyruvate dehydrogenase phosphatase.

PubMed

Guo, Xiao; Niemi, Natalie M; Coon, Joshua J; Pagliarini, David J

2017-07-14

The pyruvate dehydrogenase complex (PDC) is the primary metabolic checkpoint connecting glycolysis and mitochondrial oxidative phosphorylation and is important for maintaining cellular and organismal glucose homeostasis. Phosphorylation of the PDC E1 subunit was identified as a key inhibitory modification in bovine tissue ∼50 years ago, and this regulatory process is now known to be conserved throughout evolution. Although Saccharomyces cerevisiae is a pervasive model organism for investigating cellular metabolism and its regulation by signaling processes, the phosphatase(s) responsible for activating the PDC in S. cerevisiae has not been conclusively defined. Here, using comparative mitochondrial phosphoproteomics, analyses of protein-protein interactions by affinity enrichment-mass spectrometry, and in vitro biochemistry, we define Ptc6p as the primary PDC phosphatase in S. cerevisiae Our analyses further suggest additional substrates for related S. cerevisiae phosphatases and describe the overall phosphoproteomic changes that accompany mitochondrial respiratory dysfunction. In summary, our quantitative proteomics and biochemical analyses have identified Ptc6p as the primary-and likely sole- S. cerevisiae PDC phosphatase, closing a key knowledge gap about the regulation of yeast mitochondrial metabolism. Our findings highlight the power of integrative omics and biochemical analyses for annotating the functions of poorly characterized signaling proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

[Phosphatase activity in Amoeba proteus at pH 9.0].

PubMed

Sopina, V A

2007-01-01

In the free-living amoeba Amoeba proteus (strain B), after PAAG disk-electrophoresis of the homogenate supernatant, at using 1-naphthyl phosphate as a substrate and pH 9.0, three forms of phosphatase activity were revealed; they were arbitrarily called "fast", "intermediate", and "slow" phosphatases. The fast phosphatase has been established to be a fraction of lysosomal acid phosphatase that preserves some low activity at alkaline pH. The question as to which particular class the intermediate phosphatase belongs to has remained unanswered: it can be both acid phosphatase and protein tyrosine phosphatase (PTP). Based on data of inhibitor analysis, large substrate specificity, results of experiments with reactivation by Zn ions after inactivation with EDTA, other than in the fast and intermediate phosphatases localization in the amoeba cell, it is concluded that only slow phosphatase can be classified as alkaline phosphatase (EC 3.1.3.1).

The MAP Kinase Cascade Is Activated prior to the Induction of Gliosis in the 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Model of Dopaminergic Neurotoxicity.

PubMed

Callaghan, James P; Martin, Parthena M; Mass, Marc J

1998-05-01

Injury to the central nervous system (CNS) provokes microglial activation and astrocytic hypertrophy at the site of damage. The signaling events that underlie these cellular responses remain unknown. Recent evidence has implicated tyrosine phosphorylation systems, in general, and the mitogen-activated protein kinase (MAP kinase) cascade, in particular, in the mediation of growth-associated events linked to neural degeneration, such as glial activation. 1 Moreover, an increase in the mRNA coding for the 14.3.3 protein, a known regulator of the MAP kinase pathway, 2 appears to be involved in methamphetamine neurotoxicity. 3 To examine the potential role of these protein kinase pathways in drug-induced damage to the CNS, we used the dopaminergic neurotoxicant, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), to damage nerve terminals in the mouse neostriatum and elicit a glial reaction. The onset of reactive gliosis then was verified by Northern blot analysis of glial fibrillary acidic protein (GFAP) mRNA and qualified by enzyme-linked immunosorbent assay (ELISA) of GFAP (protein). A single administration of MPTP (12.5 mg/kg, subcutaneously (s.c.)) to the C57B1/6J mouse resulted in a 10-fold increase in GFAP mRNA by 1 day and a 4-fold increase in GFAP (protein) by 2 days. To determine the potential role of protein tyrosine phosphorylation and MAP kinase activation in these events, blots of striatal homogenates were probed with antibodies directed against phospho-tyr 204 and phospho-thr 202, residues corresponding to the active sites of p42/44 MAP kinase. After mice were sacrificed by focused microwave irradiation to preserve steady-state phosphorylation, proteins from striatal homogenates were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblots of these samples showed a number of phosphotyrosine-labeled bands, but there were no apparent differences between control and MPTP groups. In contrast, phospho-MAP kinase was elevated over 1.5-fold, 3-6hours post MPTP. These findings are suggestive of a role of the MAP kinase cascade in the early phase of injury-induced glial activation.

Carboxylesterases 1 and 2 hydrolyze phospho-nonsteroidal anti-inflammatory drugs: relevance to their pharmacological activity.

PubMed

Wong, Chi C; Cheng, Ka-Wing; Xie, Gang; Zhou, Dingying; Zhu, Cai-Hua; Constantinides, Panayiotis P; Rigas, Basil

2012-02-01

Phospho-nonsteroidal anti-inflammatory drugs (phospho-NSAIDs) are novel NSAID derivatives with improved anticancer activity and reduced side effects in preclinical models. Here, we studied the metabolism of phospho-NSAIDs by carboxylesterases and assessed the impact of carboxylesterases on the anticancer activity of phospho-NSAIDs in vitro and in vivo. The expression of human liver carboxylesterase (CES1) and intestinal carboxylesterase (CES2) in human embryonic kidney 293 cells resulted in the rapid intracellular hydrolysis of phospho-NSAIDs. Kinetic analysis revealed that CES1 is more active in the hydrolysis of phospho-sulindac, phospho-ibuprofen, phospho-naproxen, phospho-indomethacin, and phospho-tyrosol-indomethacin that possessed a bulky acyl moiety, whereas the phospho-aspirins are preferentially hydrolyzed by CES2. Carboxylesterase expression leads to a significant attenuation of the in vitro cytotoxicity of phospho-NSAIDs, suggesting that the integrity of the drug is critical for anticancer activity. Benzil and bis-p-nitrophenyl phosphate (BNPP), two carboxylesterase inhibitors, abrogated the effect of carboxylesterases and resensitized carboxylesterase-expressing cells to the potent cytotoxic effects of phospho-NSAIDs. In mice, coadministration of phospho-sulindac and BNPP partially protected the former from esterase-mediated hydrolysis, and this combination more effectively inhibited the growth of AGS human gastric xenografts in nude mice (57%) compared with phospho-sulindac alone (28%) (p = 0.037). Our results show that carboxylesterase mediates that metabolic inactivation of phospho-NSAIDs, and the inhibition of carboxylesterases improves the efficacy of phospho-NSAIDs in vitro and in vivo.

Carboxylesterases 1 and 2 Hydrolyze Phospho-Nonsteroidal Anti-Inflammatory Drugs: Relevance to Their Pharmacological Activity

PubMed Central

Wong, Chi C.; Cheng, Ka-Wing; Xie, Gang; Zhou, Dingying; Zhu, Cai-Hua; Constantinides, Panayiotis P.

2012-01-01

Phospho-nonsteroidal anti-inflammatory drugs (phospho-NSAIDs) are novel NSAID derivatives with improved anticancer activity and reduced side effects in preclinical models. Here, we studied the metabolism of phospho-NSAIDs by carboxylesterases and assessed the impact of carboxylesterases on the anticancer activity of phospho-NSAIDs in vitro and in vivo. The expression of human liver carboxylesterase (CES1) and intestinal carboxylesterase (CES2) in human embryonic kidney 293 cells resulted in the rapid intracellular hydrolysis of phospho-NSAIDs. Kinetic analysis revealed that CES1 is more active in the hydrolysis of phospho-sulindac, phospho-ibuprofen, phospho-naproxen, phospho-indomethacin, and phospho-tyrosol-indomethacin that possessed a bulky acyl moiety, whereas the phospho-aspirins are preferentially hydrolyzed by CES2. Carboxylesterase expression leads to a significant attenuation of the in vitro cytotoxicity of phospho-NSAIDs, suggesting that the integrity of the drug is critical for anticancer activity. Benzil and bis-p-nitrophenyl phosphate (BNPP), two carboxylesterase inhibitors, abrogated the effect of carboxylesterases and resensitized carboxylesterase-expressing cells to the potent cytotoxic effects of phospho-NSAIDs. In mice, coadministration of phospho-sulindac and BNPP partially protected the former from esterase-mediated hydrolysis, and this combination more effectively inhibited the growth of AGS human gastric xenografts in nude mice (57%) compared with phospho-sulindac alone (28%) (p = 0.037). Our results show that carboxylesterase mediates that metabolic inactivation of phospho-NSAIDs, and the inhibition of carboxylesterases improves the efficacy of phospho-NSAIDs in vitro and in vivo. PMID:22085648

Posttranslationally modified progesterone receptors direct ligand-specific expression of breast cancer stem cell-associated gene programs.

PubMed

Knutson, Todd P; Truong, Thu H; Ma, Shihong; Brady, Nicholas J; Sullivan, Megan E; Raj, Ganesh; Schwertfeger, Kathryn L; Lange, Carol A

2017-04-17

Estrogen and progesterone are potent breast mitogens. In addition to steroid hormones, multiple signaling pathways input to estrogen receptor (ER) and progesterone receptor (PR) actions via posttranslational events. Protein kinases commonly activated in breast cancers phosphorylate steroid hormone receptors (SRs) and profoundly impact their activities. To better understand the role of modified PRs in breast cancer, we measured total and phospho-Ser294 PRs in 209 human breast tumors represented on 2754 individual tissue spots within a tissue microarray and assayed the regulation of this site in human tumor explants cultured ex vivo. To complement this analysis, we assayed PR target gene regulation in T47D luminal breast cancer models following treatment with progestin (promegestone; R5020) and antiprogestins (mifepristone, onapristone, or aglepristone) in conditions under which the receptor is regulated by Lys388 SUMOylation (K388 intact) or is SUMO-deficient (via K388R mutation to mimic persistent Ser294 phosphorylation). Selected phospho-PR-driven target genes were validated by qRT-PCR and following RUNX2 shRNA knockdown in breast cancer cell lines. Primary and secondary mammosphere assays were performed to implicate phospho-Ser294 PRs, epidermal growth factor signaling, and RUNX2 in breast cancer stem cell biology. Phospho-Ser294 PR species were abundant in a majority (54%) of luminal breast tumors, and PR promoter selectivity was exquisitely sensitive to posttranslational modifications. Phospho-PR expression and target gene programs were significantly associated with invasive lobular carcinoma (ILC). Consistent with our finding that activated phospho-PRs undergo rapid ligand-dependent turnover, unique phospho-PR gene signatures were most prevalent in breast tumors clinically designated as PR-low to PR-null (luminal B) and included gene sets associated with cancer stem cell biology (HER2, PAX2, AHR, AR, RUNX). Validation studies demonstrated a requirement for RUNX2 in the regulation of selected phospho-PR target genes (SLC37A2). In vitro mammosphere formation assays support a role for phospho-Ser294-PRs via growth factor (EGF) signaling as well as RUNX2 as potent drivers of breast cancer stem cell fate. We conclude that PR Ser294 phosphorylation is a common event in breast cancer progression that is required to maintain breast cancer stem cell fate, in part via cooperation with growth factor-initiated signaling pathways and key phospho-PR target genes including SLC37A2 and RUNX2. Clinical measurement of phosphorylated PRs should be considered a useful marker of breast tumor stem cell potential. Alternatively, unique phospho-PR target gene sets may provide useful tools with which to identify patients likely to respond to selective PR modulators that block PR Ser294 phosphorylation as part of rational combination (i.e., with antiestrogens) endocrine therapies designed to durably block breast cancer recurrence.

Disruption of striatal-enriched protein tyrosine phosphatase (STEP) function in neuropsychiatric disorders

PubMed Central

Karasawa, Takatoshi; Lombroso, Paul J.

2014-01-01

Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific tyrosine phosphatase that plays a major role in the development of synaptic plasticity. Recent findings have implicated STEP in several psychiatric and neurological disorders, including Alzheimer’s disease, schizophrenia, fragile X syndrome, Huntington’s disease, stroke/ischemia, and stress-related psychiatric disorders. In these disorders, STEP protein expression levels and activity are dysregulated, contributing to the cognitive deficits that are present. In this review, we focus on the most recent findings on STEP, discuss how STEP expression and activity are maintained during normal cognitive function, and how disruptions in STEP activity contribute to a number of illnesses. PMID:25218562

PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE FROM SPIRODELA OLIGORRHIZA AND ITS AFFINITY FOR SELECTED ORGANOPHOSPHATE PESTICIDES

EPA Science Inventory

An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fast protein liquid chromatography (FPLC) and partially characterized. The enzyme was purified 1871-fold with a total yield of 40%. SDS-PAGE electrophoresis of the pure acid phosphatase ...

Enzyme domain affects the movement of the voltage sensor in ascidian and zebrafish voltage-sensing phosphatases.

PubMed

Hossain, Md Israil; Iwasaki, Hirohide; Okochi, Yoshifumi; Chahine, Mohamed; Higashijima, Shinichi; Nagayama, Kuniaki; Okamura, Yasushi

2008-06-27

The ascidian voltage-sensing phosphatase (Ci-VSP) consists of the voltage sensor domain (VSD) and a cytoplasmic phosphatase region that has significant homology to the phosphatase and tensin homolog deleted on chromosome TEN (PTEN). The phosphatase activity of Ci-VSP is modified by the conformational change of the VSD. In many proteins, two protein modules are bidirectionally coupled, but it is unknown whether the phosphatase domain could affect the movement of the VSD in VSP. We addressed this issue by whole-cell patch recording of gating currents from a teleost VSP (Dr-VSP) cloned from Danio rerio expressed in tsA201 cells. Replacement of a critical cysteine residue, in the phosphatase active center of Dr-VSP, by serine sharpened both ON- and OFF-gating currents. Similar changes were produced by treatment with phosphatase inhibitors, pervanadate and orthovanadate, that constitutively bind to cysteine in the active catalytic center of phosphatases. The distinct kinetics of gating currents dependent on enzyme activity were not because of altered phosphatidylinositol 4,5-bisphosphate levels, because the kinetics of gating current did not change by depletion of phosphatidylinositol 4,5-bisphosphate, as reported by coexpressed KCNQ2/3 channels. These results indicate that the movement of the VSD is influenced by the enzymatic state of the cytoplasmic domain, providing an important clue for understanding mechanisms of coupling between the VSD and its effector.

Amyloid formation reduces protein kinase B phosphorylation in primary islet β-cells which is improved by blocking IL-1β signaling

PubMed Central

Zhang, Yun; Warnock, Garth L.; Ao, Ziliang; Park, Yoo Jin; Safikhan, Nooshin; Ghahary, Aziz

2018-01-01

Amyloid formation in the pancreatic islets due to aggregation of human islet amyloid polypeptide (hIAPP) contributes to reduced β-cell mass and function in type 2 diabetes (T2D) and islet transplantation. Protein kinase B (PKB) signaling plays a key role in the regulation of β-cell survival, function and proliferation. In this study, we used human and hIAPP-expressing transgenic mouse islets in culture as two ex vivo models of human islet amyloid formation to: 1. Investigate the effects of amyloid formation on PKB phosphorylation in primary islet β-cells; 2. Test if inhibition of amyloid formation and/or interleukin-1β (IL-1β) signaling in islets can restore the changes in β-cell phospho-PKB levels mediated by amyloid formation. Human and hIAPP-expressing mouse islets were cultured in elevated glucose with an amyloid inhibitor (Congo red) or embedded within collagen matrix to prevent amyloid formation. To block the IL-1β signaling, human islets were treated with an IL-1 receptor antagonist (anakinra) or a glucagon-like peptide-1 agonist (exenatide). β-cell phospho-PKB levels, proliferation, apoptosis, islet IL-1β levels and amyloid formation were assessed. Amyloid formation in both cultured human and hIAPP-expressing mouse islets reduced β-cell phospho-PKB levels and increased islet IL-1β levels, both of which were restored by prevention of amyloid formation either by the amyloid inhibitor or embedding islets in collagen matrix, resulting in improved β-cell survival. Furthermore, inhibition of IL-1β signaling by treatment with anakinra or exenatide increased β-cell phospho-PKB levels, enhanced proliferation and reduced apoptosis in amyloid forming human islets during 7-day culture. These data suggest that amyloid formation leads to reduced PKB phosphorylation in β-cells which is associated with elevated islet IL-1β levels. Inhibitors of amyloid or amyloid-induced IL-1β production may provide a new approach to restore phospho-PKB levels thereby enhance β-cell survival and proliferation in conditions associated with islet amyloid formation such as T2D and clinical islet transplantation. PMID:29474443

Miklós Bodanszky Award Lecture: Advances in the selective targeting of protein phosphatase-1 and phosphatase-2A with peptides.

PubMed

Köhn, Maja

2017-10-01

Protein phosphatase-1 and phosphatase-2A are two ubiquitously expressed enzymes known to catalyze the majority of dephosphorylation reactions on serine and threonine inside cells. They play roles in most cellular processes and are tightly regulated by regulatory subunits in holoenzymes. Their misregulation and malfunction contribute to disease development and progression, such as in cancer, diabetes, viral infections, and neurological as well as heart diseases. Therefore, targeting these phosphatases for therapeutic use would be highly desirable; however, their complex regulation and high conservation of the active site have been major hurdles for selectively targeting them in the past. In the last decade, new approaches have been developed to overcome these hurdles and have strongly revived the field. I will focus here on peptide-based approaches, which contributed to showing that these phosphatases can be targeted selectively and aided in rethinking the design of selective phosphatase modulators. Finally, I will give a perspective on www.depod.org, the human dephosphorylation database, and how it can aid phosphatase modulator design. © 2017 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd. © 2017 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.

The Zds proteins control entry into mitosis and target protein phosphatase 2A to the Cdc25 phosphatase

PubMed Central

Wicky, Sidonie; Tjandra, Hendri; Schieltz, David; Yates, John; Kellogg, Douglas R.

2011-01-01

The Wee1 kinase restrains entry into mitosis by phosphorylating and inhibiting cyclin-dependent kinase 1 (Cdk1). The Cdc25 phosphatase promotes entry into mitosis by removing Cdk1 inhibitory phosphorylation. Experiments in diverse systems have established that Wee1 and Cdc25 are regulated by protein phosphatase 2A (PP2A), but a full understanding of the function and regulation of PP2A in entry into mitosis has remained elusive. In budding yeast, entry into mitosis is controlled by a specific form of PP2A that is associated with the Cdc55 regulatory subunit (PP2ACdc55). We show here that related proteins called Zds1 and Zds2 form a tight stoichiometric complex with PP2ACdc55 and target its activity to Cdc25 but not to Wee1. Conditional inactivation of the Zds proteins revealed that their function is required primarily at entry into mitosis. In addition, Zds1 undergoes cell cycle–dependent changes in phosphorylation. Together, these observations define a role for the Zds proteins in controlling specific functions of PP2ACdc55 and suggest that upstream signals that regulate PP2ACdc55 may play an important role in controlling entry into mitosis. PMID:21119008

The Zds proteins control entry into mitosis and target protein phosphatase 2A to the Cdc25 phosphatase.

PubMed

Wicky, Sidonie; Tjandra, Hendri; Schieltz, David; Yates, John; Kellogg, Douglas R

2011-01-01

The Wee1 kinase restrains entry into mitosis by phosphorylating and inhibiting cyclin-dependent kinase 1 (Cdk1). The Cdc25 phosphatase promotes entry into mitosis by removing Cdk1 inhibitory phosphorylation. Experiments in diverse systems have established that Wee1 and Cdc25 are regulated by protein phosphatase 2A (PP2A), but a full understanding of the function and regulation of PP2A in entry into mitosis has remained elusive. In budding yeast, entry into mitosis is controlled by a specific form of PP2A that is associated with the Cdc55 regulatory subunit (PP2A(Cdc55)). We show here that related proteins called Zds1 and Zds2 form a tight stoichiometric complex with PP2A(Cdc55) and target its activity to Cdc25 but not to Wee1. Conditional inactivation of the Zds proteins revealed that their function is required primarily at entry into mitosis. In addition, Zds1 undergoes cell cycle-dependent changes in phosphorylation. Together, these observations define a role for the Zds proteins in controlling specific functions of PP2A(Cdc55) and suggest that upstream signals that regulate PP2A(Cdc55) may play an important role in controlling entry into mitosis.

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

PubMed Central

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

2008-01-01

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

Phospho-Regulation of DDA3 Function in Mitosis

PubMed Central

Jang, Chang-Young; Coppinger, Judith A.; Yates, John R.; Fang, Guowei

2010-01-01

DDA3 is a microtubule-associated protein that controls chromosome congression and segregation by regulating the mitotic spindle. Depletion of DDA3 alters spindle structure, generates unaligned chromosomes at metaphase, and delays the mitotic progression. Through a mass spectrometry analysis, we found that DDA3 is phosphorylated on Ser225 during mitosis. Phosphorylation of this residue is important for the mitotic function of DDA3, as the phospho-mimicking DDA3-S225D variant, but not the nonphosphorable DDA3-S225A mutant, rescues the DDA3-knockdown phenotype. We conclude that the mitotic function of DDA3 is regulated by phosphorylation on the Ser225 residue. PMID:20117088

Antisense protein tyrosine phosphatase 1B reverses activation of p38 mitogen-activated protein kinase in liver of ob/ob mice.

PubMed

Gum, Rebecca J; Gaede, Lori L; Heindel, Matthew A; Waring, Jeffrey F; Trevillyan, James M; Zinker, Bradley A; Stark, Margery E; Wilcox, Denise; Jirousek, Michael R; Rondinone, Cristina M; Ulrich, Roger G

2003-06-01

Phosphorylation of stress-activated kinase p38, a MAPK family member, was increased in liver of ob/ob diabetic mice relative to lean littermates. Treatment of ob/ob mice with protein tyrosine phosphatase 1B (PTP1B) antisense oligonucleotides (ASO) reduced phosphorylation of p38 in liver-to below lean littermate levels-and normalized plasma glucose while reducing plasma insulin. Phosphorylation of ERK, but not JNK, was also decreased in ASO-treated mice. PTP1B ASO decreased TNFalpha protein levels and phosphorylation of the transcription factor cAMP response element binding protein (CREB) in liver, both of which can occur through decreased phosphorylation of p38 and both of which have been implicated in insulin resistance or hyperglycemia. Decreased p38 phosphorylation was not directly due to decreased phosphorylation of the kinases that normally phosphorylate p38-MKK3 and MKK6. Additionally, p38 phosphorylation was not enhanced in liver upon insulin stimulation of ASO-treated ob/ob mice (despite increased activation of other signaling molecules) corroborating that p38 is not directly affected via the insulin receptor. Instead, decreased phosphorylation of p38 may be due to increased expression of MAPK phosphatases, particularly the p38/ERK phosphatase PAC1 (phosphatase of activated cells). This study demonstrates that reduction of PTP1B protein using ASO reduces activation of p38 and its substrates TNFalpha and CREB in liver of diabetic mice, which correlates with decreased hyperglycemia and hyperinsulinemia.

Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome.

PubMed

Panizza, Elena; Branca, Rui M M; Oliviusson, Peter; Orre, Lukas M; Lehtiö, Janne

2017-07-03

Protein phosphorylation is involved in the regulation of most eukaryotic cells functions and mass spectrometry-based analysis has made major contributions to our understanding of this regulation. However, low abundance of phosphorylated species presents a major challenge in achieving comprehensive phosphoproteome coverage and robust quantification. In this study, we developed a workflow employing titanium dioxide phospho-enrichment coupled with isobaric labeling by Tandem Mass Tags (TMT) and high-resolution isoelectric focusing (HiRIEF) fractionation to perform in-depth quantitative phosphoproteomics starting with a low sample quantity. To benchmark the workflow, we analyzed HeLa cells upon pervanadate treatment or cell cycle arrest in mitosis. Analyzing 300 µg of peptides per sample, we identified 22,712 phosphorylation sites, of which 19,075 were localized with high confidence and 1,203 are phosphorylated tyrosine residues, representing 6.3% of all detected phospho-sites. HiRIEF fractions with the most acidic isoelectric points are enriched in multiply phosphorylated peptides, which represent 18% of all the phospho-peptides detected in the pH range 2.5-3.7. Cross-referencing with the PhosphoSitePlus database reveals 1,264 phosphorylation sites that have not been previously reported and kinase association analysis suggests that a subset of these may be functional during the mitotic phase.

Protein Phosphatase 2A Signaling in Human Prostate Cancer

DTIC Science & Technology

2013-06-01

family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J 2001;353:417-39. (6) Eto M, Bennouna J, Hunter OC...phosphatidylinositol 3’-kinase and Akt/protein kinase B. Cancer Res 1999;59:1449-53. (14) Grethe S, Porn -Ares MI. p38 MAPK regulates phosphorylation of Bad

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

PubMed

Catarsi, S; Drapeau, P

1997-08-01

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

Mitogen-activated protein kinase phosphatase-1: a critical phosphatase manipulating mitogen-activated protein kinase signaling in cardiovascular disease (review).

PubMed

Li, Chang-Yi; Yang, Ling-Chao; Guo, Kai; Wang, Yue-Peng; Li, Yi-Gang

2015-04-01

Mitogen-activated protein kinase (MAPK) cascades are important players in the overall representation of cellular signal transduction pathways, and the deregulation of MAPKs is involved in a variety of diseases. The activation of MAPK signals occurs through phosphorylation by MAPK kinases at conserved threonine and tyrosine (Thr-Xaa-Tyr) residues. The mitogen-activated protein kinase phosphatases (MKPs) are a major part of the dual-specificity family of phosphatases and specifically inactivate MAPKs by dephosphorylating both phosphotyrosine and phosphoserine/phosphothreonine residues within the one substrate. MAPKs binding to MKPs can enhance MKP stability and activity, providing an important negative-feedback control mechanism that limits the MAPK cascades. In recent years, accumulating and compelling evidence from studies mainly employing cultured cells and mouse models has suggested that the archetypal MKP family member, MKP-1, plays a pivotal role in cardiovascular disease as a major negative modulator of MAPK signaling pathways. In the present review, we summarize the current knowledge on the pathological properties and the regulation of MKP-1 in cardiovascular disease, which may provide valuable therapeutic options.

Actin realignment and cofilin regulation are essential for barrier integrity during shear stress

PubMed Central

Slee, Joshua B.; Lowe-Krentz, Linda J.

2014-01-01

Vascular endothelial cells and their actin microfilaments align in the direction of fluid shear stress (FSS) in vitro and in vivo. To determine whether cofilin, an actin severing protein, is required in this process, the levels of phospho-cofilin (serine-3) were evaluated in cells exposed to FSS. Phospho-cofilin levels decreased in the cytoplasm and increased in the nucleus during FSS exposure. This was accompanied by increased nuclear staining for activated LIMK, a cofilin kinase. Blocking stress kinases JNK and p38, known to play roles in actin realignment during FSS, decreased cofilin phosphorylation under static conditions, and JNK inhibition also resulted in decreased phospho-cofilin during FSS exposure. Inhibition of dynamic changes in cofilin phosphorylation through cofilin mutants decreased correct actin realignment. The mutants also decreased barrier integrity as did inhibition of the stress kinases. These results identify the importance of cofilin in the process of actin alignment and the requirement for actin realignment in endothelial barrier integrity during FSS. PMID:23060131

Protein phosphatase 5 promotes hepatocarcinogenesis through interaction with AMP-activated protein kinase.

PubMed

Chen, Yao-Li; Hung, Man-Hsin; Chu, Pei-Yi; Chao, Tzu-I; Tsai, Ming-Hsien; Chen, Li-Ju; Hsiao, Yung-Jen; Shih, Chih-Ting; Hsieh, Feng-Shu; Chen, Kuen-Feng

2017-08-15

The serine-threonine protein phosphatase family members are known as critical regulators of various cellular functions, such as survival and transformation. Growing evidence suggests that pharmacological manipulation of phosphatase activity exhibits therapeutic benefits. Ser/Thr protein phosphatase 5 (PP5) is known to participate in glucocorticoid receptor (GR) and stress-induced signaling cascades that regulate cell growth and apoptosis, and has been shown to be overexpressed in various human malignant diseases. However, the role of PP5 in hepatocellular carcinoma (HCC) and whether PP5 may be a viable therapeutic target for HCC treatment are unknown. Here, by analyzing HCC clinical samples obtained from 215 patients, we found that overexpression of PP5 is tumor specific and associated with worse clinical outcomes. We further characterized the oncogenic properties of PP5 in HCC cells. Importantly, both silencing of PP5 with lentiviral-mediated short hairpin RNA (shRNA) and chemical inhibition of PP5 phosphatase activity using the natural compound cantharidin/norcantharidin markedly suppressed the growth of HCC cells and tumors in vitro and in vivo. Moreover, we identified AMP-activated protein kinase (AMPK) as a novel downstream target of oncogenic PP5 and demonstrated that the antitumor mechanisms underlying PP5 inhibition involve activation of AMPK signaling. Overall, our results establish a pathological function of PP5 in hepatocarcinogenesis via affecting AMPK signaling and suggest that PP5 inhibition is an attractive therapeutic approach for HCC. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of C-terminal phosphorylation sites of N-formyl peptide receptor-1 (FPR1) in human blood neutrophils.

PubMed

Maaty, Walid S; Lord, Connie I; Gripentrog, Jeannie M; Riesselman, Marcia; Keren-Aviram, Gal; Liu, Ting; Dratz, Edward A; Bothner, Brian; Jesaitis, Algirdas J

2013-09-20

Accumulation, activation, and control of neutrophils at inflammation sites is partly driven by N-formyl peptide chemoattractant receptors (FPRs). Occupancy of these G-protein-coupled receptors by formyl peptides has been shown to induce regulatory phosphorylation of cytoplasmic serine/threonine amino acid residues in heterologously expressed recombinant receptors, but the biochemistry of these modifications in primary human neutrophils remains relatively unstudied. FPR1 and FPR2 were partially immunopurified using antibodies that recognize both receptors (NFPRa) or unphosphorylated FPR1 (NFPRb) in dodecylmaltoside extracts of unstimulated and N-formyl-Met-Leu-Phe (fMLF) + cytochalasin B-stimulated neutrophils or their membrane fractions. After deglycosylation and separation by SDS-PAGE, excised Coomassie Blue-staining bands (∼34,000 Mr) were tryptically digested, and FPR1, phospho-FPR1, and FPR2 content was confirmed by peptide mass spectrometry. C-terminal FPR1 peptides (Leu(312)-Arg(322) and Arg(323)-Lys(350)) and extracellular FPR1 peptide (Ile(191)-Arg(201)) as well as three similarly placed FPR2 peptides were identified in unstimulated and fMLF + cytochalasin B-stimulated samples. LC/MS/MS identified seven isoforms of Ala(323)-Lys(350) only in the fMLF + cytochalasin B-stimulated sample. These were individually phosphorylated at Thr(325), Ser(328), Thr(329), Thr(331), Ser(332), Thr(334), and Thr(339). No phospho-FPR2 peptides were detected. Cytochalasin B treatment of neutrophils decreased the sensitivity of fMLF-dependent NFPRb recognition 2-fold, from EC50 = 33 ± 8 to 74 ± 21 nM. Our results suggest that 1) partial immunopurification, deglycosylation, and SDS-PAGE separation of FPRs is sufficient to identify C-terminal FPR1 Ser/Thr phosphorylations by LC/MS/MS; 2) kinases/phosphatases activated in fMLF/cytochalasin B-stimulated neutrophils produce multiple C-terminal tail FPR1 Ser/Thr phosphorylations but have little effect on corresponding FPR2 sites; and 3) the extent of FPR1 phosphorylation can be monitored with C-terminal tail FPR1-phosphospecific antibodies.

[IL-1β-Treated Bone Marrow Mesenchymal Stem Cells Enhances Osteogenetic Potential via NF-κB Pathway].

PubMed

Ye, Wei; Fei, Xiao-Ming; Tang, Yu; Xu, Xin-Xin; Zhu, Yan

2017-06-01

Obsjective:To investigate the effects of differentaction time of IL-1β on the osteogenic capacity of bone marrow mensenchymal cells(BMMSC) and the role of nuclear factor-κB (NF-kB) pathway. BMMSC isolated from normal donors was treated with IL-1β for 1 or 7 days, respectively. Alkaline phosphatase (ALP) and alizarin red(AR) stainings were used to detect the osteogenic differentiation potential of BMMSC. The mRNA expression of EphB4, IGF-1 and OPG in BMMSC was measured by real-time PCR. The immunohistochemistry was employed to measure the expression of bone morphgenetic protein-2(BMP-2) and p-Smad1/5/8 in BMMSC. Furthermore, the Western blot was used for the detection of iκBα and phospho-iκBα (p-ikBα) in IL-1β-treated BMMSC. And the results of IL-1β-treated BMMSC were compared with control group. Compared with control group, the osteogenetic potential of IL-1β-treated BMMSC was enhanced, but the pro-osteogenic differentiation effect of IL-1β was remarkedly inhibited in the presence of NF-kB pathway inhibitor PDTC. The total ikBα level of IL-1β-treated BMMSC was lower (P<0.05), and phospho-iκBα (p-iκBα) level was higher (P<0.05). Besides, BMP-2 expression was higher (P<0.05) in the IL-1β-treated BMMSC, however, p-Smad1/5/8 protien level was not significantly different among IL-1β-treated for 1 d, 7 d and control groups (P<0.05). And the mRNA expression levels of IGF-1, EphB4 and OPG in BMMSC were up-regulated after IL-1β treatment (P<0.05). In addition, the osteoblastogenesis of BMMSC treated with IL-1β for 7 days was significantly different from those treated only for 1 day. Prolonging IL-1β treatment can enhance the osteogenetic differentiation of BMMSC more significantly. And this osteogenetic alteration of BMMSC occurs via its NF-κB pathway, but not via BMP-2/Smad pathway.

2-D Difference in gel electrophoresis combined with Pro-Q Diamond staining: a successful approach for the identification of kinase/phosphatase targets.

PubMed

Orsatti, Laura; Forte, Eleonora; Tomei, Licia; Caterino, Marianna; Pessi, Antonello; Talamo, Fabio

2009-07-01

The protein tyrosine phosphatase PRL-3 is an appealing therapeutic cancer target for its well described involvement in the metastasis progression. Nevertheless, very little is known about PRL-3 role in tumorigenesis. In the attempt to identify the protein target of this phosphatase we have devised a model system based on the use of highly invasive HCT116 colon cancer cells over-expressing PRL-3. We used 2-D difference gel electrophoresis combined with the fluorescence staining Pro-Q Diamond selective for phosphorylated proteins to monitor changes in the phosphorylation status of possible substrates. Proteins whose phosphorylation level was negatively affected by PRL-3 over-expression were identified by MS. Two proteins were found to be significantly dephosphorylated in this condition, the cytoskeletal protein ezrin and elongation factor 2. Ezrin has already been described as having a proactive role in cancer metastasis through control of its phosphorylation status, and the PRL-3-induced modulation of ezrin phosphorylation in HCT116 and human umblical vascular endothelial cells is the subject of a separate paper by Forte et al. [Biochim. Biophys. Acta 2008, 1783, 334-344]. The combination of 2-D difference in gel electrophoresis and Pro-Q Diamond was hence confirmed successful in analyzing changes of protein phosphorylation which enable the identification of kinase/phosphatase targets.

Inhibition of EphA2/EphrinA1 signal attenuates lipopolysaccharide-induced lung injury.

PubMed

Hong, Ji Young; Shin, Mi Hwa; Douglas, Ivor S; Chung, Kyung Soo; Kim, Eun Young; Jung, Ji Ye; Kang, Young Ae; Kim, Se Kyu; Chang, Joon; Kim, Young Sam; Park, Moo Suk

2016-11-01

Eph-Ephrin signalling mediates various cellular processes, including vasculogenesis, angiogenesis, cell migration, axon guidance, fluid homoeostasis and repair after injury. Although previous studies have demonstrated that stimulation of the EphA receptor induces increased vascular permeability and inflammatory response in lung injury, the detailed mechanisms of EphA2 signalling are unknown. In the present study, we evaluated the role of EphA2 signalling in mice with lipopolysaccharide (LPS)-induced lung injury. Acute LPS exposure significantly up-regulated EphA2 and EphrinA1 expression. Compared with LPS+IgG mice (IgG instillation after LPS exposure), LPS+EphA2 mAb mice [EphA2 monoclonal antibody (mAb) instillation posttreatment after LPS exposure] had attenuated lung injury and reduced cell counts and protein concentration of bronchoalveolar lavage fluid (BALF). EphA2 mAb posttreatment down-regulated the expression of phosphoinositide 3-kinases (PI3K) 110γ, phospho-Akt, phospho-NF-κB p65, phospho-Src and phospho-S6K in lung lysates. In addition, inhibiting the EphA2 receptor augmented the expression of E-cadherin, which is involved in cell-cell adhesion. Our study identified EphA2 receptor as an unrecognized modulator of several signalling pathways-including PI3K-Akt-NF-kB, Src-NF-κB, E-cadherin and mTOR-in LPS-induced lung injury. These results suggest that EphA2 receptor inhibitors may function as novel therapeutic agents for LPS-induced lung injury. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Iron Enhances Hepatic Fibrogenesis and Activates Transforming Growth Factor-β Signaling in Murine Hepatic Stellate Cells.

PubMed

Mehta, Kosha J; Coombes, Jason D; Briones-Orta, Marco; Manka, Paul P; Williams, Roger; Patel, Vinood B; Syn, Wing-Kin

2018-02-01

Although excess iron induces oxidative stress in the liver, it is unclear whether it directly activates the hepatic stellate cells (HSC). We evaluated the effects of excess iron on fibrogenesis and transforming growth factor beta (TGF-β) signaling in murine HSC. Cells were treated with holotransferrin (0.005-5g/L) for 24 hours, with or without the iron chelator deferoxamine (10µM). Gene expressions (α-SMA, Col1-α1, Serpine-1, TGF-β, Hif1-α, Tfrc and Slc40a1) were analyzed by quantitative real time-polymerase chain reaction, whereas TfR1, ferroportin, ferritin, vimentin, collagen, TGF-β RII and phospho-Smad2 proteins were evaluated by immunofluorescence, Western blot and enzyme-linked immunosorbent assay. HSC expressed the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-export protein ferroportin. Holotransferrin upregulated TfR1 expression by 1.8-fold (P < 0.03) and ferritin accumulation (iron storage) by 2-fold (P < 0.01), and activated HSC with 2-fold elevations (P < 0.03) in α-SMA messenger RNA and collagen secretion, and a 1.6-fold increase (P < 0.01) in vimentin protein. Moreover, holotransferrin activated the TGF-β pathway with TGF-β messenger RNA elevated 1.6-fold (P = 0.05), and protein levels of TGF-β RII and phospho-Smad2 increased by 1.8-fold (P < 0.01) and 1.6-fold (P < 0.01), respectively. In contrast, iron chelation decreased ferritin levels by 30% (P < 0.03), inhibited collagen secretion by 60% (P < 0.01), repressed fibrogenic genes α-SMA (0.2-fold; P < 0.05) and TGF-β (0.4-fold; P < 0.01) and reduced levels of TGF-β RII and phospho-Smad2 proteins. HSC express iron-transport proteins. Holotransferrin (iron) activates HSC fibrogenesis and the TGF-β pathway, whereas iron depletion by chelation reverses this, suggesting that this could be a useful adjunct therapy for patients with fibrosis. Further studies in primary human HSC and animal models are necessary to confirm this. Published by Elsevier Inc.

A family of metal-dependent phosphatases implicated in metabolite damage-control

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

Huang, Lili; Khusnutdinova, Anna; Nocek, Boguslaw

DUF89 family proteins occur widely in both prokaryotes and eukaryotes, but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), with subfamily II being split into stand-alone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metal-dependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027Wmore » revealed a novel phosphatase active site with fructose 6-phosphate and Mg2+ bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.« less

A family of metal-dependent phosphatases implicated in metabolite damage-control

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

Huang, Lili; Shanklin, John; Khusnutdinova, Anna

DUF89 family proteins occur widely in pro- and eukaryotes but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), subfamily II being split into standalone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metaldependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027W revealed amore » novel phosphatase active site with fructose 6-phosphate and Mg 2+ bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.« less

A family of metal-dependent phosphatases implicated in metabolite damage-control

DOE PAGES

Huang, Lili; Shanklin, John; Khusnutdinova, Anna; ...

2016-06-20

DUF89 family proteins occur widely in pro- and eukaryotes but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), subfamily II being split into standalone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metaldependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027W revealed amore » novel phosphatase active site with fructose 6-phosphate and Mg 2+ bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.« less

Activation of ferret erythrocyte Na+–K+–2Cl− cotransport by deoxygenation

PubMed Central

Flatman, Peter W

2005-01-01

Deoxygenation of ferret erythrocytes stimulates Na+–K+–2Cl− cotransport by 111% (s.d., 46) compared to controls in air. Half-maximal activation occurs at a PO2 of 24 mmHg (s.d., 2) indicating that physiological changes in oxygen tension can influence cotransport function. Approximately 25–35% of this stimulation can be attributed to the rise of intracellular free magnesium concentration that occurs on deoxygenation (from 0.82 (s.d., 0.07) to 1.40 mm (s.d., 0.17)). Most of the stimulation is probably caused by activation of a kinase which can be prevented or reversed by treating cells with the kinase inhibitors PP1 or staurosporine, or by reducing cell magnesium content to submicromolar levels. Stimulation by deoxygenation is comparable with that caused by calyculin A or sodium arsenite, compounds that cause a 2- to 3-fold increase in threonine phosphorylation of the cotransporter which can be detected with phospho-specific antibodies. However, the same approach failed to detect significant changes in threonine phosphorylation following deoxygenation. The results suggest that deoxygenation causes activation of a kinase that either phosphorylates the transporter, but probably not on threonine, or phosphorylates another protein that in turn influences cotransporter behaviour. They also indicate that more than one kinase and phosphatase are involved in cotransporter phosphorylation. PMID:15618270

Crataegus oxycantha extract attenuates apoptotic incidence in myocardial ischemia-reperfusion injury by regulating Akt and HIF-1 signaling pathways.

PubMed

Jayachandran, Kesavan S; Khan, Mahmood; Selvendiran, Karuppaiyah; Devaraj, S Niranjali; Kuppusamy, Periannan

2010-11-01

The objective of the present study was to evaluate the efficacy and mechanism of Crataegus oxycantha (COC) extract in preventing ischemia-reperfusion (IR) injury in an in vivo rat model of acute myocardial infarction induced by a 30-minute regional ischemia followed by 72 hours of reperfusion. The COC extract [100 mg/(kg body weight)] was administered 12 hours after the surgical procedure and then at 24-hour intervals for 3 days. Animals treated with COC extract showed a significant decrease in creatine kinase activity and infarct size. At the molecular level, COC administration resulted in a significant attenuation of PTEN (phosphatase and tensin homolog deleted on chromosome 10) and upregulation of phospho-Akt and c-Raf levels in the heart. As a consequence, cleaved caspase-9 and cleaved caspase-7 levels were significantly downregulated, indicating negative regulation of apoptosis by COC extract. In part with the hypoxia-inducible factor (HIF) signaling pathway, COC extract administration significantly upregulated the prolyl hydroxylase-2 level. In contrast, other proapoptotic proteins such as nuclear factor-κB, cytochrome c, apoptosis-inducing factor, and cleaved poly(adenosine diphosphate-ribose) polymerase levels were significantly downregulated in the COC-treated group when compared with the untreated control group. The results suggested that COC extract attenuated apoptotic incidence in the experimental myocardial ischemia-reperfusion model by regulating Akt and HIF-1 signaling pathways.

Enzymatic characteristics of an ApaH-like phosphatase, PrpA, and a diadenosine tetraphosphate hydrolase, ApaH, from Myxococcus xanthus.

PubMed

Sasaki, Masashi; Takegawa, Kaoru; Kimura, Yoshio

2014-09-17

We characterized the activities of the Myxococcus xanthus ApaH-like phosphatases PrpA and ApaH, which share homologies with both phosphoprotein phosphatases and diadenosine tetraphosphate (Ap4A) hydrolases. PrpA exhibited a phosphatase activity towards p-nitrophenyl phosphate (pNPP), tyrosine phosphopeptide and tyrosine-phosphorylated protein, and a weak hydrolase activity towards ApnA and ATP. In the presence of Mn(2+), PrpA hydrolyzed Ap4A into AMP and ATP, whereas in the presence of Co(2+) PrpA hydrolyzed Ap4A into two molecules of ADP. ApaH exhibited high phosphatase activity towards pNPP, and hydrolase activity towards ApnA and ATP. Mn(2+) was required for ApaH-mediated pNPP dephosphorylation and ATP hydrolysis, whereas Co(2+) was required for ApnA hydrolysis. Thus, PrpA and ApaH may function mainly as a tyrosine protein phosphatase and an ApnA hydrolase, respectively. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Mining the Immune Cell Proteome to Identify Ovarian Cancer-Specific Biomarkers

DTIC Science & Technology

2013-11-01

transcription 4 (STAT4) Ras-related C3 botulinum toxin substrate 3 (RAC3) Serine/ threonine -protein phosphatase 2A catalytic subunit beta isoform (PP2AB...Mitogen-activated protein kinase 14 (MK14) Wnt signaling pathway (6) Beta-arrestin-1 (ARRB1) Serine/ threonine -protein phosphatase 2A catalytic...carried the diagnosis of chronic hypertension, diabetes , anti-phospholipid lipid antibody syndrome, or systemic lupus erythematous. Subjects were also

ERK5 signaling gets XIAPed: a role for ubiquitin in the disassembly of a MAPK cascade

PubMed Central

Klein, Aileen M; Cobb, Melanie H

2014-01-01

Mitogen-activated protein kinase (MAPK) cascades are tightly controlled through a series of well-characterized phospho-regulatory events. In this issue, Takeda et al (2014) identify the inhibitor of apoptosis protein, XIAP, as a key regulator of ERK5 activation via uncoupling of upstream kinase activity by non-degradative ubiquitination. PMID:25012518

The structure of the Tiam1 PDZ domain/ phospho-syndecan1 complex reveals a ligand conformation that modulates protein dynamics.

PubMed

Liu, Xu; Shepherd, Tyson R; Murray, Ann M; Xu, Zhen; Fuentes, Ernesto J

2013-03-05

PDZ (PSD-95/Dlg/ZO-1) domains are protein-protein interaction modules often regulated by ligand phosphorylation. Here, we investigated the specificity, structure, and dynamics of Tiam1 PDZ domain/ligand interactions. We show that the PDZ domain specifically binds syndecan1 (SDC1), phosphorylated SDC1 (pSDC1), and SDC3 but not other syndecan isoforms. The crystal structure of the PDZ/SDC1 complex indicates that syndecan affinity is derived from amino acids beyond the four C-terminal residues. Remarkably, the crystal structure of the PDZ/pSDC1 complex reveals a binding pocket that accommodates the phosphoryl group. Methyl relaxation experiments of PDZ/SCD1 and PDZ/pSDC1 complexes reveal that PDZ-phosphoryl interactions dampen dynamic motions in a distal region of the PDZ domain by decoupling them from the ligand-binding site. Our data are consistent with a selection model by which specificity and phosphorylation regulate PDZ/syndecan interactions and signaling events. Importantly, our relaxation data demonstrate that PDZ/phospho-ligand interactions regulate protein dynamics and their coupling to distal sites. Copyright © 2013 Elsevier Ltd. All rights reserved.

Dihydroartemisinin-induced apoptosis in human acute monocytic leukemia cells

PubMed Central

Cao, Jia-Tian; Mo, Hui-Min; Wang, Yue; Zhao, Kai; Zhang, Tian-Tian; Wang, Chang-Qian; Xu, Kai-Lin; Han, Zhi-Hua

2018-01-01

Dihydroartemisinin (DHA) is a derivative of artemisinin. The present study aimed to investigate whether DHA induces apoptosis in the THP-1 human acute monocytic leukemia cell line (AMoL), and to identify the relative molecular mechanisms. The results of the present study demonstrated that the viability of THP-1 cells were inhibited by DHA in a dose- and time-dependent manner, which was accompanied by morphological characteristics associated with apoptosis. After 24 h of 200 µM DHA treatment, the proportion of apoptotic cells was significantly increased compared with the untreated controls (P<0.01). In addition, DHA downregulated the levels of B-cell lymphoma (Bcl)-2, protein kinase B (Akt)1, Akt2 and Akt3 gene expression, and increased the expression of the Bcl-2-associated X protein apoptosis regulator. The protein expression of phospho-Akt and phospho-extracellular signal-regulated kinase (ERK) was also decreased, and the protein expression level of cleaved caspase-3 was increased following treatment with DHA. Therefore, DHA may induce apoptosis in the AMoL THP-1 cell line via currently unknown underlying molecular mechanisms, including the downregulation of ERK and Akt, and the activation of caspase-3. PMID:29435054

Expression and Purification of Soluble STAT5b/STAT3 Proteins for SH2 Domain Binding Assay.

PubMed

Asai, Akira; Takakuma, Kazuyuki

2017-01-01

When a large hydrophobic full-length protein is expressed in bacteria, it is often challenging to obtain recombinant proteins in the soluble fraction. One way to overcome this challenge is expression of deletion mutants that have improved solubility while maintaining biological activity. In this chapter, we describe a protocol for expression of truncated forms of STAT5b and STAT3 proteins that are soluble and retain SH2-mediated activity for phospho-Tyr peptide recognition.

Complex regulation of Arabidopsis AGR1/PIN2-mediated root gravitropic response and basipetal auxin transport by cantharidin-sensitive protein phosphatases

NASA Technical Reports Server (NTRS)

Shin, Heungsop; Shin, Hwa-Soo; Guo, Zibiao; Blancaflor, Elison B.; Masson, Patrick H.; Chen, Rujin

2005-01-01

Polar auxin transport, mediated by two distinct plasma membrane-localized auxin influx and efflux carrier proteins/complexes, plays an important role in many plant growth and developmental processes including tropic responses to gravity and light, development of lateral roots and patterning in embryogenesis. We have previously shown that the Arabidopsis AGRAVITROPIC 1/PIN2 gene encodes an auxin efflux component regulating root gravitropism and basipetal auxin transport. However, the regulatory mechanism underlying the function of AGR1/PIN2 is largely unknown. Recently, protein phosphorylation and dephosphorylation mediated by protein kinases and phosphatases, respectively, have been implicated in regulating polar auxin transport and root gravitropism. Here, we examined the effects of chemical inhibitors of protein phosphatases on root gravitropism and basipetal auxin transport, as well as the expression pattern of AGR1/PIN2 gene and the localization of AGR1/PIN2 protein. We also examined the effects of inhibitors of vesicle trafficking and protein kinases. Our data suggest that protein phosphatases, sensitive to cantharidin and okadaic acid, are likely involved in regulating AGR1/PIN2-mediated root basipetal auxin transport and gravitropism, as well as auxin response in the root central elongation zone (CEZ). BFA-sensitive vesicle trafficking may be required for the cycling of AGR1/PIN2 between plasma membrane and the BFA compartment, but not for the AGR1/PIN2-mediated root basipetal auxin transport and auxin response in CEZ cells.

Dragmacidins: new protein phosphatase inhibitors from a southern australian deep-water marine sponge, spongosorites sp

PubMed

Capon; Rooney; Murray; Collins; Sim; Rostas; Butler; Carroll

1998-05-01

A Spongosorites sp. collected during trawling operations off the southern coast of Australia returned the new alkaloid dragmacidin E (3), the structure of which was secured by detailed spectroscopic analysis. Dragmacidin E (3), and its co-metabolite dragmacidin D (1) have been identified as potent inhibitors of serine-threonine protein phosphatases.

All roads lead to PP2A: Exploiting the therapeutic potential of this phosphatase

PubMed Central

Sangodkar, Jaya; Farrington, Caroline; McClinch, Kimberly; Galsky, Matthew D.; Kastrinsky, David B.; Narla, Goutham

2015-01-01

Protein phosphatase 2A is a serine/threonine phosphatase involved in the regulation of many cellular processes. A confirmed tumor suppressor protein, PP2A is genetically altered or functionally inactivated in many cancers highlighting a need for its therapeutic reactivation. In this review we will discuss recent literature on PP2A: the elucidation of its structure and the functions of its subunits, and the identification of molecular lesions and post-translational modifications leading to its dysregulation in cancer. A final section will discuss the proteins and small molecules that modulate PP2A and how these might be used to target dysregulated forms of PP2A to treat cancers and other diseases. PMID:26507691

The interaction between early life epilepsy and autistic-like behavioral consequences: a role for the mammalian target of rapamycin (mTOR) pathway.

PubMed

Talos, Delia M; Sun, Hongyu; Zhou, Xiangping; Fitzgerald, Erin C; Jackson, Michele C; Klein, Peter M; Lan, Victor J; Joseph, Annelise; Jensen, Frances E

2012-01-01

Early life seizures can result in chronic epilepsy, cognitive deficits and behavioral changes such as autism, and conversely epilepsy is common in autistic children. We hypothesized that during early brain development, seizures could alter regulators of synaptic development and underlie the interaction between epilepsy and autism. The mammalian Target of Rapamycin (mTOR) modulates protein translation and is dysregulated in Tuberous Sclerosis Complex, a disorder characterized by epilepsy and autism. We used a rodent model of acute hypoxia-induced neonatal seizures that results in long term increases in neuronal excitability, seizure susceptibility, and spontaneous seizures, to determine how seizures alter mTOR Complex 1 (mTORC1) signaling. We hypothesized that seizures occurring at a developmental stage coinciding with a critical period of synaptogenesis will activate mTORC1, contributing to epileptic networks and autistic-like behavior in later life. Here we show that in the rat, baseline mTORC1 activation peaks during the first three postnatal weeks, and induction of seizures at postnatal day 10 results in further transient activation of its downstream targets phospho-4E-BP1 (Thr37/46), phospho-p70S6K (Thr389) and phospho-S6 (Ser235/236), as well as rapid induction of activity-dependent upstream signaling molecules, including BDNF, phospho-Akt (Thr308) and phospho-ERK (Thr202/Tyr204). Furthermore, treatment with the mTORC1 inhibitor rapamycin immediately before and after seizures reversed early increases in glutamatergic neurotransmission and seizure susceptibility and attenuated later life epilepsy and autistic-like behavior. Together, these findings suggest that in the developing brain the mTORC1 signaling pathway is involved in epileptogenesis and altered social behavior, and that it may be a target for development of novel therapies that eliminate the progressive effects of neonatal seizures.

The Interaction between Early Life Epilepsy and Autistic-Like Behavioral Consequences: A Role for the Mammalian Target of Rapamycin (mTOR) Pathway

PubMed Central

Fitzgerald, Erin C.; Jackson, Michele C.; Klein, Peter M.; Lan, Victor J.; Joseph, Annelise; Jensen, Frances E.

2012-01-01

Early life seizures can result in chronic epilepsy, cognitive deficits and behavioral changes such as autism, and conversely epilepsy is common in autistic children. We hypothesized that during early brain development, seizures could alter regulators of synaptic development and underlie the interaction between epilepsy and autism. The mammalian Target of Rapamycin (mTOR) modulates protein translation and is dysregulated in Tuberous Sclerosis Complex, a disorder characterized by epilepsy and autism. We used a rodent model of acute hypoxia-induced neonatal seizures that results in long term increases in neuronal excitability, seizure susceptibility, and spontaneous seizures, to determine how seizures alter mTOR Complex 1 (mTORC1) signaling. We hypothesized that seizures occurring at a developmental stage coinciding with a critical period of synaptogenesis will activate mTORC1, contributing to epileptic networks and autistic-like behavior in later life. Here we show that in the rat, baseline mTORC1 activation peaks during the first three postnatal weeks, and induction of seizures at postnatal day 10 results in further transient activation of its downstream targets phospho-4E-BP1 (Thr37/46), phospho-p70S6K (Thr389) and phospho-S6 (Ser235/236), as well as rapid induction of activity-dependent upstream signaling molecules, including BDNF, phospho-Akt (Thr308) and phospho-ERK (Thr202/Tyr204). Furthermore, treatment with the mTORC1 inhibitor rapamycin immediately before and after seizures reversed early increases in glutamatergic neurotransmission and seizure susceptibility and attenuated later life epilepsy and autistic-like behavior. Together, these findings suggest that in the developing brain the mTORC1 signaling pathway is involved in epileptogenesis and altered social behavior, and that it may be a target for development of novel therapies that eliminate the progressive effects of neonatal seizures. PMID:22567115

Prenatal stress decreases glycogen synthase kinase-3 phosphorylation in the rat frontal cortex.

PubMed

Szymańska, Magdalena; Suska, Anna; Budziszewska, Bogusława; Jaworska-Feil, Lucylla; Basta-Kaim, Agnieszka; Leśkiewicz, Monika; Kubera, Marta; Gergont, Aleksandra; Kroczka, Sławomir; Kaciński, Marek; Lasoń, Władysław

2009-01-01

It has been postulated that hyperactive glycogen synthase kinase-3 (GSK-3) is an important factor in the pathogenesis of depression, and that this enzyme also contributes to the mechanism of antidepressant drug action. In the present study, we investigated the effect of prenatal stress (an animal model of depression) and long-term treatment with antidepressant drugs on the concentration of GSK-3beta and its main regulating protein kinase B (PKB, Akt). The concentration of GSK-3beta, its inactive form (phospho-Ser9-GSK-3beta), and the amounts of active (phospho-Akt) and total Akt were determined in the hippocampus and frontal cortex in rats. In order to verify our animal model of depression, immobility time in the forced swim test (Porsolt test) was also determined.We found that prenatally stressed rats display a high level of immobility in the Porsolt test and chronic treatment with imipramine, fluoxetine, mirtazapine and tianeptine normalize this change. Western blot analysis demonstrated that GSK-3beta levels were significantly elevated in the frontal cortex, but not in the hippocampus, of prenatally stressed rats. The concentration of its non-active form (phospho-Ser9-GSK-3beta) was decreased only in the former brain structure. No changes were found in the amounts of active (phospho-Akt) and total Akt in both studied brain structures. Chronic treatment with antidepressant drugs diminished stress-induced alterations in GSK-3beta and phospho-GSK-3beta the frontal cortex, but had no effect on the concentration of these enzymes in the hippocampus. Moreover, levels of Akt and phospho-Akt in all experimental groups remained unchanged. Since our animal model of depression is connected with hyperactivity of the HPA axis, our results suggest that GSK-3beta is an important intracellular target for maladaptive glucocorticoid action on frontal cortex neurons and in antidepressant drug effects. Furthermore, the influence of stress and antidepressant drugs on GSK-3beta does not appear to impact the kinase activity of Akt.

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

PubMed

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

1991-08-22

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

Human cytomegalovirus phosphoproteins are hypophosphorylated and intrinsically disordered.

PubMed

Rieder, Franz J J; Kastner, Marie-Theres; Hartl, Markus; Puchinger, Martin G; Schneider, Martina; Majdic, Otto; Britt, William J; Djinović-Carugo, Kristina; Steininger, Christoph

2017-03-01

Protein phosphorylation has important regulatory functions in cell homeostasis and is tightly regulated by kinases and phosphatases. The tegument of human cytomegalovirus (CMV) contains not only several proteins reported to be extensively phosphorylated but also cellular protein phosphatases (PP1 and PP2A). To investigate this apparent inconsistency, we evaluated the phosphorylation status of the tegument proteins pUL32 and pp65 by enzymatic dephosphorylation and MS. Enzymatic dephosphorylation with bacterial λ phosphatase, but not with PP1, shifted the pUL32-specific signal on reducing SDS-PAGE from ~150 to ~148 kDa, a mass still much larger than the ~118 kDa obtained from our diffusion studies and from the calculated protein mass of ~113 kDa. Remarkably, inhibition of phosphatases through treatment with the phosphatase inhibitors calyculin A and okadaic acid resulted in a shift to ~190 or ~180 kDa, respectively, indicating that a considerable number of potential phosphorylated residues on pUL32 are not phosphorylated under normal conditions. MS revealed a general state of hypophosphorylation of CMV phosphoproteins with only 17 phosphorylated residues detected on pUL32 and 19 on pp65, respectively. Moreover, bioinformatics analysis shows that the C-terminal two-thirds of pUL32 are intrinsically disordered and that most phosphorylations map to this region. In conclusion, we show that important CMV tegument proteins are indeed phosphorylated, though to a lesser extent than previously reported, and the difference in mobility on SDS-PAGE and calculated mass of pUL32 may not be attributed to phosphorylation but more likely due to the partially intrinsically disordered nature of pUL32.

Plants Actively Avoid State Transitions upon Changes in Light Intensity: Role of Light-Harvesting Complex II Protein Dephosphorylation in High Light1[OPEN

PubMed Central

Suorsa, Marjaana; Rantala, Marjaana; Aro, Eva-Mari

2015-01-01

Photosystem II (PSII) core and light-harvesting complex II (LHCII) proteins in plant chloroplasts undergo reversible phosphorylation upon changes in light intensity (being under control of redox-regulated STN7 and STN8 kinases and TAP38/PPH1 and PSII core phosphatases). Shift of plants from growth light to high light results in an increase of PSII core phosphorylation, whereas LHCII phosphorylation concomitantly decreases. Exactly the opposite takes place when plants are shifted to lower light intensity. Despite distinct changes occurring in thylakoid protein phosphorylation upon light intensity changes, the excitation balance between PSII and photosystem I remains unchanged. This differs drastically from the canonical-state transition model induced by artificial states 1 and 2 lights that concomitantly either dephosphorylate or phosphorylate, respectively, both the PSII core and LHCII phosphoproteins. Analysis of the kinase and phosphatase mutants revealed that TAP38/PPH1 phosphatase is crucial in preventing state transition upon increase in light intensity. Indeed, tap38/pph1 mutant revealed strong concomitant phosphorylation of both the PSII core and LHCII proteins upon transfer to high light, thus resembling the wild type under state 2 light. Coordinated function of thylakoid protein kinases and phosphatases is shown to secure balanced excitation energy for both photosystems by preventing state transitions upon changes in light intensity. Moreover, PROTON GRADIENT REGULATION5 (PGR5) is required for proper regulation of thylakoid protein kinases and phosphatases, and the pgr5 mutant mimics phenotypes of tap38/pph1. This shows that there is a close cooperation between the redox- and proton gradient-dependent regulatory mechanisms for proper function of the photosynthetic machinery. PMID:25902812

The KIM-family protein-tyrosine phosphatases use distinct reversible oxidation intermediates: Intramolecular or intermolecular disulfide bond formation.

PubMed

Machado, Luciana E S F; Shen, Tun-Li; Page, Rebecca; Peti, Wolfgang

2017-05-26

The kinase interaction motif (KIM) family of protein-tyrosine phosphatases (PTPs) includes hematopoietic protein-tyrosine phosphatase (HePTP), striatal-enriched protein-tyrosine phosphatase (STEP), and protein-tyrosine phosphatase receptor type R (PTPRR). KIM-PTPs bind and dephosphorylate mitogen-activated protein kinases (MAPKs) and thereby critically modulate cell proliferation and differentiation. PTP activity can readily be diminished by reactive oxygen species (ROS), e.g. H 2 O 2 , which oxidize the catalytically indispensable active-site cysteine. This initial oxidation generates an unstable sulfenic acid intermediate that is quickly converted into either a sulfinic/sulfonic acid (catalytically dead and irreversible inactivation) or a stable sulfenamide or disulfide bond intermediate (reversible inactivation). Critically, our understanding of ROS-mediated PTP oxidation is not yet sufficient to predict the molecular responses of PTPs to oxidative stress. However, identifying distinct responses will enable novel routes for PTP-selective drug design, important for managing diseases such as cancer and Alzheimer's disease. Therefore, we performed a detailed biochemical and molecular study of all KIM-PTP family members to determine their H 2 O 2 oxidation profiles and identify their reversible inactivation mechanism(s). We show that despite having nearly identical 3D structures and sequences, each KIM-PTP family member has a unique oxidation profile. Furthermore, we also show that whereas STEP and PTPRR stabilize their reversibly oxidized state by forming an intramolecular disulfide bond, HePTP uses an unexpected mechanism, namely, formation of a reversible intermolecular disulfide bond. In summary, despite being closely related, KIM-PTPs significantly differ in oxidation profiles. These findings highlight that oxidation protection is critical when analyzing PTPs, for example, in drug screening. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.

PubMed

Leis, J F; Kaplan, N O

1982-11-01

The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid phosphatases tested from potato, wheat germ, milk, and bovine prostate did not show this degree of specificity. The plasma membrane activity also dephosphorylated phosphotyrosine histone at a much greater rate than did the other acid phosphatases. pH profiles for free O-phosphotyrosine and phosphotyrosine histone showed a shift toward physiological pH, indicating possible physiological significance. Phosphotyrosine histone dephosphorylation activity was nearly 10 times greater than that seen for phosphoserine histone dephosphorylation, and Km values were much lower for phosphotyrosine histone dephosphorylation (0.5 microM vs. 10 microM). Fluoride and zinc significantly inhibited phosphoserine histone dephosphorylation. Vanadate, on the other hand, was a potent inhibitor of phosphotyrosine histone dephosphorylation (50% inhibition at 0.5 microM) but not of phosphoserine histone. ATP stimulated phosphotyrosine histone dephosphorylation (160-250%) but inhibited phosphoserine histone dephosphorylation (95%). These results suggest the existence of a highly specific phosphotyrosine protein phosphatase activity associated with the plasma membrane of human astrocytoma.

Regulation of cardiac excitation and contraction by p21 activated kinase-1.

PubMed

Ke, Yunbo; Lei, Ming; Solaro, R John

2008-01-01

Cardiac excitation and contraction are regulated by a variety of signaling molecules. Central to the regulatory scheme are protein kinases and phosphatases that carry out reversible phosphorylation of different effectors. The process of beta-adrenergic stimulation mediated by cAMP dependent protein kinase (PKA) forms a well-known pathway considered as the most significant control mechanism in excitation and contraction as well as many other regulatory mechanisms in cardiac function. However, although dephosphorylation pathways are critical to these regulatory processes, signaling to phosphatases is relatively poorly understood. Emerging evidence indicates that regulation of phosphatases, which dampen the effect of beta-adrenergic stimulation, is also important. We review here functional studies of p21 activated kinase-1 (Pak1) and its potential role as an upstream signal for protein phosphatase PP2A in the heart. Pak1 is a serine/threonine protein kinase directly activated by the small GTPases Cdc42 and Rac1. Pak1 is highly expressed in different regions of the heart and modulates the activities of ion channels, sarcomeric proteins, and other phosphoproteins through up-regulation of PP2A activity. Coordination of Pak1 and PP2A activities is not only potentially involved in regulation of normal cardiac function, but is likely to be important in patho-physiological conditions.

Rapid detection of protein phosphatase activity using Zn(II)-coordinated gold nanosensors based on His-tagged phosphopeptides.

PubMed

Lee, Jin Oh; Kim, Eun-Ji; Lim, Butaek; Kim, Tae-Wuk; Kim, Young-Pil

2015-01-20

We report a rapid colorimetric assay to detect protein phosphatase (PP) activity based on the controlled assembly and disassembly of gold nanoparticles (AuNPs) via Zn(II)-specific coordination in the presence of His6-tagged phosphopeptides. Among divalent metal ions including Ni(II), Cu(II), Co(II), Mg(II), Mn(II), and Zn(II), only Zn(II) triggered a strong association between phosphopeptides with hexahistidine at a single end and nitrilotriacetic acid (NTA)-modified AuNPs (21.3 nm in core diameter), leading to the self-assembly of AuNPs and consequently changes in color of the AuNP solution. In contrast, unphosphorylated peptides and His6-deficient phosphopeptides did not change the color of the AuNP solution. As a result, protein phosphatase 1 (PP1) activity and its inhibition were easily quantified with high sensitivity by determining the extinction ratio (E520/E700) of colloidal AuNPs. Most importantly, this method was capable of detecting protein phosphatase 2A (PP2A) activity in immunoprecipitated plant extracts. Because PPs play pivotal roles in mediating diverse signal transduction pathways as primary effectors of protein dephosphorylation, we anticipate that our method will be applied as a rapid format method to analyze the activities of various PPs and their inhibition.

NMDA-induced potentiation of mGluR5 is mediated by activation of protein phosphatase 2B/calcineurin

PubMed Central

Alagarsamy, Sudar; Saugstad, Julie; Warren, Lee; Mansuy, Isabelle M.; Gereau, Robert W.; Conn, P. Jeffrey

2010-01-01

Previous reports have shown that activation of N-methyl-D-aspartate (NMDA) receptors potentiates responses to activation of the group I metabotropic glutamate receptor mGluR5 by reversing PKC-mediated desensitization of this receptor. NMDA-induced reversal of mGluR5 desensitization is dependent on activation of protein phosphatases. However, the specific protein phosphatase involved and the precise mechanism by which NMDA receptor activation reduces mGluR desensitization are not known. We have performed a series of molecular, biochemical, and genetic studies to show that NMDA-induced regulation of mGluR5 is dependent on activation of calcium-dependent protein phosphatase 2B/calcineurin (PP2B/CaN). Furthermore, we report that purified calcineurin directly dephosphorylates the C-terminal tail of mGluR5 at sites that are phosphorylated by PKC. Finally, immunoprecipitation and GST fusion protein pull-down experiments reveal that calcineurin interacts with mGluR5, suggesting that these proteins could be colocalized in a signaling complex. Taken together with previous studies, these data suggest that activation of NMDA receptors leads to activation of calcineurin and that calcineurin modulates mGluR5 function by directly dephosphorylating mGluR5 at PKC sites that are involved in desensitization of this receptor. 2005 Elsevier Ltd. All rights reserved. PMID:16005030

Rhodanine derivatives as inhibitors of JSP-1.

PubMed

Cutshall, Neil S; O'Day, Christine; Prezhdo, Marina

2005-07-15

Dual-specificity phosphatases (DSPs) are a subclass within the protein tyrosine phosphatase family (PTPs). A series of rhodanine-based inhibitors was synthesized and shown to be novel, potent, and selective inhibitors against the DSP family member JNK-stimulating phosphatase-1 (JSP-1). Compounds of this class may be useful for the treatment of inflammatory and proliferative disorders.

A protein phosphatase network controls the temporal and spatial dynamics of differentiation commitment in human epidermis

PubMed Central

Walko, Gernot; Viswanathan, Priyalakshmi; Tihy, Matthieu; Nijjher, Jagdeesh; Dunn, Sara-Jane; Lamond, Angus I

2017-01-01

Epidermal homeostasis depends on a balance between stem cell renewal and terminal differentiation. The transition between the two cell states, termed commitment, is poorly understood. Here, we characterise commitment by integrating transcriptomic and proteomic data from disaggregated primary human keratinocytes held in suspension to induce differentiation. Cell detachment induces several protein phosphatases, five of which - DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA – promote differentiation by negatively regulating ERK MAPK and positively regulating AP1 transcription factors. Conversely, DUSP10 expression antagonises commitment. The phosphatases form a dynamic network of transient positive and negative interactions that change over time, with DUSP6 predominating at commitment. Boolean network modelling identifies a mandatory switch between two stable states (stem and differentiated) via an unstable (committed) state. Phosphatase expression is also spatially regulated in vivo and in vitro. We conclude that an auto-regulatory phosphatase network maintains epidermal homeostasis by controlling the onset and duration of commitment. PMID:29043977

Effect of aluminum phosphate on alkaline phosphatase activity of polyurethane foam immobilized cyanobacteria.

PubMed

Ramalingam, N; Prasanna, B Gowtham

2006-09-01

The impact of insoluble phosphorus such as aluminum and rock phosphate on alkaline phosphatase activity of polyurethane foam immobilized cyanobacteria was assessed. Polyurethane foam immobilized Nodularia recorded the highest alkaline phosphatase activity of 9.04 (m. mol p-nitrophenol released h(-1) mg(-1) protein) in vitro. A higher concentration of aluminum phosphate was recorded a 25% reduction in alkaline phosphatase activity, ammonia content, and available phosphorus in culture filtrate of polyurethane foam immobilized cyanobacteria. In general, immobilized cyanobacteria exhibited a higher alkaline phosphatase activity in rock phosphate than aluminum phosphate.

Unique carbohydrate binding platforms employed by the glucan phosphatases

PubMed Central

MEEKINS, David A.; GENTRY, Matthew S.

2016-01-01

Glucan phosphatases are a family of enzymes that are functionally conserved at the enzymatic level in animals and plants. These enzymes bind and dephosphorylate glycogen in animals and starch in plants. While the enzymatic function is conserved, the glucan phosphatases employ distinct mechanisms to bind and dephosphorylate glycogen or starch. The founding member of the family is a bimodular human protein called laforin that is comprised of a carbohydrate binding module 20 (CBM20) followed by a dual specificity phosphatase domain. Plants contain two glucan phosphatases: Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2). SEX4 contains a chloroplast targeting peptide, dual specificity phosphatase (DSP) domain, a CBM45, and a carboxy-terminal motif. LSF2 is comprised of simply a chloroplast targeting peptide, DSP domain, and carboxy-terminal motif. SEX4 employs an integrated DSP-CBM glucan-binding platform to engage and dephosphorylate starch. LSF2 lacks a CBM and instead utilizes two surface binding sites to bind and dephosphorylate starch. Laforin is a dimeric protein in solution and it utilizes a tetramodular architecture and cooperativity to bind and dephosphorylate glycogen. This chapter describes the biological role of glucan phosphatases in glycogen and starch metabolism and compares and contrasts their ability to bind and dephosphorylate glucans. PMID:27147465

Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris

NASA Technical Reports Server (NTRS)

Karr, Laurel; Malone, Christine, C.; Rose, M. Franklin (Technical Monitor)

2000-01-01

The Pichiapastoris expression system was utilized to produce functionally active human bone alkaline phosphatase in gram quantities. Bone alkaline phosphatase is a key enzyme in bone formation and biomineralization, yet important questions about its structural chemistry and interactions with other cellular enzymes in mineralizing tissues remain unanswered. A soluble form of human bone alkaline phosphatase was constructed by deletion of the 25 amino acid hydrophobic C-terminal region of the encoding cDNA and inserted into the X-33 Pichiapastoris strain. An overexpression system was developed in shake flasks and converted to large-scale fermentation. Alkaline phosphatase was secreted into the medium to a level of 32mgAL when cultured in shake flasks. Enzyme activity was 12U/mg measured by a spectrophotometric assay. Fermentation yielded 880mgAL with enzymatic activity of 968U/mg. Gel electrophoresis analysis indicates that greater than 50% of the total protein in the fermentation is alkaline phosphatase. A purification scheme has been developed using ammonium sulfate precipitation followed by hydrophobic interaction chromatography. We are currently screening crystallization conditions of the purified recombinant protein for subsequent X-ray diffraction analyses. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

The activation of G protein-coupled estrogen receptor induces relaxation via cAMP as well as potentiates contraction via EGFR transactivation in porcine coronary arteries.

PubMed

Yu, Xuan; Stallone, John N; Heaps, Cristine L; Han, Guichun

2018-01-01

Estrogen exerts protective effects against cardiovascular diseases in premenopausal women, but is associated with an increased risk of both coronary heart disease and stroke in older postmenopausal women. Studies have shown that activation of the G-protein-coupled estrogen receptor 1 (GPER) can cause either relaxation or contraction of arteries. It is highly likely that these dual actions of GPER may contribute to the seemingly paradoxical effects of estrogen in regulating coronary artery function. The objective of this study was to test the hypothesis that activation of GPER enhances agonist-stimulated porcine coronary artery contraction via epidermal growth factor receptor (EGFR) transactivation and its downstream extracellular signal-regulated kinases (ERK1/2) pathway. Isometric tension studies and western blot were performed to determine the effect of GPER activation on coronary artery contraction. Our findings demonstrated that G-1 caused concentration-dependent relaxation of ET-1-induced contraction, while pretreatment of arterial rings with G-1 significantly enhanced ET-1-induced contraction. GPER antagonist, G-36, significantly inhibited both the G-1-induced relaxation effect and G-1-enhanced ET-1 contraction. Gallein, a Gβγ inhibitor, significantly increased G-1-induced relaxation, yet inhibited G-1-enhanced ET-1-mediated contraction. Similarly, inhibition of EGFR with AG1478 or inhibition of Src with phosphatase 2 further increased G-1-induced relaxation responses in coronary arteries, but decreased G-1-enhanced ET-1-induced contraction. Western blot experiments in porcine coronary artery smooth muscle cells (PCASMC) showed that G-1 increased tyrosine phosphorylation of EGFR, which was inhibited by AG-1478. Furthermore, enzyme-linked immunosorbent assays showed that the level of heparin-binding EGF (HB-EGF) released by ET-1 treatment increased two-fold; whereas pre-incubation with G-1 further increased ET-1-induced HB-EGF release to four-fold over control conditions. Lastly, the role of ERK1/2 was determined by applying the MEK inhibitor, PD98059, in isometric tension studies and detecting phospho-ERK1/2 in immunoblotting. PD98059 potentiated G-1-induced relaxation response, but blocked G-1-enhanced ET-1-induced contraction. By western blot, G-1 treatment decreased phospho-ERK1/2, however, in the presence of the adenylyl cyclase inhibitor, SQ22536, G-1 significantly increased ERK1/2 phosphorylation in PCASMC. These data demonstrate that activation of GPER induces relaxation via cAMP as well as contraction via a mechanism involving transactivation of EGFR and the phosphorylation of ERK1/2 in porcine coronary arteries.

PhosphoregDB: The tissue and sub-cellular distribution of mammalian protein kinases and phosphatases

PubMed Central

Forrest, Alistair RR; Taylor, Darrin F; Fink, J Lynn; Gongora, M Milena; Flegg, Cameron; Teasdale, Rohan D; Suzuki, Harukazu; Kanamori, Mutsumi; Kai, Chikatoshi; Hayashizaki, Yoshihide; Grimmond, Sean M

2006-01-01

Background Protein kinases and protein phosphatases are the fundamental components of phosphorylation dependent protein regulatory systems. We have created a database for the protein kinase-like and phosphatase-like loci of mouse that integrates protein sequence, interaction, classification and pathway information with the results of a systematic screen of their sub-cellular localization and tissue specific expression data mined from the GNF tissue atlas of mouse. Results The database lets users query where a specific kinase or phosphatase is expressed at both the tissue and sub-cellular levels. Similarly the interface allows the user to query by tissue, pathway or sub-cellular localization, to reveal which components are co-expressed or co-localized. A review of their expression reveals 30% of these components are detected in all tissues tested while 70% show some level of tissue restriction. Hierarchical clustering of the expression data reveals that expression of these genes can be used to separate the samples into tissues of related lineage, including 3 larger clusters of nervous tissue, developing embryo and cells of the immune system. By overlaying the expression, sub-cellular localization and classification data we examine correlations between class, specificity and tissue restriction and show that tyrosine kinases are more generally expressed in fewer tissues than serine/threonine kinases. Conclusion Together these data demonstrate that cell type specific systems exist to regulate protein phosphorylation and that for accurate modelling and for determination of enzyme substrate relationships the co-location of components needs to be considered. PMID:16504016

Structural Basis for the Catalytic Activity of Human SER/THR Protein Phosphatase-5

NASA Technical Reports Server (NTRS)

Swingle, M. R.; Honkanen, R.; Ciszak, E.

2004-01-01

Serinekhreonine protein phosphatase-5 (PP5) affects many signaling networks that regulate cell growth. Here we report the 1.6 Angstrom resolution crystal structure of PP5 catalytic domain with metal and phosphate ions in the active site. The structure reveals a mechanism for PPS-mediated catalysis that requires the precise positioning of two metal ions within a conserved Asp(sup 271)-M(sub 1),-M(sub 2)-His(sup 427)-W(sup 2)-His(sup 304)-Asp(sup 274) catalytic motif, and provides a structural basis for the exceptional catalytic proficiency of protein phosphatases placing them among the most powerful catalysts. Resolution of the entire C-terminus revealed a novel subdomain, and the structure of PP5 should aid development of specific inhibitors.

Characterization of protein phosphatase 2A acting on phosphorylated plasma membrane aquaporin of tulip petals.

PubMed

Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

2004-05-01

A protein phosphatase holo-type enzyme (38, 65, and 75 kDa) preparation and a free catalytic subunit (38 kDa) purified from tulip petals were characterized as protein phosphatase 2A (PP2A) by immunological and biochemical approaches. The plasma membrane containing the putative plasma membrane aquaporin (PM-AQP) was prepared from tulip petals, phosphorylated in vitro, and used as the substrate for both of the purified PP2A preparations. Although both preparations dephosphorylated the phosphorylated PM-AQP at 20 degrees C, only the holo-type enzyme preparation acted at 5 degrees C on the phosphorylated PM-AQP with higher substrate specificity, suggesting that regulatory subunits are required for low temperature-dependent dephosphorylation of PM-AQP in tulip petals.

The immunoglobulin-like domains 1 and 2 of the protein tyrosine phosphatase LAR adopt an unusual horseshoe-like conformation

PubMed Central

Biersmith, Bridget H.; Hammel, Michal; Geisbrecht, Erika R.; Bouyain, Samuel

2011-01-01

Neurogenesis depends on exquisitely regulated interactions between macromolecules on the cell surface and in the extracellular matrix. In particular, interactions between proteoglycans and members of the type IIa subgroup of receptor protein tyrosine phosphatases underlie critical developmental processes such as the formation of synapses at the neuromuscular junction and the migration of axons to their appropriate targets. We report here the crystal structures of the first and second immunoglobulin-like domains of the Drosophila type IIa receptor Dlar and its mouse homologue LAR. These two domains adopt an unusual antiparallel arrangement that has not been previously observed in tandem repeats of immunoglobulin-like domains and that is presumably conserved in all type IIa receptor protein tyrosine phosphatases. PMID:21402080

The E3 ubiquitin ligase Mule acts through the ATM-p53 axis to maintain B lymphocyte homeostasis.

PubMed

Hao, Zhenyue; Duncan, Gordon S; Su, Yu-Wen; Li, Wanda Y; Silvester, Jennifer; Hong, Claire; You, Han; Brenner, Dirk; Gorrini, Chiara; Haight, Jillian; Wakeham, Andrew; You-Ten, Annick; McCracken, Susan; Elia, Andrew; Li, Qinxi; Detmar, Jacqui; Jurisicova, Andrea; Hobeika, Elias; Reth, Michael; Sheng, Yi; Lang, Philipp A; Ohashi, Pamela S; Zhong, Qing; Wang, Xiaodong; Mak, Tak W

2012-01-16

Cellular homeostasis is controlled by pathways that balance cell death with survival. Mcl-1 ubiquitin ligase E3 (Mule) is an E3 ubiquitin ligase that targets the proapoptotic molecule p53 for polyubiquitination and degradation. To elucidate the role of Mule in B lymphocyte homeostasis, B cell-specific Mule knockout (BMKO) mice were generated using the Cre-LoxP recombination system. Analysis of BMKO mice showed that Mule was essential for B cell development, proliferation, homeostasis, and humoral immune responses. p53 transactivation was increased by two- to fourfold in Mule-deficient B cells at steady state. Genetic ablation of p53 in BMKO mice restored B cell development, proliferation, and homeostasis. p53 protein was increased in resting Mule-deficient mouse embryonic fibroblasts (MEFs) and embryonic stem (ES) cells. Loss of Mule in both MEFs and B cells at steady state resulted in increased levels of phospho-ataxia telangiectasia mutated (ATM) and the ATM substrate p53. Under genotoxic stress, BMKO B cells were resistant to apoptosis, and control MEFs exhibited evidence of a physical interaction between Mule and phospho-ATM. Phospho-ATM, phospho-p53, and Brca1 levels were reduced in Mule-deficient B cells and MEFs subjected to genotoxic stress. Thus, Mule regulates the ATM-p53 axis to maintain B cell homeostasis under both steady-state and stress conditions.

Regulation of mouse brain glycogen synthase kinase-3 by atypical antipsychotics.

PubMed

Li, Xiaohua; Rosborough, Kelley M; Friedman, Ari B; Zhu, Wawa; Roth, Kevin A

2007-02-01

Glycogen synthase kinase-3 (GSK3) has been recognized as an important enzyme that modulates many aspects of neuronal function. Accumulating evidence implicates abnormal activity of GSK3 in mood disorders and schizophrenia, and GSK3 is a potential protein kinase target for psychotropics used in these disorders. We previously reported that serotonin, a major neurotransmitter involved in mood disorders, regulates GSK3 by acutely increasing its N-terminal serine phosphorylation. The present study was undertaken to further determine if atypical antipsychotics, which have therapeutic effects in both mood disorders and schizophrenia, can regulate phospho-Ser-GSK3 and inhibit its activity. The results showed that acute treatment of mice with risperidone rapidly increased the level of brain phospho-Ser-GSK3 in the cortex, hippocampus, striatum, and cerebellum in a dose-dependent manner. Regulation of phospho-Ser-GSK3 was a shared effect among several atypical antipsychotics, including olanzapine, clozapine, quetiapine, and ziprasidone. In addition, combination treatment of mice with risperidone and a monoamine reuptake inhibitor antidepressant imipramine or fluoxetine elicited larger increases in brain phospho-Ser-GSK3 than each agent alone. Taken together, these results provide new information suggesting that atypical antipsychotics, in addition to mood stabilizers and antidepressants, can inhibit the activity of GSK3. These findings may support the pharmacological mechanisms of atypical antipsychotics in the treatment of mood disorders.

Angiotensin II receptor blocker telmisartan enhances running endurance of skeletal muscle through activation of the PPAR-δ/AMPK pathway

PubMed Central

Feng, Xiaoli; Luo, Zhidan; Ma, Liqun; Ma, Shuangtao; Yang, Dachun; Zhao, Zhigang; Yan, Zhencheng; He, Hongbo; Cao, Tingbing; Liu, Daoyan; Zhu, Zhiming

2011-01-01

Abstract Clinical trials have shown that angiotensin II receptor blockers reduce the new onset of diabetes in hypertensives; however, the underlying mechanisms remain unknown. We investigated the effects of telmisartan on peroxisome proliferator activated receptor γ (PPAR-δ) and the adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway in cultured myotubes, as well as on the running endurance of wild-type and PPAR-δ-deficient mice. Administration of telmisartan up-regulated levels of PPAR-δ and phospho-AMPKα in cultured myotubes. However, PPAR-δ gene deficiency completely abolished the telmisartan effect on phospho-AMPKαin vitro. Chronic administration of telmisartan remarkably prevented weight gain, enhanced running endurance and post-exercise oxygen consumption, and increased slow-twitch skeletal muscle fibres in wild-type mice, but these effects were absent in PPAR-δ-deficient mice. The mechanism is involved in PPAR-δ-mediated stimulation of the AMPK pathway. Compared to the control mice, phospho-AMPKα level in skeletal muscle was up-regulated in mice treated with telmisartan. In contrast, phospho-AMPKα expression in skeletal muscle was unchanged in PPAR-δ-deficient mice treated with telmisartan. These findings highlight the ability of telmisartan to improve skeletal muscle function, and they implicate PPAR-δ as a potential therapeutic target for the prevention of type 2 diabetes. PMID:20477906

Tanreqing Injection Attenuates Lipopolysaccharide-Induced Airway Inflammation through MAPK/NF-κB Signaling Pathways in Rats Model

PubMed Central

Liu, Wei; Jiang, Hong-li; Cai, Lin-li; Yan, Min; Dong, Shou-jin; Mao, Bing

2016-01-01

Background. Tanreqing injection (TRQ) is a commonly used herbal patent medicine for treating inflammatory airway diseases in view of its outstanding anti-inflammatory properties. In this study, we explored the signaling pathways involved in contributions of TRQ to LPS-induced airway inflammation in rats. Methods/Design. Adult male Sprague Dawley (SD) rats randomly divided into different groups received intratracheal instillation of LPS and/or intraperitoneal injection of TRQ. Bronchoalveolar Lavage Fluid (BALF) and lung samples were collected at 24 h, 48 h, and 96 h after TRQ administration. Protein and mRNA levels of tumor necrosis factor- (TNF-) α, Interleukin- (IL-) 1β, IL-6, and IL-8 in BALF and lung homogenate were observed by ELISA and real-time PCR, respectively. Lung sections were stained for p38 MAPK and NF-κB detection by immunohistochemistry. Phospho-p38 MAPK, phosphor-extracellular signal-regulated kinases ERK1/2, phospho-SAPK/JNK, phospho-NF-κB p65, phospho-IKKα/β, and phospho-IκB-α were measured by western blot analysis. Results. The results showed that TRQ significantly counteracted LPS-stimulated release of TNF-α, IL-1β, IL-6, and IL-8, attenuated cells influx in BALF, mitigated mucus hypersecretion, suppressed phosphorylation of NF-κB p65, IκB-α, ΙKKα/β, ERK1/2, JNK, and p38 MAPK, and inhibited p38 MAPK and NF-κB p65 expression in rat lungs. Conclusions. Results of the current research indicate that TRQ possesses potent exhibitory effects in LPS-induced airway inflammation by, at least partially, suppressing the MAPKs and NF-κB signaling pathways, in a general dose-dependent manner. PMID:27366191

Phospho-Bcl-x(L)(Ser62) plays a key role at DNA damage-induced G(2) checkpoint.

PubMed

Wang, Jianfang; Beauchemin, Myriam; Bertrand, Richard

2012-06-01

Accumulating evidence suggests that Bcl-xL, an anti-apoptotic member of the Bcl-2 family, also functions in cell cycle progression and cell cycle checkpoints. Analysis of a series of phosphorylation site mutants reveals that cells expressing Bcl-xL(Ser62Ala) mutant are less stable at the G 2 checkpoint and enter mitosis more rapidly than cells expressing wild-type Bcl-xL or Bcl-xL phosphorylation site mutants, including Thr41Ala, Ser43Ala, Thr47Ala, Ser56Ala and Thr115Ala. Analysis of the dynamic phosphorylation and location of phospho-Bcl-xL(Ser62) in unperturbed, synchronized cells and during DNA damage-induced G 2 arrest discloses that a pool of phospho-Bcl-xL(Ser62) accumulates into nucleolar structures in etoposide-exposed cells during G 2 arrest. In a series of in vitro kinase assays, pharmacological inhibitors and specific siRNAs experiments, we found that Polo kinase 1 and MAPK9/JNK2 are major protein kinases involved in Bcl-xL(Ser62) phosphorylation and accumulation into nucleolar structures during the G 2 checkpoint. In nucleoli, phospho-Bcl-xL(Ser62) binds to and co-localizes with Cdk1(cdc2), the key cyclin-dependent kinase required for entry into mitosis. These data indicate that during G 2 checkpoint, phospho-Bcl-xL(Ser62) stabilizes G 2 arrest by timely trapping of Cdk1(cdc2) in nucleolar structures to slow mitotic entry. It also highlights that DNA damage affects the dynamic composition of the nucleolus, which now emerges as a piece of the DNA damage response.

Injury-induced rapid activation of MAPK signaling in dechorionated eggs and larvae of the silkworm Bombyx mori.

PubMed

Gu, Shi-Hong; Chen, Chien-Hung

2017-04-01

Previous study showed that diapause in Bombyx mori eggs can be terminated by dechorionation and that activation in the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) in dechorionated cultured eggs is involved in diapause termination. In the present study, the possible mechanism underlying activation of ERK upon dechorionation was further investigated. Results showed that mechanical injury of diapause eggs without medium incubation also resulted in rapid increase in the phospho-ERK levels and that injury increased the phospho-ERK levels at different stages of both diapause eggs and eggs in which diapause initiation was prevented by HCl. Effects of anaerobiosis on dechorionation-stimulated phospho-ERK levels showed that the mechanical injury itself but not the dramatic increase in oxygen uptake upon injury is involved in a rapid activation of ERK. Chemical anaerobiosis on dechorionation-stimulated phospho-ERK levels and the in vivo effect of anaerobiosis showed that the supply of oxygen also plays a role in ERK signaling. In addition, injury induced the phosphorylation of c-jun N-terminal kinases (JNKs) and p38 kinase, components of two parallel MAPK pathways. A kinase assay showed a dramatic increase in JNK kinase activity in egg lysates upon injury. When newly hatched first instar larvae were injured, an increase in the phospho-ERK levels similar to that in dechorionated eggs was observed. From the results, we hypothesize that the injury-induced rapid activation of MAPK signaling, which serves as a natural signal for embryonic development, is related to diapause termination in dechorionated eggs. © 2015 Institute of Zoology, Chinese Academy of Sciences.

2,4-Dihydroxychalcone derivatives as novel potent cell division cycle 25B phosphatase inhibitors and protein tyrosine phosphatase 1B inhibitors.

PubMed

Xie, Chao; Sun, Yuan; Pan, Cheng-Yan; Tang, Li-Ming; Guan, Li-Ping

2014-04-01

Eleven 2,4-dihydroxychalcone compounds were synthesized and identified as reversible and competitive cell division cycle 25 (CDC25) B and protein tyrosine phosphatase (PTP) 1B inhibitors with inhibition values in the micromolar range. The results showed that nine compounds significantly inhibited CDC25B phosphatase, whereas seven compounds inhibited the activity against PTP1B in vitro. Compound 8 had the greatest inhibition activity against CDC25B and PTP1B in vitro, with percentage inhibition values of 97.5% and 96.3% at a dose of 20 microg/mL, respectively. Cytotoxic activity assays revealed that compound 8 was the most potent against HCT116, HeLa, and A549 cells. Furthermore, compound 8 exhibited potent antitumor activity in a colo205 xenograft model.

Phosphatidic acid (PA) binds PP2AA1 to regulate PP2A activity and PIN1 polar localization.

PubMed

Gao, Hong-Bo; Chu, Yu-Jia; Xue, Hong-Wei

2013-09-01

Phospholipase D (PLD) exerts broad biological functions in eukaryotes through regulating downstream effectors by its product, phosphatidic acid (PA). Protein kinases and phosphatases, such as mammalian target of rapamycin (mTOR), Protein Phosphatase 1 (PP1) and Protein Phosphatase 2C (PP2C), are PA-binding proteins that execute crucial regulatory functions in both animals and plants. PA participates in many signaling pathways by modulating the enzymatic activity and/or subcellular localization of bound proteins. In this study, we demonstrated that PLD-derived PA interacts with the scaffolding A1 subunit of Protein Phosphatase 2A (PP2A) and regulates PP2A-mediated PIN1 dephosphorylation in Arabidopsis. Genetic and pharmacological studies showed that both PA and PP2A participate in the regulation of auxin distribution. In addition, both the phosphorylation status and polar localization of PIN1 protein were affected by PLD inhibitors. Exogenous PA triggered the membrane accumulation of PP2AA1 and enhanced the PP2A activity at membrane, while PLD inhibition resulted in the reduced endosomal localization and perinuclear aggregation of PP2AA1. These results demonstrate the important role of PLD-derived PA in normal PP2A-mediated PIN dephosphorylation and reveal a novel mechanism, in which PA recruits PP2AA1 to the membrane system and regulates PP2A function on membrane-targeted proteins. As PA and PP2A are conserved among eukaryotes, other organisms might use similar mechanisms to mediate multiple biological processes.

Identification and characterization of a gene encoding for a nucleotidase from Phaseolus vulgaris.

PubMed

Cabello-Díaz, Juan Miguel; Gálvez-Valdivieso, Gregorio; Caballo, Cristina; Lambert, Rocío; Quiles, Francisco Antonio; Pineda, Manuel; Piedras, Pedro

2015-08-01

Nucleotidases are phosphatases that catalyze the removal of phosphate from nucleotides, compounds with an important role in plant metabolism. A phosphatase enzyme, with high affinity for nucleotides monophosphate previously identified and purified in embryonic axes from French bean, has been analyzed by MALDI TOF/TOF and two internal peptides have been obtained. The information of these peptide sequences has been used to search in the genome database and only a candidate gene that encodes for the phosphatase was identified (PvNTD1). The putative protein contains the conserved domains (motif I-IV) for haloacid dehalogenase-like hydrolases superfamily. The residues involved in the catalytic activity are also conserved. A recombinant protein overexpressed in Escherichia coli has shown molybdate resistant phosphatase activity with nucleosides monophosphate as substrate, confirming that the identified gene encodes for the phosphatase with high affinity for nucleotides purified in French bean embryonic axes. The activity of the purified protein was inhibited by adenosine. The expression of PvNTD1 gene was induced at the specific moment of radicle protrusion in embryonic axes. The gene was also highly expressed in young leaves whereas the level of expression in mature tissues was minimal. Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.

Multiple myeloma phosphotyrosine proteomic profile associated with FGFR3 expression, ligand activation, and drug inhibition

PubMed Central

St-Germain, Jonathan R.; Taylor, Paul; Tong, Jiefei; Jin, Lily L.; Nikolic, Ana; Stewart, Ian I.; Ewing, Robert M.; Dharsee, Moyez; Li, Zhihua; Trudel, Suzanne; Moran, Michael F.

2009-01-01

Signaling by growth factor receptor tyrosine kinases is manifest through networks of proteins that are substrates and/or bind to the activated receptors. FGF receptor-3 (FGFR3) is a drug target in a subset of human multiple myelomas (MM) and is mutationally activated in some cervical and colon and many bladder cancers and in certain skeletal dysplasias. To define the FGFR3 network in multiple myeloma, mass spectrometry was used to identify and quantify phosphotyrosine (pY) sites modulated by FGFR3 activation and inhibition in myeloma-derived KMS11 cells. Label-free quantification of peptide ion currents indicated the activation of FGFR3 by phosphorylation of tandem tyrosines in the kinase domain activation loop when cellular pY phosphatases were inhibited by pervanadate. Among the 175 proteins that accumulated pY in response to pervanadate was a subset of 52 including FGFR3 that contained a total of 61 pY sites that were sensitive to inhibition by the FGFR3 inhibitor PD173074. The FGFR3 isoform containing the tandem pY motif in its activation loop was targeted by PD173074. Forty of the drug-sensitive pY sites, including two located within the 35-residue cytoplasmic domain of the transmembrane growth factor binding proteoglycan (and multiple myeloma biomarker) Syndecan-1/CD138, were also stimulated in cells treated with the ligand FGF1, providing additional validation of their link to FGFR3. The identification of these overlapping sets of co-modulated tyrosine phosphorylations presents an outline of an FGFR3 network in the MM model and demonstrates the potential for pharmacodynamic monitoring by label-free quantitative phospho-proteomics. PMID:19901323

Pulsed electromagnetic field inhibits RANKL-dependent osteoclastic differentiation in RAW264.7 cells through the Ca{sup 2+}-calcineurin-NFATc1 signaling pathway

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

Zhang, Jie; Xu, Haixia; Han, Zhongyu

Pulsed electromagnetic field (PEMF) has been reported to improve bone healing in osteoporosis patients. However, the precise mechanism has remained largely unknown. This study aimed to investigate the effects of PEMF on nuclear factor κB ligand (RANKL)-dependent osteoclastic differentiation and the Ca{sup 2+}-calcineurin-NFATc1 signaling pathway in RAW264.7 cells in vitro. Treating RAW264.7 cells with RANKL for 4 days induced osteoclastic differentiation in vitro, and the formation of multinucleated osteoclasts, bone resorption-pit formation, tartrate-resistant acid phosphatase (TRAP) activity and the protein levels of cathepsin K, TRAP, Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and matrix metalloproteinase 9 (MMP-9) were significantly decreased. The mRNA levelsmore » of specific genes related to osteoclastogenesis (TRAP, NFATc1, CTSK and MMP-9) were also reduced. Moreover, the oscillations of intracellular Ca{sup 2+} in RANKL-dependent RAW264.7 cells were suppressed by PEMF, as well as by inhibitors of membrane and store-operated Ca{sup 2+} channels. Meanwhile, calcineurin activity was increased, although its protein level was not changed. PEMF increased phospho-NFATc1 in the cytosol while suppressing the nuclear translocation of NFATc1, thus inhibiting osteoclastic differentiation by suppressing the Ca{sup 2+}-calcineurin-NFATc1 signaling pathway. Although many questions remain unresolved, to our knowledge, this is the first report demonstrating that PEMF is beneficial against RANKL-dependent osteoclastic differentiation in RAW264.7 cells in vitro via inhibiting the Ca{sup 2+}-calcineurin-NFATc1 signaling pathway.« less

Pulsed electromagnetic field inhibits RANKL-dependent osteoclastic differentiation in RAW264.7 cells through the Ca2+-calcineurin-NFATc1 signaling pathway.

PubMed

Zhang, Jie; Xu, Haixia; Han, Zhongyu; Chen, Ping; Yu, Qiang; Lei, Yutian; Li, Zongze; Zhao, Ming; Tian, Jing

2017-01-08

Pulsed electromagnetic field (PEMF) has been reported to improve bone healing in osteoporosis patients. However, the precise mechanism has remained largely unknown. This study aimed to investigate the effects of PEMF on nuclear factor κB ligand (RANKL)-dependent osteoclastic differentiation and the Ca 2+ -calcineurin-NFATc1 signaling pathway in RAW264.7 cells in vitro. Treating RAW264.7 cells with RANKL for 4 days induced osteoclastic differentiation in vitro, and the formation of multinucleated osteoclasts, bone resorption-pit formation, tartrate-resistant acid phosphatase (TRAP) activity and the protein levels of cathepsin K, TRAP, Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and matrix metalloproteinase 9 (MMP-9) were significantly decreased. The mRNA levels of specific genes related to osteoclastogenesis (TRAP, NFATc1, CTSK and MMP-9) were also reduced. Moreover, the oscillations of intracellular Ca 2+ in RANKL-dependent RAW264.7 cells were suppressed by PEMF, as well as by inhibitors of membrane and store-operated Ca 2+ channels. Meanwhile, calcineurin activity was increased, although its protein level was not changed. PEMF increased phospho-NFATc1 in the cytosol while suppressing the nuclear translocation of NFATc1, thus inhibiting osteoclastic differentiation by suppressing the Ca 2+ -calcineurin-NFATc1 signaling pathway. Although many questions remain unresolved, to our knowledge, this is the first report demonstrating that PEMF is beneficial against RANKL-dependent osteoclastic differentiation in RAW264.7 cells in vitro via inhibiting the Ca 2+ -calcineurin-NFATc1 signaling pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

Anticancer effect of curcumin inhibits cell growth through miR-21/PTEN/Akt pathway in breast cancer cell.

PubMed

Wang, Xinzheng; Hang, Yakai; Liu, Jinbiao; Hou, Yongqiang; Wang, Ning; Wang, Mingjun

2017-06-01

Curcumin is a polyphenol extracted from turmeric, which that belongs to the Zingiberaceae family. Curcumin has numerous effects, including anti-inflammatory, antitumor, anti-oxidative and antimicrobial effects. However, the effects of curcumin on human breast cancer cells remain largely unknown. The aim of the present study was to investigate the anticancer effects and the mechanisms by which curcumin affects breast cancer cells. The anticancer effect of curcumin on cell viability and cytotoxicity on human breast cancer MCF-7 cells was analyzed using 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide and lactate dehydrogenase assays, respectively. Cell apoptosis of MCF-7 cells was detected using flow cytometry, 4',6-diamidino-2-phenylindolestaining assay and caspase-3/9 activity kits. Reverse transcription-quantitative polymerase chain reaction was used to analyze microRNA-21 (miR-21) expression in MCF-7 cells. The protein expression of phosphatase and tensin homolog (PTEN) and phospho-protein kinase B (pAkt) was determined by western blot analysis. miR-21 was transfected into MCF-7 cells and the anticancer effect of curcumin on cell viability and the expression of PTEN and pAkt was analyzed. The present results demonstrated that curcumin inhibited cell viability and induced cytotoxicity of MCF-7 cells in a concentration- and time-dependent manner, by inducing apoptosis and increasing caspase-3/9 activities. In addition, curcumin downregulated miR-21 expression in MCF-7 cells by upregulating the PTEN/Akt signaling pathway. The present study has for the first time, to the best of our knowledge, revealed the anticancer effect of curcumin in suppressing breast cancer cell growth, and has elucidated that the miR-21/PTEN/Akt signaling pathway is a key mechanism for the anticancer effects of curcumin.

Berberine-induced Inactivation of Signal Transducer and Activator of Transcription 5 Signaling Promotes Male-specific Expression of a Bile Acid Uptake Transporter*

PubMed Central

Bu, Pengli; Le, Yuan; Zhang, Yue; Zhang, Youcai; Cheng, Xingguo

2017-01-01

Sodium-taurocholate co-transporting polypeptide (Ntcp/NTCP) is the major uptake transporter of bile salts in mouse and human livers. In certain diseases, including endotoxemia, cholestasis, diabetes, and hepatocarcinoma, Ntcp/NTCP expression is markedly reduced, which interferes with enterohepatic circulation of bile salts, impairing the absorption of lipophilic compounds. Therefore, normal Ntcp/NTCP expression in the liver is physiologically important. Berberine is an herbal medicine used historically to improve liver function and has recently been shown to repress STAT signaling. However, berberine effects on Ntcp/NTCP expression are unknown, prompting use to investigate this possible connection. Our results showed that berberine dose-dependently increased Ntcp expression in male mouse liver and decreased taurocholic acid levels in serum but increased them in the liver. In mouse and human hepatoma cells, berberine induced Ntcp/NTCP mRNA and protein expression and increased cellular uptake of [3H] taurocholate. Mechanistically, berberine decreased nuclear protein levels of phospho-JAK2 and phospho-STAT5, thus disrupting the JAK2-STAT5 signaling. Moreover, berberine stimulated luciferase reporter expression from the mouse Ntcp promoter when one putative STAT5 response element (RE) (−1137 bp) was deleted and from the human NTCP promoter when three putative STAT5REs (−2898, −2164, and −691 bp) were deleted. Chromatin immunoprecipitation demonstrated that berberine decreased binding of phospho-STAT5 protein to the−2164 and −691 bp STAT5REs in the human NTCP promoter. In summary, berberine-disrupted STAT5 signaling promoted mouse and human Ntcp/NTCP expression, resulting in enhanced bile acid uptake. Therefore, berberine may be a therapeutic candidate compound for maintaining bile acid homeostasis. PMID:28154180

Berberine-induced Inactivation of Signal Transducer and Activator of Transcription 5 Signaling Promotes Male-specific Expression of a Bile Acid Uptake Transporter.

PubMed

Bu, Pengli; Le, Yuan; Zhang, Yue; Zhang, Youcai; Cheng, Xingguo

2017-03-17

Sodium-taurocholate co-transporting polypeptide (Ntcp/NTCP) is the major uptake transporter of bile salts in mouse and human livers. In certain diseases, including endotoxemia, cholestasis, diabetes, and hepatocarcinoma, Ntcp/NTCP expression is markedly reduced, which interferes with enterohepatic circulation of bile salts, impairing the absorption of lipophilic compounds. Therefore, normal Ntcp/NTCP expression in the liver is physiologically important. Berberine is an herbal medicine used historically to improve liver function and has recently been shown to repress STAT signaling. However, berberine effects on Ntcp/NTCP expression are unknown, prompting use to investigate this possible connection. Our results showed that berberine dose-dependently increased Ntcp expression in male mouse liver and decreased taurocholic acid levels in serum but increased them in the liver. In mouse and human hepatoma cells, berberine induced Ntcp/NTCP mRNA and protein expression and increased cellular uptake of [3H] taurocholate. Mechanistically, berberine decreased nuclear protein levels of phospho-JAK2 and phospho-STAT5, thus disrupting the JAK2-STAT5 signaling. Moreover, berberine stimulated luciferase reporter expression from the mouse Ntcp promoter when one putative STAT5 response element (RE) (-1137 bp) was deleted and from the human NTCP promoter when three putative STAT5REs (-2898, -2164, and -691 bp) were deleted. Chromatin immunoprecipitation demonstrated that berberine decreased binding of phospho-STAT5 protein to the-2164 and -691 bp STAT5REs in the human NTCP promoter. In summary, berberine-disrupted STAT5 signaling promoted mouse and human Ntcp/NTCP expression, resulting in enhanced bile acid uptake. Therefore, berberine may be a therapeutic candidate compound for maintaining bile acid homeostasis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Identifying protein kinase target preferences using mass spectrometry

PubMed Central

Douglass, Jacqueline; Gunaratne, Ruwan; Bradford, Davis; Saeed, Fahad; Hoffert, Jason D.; Steinbach, Peter J.; Pisitkun, Trairak

2012-01-01

A general question in molecular physiology is how to identify candidate protein kinases corresponding to a known or hypothetical phosphorylation site in a protein of interest. It is generally recognized that the amino acid sequence surrounding the phosphorylation site provides information that is relevant to identification of the cognate protein kinase. Here, we present a mass spectrometry-based method for profiling the target specificity of a given protein kinase as well as a computational tool for the calculation and visualization of the target preferences. The mass spectrometry-based method identifies sites phosphorylated in response to in vitro incubation of protein mixtures with active recombinant protein kinases followed by standard phosphoproteomic methodologies. The computational tool, called “PhosphoLogo,” uses an information-theoretic algorithm to calculate position-specific amino acid preferences and anti-preferences from the mass-spectrometry data (http://helixweb.nih.gov/PhosphoLogo/). The method was tested using protein kinase A (catalytic subunit α), revealing the well-known preference for basic amino acids in positions −2 and −3 relative to the phosphorylated amino acid. It also provides evidence for a preference for amino acids with a branched aliphatic side chain in position +1, a finding compatible with known crystal structures of protein kinase A. The method was also employed to profile target preferences and anti-preferences for 15 additional protein kinases with potential roles in regulation of epithelial transport: CK2, p38, AKT1, SGK1, PKCδ, CaMK2δ, DAPK1, MAPKAPK2, PKD3, PIM1, OSR1, STK39/SPAK, GSK3β, Wnk1, and Wnk4. PMID:22723110

Protein Phosphatase 1-α Regulates AS160 Ser588 and Thr642 Dephosphorylation in Skeletal Muscle.

PubMed

Sharma, Pragya; Arias, Edward B; Cartee, Gregory D

2016-09-01

Akt substrate of 160 kDa (AS160) phosphorylation on Thr(642) and Ser(588) by Akt is essential for insulin's full effect on glucose transport. However, protein phosphorylation is determined by the balance of actions by kinases and phosphatases, and the specific phosphatase(s) controlling AS160 dephosphorylation is (are) unknown. Accordingly, we assessed roles of highly expressed skeletal muscle serine/threonine phosphatases (PP1, PP2A, PP2B, and PP2C) on AS160 dephosphorylation. Preliminary screening of candidate phosphatases used an AS160 dephosphorylation assay. Lysates from insulin-stimulated skeletal muscle were treated with pharmacological phosphatase inhibitors and assessed for AS160 Ser(588) and Thr(642) dephosphorylation. AS160 dephosphorylation on both phosphorylation sites was unaltered by PP2B or PP2C inhibitors. Okadaic acid (low dose inhibits PP2A; high dose inhibits PP1) delayed AS160 Ser(588) (both doses) and Thr(642) (high dose only) dephosphorylation concomitant with greater Akt phosphorylation (both doses). AS160 was coimmunoprecipitated with PP1-α but not with PP1-β, PP1-γ1, or PP2A. Recombinant inhibitor-2 protein (a selective PP1 inhibitor) delayed AS160 dephosphorylation on both phosphorylation sites without altering Akt phosphorylation. Furthermore, knockdown of PP1-α but not PP1-β or PP1-γ1 by small interfering RNA caused greater AS160 Ser(588) and Thr(642) phosphorylation concomitant with unaltered Akt phosphorylation. Together, these results identified PP1-α as a regulator of AS160 Thr(642) and Ser(588) dephosphorylation in skeletal muscle. © 2016 by the American Diabetes Association.

A paralogue of the phosphomutase-like gene family in Candida glabrata, CgPmu2, gained broad-range phosphatase activity due to a small number of clustered substitutions.

PubMed

Orlando, Kelly A; Iosue, Christine L; Leone, Sarah G; Davies, Danielle L; Wykoff, Dennis D

2015-10-15

Inorganic phosphate is required for a range of cellular processes, such as DNA/RNA synthesis and intracellular signalling. The phosphate starvation-inducible phosphatase activity of Candida glabrata is encoded by the gene CgPMU2 (C. glabrata phosphomutase-like protein). CgPMU2 is part of a three-gene family (∼75% identical) created through gene duplication in the C. glabrata clade; only CgPmu2 is a PHO-regulated broad range acid phosphatase. We identified amino acids that confer broad range phosphatase activity on CgPmu2 by creating fusions of sections of CgPMU2 with CgPMU1, a paralogue with little broad range phosphatase activity. We used site-directed mutagenesis on various fusions to sequentially convert CgPmu1 to CgPmu2. Based on molecular modelling of the Pmu proteins on to a histidine phosphatase crystal structure, clusters of amino acids were found in two distinct regions that were able to confer phosphatase activity. Substitutions in these two regions together conferred broad phosphatase activity on CgPmu1. Interestingly, one change is a histidine adjacent to the active site histidine of CgPmu2 and it exhibits a novel ability to partially replace the conserved active site histidine in CgPmu2. Additionally, a second amino acid change was able to confer nt phosphatase activity to CgPmu1, suggesting single amino acid changes neofunctionalize CgPmu2. © 2015 Authors; published by Portland Press Limited.

Stress Induces AMP-Dependent Loss of Potency Factors Id2 and Cdx2 in Early Embryos and Stem Cells

PubMed Central

Xie, Yufen; Awonuga, Awoniyi; Liu, Jian; Rings, Edmond; Puscheck, Elizabeth Ella

2013-01-01

The AMP-activated protein kinase (AMPK) mediates rapid, stress-induced loss of the inhibitor of differentiation (Id)2 in blastocysts and trophoblast stem cells (TSC), and a lasting differentiation in TSC. However, it is not known if AMPK regulates other potency factors or regulates them before the blastocyst stage. The caudal-related homeodomain protein (Cdx)2 is a regulatory gene for determining TSC, the earliest placental lineage in the preimplantation mouse embryo, but is expressed in the oocyte and in early cleavage stage embryos before TSC arise. We assayed the expression of putative potency-maintaining phosphorylated Cdx2 ser60 in the oocyte, two-cell stage embryo, blastocyst, and in TSC. We studied the loss of Cdx2 phospho ser60 expression induced by hyperosmolar stress and its underlying mechanisms. Hyperosmolar stress caused rapid loss of nuclear Cdx2 phospho ser60 and Id2 in the two-cell stage embryo by 0.5 h. Stress-induced Cdx2 phospho ser60 and Id2 loss is reversed by the AMPK inhibitor compound C and is induced by the AMPK agonist 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide in the absence of stress. In the two-cell stage embryo and TSC hyperosmolar, stress caused AMPK-mediated loss of Cdx2 phospho ser60 as detected by immunofluorescence and immunoblot. We propose that AMPK may be the master regulatory enzyme for mediating stress-induced loss of potency as AMPK is also required for stress-induced loss of Id2 in blastocysts and TSC. Since AMPK mediates potency loss in embryos and stem cells it will be important to measure, test mechanisms for, and manage the AMPK function to optimize the stem cell and embryo quality in vitro and in vivo. PMID:23316940

Regulation of the DNA damage response by DNA-PKcs inhibitory phosphorylation of ATM

PubMed Central

Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T.

2017-01-01

SUMMARY Ataxia-Telangiectasia Mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice. PMID:27939942

Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM.

PubMed

Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T

2017-01-05

Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Mogi, Makio, E-mail: makio@dpc.aichi-gakuin.ac.jp; Kondo, Ayami

Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor regulates bone mass by inhibiting osteoclastic bone resorption. mTOR, which is the mammalian target of rapamycin, is a kinase and central regulator of cell growth, proliferation, and survival. By using Rapamycin, we studied whether mTOR pathway is associated with OPG protein production in the mouse bone marrow-derived stromal cell line ST2. Rapamycin markedly increased the level of soluble OPG in ST2 cells. This antibiotic treatment resulted in the suppression of phosphorylation of mTOR. Rapamycin had no effects on the proliferation, differentiation, or apoptosis of the cells. Treatment with bone morphogenetic protein-4, which can induce OPG proteinmore » in ST2 cells, also resulted in a decrease in the density of the phospho-mTOR-band, suggesting that the suppression of the phospho-mTOR pathway is necessary for OPG production in ST2 cells. Thus, suitable suppression of mTOR phosphorylation is a necessary requirement for OPG production in bone marrow stromal cells.« less

Impairment of Hepcidin Upregulation by Lipopolysaccharide in the Interleukin-6 Knockout Mouse Brain.

PubMed

Zhang, Fa-Li; Hou, Hui-Min; Yin, Zhi-Nan; Chang, Lan; Li, Fe-Mi; Chen, Y-J; Ke, Ya; Qian, Zhong-Ming

2017-01-01

To find out whether the Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signaling pathway is involved in the expression of hepcidin in the mouse brain in vivo , we investigated the phosphorylation of STAT3, as well as the expression of hepcidin mRNA, ferroportin 1 (Fpn1) and ferritin light chain (Ft-L) proteins in the cortex and hippocampus of LPS-treated wild type (IL-6+/+) and IL-6 knockout (IL-6-/-) mice. We demonstrated that IL-6 knockout could significantly reduce the response of hepcidin mRNA, phospho-STAT3, Fpn1 and Ft-L protein expression to LPS treatment, in both the cortex and hippocampus of mice. Also, Stattic, an inhibitor of STAT3, significantly reduced the expression of phospho-STAT3 and hepcidin mRNA in the cortex and hippocampus of the LPS-treated wild type mice. These findings provide in vivo evidence for the involvement of the IL-6/STAT3 signaling pathway in the expression of hepcidin.

A novel function of twins, B subunit of protein phosphatase 2A, in regulating actin polymerization.

PubMed

Yeh, Po-An; Chang, Ching-Jin

2017-01-01

Actin is an important component of the cytoskeleton and its polymerization is delicately regulated by several kinases and phosphatases. Heterotrimeric protein phosphatase 2A (PP2A) is a potent phosphatase that is crucial for cell proliferation, apoptosis, tumorigenesis, signal transduction, cytoskeleton arrangement, and neurodegeneration. To facilitate these varied functions, different regulators determine the different targets of PP2A. Among these regulators of PP2A, the B subunits in particular may be involved in cytoskeleton arrangement. However, little is known about the role of PP2A in actin polymerization in vivo. Using sophisticated fly genetics, we demonstrated a novel function for the fly B subunit, twins, to promote actin polymerization in varied tissue types, suggesting a broad and conserved effect. Furthermore, our genetic data suggest that twins may act upstream of the actin-polymerized-proteins, Moesin and Myosin-light-chain, and downstream of Rho to promote actin polymerization. This work opens a new avenue for exploring the biological functions of a PP2A regulator, twins, in cytoskeleton regulation.

A novel function of twins, B subunit of protein phosphatase 2A, in regulating actin polymerization

PubMed Central

Chang, Ching-Jin

2017-01-01

Actin is an important component of the cytoskeleton and its polymerization is delicately regulated by several kinases and phosphatases. Heterotrimeric protein phosphatase 2A (PP2A) is a potent phosphatase that is crucial for cell proliferation, apoptosis, tumorigenesis, signal transduction, cytoskeleton arrangement, and neurodegeneration. To facilitate these varied functions, different regulators determine the different targets of PP2A. Among these regulators of PP2A, the B subunits in particular may be involved in cytoskeleton arrangement. However, little is known about the role of PP2A in actin polymerization in vivo. Using sophisticated fly genetics, we demonstrated a novel function for the fly B subunit, twins, to promote actin polymerization in varied tissue types, suggesting a broad and conserved effect. Furthermore, our genetic data suggest that twins may act upstream of the actin-polymerized-proteins, Moesin and Myosin-light-chain, and downstream of Rho to promote actin polymerization. This work opens a new avenue for exploring the biological functions of a PP2A regulator, twins, in cytoskeleton regulation. PMID:28977036

Identification, characterization and purification to near-homogeneity of a novel 67 kDa phosphotyrosyl protein phosphatase associated with pig lung annexin extract.

PubMed Central

Vicendo, P; Fauvel, J; Ragab-Thomas, J M; Chap, H

1991-01-01

During the purification of annexin VI from pig lung, we previously reported the isolation of another 67 kDa protein (protein 67E) differing from the former by immunological reactivity, amino acid composition, inability to interact with anionic phospholipids in the presence of Ca2+ and inability to inhibit phospholipase A2 [Fauvel, Vicendo, Roques, Ragab-Thomas, Granier, Vilgrain, Chambaz, Rochat, Chap & Douste-Blazy (1987) FEBS Lett. 221, 397-402]. Attempts to phosphorylate protein 67E by the protein tyrosine kinase of epidermal-growth-factor receptor revealed a dramatic inhibition of receptor autophosphorylation, which was also observed with insulin receptor. This inhibitory effect was found to be supported by a phosphatase active towards p-nitrophenyl phosphate, phosphotyrosine, [32P]phosphotyrosyl histones and [32P]phosphotyrosyl poly(Glu,Tyr), but inactive towards phosphoserine, phosphothreonine and [32P]phosphoseryl histones. Although not purified to complete homogeneity, the enzyme was purified 273-fold over EGTA extracts from pig lung and corresponded to a monomeric protein displaying an apparent molecular mass of 67 kDa. With [32P]phosphotyrosyl poly(Glu,Tyr) as substrate, the purified enzyme displayed Km and Vmax. values of 10 microM and 1.93 mumol/min per mg respectively, which compare reasonably well with other recently described phosphotyrosyl protein phosphatases. From these data and from its sensitivity to various inhibitors, it is concluded that protein fraction 67E contains a novel phosphotyrosyl protein phosphatase, the association of which with annexin extract might offer a clue to the understanding of its possible targeting to membrane substrates. Images Fig. 1. Fig. 3. Fig. 5. PMID:1654882

Exercise activates the phosphatidylinositol 3-kinase pathway.

PubMed

Chen, Michael J; Russo-Neustadt, Amelia A

2005-04-27

Physical exercise is known to enhance psychological well-being and coping capacity. Voluntary physical exercise in rats also robustly and rapidly up-regulates hippocampal brain-derived neurotrophic factor (BDNF) mRNA levels, which are potentiated following a regimen of chronic antidepressant treatment. Increased BDNF levels are associated with enhanced activity of cyclic AMP response element binding protein (CREB). So far, relatively little is known about the intracellular signaling mechanisms mediating this effect of exercise. We wished to explore the possibility that exercise and/or antidepressant treatment activate the hippocampal phosphatidylinositol-3 (PI-3) kinase pathway, which mediates cellular survival. In young male Sprague-Dawley rats, we examined the effects of 2 weeks of daily voluntary wheel-running activity and/or tranylcypromine (n = 7 per group) on the levels of the active forms of protein-dependent kinase-1 (PDK-1), PI-3 kinase, phospho-thr308-Akt, phospho-ser473-Akt, and phospho-glycogen synthase kinase-3beta (GSK3beta; inactive form), as well as BDNF, activated CREB, and the phospho-Trk receptor, in the rat hippocampus, and compared these with sedentary saline-treated controls. Immunoblotting analyses revealed that in exercising rats, there was a significant increase in PI-3 kinase expression (4.61 times that of controls, P = 0.0161) and phosphorylation of PDK-1 (2.73 times that of controls, P = 0.0454), thr308-Akt (2.857 times that of controls, P = 0.0082), CREB (60.27 times that of controls, P = 0.05), and Trk (35.3 times that of controls, P < 0.0001) in the hippocampi of exercising animals; BDNF was also increased (3.2 times that of controls), but this was not statistically significant. In rats receiving both exercise and tranylcypromine, BDNF (4.51 times that of controls, P = 0.0068) and PI-3 kinase (4.88 times that of controls, P = 0.0103), and the phospho- forms of Trk (13.67 times that of controls, P = 0.0278), thr308-Akt (3.644 times that of controls, P = 0.0004), GSK-3beta (2.93 times that of controls, P = 0.026), and CREB (88.97 times that of controls, P = 0.0053) were significantly increased. These results suggest that the exercise-induced expression of BDNF is associated with the increased expression of several key intermediates of the PI-3 kinase/Akt pathway, which is known for its role in enhancing neuronal survival.

Role of serine threonine protein phosphatase type 5 (PP5) in the regulation of stress induced signaling networks and cancer

PubMed Central

Golden, Teresa; Swingle, Mark; Honkanen, Richard E.

2008-01-01

Although the aberrant actions of protein kinases have long been known to contribute to tumor promotion and carcinogenesis, roles for proteins phosphatases in the development of human cancer have only emerged in the last decade. In this review, we discuss the data obtained from studies examining the biological and pathological roles of a serine/threonine protein phosphate, PP5, which suggest that PP5 is a potentially important regulator of both hormone- and stress-induced networks that enable a cell to respond appropriately to genomic stress. PMID:18253812

Protein Kinase A (PKA) Phosphorylation of Shp2 Protein Inhibits Its Phosphatase Activity and Modulates Ligand Specificity

PubMed Central

Burmeister, Brian T.; Wang, Li; Gold, Matthew G.; Skidgel, Randal A.; O'Bryan, John P.; Carnegie, Graeme K.

2015-01-01

Pathological cardiac hypertrophy (an increase in cardiac mass resulting from stress-induced cardiac myocyte growth) is a major factor underlying heart failure. Src homology 2 domain-containing phosphatase (Shp2) is critical for cardiac function because mutations resulting in loss of Shp2 catalytic activity are associated with congenital cardiac defects and hypertrophy. We identified a novel mechanism of Shp2 inhibition that may promote cardiac hypertrophy. We demonstrate that Shp2 is a component of the protein kinase A anchoring protein (AKAP)-Lbc complex. AKAP-Lbc facilitates PKA phosphorylation of Shp2, which inhibits Shp2 phosphatase activity. We identified two key amino acids in Shp2 that are phosphorylated by PKA. Thr-73 contributes a helix cap to helix αB within the N-terminal SH2 domain of Shp2, whereas Ser-189 occupies an equivalent position within the C-terminal SH2 domain. Utilizing double mutant PKA phosphodeficient (T73A/S189A) and phosphomimetic (T73D/S189D) constructs, in vitro binding assays, and phosphatase activity assays, we demonstrate that phosphorylation of these residues disrupts Shp2 interaction with tyrosine-phosphorylated ligands and inhibits its protein-tyrosine phosphatase activity. Overall, our data indicate that AKAP-Lbc integrates PKA and Shp2 signaling in the heart and that AKAP-Lbc-associated Shp2 activity is reduced in hypertrophic hearts in response to chronic β-adrenergic stimulation and PKA activation. Therefore, although induction of cardiac hypertrophy is a multifaceted process, inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote this compensatory response. PMID:25802336

Protein Kinase A (PKA) Phosphorylation of Shp2 Protein Inhibits Its Phosphatase Activity and Modulates Ligand Specificity.

PubMed

Burmeister, Brian T; Wang, Li; Gold, Matthew G; Skidgel, Randal A; O'Bryan, John P; Carnegie, Graeme K

2015-05-08

Pathological cardiac hypertrophy (an increase in cardiac mass resulting from stress-induced cardiac myocyte growth) is a major factor underlying heart failure. Src homology 2 domain-containing phosphatase (Shp2) is critical for cardiac function because mutations resulting in loss of Shp2 catalytic activity are associated with congenital cardiac defects and hypertrophy. We identified a novel mechanism of Shp2 inhibition that may promote cardiac hypertrophy. We demonstrate that Shp2 is a component of the protein kinase A anchoring protein (AKAP)-Lbc complex. AKAP-Lbc facilitates PKA phosphorylation of Shp2, which inhibits Shp2 phosphatase activity. We identified two key amino acids in Shp2 that are phosphorylated by PKA. Thr-73 contributes a helix cap to helix αB within the N-terminal SH2 domain of Shp2, whereas Ser-189 occupies an equivalent position within the C-terminal SH2 domain. Utilizing double mutant PKA phosphodeficient (T73A/S189A) and phosphomimetic (T73D/S189D) constructs, in vitro binding assays, and phosphatase activity assays, we demonstrate that phosphorylation of these residues disrupts Shp2 interaction with tyrosine-phosphorylated ligands and inhibits its protein-tyrosine phosphatase activity. Overall, our data indicate that AKAP-Lbc integrates PKA and Shp2 signaling in the heart and that AKAP-Lbc-associated Shp2 activity is reduced in hypertrophic hearts in response to chronic β-adrenergic stimulation and PKA activation. Therefore, although induction of cardiac hypertrophy is a multifaceted process, inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote this compensatory response. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Inhibitors of Serine/Threonine Protein Phosphatases: Biochemical and Structural Studies Provide Insight for Further Development.

PubMed

Swingle, Mark R; Honkanen, Richard Eric

2018-05-07

The reversible phosphorylation of proteins regulates many key functions in eukaryotic cells. Phosphorylation is catalyzed by protein kinases, with the majority of phosphorylation occurring on side chains of serine and threonine residues. The phosphomonoesters generated by protein kinases are hydrolyzed by protein phosphatases. In the absence of a phosphatase the half-time for the hydrolysis of alkyl phosphate dianions at 25º C is over 1 trillion years; knon ~2 x 10-20 sec-1. Therefore, ser/thr phosphatases are critical for processes controlled by reversible phosphorylation. This review is based on a search of the literature in available databases. We compare the catalytic mechanism of PPP-family phosphatases (PPPases) and the interactions of inhibitors that target these enzymes. PPPases are metal-dependent hydrolases that enhance the rate of hydrolysis ([kcat/kM]/knon ) by a factor of ~1021, placing them among the most powerful known catalysts on earth. Biochemical and structural studies indicate the remarkable catalytic proficiencies of PPPases are achieved by 10 conserved amino acids, DXH(X)~26DXXDR(X)~20-26NH(X)~50H(X)~25-45R(X)~30-40H. Six act as metal-coordinating residues. Four position and orient the substrate phosphate. Together, two metal ions and the 10 catalytic residues position the phosphoryl group and an activated bridging water/hydroxide nucleophile for inline attack upon the substrate phosphorous atom. The PPPases are conserved among species, and many structurally diverse natural toxins co-evolved to target these enzymes. Although the catalytic site is conserved, opportunities for the development of selective inhibitors of this important group of metalloenzymes exist. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) regulates G-protein-coupled receptor kinase 5 (GRK5)-induced cardiac hypertrophy in vitro.

PubMed

Yeh, Szu-Tsen; Zambrano, Cristina M; Koch, Walter J; Purcell, Nicole H

2018-05-25

PH domain leucine-rich repeat protein phosphatase (PHLPP) is a serine/threonine phosphatase that has been shown to regulate cell growth and survival through dephosphorylation of several members of the AGC family of kinases. G-protein-coupled receptor kinase 5 (GRK5) is an AGC kinase that regulates phenylephrine (PE)-induced cardiac hypertrophy through its noncanonical function of directly targeting proteins to the nucleus to regulate transcription. Here we investigated the possibility that the PHLPP2 isoform can regulate GRK5-induced cardiomyocyte hypertrophy in neonatal rat ventricular myocytes (NRVMs). We show that removal of PHLPP2 by siRNA induces hypertrophic growth of NRVMs as measured by cell size changes at baseline, potentiated PE-induced cell size changes, and re-expression of fetal genes atrial natriuretic factor and brain natriuretic peptide. Endogenous GRK5 and PHLPP2 were found to interact in NRVMs, and PE-induced nuclear accumulation of GRK5 was enhanced upon down-regulation of PHLPP2. Conversely, overexpression of PHLPP2 blocked PE-induced hypertrophic growth, re-expression of fetal genes, and nuclear accumulation of GRK5, which depended on its phosphatase activity. Finally, using siRNA against GRK5, we found that GRK5 was necessary for the hypertrophic response induced by PHLPP2 knockdown. Our findings demonstrate for the first time a novel regulation of GRK5 by the phosphatase PHLPP2, which modulates hypertrophic growth. Understanding the signaling pathways affected by PHLPP2 has potential for new therapeutic targets in the treatment of cardiac hypertrophy and failure. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Human Protein Kinases and Obesity.

PubMed

Engin, Atilla

2017-01-01

The action of protein kinases and protein phosphatases is essential for multiple physiological responses. Each protein kinase displays its own unique substrate specificity, and a regulatory mechanism that may be modulated by association with other proteins. Protein kinases are classified by the target amino acid in their substrates. Some protein kinases can phosphorylate both serine/threonine, as well as tyrosine residues. This group of kinases has been known as dual specificity kinases. Unlike the dual specificity kinases, a heterogeneous group of protein phosphatases are known as dual-specificity phosphatases. These phosphatases remove phosphate groups from tyrosine and serine/threonine residues on their substrate. Dual-specificity phosphatases are important signal transduction enzymes that regulate various cellular processes in coordination with protein kinases. The protein kinase-phosphoproteins interactions play an important role in obesity . In obesity, the pro- and anti-inflammatory effects of adipokines and cytokines through intracellular signaling pathways mainly involve the nuclear factor kappa B (NF-kappaB) and the c-Jun N-terminal kinase (JNK) systems as well as the inhibitor of kappaB-kinase beta (IKK beta). Impairment of insulin signaling in obesity is largely mediated by the activation of the IKKbeta and the JNK. Furthermore, oxidative stress and endoplasmic reticulum (ER) stress activate the JNK pathway which suppresses insulin biosynthesis. Additionally, obesity-activated calcium/calmodulin dependent-protein kinase II/p38 suppresses insulin-induced protein kinase B phosphorylation by activating the ER stress effector, activating transcription factor-4. Obese adults with vascular endothelial dysfunction have greater endothelial cells activation of unfolded protein response stress sensors, RNA-dependent protein kinase-like ER eukaryotic initiation factor-2alpha kinase (PERK) and activating transcription factor-6. The transcriptional regulation of adipogenesis in obesity is influenced by AGC (protein kinase A (PKA), PKG, PKC) family signaling kinases. Obesity may induce systemic oxidative stress and increase reactive oxygen species in adipocytes. Increase in intracellular oxidative stress can promote PKC-beta activation. Activated PKC-beta induces growth factor adapter Shc phosphorylation. Shc-generated peroxides reduce mitochondrial oxygen consumption and enhances triglyceride accumulation. Obesity is fundamentally caused by cellular energy imbalance and dysregulation. Like adenosine monophosphate (AMP)-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), N-terminal Per-ARNT-Sim (PAS) kinase are nutrient responsive protein kinases and important for proper regulation of glucose metabolism in mammals at both the hormonal and cellular level. Defective responses of AMPK to leptin may contribute to resistance to leptin action on food intake and energy expenditure in obese states.

THE UNCOVERING OF A NOVEL REGULATORY MECHANISM FOR PLD2: FORMATION OF A TERNARY COMPLEX WITH PROTEIN TYROSINE PHOSPHATASE PTP1B AND GROWTH FACTOR RECEPTOR-BOUND PROTEIN GRB2

PubMed Central

Horn, Jeff; Lopez, Isabel; Miller, Mill; Gomez-Cambronero, Julian

2011-01-01

The regulation of PLD2 activation is poorly understood at present. Transient transfection of COS-7 with a mycPLD2 construct results in elevated levels of PLD2 enzymatic activity and tyrosyl phosphorylation. To investigate whether this phosphorylation affects PLD2 enzymatic activity, anti-myc immunoprecipitates were treated with recombinant protein tyrosine phosphatase PTP1B. Surprisingly, lipase activity and PY levels both increased over a range of PTP1B concentrations. These increases occurred in parallel to a measurable PTP1B-associated phosphatase activity. Inhibitor studies demonstrated that an EGF-receptor type kinase is involved in phosphorylation. In a COS-7 cell line created in the laboratory that stably expressed myc-PLD2, PTP1B induced a robust (>6-fold) augmentation of myc-PLD2 phosphotyrosine content. The addition of growth factor receptor-bound protein 2 (Grb2) to cell extracts also elevated PY levels of myc-PLD (>10-fold). Systematic co-immunoprecipitation-immunoblotting experiments pointed at a physical association between PLD2, Grb2 and PTP1B in both physiological conditions and in overexpressed cells. This is the first report of a demonstration of the mammalian isoform PLD2 existing in a ternary complex with a protein tyrosine phosphatase, PTP1b, and the docking protein Grb2 which greatly enhances tyrosyl phosphorylation of the lipase. PMID:15896299

ABA signaling in guard cells entails a dynamic protein-protein interaction relay from the PYL-RCAR family receptors to ion channels.

PubMed

Lee, Sung Chul; Lim, Chae Woo; Lan, Wenzhi; He, Kai; Luan, Sheng

2013-03-01

Plant hormone abscisic acid (ABA) serves as an integrator of environmental stresses such as drought to trigger stomatal closure by regulating specific ion channels in guard cells. We previously reported that SLAC1, an outward anion channel required for stomatal closure, was regulated via reversible protein phosphorylation events involving ABA signaling components, including protein phosphatase 2C members and a SnRK2-type kinase (OST1). In this study, we reconstituted the ABA signaling pathway as a protein-protein interaction relay from the PYL/RCAR-type receptors, to the PP2C-SnRK2 phosphatase-kinase pairs, to the ion channel SLAC1. The ABA receptors interacted with and inhibited PP2C phosphatase activity against the SnRK2-type kinase, releasing active SnRK2 kinase to phosphorylate, and activate the SLAC1 channel, leading to reduced guard cell turgor and stomatal closure. Both yeast two-hybrid and bimolecular fluorescence complementation assays were used to verify the interactions among the components in the pathway. These biochemical assays demonstrated activity modifications of phosphatases and kinases by their interaction partners. The SLAC1 channel activity was used as an endpoint readout for the strength of the signaling pathway, depending on the presence of different combinations of signaling components. Further study using transgenic plants overexpressing one of the ABA receptors demonstrated that changing the relative level of interacting partners would change ABA sensitivity.

Developmental expression and function analysis of protein tyrosine phosphatase receptor type D in oligodendrocyte myelination

PubMed Central

Zhu, Qiang; Tan, Zhou; Zhao, Shufang; Huang, Hao; Zhao, Xiaofeng; Hu, Xuemei; Zhang, Yiping; Shields, Christopher B; Uetani, Noriko; Qiu, Mengsheng

2015-01-01

Receptor protein tyrosine phosphatases (RPTPs) are extensively expressed in the central nervous system (CNS), and have distinct spatial and temporal patterns in different cell types during development. Previous studies have demonstrated possible roles for RPTPs in axon outgrowth, guidance, and synaptogenesis. In the present study, our results revealed that protein tyrosine phosphatase, receptor type D (PTPRD) was initially expressed in mature neurons in embryonic CNS, and later in oligodendroglial cells at postnatal stages when oligodendrocyte undergo active axonal myelination process. In PTPRD mutants, oligodendrocyte differentiation was normal and a transient myelination delay occurred at early postnatal stages, indicating the contribution of PTPRD to the initiation of axonal myelination. Our results also showed that the remyelination process was not affected in the absence of PTPRD function after a cuprizone-induced demyelination in adult animals. PMID:26341907

Resveratrol protects bupivacaine-induced neuro-apoptosis in dorsal root ganglion neurons via activation on tropomyosin receptor kinase A.

PubMed

Guo, Zhiliang; Liu, Yuanyuan; Cheng, Min

2018-07-01

General anesthesia in spinal cord may lead to unexpected but irreversible neurotoxicity. We investigated whether resveratrol (RSV) may protect bupivacaine (BUP)-induced neuro-apoptosis in spinal cord dorsal root ganglia (DRG). Mouse DRG cells were cultured in vitro, pre-treated with RSV and then 5 mM BUP. A concentration-dependent effect of RSV on reducing BUP-induced apoptosis of DRG neurons (DRGNs) was evaluated using a TUNEL assay. QRT-PCR and western blot assays were also conducted to evaluate gene and protein expressions of tropomyosin receptor kinase A/B/C (TrkA/B/C) and activated (phosphorylated) Trk receptors, phospho-TrkA/B/C. In addition, a functional TrkA blocking antibody MNAC13 was applied in DRG culture to further measure the functional role of Trk receptor in RSV-initiated apoptotic protection on BUP-damaged DRGNs. BUP promoted significant apoptosis in DRG. RSV exhibited protective effects against BUP-induced neuro-apoptosis in a concentration-dependent manner. qRT-PCR and western blot showed that RSV did not alter TrkA/B/C gene or protein expression, but significantly upregulated phospho-TrkA. Conversely, application of MNAC13 decreased phospho-TrkA and reversed RSV-initiated neuro-protection on BUP-induced DRGN apoptosis. Resveratrol may protect anesthesia-induced DRG neuro-apoptosis, and activation of TrkA signaling pathway may be the underlying mechanism in this process. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Impaired Ca2+ cycling of nonischemic myocytes contributes to sarcomere dysfunction early after myocardial infarction.

PubMed

Kronenbitter, Annette; Funk, Florian; Hackert, Katarzyna; Gorreßen, Simone; Glaser, Dennis; Boknik, Peter; Poschmann, Gereon; Stühler, Kai; Isić, Malgorzata; Krüger, Martina; Schmitt, Joachim P

2018-06-01

Changes in the nonischemic remote myocardium of the heart contribute to left ventricular dysfunction after ischemia and reperfusion (I/R). Understanding the underlying mechanisms early after I/R is crucial to improve the adaptation of the viable myocardium to increased mechanical demands. Here, we investigated the role of myocyte Ca 2+ handling in the remote myocardium 24 h after 60 min LAD occlusion. Cardiomyocytes isolated from the basal noninfarct-related parts of wild type mouse hearts demonstrated depressed beat-to-beat Ca 2+ handling. The amplitude of the Ca 2+ transients as well as the kinetics of Ca 2+ transport were reduced by up to 25%. These changes were associated with impaired sarcomere contraction. While expression levels of Ca 2+ regulatory proteins were unchanged in remote myocardium compared to the corresponding regions of sham-operated hearts, mobility shift analyses of phosphorylated protein showed 2.9 ± 0.4-fold more unphosphorylated phospholamban (PLN) monomers, the PLN species that inhibits the Ca 2+ ATPase SERCA2a (P ≤ 0.001). Phospho-specific antibodies revealed normal phosphorylation of PLN at T17 in remote myocardium, but markedly reduced phosphorylation at its PKA-dependent phosphorylation site, S16 (P ≤ 0.01). The underlying cause involved enhanced activity of protein phosphatases, particularly PP2A (P ≤ 0.01). In contrast, overall PKA activity was normal. The PLN interactome, as determined by co-immunoprecipitation and mass spectrometry, and the phosphorylation state of PKA targets other than PLN were also unchanged. Isoproterenol enhanced cellular Ca 2+ cycling much stronger in remote myocytes than in healthy controls and improved sarcomere function. We conclude that the reduced phosphorylation state of PLN at S16 impairs myocyte Ca 2+ cycling in the remote myocardium 24 h after I/R and contributes to contractile dysfunction. Copyright © 2018 Elsevier Ltd. All rights reserved.

Roux-en-Y Esophagojejunostomy Ameliorates Renal Function Through Reduction of Renal Inflammatory and Fibrotic Markers in Diabetic Nephropathy.

PubMed

Wang, Cuifang; He, Bing; Piao, Dongxu; Han, Ping

2016-07-01

Roux-en-Y bariatric surgery has been shown to have a remarkable and sustainable improvement in type 2 diabetes. Recent clinical studies have shown that bariatric surgery can improve or halt the development of diabetic microvascular complications such as nephropathy. However, the exact underlying mechanisms of surgical procedures are unknown. Here, we have investigated the effects of Roux-en-Y esophagojejunostomy (RYEJ) on renal function and inflammation and fibrosis biomarkers for renal injury in type 2 diabetic rats. Sprague-Dawley rats with high fat diet and streptozotocin (STZ)-induced diabetes were randomly assigned into four groups: diabetic nephropathy (DN), DN treated with food restriction (DN-FR), DN treated with RYEJ surgery (DN-RYEJ), and DN-RYEJ sham (n = 6/group). Age-matched normal rats were assigned as control group. RYEJ and sham surgeries were performed. Hyperinsulinemic-euglycemic clamps with tracer infusion were completed to assess insulin sensitivity. Twenty-four hour urine albumin excretion rate (UAER) and glomerular filtration rate (GFR) were measured. The renal pathological injury was assessed by hematoxylin and eosin (HE) staining. Kidney messenger RNA (mRNA) and/or protein content/distribution of phospho-c-Jun NH2-terminal kinase (JNK), monocyte chemoattractant protein (MCP)-1, transforming growth factor (TGF)-β1, and mitogen-activated protein kinase phosphatase 5 (MKP5) were evaluated by real-time PCR and/or Western blotting/immunohistochemistry. Roux-en-Y esophagojejunostomy improved insulin sensitivity. RYEJ ameliorated renal function by improving UAER and GFR and attenuated glomerular hypertrophy after surgery. RYEJ also significantly downregulated the levels of JNK-mediated inflammatory response and upregulated the level of the anti-inflammatory mediator MKP5. Roux-en-Y esophagojejunostomy alleviates insulin resistance. RYEJ surgery ameliorated renal function and attenuated glomerular hypertrophy in a DN rat model. The considerable nephroprotective function may be mainly attributed to the reduced inflammatory and fibrotic biomarkers after RYEJ. The improvements in renal function and inflammation are not wholly dependent on the magnitude of weight loss.

Bioengineered human IAS reconstructs with functional and molecular properties similar to intact IAS

PubMed Central

Singh, Jagmohan

2012-01-01

Because of its critical importance in rectoanal incontinence, we determined the feasibility to reconstruct internal anal sphincter (IAS) from human IAS smooth muscle cells (SMCs) with functional and molecular attributes similar to the intact sphincter. The reconstructs were developed using SMCs from the circular smooth muscle layer of the human IAS, grown in smooth muscle differentiation media under sterile conditions in Sylgard-coated tissue culture plates with central Sylgard posts. The basal tone in the reconstructs and its changes were recorded following 0 Ca2+, KCl, bethanechol, isoproterenol, protein kinase C (PKC) activator phorbol 12,13-dibutyrate, and Rho kinase (ROCK) and PKC inhibitors Y-27632 and Gö-6850, respectively. Western blot (WB), immunofluorescence (IF), and immunocytochemical (IC) analyses were also performed. The reconstructs developed spontaneous tone (0.68 ± 0.26 mN). Bethanechol (a muscarinic agonist) and K+ depolarization produced contraction, whereas isoproterenol (β-adrenoceptor agonist) and Y-27632 produced a concentration-dependent decrease in the tone. Maximal decrease in basal tone with Y-27632 and Gö-6850 (each 10−5 M) was 80.45 ± 3.29 and 17.76 ± 3.50%, respectively. WB data with the IAS constructs′ SMCs revealed higher levels of RhoA/ROCK, protein kinase C-potentiated inhibitor or inhibitory phosphoprotein for myosin phosphatase (CPI-17), phospho-CPI-17, MYPT1, and 20-kDa myosin light chain vs. rectal smooth muscle. WB, IF, and IC studies of original SMCs and redispersed from the reconstructs for the relative distribution of different signal transduction proteins confirmed the feasibility of reconstruction of IAS with functional properties similar to intact IAS and demonstrated the development of myogenic tone with critical dependence on RhoA/ROCK. We conclude that it is feasible to bioengineer IAS constructs using human IAS SMCs that behave like intact IAS. PMID:22790596

Bone morphogenetic protein 4 induces differentiation of colorectal cancer stem cells and increases their response to chemotherapy in mice.

PubMed

Lombardo, Ylenia; Scopelliti, Alessandro; Cammareri, Patrizia; Todaro, Matilde; Iovino, Flora; Ricci-Vitiani, Lucia; Gulotta, Gaspare; Dieli, Francesco; de Maria, Ruggero; Stassi, Giorgio

2011-01-01

The limited clinical response observed in many patients with colorectal cancer may be related to the presence of chemoresistant colorectal cancer stem cells (CRC-SCs). Bone morphogenetic protein 4 (BMP4) promotes the differentiation of normal colonic stem cells. We investigated whether BMP4 might be used to induce differentiation of CRC-SCs and for therapeutic purposes. CRC-SCs were isolated from 25 tumor samples based on expression of CD133 or using a selection culture medium. BMP4 expression and activity on CRC-SCs were evaluated in vitro; progeny of the stem cells were evaluated by immunofluorescence, immunoblot, and flow cytometry analyses. The potential therapeutic effect of BMP4 was assessed in immunocompromised mice after injection of CRC-SCs that responded to chemotherapy (n = 4) or that did not (n = 2). CRC-SCs did not express BMP4 whereas differentiated cells did. Recombinant BMP4 promoted differentiation and apoptosis of CRC-SCs in 12 of 15 independent experiments; this effect did not depend on Small Mothers against decapentaplegic (Smad)4 expression level or microsatellite stability. BMP4 activated the canonical and noncanonical BMP signaling pathways, including phosphoInositide 3-kinase (PI3K) and PKB (protein kinase B)/AKT. Mutations in PI3K or loss of Phosphatase and Tensin homolog (PTEN) in Smad4-defective tumors made CRC-SCs unresponsive to BMP4. Administration of BMP4 to immunocompromised mice with tumors that arose from CRC-SCs increased the antitumor effects of 5-fluorouracil and oxaliplatin. BMP4 promotes terminal differentiation, apoptosis, and chemosensitization of CRC-SCs in tumors that do not have simultaneous mutations in Smad4 and constitutive activation of PI3K. BMP4 might be developed as a therapeutic agent against cancer stem cells in advanced colorectal tumors. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

Multiplexed Imaging of Protein Phosphorylation on Membranes Based on Ti(IV) Functionalized Nanopolymers.

PubMed

Iliuk, Anton; Li, Li; Melesse, Michael; Hall, Mark C; Tao, W Andy

2016-05-17

Accurate protein phosphorylation analysis reveals dynamic cellular signaling events not evident from protein expression levels. The most dominant biochemical assay, western blotting, suffers from the inadequate availability and poor quality of phospho-specific antibodies for phosphorylated proteins. Furthermore, multiplexed assays based on antibodies are limited by steric interference between the antibodies. Here we introduce a multifunctionalized nanopolymer for the universal detection of phosphoproteins that, in combination with regular antibodies, allows multiplexed imaging and accurate determination of protein phosphorylation on membranes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phosphatase activity of the voltage-sensing phosphatase, VSP, shows graded dependence on the extent of activation of the voltage sensor

PubMed Central

Sakata, Souhei; Okamura, Yasushi

2014-01-01

The voltage-sensing phosphatase (VSP) consists of a voltage sensor and a cytoplasmic phosphatase region, and the movement of the voltage sensor is coupled to the phosphatase activity. However, its coupling mechanisms still remain unclear. One possible scenario is that the phosphatase is activated only when the voltage sensor is in a fully activated state. Alternatively, the enzymatic activity of single VSP proteins could be graded in distinct activated states of the voltage sensor, and partial activation of the voltage sensor could lead to partial activation of the phosphatase. To distinguish between these two possibilities, we studied a voltage sensor mutant of zebrafish VSP, where the voltage sensor moves in two steps as evidenced by analyses of charge movements of the voltage sensor and voltage clamp fluorometry. Measurements of the phosphatase activity toward phosphatidylinositol 4,5-bisphosphate revealed that both steps of voltage sensor activation are coupled to the tuning of phosphatase activities, consistent with the idea that the phosphatase activity is graded by the magnitude of the movement of the voltage sensor. PMID:24277865

Phosphatase activity of the voltage-sensing phosphatase, VSP, shows graded dependence on the extent of activation of the voltage sensor.

PubMed

Sakata, Souhei; Okamura, Yasushi

2014-03-01

The voltage-sensing phosphatase (VSP) consists of a voltage sensor and a cytoplasmic phosphatase region, and the movement of the voltage sensor is coupled to the phosphatase activity. However, its coupling mechanisms still remain unclear. One possible scenario is that the phosphatase is activated only when the voltage sensor is in a fully activated state. Alternatively, the enzymatic activity of single VSP proteins could be graded in distinct activated states of the voltage sensor, and partial activation of the voltage sensor could lead to partial activation of the phosphatase. To distinguish between these two possibilities, we studied a voltage sensor mutant of zebrafish VSP, where the voltage sensor moves in two steps as evidenced by analyses of charge movements of the voltage sensor and voltage clamp fluorometry. Measurements of the phosphatase activity toward phosphatidylinositol 4,5-bisphosphate revealed that both steps of voltage sensor activation are coupled to the tuning of phosphatase activities, consistent with the idea that the phosphatase activity is graded by the magnitude of the movement of the voltage sensor.

Altered binding of /sup 125/I-labeled calmodulin to a 46. 5-kilodalton protein in skin fibroblasts cultured from patients with cystic fibrosis

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

Tallant, E.A.; Wallace, R.W.

1987-02-01

The levels of calmodulin and calmodulin-binding proteins have been determined in cultured skin fibroblasts from patients with cystic fibrosis (CF) and age- and sex-matched controls. Calmodulin ranged from 0.20 to 0.76 microgram/mg protein; there was no difference between calmodulin concentration in fibroblasts from CF patients and controls. Calmodulin-binding proteins of 230, 212, 204, 164, 139, 70, 59, 46.5, and 41 kD were identified. A protein with a mobility identical to the 59-kD calmodulin-binding protein was labeled by antiserum against calmodulin-dependent phosphatase. Although Ca/sup 2 +//calmodulin-dependent phosphatase activity was detected, there was no different in activity between control and CF fibroblastsmore » or in the level of phosphatase protein as determined by radioimmunoassay. Lower amounts of /sup 125/I-calmodulin were bound to the 46.5-kD calmodulin-binding protein in CF fibroblasts as compared with controls. The 46.5-kD calmodulin-binding protein may be reduced in CF fibroblasts or its structure may be altered resulting in a reduced binding capacity and/or affinity for calmodulin and perhaps reflecting, either directly or indirectly, the genetic defect responsible for cystic fibrosis.« less

A Loss-of-Function Screen for Phosphatases that Regulate Neurite Outgrowth Identifies PTPN12 as a Negative Regulator of TrkB Tyrosine Phosphorylation

PubMed Central

Ambjørn, Malene; Dubreuil, Véronique; Miozzo, Federico; Nigon, Fabienne; Møller, Bente; Issazadeh-Navikas, Shohreh; Berg, Jacob; Lees, Michael; Sap, Jan

2013-01-01

Alterations in function of the neurotrophin BDNF are associated with neurodegeneration, cognitive decline, and psychiatric disorders. BDNF promotes axonal outgrowth and branching, regulates dendritic tree morphology and is important for axonal regeneration after injury, responses that largely result from activation of its tyrosine kinase receptor TrkB. Although intracellular neurotrophin (NT) signaling presumably reflects the combined action of kinases and phosphatases, little is known about the contributions of the latter to TrkB regulation. The issue is complicated by the fact that phosphatases belong to multiple independently evolved families, which are rarely studied together. We undertook a loss-of-function RNA-interference-based screen of virtually all known (254) human phosphatases to understand their function in BDNF/TrkB-mediated neurite outgrowth in differentiated SH-SY5Y cells. This approach identified phosphatases from diverse families, which either positively or negatively modulate BDNF-TrkB-mediated neurite outgrowth, and most of which have little or no previously established function related to NT signaling. “Classical” protein tyrosine phosphatases (PTPs) accounted for 13% of the candidate regulatory phosphatases. The top classical PTP identified as a negative regulator of BDNF-TrkB-mediated neurite outgrowth was PTPN12 (also called PTP-PEST). Validation and follow-up studies showed that endogenous PTPN12 antagonizes tyrosine phosphorylation of TrkB itself, and the downstream activation of ERK1/2. We also found PTPN12 to negatively regulate phosphorylation of p130cas and FAK, proteins with previously described functions related to cell motility and growth cone behavior. Our data provide the first comprehensive survey of phosphatase function in NT signaling and neurite outgrowth. They reveal the complexity of phosphatase control, with several evolutionarily unrelated phosphatase families cooperating to affect this biological response, and hence the relevance of considering all phosphatase families when mining for potentially druggable targets. PMID:23785422

Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

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

Chen, Yi; Pirisi, Lucia; Creek, Kim E., E-mail: creekk@sccp.sc.edu

2013-09-15

We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cellmore » proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer. - Highlights: • Ski oncoprotein levels increase during progression of HPV16-transformed cells. • Ski and phospho-Ski protein levels are cell cycle dependent. • Ski knock-down in HPV16-transformed keratinocytes inhibited cell proliferation. • Cervical cancer samples overexpress Ski.« less

Quantitative and Dynamic Imaging of ATM Kinase Activity by Bioluminescence Imaging.

PubMed

Nyati, Shyam; Young, Grant; Ross, Brian Dale; Rehemtulla, Alnawaz

2017-01-01

Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA damage response, including DNA double strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter-expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

Quantitative and Dynamic Imaging of ATM Kinase Activity.

PubMed

Nyati, Shyam; Young, Grant; Ross, Brian Dale; Rehemtulla, Alnawaz

2017-01-01

Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including DNA double-strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

Production and characterization of a single-chain variable fragment linked alkaline phosphatase fusion protein for detection of O,O-diethyl organophosphorus pesticides in a one-step enzyme-linked immunosorbent assay

USDA-ARS?s Scientific Manuscript database

A single-chain variable fragment (scFv) and alkaline phosphatase (AP) fusion protein for detection of O, O-diethyl organophosphorus pesticides (OPs) was produced and characterized. The scFv gene was prepared by cloning VL and VH genes from a hybridoma cell secreting monoclonal antibody with broad-s...

Mutational analysis of the SRC homology 2 domain protein-tyrosine phosphatase Corkscrew.

PubMed

Allard, J D; Herbst, R; Carroll, P M; Simon, M A

1998-05-22

The SRC homology 2 (SH2) domain protein-tyrosine phosphatase, Corkscrew (CSW) is required for signaling by receptor tyrosine kinases, including the Sevenless receptor tyrosine kinase (SEV), which directs Drosophila R7 photoreceptor cell development. To investigate the role of the different domains of CSW, we constructed domain-specific csw mutations and assayed their effects on CSW function. Our results indicate that CSW SH2 domain function is essential, but either CSW SH2 domain can fulfill this requirement. We also found that CSW and activated SEV are associated in vivo in a manner that does not require either CSW SH2 domain function or tyrosine phosphorylation of SEV. In contrast, the interaction between CSW and Daughter of Sevenless, a CSW substrate, is dependent on SH2 domain function. These results suggest that the role of the CSW SH2 domains during SEV signaling is to bind Daughter of Sevenless rather than activated SEV. We also found that although CSW protein-tyrosine phosphatase activity is required for full CSW function, a catalytically inactive CSW is capable of providing partial function. In addition, we found that deletion of either the CSW protein- tyrosine phosphatase insert or the entire CSW carboxyl terminus, which includes a conserved DRK/GRB2 SH2 domain binding sequence, does not abolish CSW function.

Identification of a human src homology 2-containing protein-tyrosine-phosphatase: a putative homolog of Drosophila corkscrew.

PubMed Central

Freeman, R M; Plutzky, J; Neel, B G

1992-01-01

src homology 2 (SH2) domains direct binding to specific phosphotyrosyl proteins. Recently, SH2-containing protein-tyrosine-phosphatases (PTPs) were identified. Using degenerate oligonucleotides and the PCR, we have cloned a cDNA for an additional PTP, SH-PTP2, which contains two SH2 domains and is expressed ubiquitously. When expressed in Escherichia coli, SH-PTP2 displays tyrosine-specific phosphatase activity. Strong sequence similarity between SH-PTP2 and the Drosophila gene corkscrew (csw) and their similar patterns of expression suggest that SH-PTP2 is the human corkscrew homolog. Sequence comparisons between SH-PTP2, SH-PTP1, corkscrew, and other SH2-containing proteins suggest the existence of a subfamily of SH2 domains found specifically in PTPs, whereas comparison of the PTP domains of the SH2-containing PTPs with other tyrosine phosphatases suggests the existence of a subfamily of PTPs containing SH2 domains. Since corkscrew, a member of the terminal class signal transduction pathway, acts in concert with D-raf to positively transduce the signal generated by the receptor tyrosine kinase torso, these findings suggest several mechanisms by which SH-PTP2 may participate in mammalian signal transduction. Images PMID:1280823

In vivo phosphorylation of WRKY transcription factor by MAPK.

PubMed

Ishihama, Nobuaki; Adachi, Hiroaki; Yoshioka, Miki; Yoshioka, Hirofumi

2014-01-01

Plants activate signaling networks in response to diverse pathogen-derived signals, facilitating transcriptional reprogramming through mitogen-activated protein kinase (MAPK) cascades. Identification of phosphorylation targets of MAPK and in vivo detection of the phosphorylated substrates are important processes to elucidate the signaling pathway in plant immune responses. We have identified a WRKY transcription factor, which is phosphorylated by defense-related MAPKs, SIPK and WIPK. Recent evidence demonstrated that some group I WRKY transcription factors, which contain a conserved motif in the N-terminal region, are activated by MAPK-dependent phosphorylation. In this chapter, we describe protocols for preparation of anti-phosphopeptide antibodies, detection of activated MAPKs using anti-phospho-MAPK antibody, and activated WRKY using anti-phospho-WRKY antibody, respectively.

The kampo medicine Daikenchuto inhibits peritoneal fibrosis in mice.

PubMed

Kitamura, Mineaki; Nishino, Tomoya; Obata, Yoko; Oka, Satoru; Abe, Shinichi; Muta, Kumiko; Ozono, Yoshiyuki; Koji, Takehiko; Kohno, Shigeru

2015-01-01

Long-term peritoneal dialysis therapy causes inflammation and histological changes in the peritoneal membrane. Inflammation generally activates fibroblasts and results in fibroblast-myofibroblast differentiation. Heat-shock protein 47 (HSP 47), a collagen-specific molecular chaperone, is localized in myofibroblasts and is involved in the progression of peritoneal fibrosis. Daikenchuto (DKT), a Kampo medicine, is used to prevent postoperative colon adhesion. It inhibits inflammation and HSP 47 expression in the gastrointestinal tract. We examined the effect of DKT on chlorhexidine gluconate (CG)-induced peritoneal fibrosis in mice injected with 0.1% CG dissolved in 15% ethanol. DKT was dissolved in the drinking water. Histological changes were assessed using Masson trichrome staining. Cells expressing α-smooth muscle actin (α-SMA), HSP 47, phospho-Smad 2/3, F4/80, and monocyte chemotactic protein-1 were examined immunohistochemically. Compared with the control group, the peritoneal tissues of the CG group were markedly thickened, and the number of cells expressing α-SMA, HSP 47, phospho-Smad 2/3, F4/80, and monocyte chemotactic protein-1 was significantly increased. However, these changes were inhibited in the DKT-treated group. These results indicate that DKT can prevent peritoneal fibrosis by inhibiting inflammation and HSP 47 expression.

Molecular Cloning and Function of FAS/APO1 Associated Protein in Breast Cancer.

DTIC Science & Technology

1996-06-01

Ariyama T, Abe T, Druck T, Ohta M, Huebner K, Yanagisawa J, Reed JC, Sato T: PTPN13, a Fas-associated protein tyrosine phosphatase, is located on...20. Yang, Q., and Tonks, N. K. (1991). Isolation of a cDNA clone encoding a human protein-tyrosine phosphatase with homology 7. Huebner, K., Druck , T...Acad. Sci. U.S.A. 91, 7477 (1994). Res. 53, 1945 (1993).(Fig. 3D ). In contrast to Jurkat cells which 13. The original description of PTP-BAS (12

Overproduction, purification, and biochemical characterization of the dual specificity H1 protein phosphatase encoded by variola major virus.

PubMed

Tropea, Joseph E; Phan, Jason; Waugh, David S

2006-11-01

Smallpox, a highly contagious infectious disease caused by the variola major virus, has an overall mortality rate of about 30%. Because there currently is no specific treatment for smallpox, and the only prevention is vaccination, there is an urgent need for the development of effective antiviral drugs. The dual specificity protein phosphatase encoded by the smallpox virus (H1) is essential for the production of infectious viral particles, making it a promising molecular target for antiviral therapeutics. Here, we report the molecular cloning, overproduction, purification, and initial biochemical characterization of H1 phosphatase, thereby paving the way for the discovery of small molecule inhibitors.

Sodium arsenite induces chromosome endoreduplication and inhibits protein phosphatase activity in human fibroblasts

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

Rong-Nan Huang; I-Ching Ho; Ling-Hui Yih

Arsenic, strongly associated with increased risks of human cancers, is a potent clastogen in a variety of mammalian cell systems. The effect of sodium arsenite (a trivalent arsenic compound) on chromatid separation was studied in human skin fibroblasts (HFW). Human fibroblasts were arrested in S phase by the aid of serum starvation and aphidicolin blocking and then these cells were allowed to synchronously progress into G2 phase. Treatment of the G2-enriched HFW cells with sodium arsenite (0-200 {mu}M) resulted in arrest of cells in the G2 phase, interference with mitotic division, inhibition of spindle assembly, and induction of chromosome endoreduplicationmore » in their second mitosis. Sodium arsenite treatment also inhibited the activities of serine/threonine protein phosphatases and enhanced phosphorylation levels of a small heat shock protein (HSP27). These results suggest that sodium arsenite may mimic okadaic acid to induce chromosome endoreduplication through its inhibitory effect on protein phosphatase activity. 61 refs., 6 figs., 2 tabs.« less

Macrophage-stimulating protein attenuates gentamicin-induced inflammation and apoptosis in human renal proximal tubular epithelial cells

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

Lee, Ko Eun; Kim, Eun Young; Kim, Chang Seong

2013-05-10

Highlights: •MSP/RON system is activated in rat kidney damaged by gentamicin. •MSP inhibits GM-induced cellular apoptosis and inflammation in HK-2 cells. •MSP attenuates GM-induced activation of MAPKs and NF-κB pathways in HK-2 cells. -- Abstract: The present study aimed to investigate whether macrophage-stimulating protein (MSP) treatment attenuates renal apoptosis and inflammation in gentamicin (GM)-induced tubule injury and its underlying molecular mechanisms. To examine changes in MSP and its receptor, recepteur d’origine nantais (RON) in GM-induced nephropathy, rats were injected with GM for 7 days. Human renal proximal tubular epithelial (HK-2) cells were incubated with GM for 24 h in themore » presence of different concentrations of MSP and cell viability was measured by MTT assay. Apoptosis was determined by flow cytometry of cells stained with fluorescein isothiocyanate-conjugated annexin V protein and propidium iodide. Expression of Bcl-2, Bax, caspase-3, cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), nuclear factor-kappa B (NF-κB), IκB-α, and mitogen-activated protein kinases (MAPKs) was analyzed by semiquantitative immunoblotting. MSP and RON expression was significantly greater in GM-treated rats, than in untreated controls. GM-treatment reduced HK-2 cell viability, an effect that was counteracted by MSP. Flow cytometry and DAPI staining revealed GM-induced apoptosis was prevented by MSP. GM reduced expression of anti-apoptotic protein Bcl-2 and induced expression of Bax and cleaved caspase 3; these effects and GM-induced expression of COX-2 and iNOS were also attenuated by MSP. GM caused MSP-reversible induction of phospho-ERK, phospho-JNK, and phospho-p38. GM induced NF-κB activation and degradation of IκB-α; the increase in nuclear NF-κB was blocked by inhibitors of ERK, JNK, p-38, or MSP pretreatment. These findings suggest that MSP attenuates GM-induced inflammation and apoptosis by inhibition of the MAPKs/NF-κB signaling pathways.« less

Insight into mechanism of oxidative DNA damage in angiomyolipomas from TSC patients

PubMed Central

Habib, Samy L

2009-01-01

Background The tuberous sclerosis complex (TSC) is caused by defects in one of two tumor suppressor genes, TSC-1 or TSC-2. TSC-2 gene encodes tuberin, a protein involved in the pathogenesis of kidney tumors, both angiomyolipomas and renal cell carcinomas. Loss of heterozygosity at the 8-oxoG-DNA glycosylase (OGG1) allele is found in human kidney clear cell carcinoma identifying loss of OGG1 function as a possible contributor to tumorigenesis in the kidney. Tuberin regulates OGG1 through the transcription factor NF-YA in cultured cells. The purpose of this study is to determine the effect of tuberin-deficiency on OGG1 protein and mRNA levels as well as on 8-oxodG levels in kidney tumors from patients with TSC. In addition we evaluated the phophorylation level of downstream targets of mTOR, phospho-S70K, in kidney tumor tissue from TSC patients. Results Kidney angiomyolipoma tissue from TSC patients expresses significant levels of phopho-tuberin and low levels of tuberin compared to control kidney tissue. The increase in tuberin phosphorylation and the decrease tuberin expression are associated with decrease in OGG1 protein and mRNA levels in tumor samples compared to normal kidney samples. The decrease OGG1 expression is also associated with significant decrease in the transcription factor, NF-YA, expression in tumor samples compared to normal tissues. In addition, the levels of 8-oxodG are 4-fold higher in tumors compared to control samples. The significant increase of phospho-tuberin expression is associated with increase phosphorylation of S6K in tumor samples compared to controls. Cyclin D1 expression is also 3-fold higher in increase in the tumor tissues compared to normal kidney tissues. Conclusion These data indicate that tuberin deficiency in angiomyolipoma enhances mTOR activation by phosphorylation of S6K and downregulation of protein and mRNA expression of OGG1 resulted in accumulation of oxidized DNA in patients with TSC. These data suggest that tuberin and OGG1 are important proteins in the pathogenesis of angiomyolipoma in TSC patients. PMID:19265534

M-phase kinases induce phospho-dependent ubiquitination of somatic Wee1 by SCFβ-TrCP

PubMed Central

Watanabe, Nobumoto; Arai, Harumi; Nishihara, Yoshifumi; Taniguchi, Makoto; Watanabe, Naoko; Hunter, Tony; Osada, Hiroyuki

2004-01-01

Wee1, the Cdc2 inhibitory kinase, needs to be down-regulated at the onset of mitosis to ensure rapid activation of Cdc2. Previously, we have shown that human somatic Wee1 (Wee1A) is down-regulated both by protein phosphorylation and degradation, but the underlying mechanisms had not been elucidated. In the present study, we have identified the β-transducin repeat-containing protein 1/2 (β-TrCP1/2) F-box protein-containing SKP1/Cul1/F-box protein (SCF) complex (SCFβ-TrCP1/2) as an E3 ubiquitin ligase for Wee1A ubiquitination. Although Wee1A lacks a consensus DS(p)GXXS(p) phospho-dependent binding motif for β-TrCP, recognition of Wee1A by β-TrCP depended on phosphorylation, and two serine residues in Wee1A, S53 and S123, were found to be the most important phosphorylation sites for β-TrCP recognition. We have found also that the major M-phase kinases polo-like kinase 1 (Plk1) and Cdc2 are responsible for the phosphorylation of S53 and S123, respectively, and that in each case phosphorylation generates an unconventional phospho-degron (signal for degradation) that can be recognized by β-TrCP. Phosphorylation of Wee1A by these kinases cooperatively stimulated the recognition and ubiquitination of Wee1A by SCFβ-TrCP1/2 in vitro. Mutation of these residues or depletion of β-TrCP by small-interfering RNA treatment increased the stability of Wee1A in HeLa cells. Moreover, our analysis indicates that β-TrCP-dependent degradation of Wee1A is important for the normal onset of M-phase in vivo. These results also establish the existence of a feedback loop between Cdc2 and Wee1A in somatic cells that depends on ubiquitination and protein degradation and ensures the rapid activation of Cdc2 when cells are ready to divide. PMID:15070733

Two Membrane-Associated Tyrosine Phosphatase Homologs Potentiate C. elegans AKT-1/PKB Signaling

PubMed Central

Hu, Patrick J; Xu, Jinling; Ruvkun, Gary

2006-01-01

Akt/protein kinase B (PKB) functions in conserved signaling cascades that regulate growth and metabolism. In humans, Akt/PKB is dysregulated in diabetes and cancer; in Caenorhabditis elegans, Akt/PKB functions in an insulin-like signaling pathway to regulate larval development. To identify molecules that modulate C. elegans Akt/PKB signaling, we performed a genetic screen for enhancers of the akt-1 mutant phenotype (eak). We report the analysis of three eak genes. eak-6 and eak-5/sdf-9 encode protein tyrosine phosphatase homologs; eak-4 encodes a novel protein with an N-myristoylation signal. All three genes are expressed primarily in the two endocrine XXX cells, and their predicted gene products localize to the plasma membrane. Genetic evidence indicates that these proteins function in parallel to AKT-1 to inhibit the FoxO transcription factor DAF-16. These results define two membrane-associated protein tyrosine phosphatase homologs that may potentiate C. elegans Akt/PKB signaling by cell autonomous and cell nonautonomous mechanisms. Similar molecules may modulate Akt/PKB signaling in human endocrine tissues. PMID:16839187

Role of G protein-regulated inducer of neurite outgrowth 3 (GRIN3) in β-arrestin 2-Akt signaling and dopaminergic behaviors.

PubMed

Mototani, Yasumasa; Okamura, Tadashi; Goto, Motohito; Shimizu, Yukiko; Yanobu-Takanashi, Rieko; Ito, Aiko; Kawamura, Naoya; Yagisawa, Yuka; Umeki, Daisuke; Nariyama, Megumi; Suita, Kenji; Ohnuki, Yoshiki; Shiozawa, Kouichi; Sahara, Yoshinori; Kozasa, Tohru; Saeki, Yasutake; Okumura, Satoshi

2018-06-01

The G protein-regulated inducer of neurite growth (GRIN) family has three isoforms (GRIN1-3), which bind to the Gαi/o subfamily of G protein that mediate signal processing via G protein-coupled receptors (GPCRs). Here, we show that GRIN3 is involved in regulation of dopamine-dependent behaviors and is essential for activation of the dopamine receptors (DAR)-β-arrestin signaling cascade. Analysis of functional regions of GRIN3 showed that a di-cysteine motif (Cys751/752) is required for plasma membrane localization. GRIN3 was co-immunoprecipitated with GPCR kinases 2/6 and β-arrestins 1/2. Among GRINs, only GRIN3, which is highly expressed in striatum, strongly interacted with β-arrestin 2. We also generated GRIN3-knockout mice (GRIN3KO). GRIN3KO exhibited reduced locomotor activity and increased anxiety-like behavior in the elevated maze test, as well as a reduced locomoter response to dopamine stimulation. We also examined the phosphorylation of Akt at threonine 308 (phospho308-Akt), which is dephosphorylated via a β-arrestin 2-mediated pathway. Dephosphorylation of phospho308-Akt via the D2R-β-arrestin 2 signaling pathway was completely abolished in striatum of GRIN3KO. Our results suggest that GRIN3 has a role in recruitment and assembly of proteins involved in β-arrestin-dependent, G protein-independent signaling.

Sprouty proteins are in vivo targets of Corkscrew/SHP-2 tyrosine phosphatases.

PubMed

Jarvis, Lesley A; Toering, Stephanie J; Simon, Michael A; Krasnow, Mark A; Smith-Bolton, Rachel K

2006-03-01

Drosophila Corkscrew protein and its vertebrate ortholog SHP-2 (now known as Ptpn11) positively modulate receptor tyrosine kinase (RTK) signaling during development, but how these tyrosine phosphatases promote tyrosine kinase signaling is not well understood. Sprouty proteins are tyrosine-phosphorylated RTK feedback inhibitors, but their regulation and mechanism of action are also poorly understood. Here, we show that Corkscrew/SHP-2 proteins control Sprouty phosphorylation and function. Genetic experiments demonstrate that Corkscrew/SHP-2 and Sprouty proteins have opposite effects on RTK-mediated developmental events in Drosophila and an RTK signaling process in cultured mammalian cells, and the genes display dose-sensitive genetic interactions. In cultured cells, inactivation of SHP-2 increases phosphorylation on the critical tyrosine of Sprouty 1. SHP-2 associates in a complex with Sprouty 1 in cultured cells and in vitro, and a purified SHP-2 protein dephosphorylates the critical tyrosine of Sprouty 1. Substrate-trapping forms of Corkscrew bind Sprouty in cultured Drosophila cells and the developing eye. These results identify Sprouty proteins as in vivo targets of Corkscrew/SHP-2 tyrosine phosphatases and show how Corkscrew/SHP-2 proteins can promote RTK signaling by inactivating a feedback inhibitor. We propose that this double-negative feedback circuit shapes the output profile of RTK signaling events.

The heterotrimeric G protein Gβ1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein-coupled receptor signaling in platelets.

PubMed

Pradhan, Subhashree; Khatlani, Tanvir; Nairn, Angus C; Vijayan, K Vinod

2017-08-11

Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein-coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gβγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gβ 1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gβ 1 revealed that Gβ 1 interacts with the PP1c α, β, and γ1 isoforms. Purified PP1c bound to recombinant Gβ 1 -GST protein, and PP1c co-immunoprecipitated with Gβ 1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gβ 1 complex, which correlated with an association of PP1c with phospholipase C β3 (PLCβ3), along with a concomitant dephosphorylation of the inhibitory Ser 1105 residue in PLCβ3. siRNA-mediated depletion of GNB1 (encoding Gβ 1 ) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα -/- murine platelets and in human platelets treated with a small-molecule inhibitor of Gβγ. Finally, disruption of PP1c-Gβ 1 complexes with myristoylated Gβ 1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gβ 1 protein enlists PP1c to modulate GPCR signaling in platelets.

Ferulic Acid Administered at Various Time Points Protects against Cerebral Infarction by Activating p38 MAPK/p90RSK/CREB/Bcl-2 Anti-Apoptotic Signaling in the Subacute Phase of Cerebral Ischemia-Reperfusion Injury in Rats.

PubMed

Cheng, Chin-Yi; Tang, Nou-Ying; Kao, Shung-Te; Hsieh, Ching-Liang

2016-01-01

This study aimed to evaluate the effects of ferulic acid (FA) administered at various time points before or after 30 min of middle cerebral artery occlusion (MCAo) followed by 7 d of reperfusion and to examine the involvement of mitogen-activated protein kinase (MAPK) signaling pathways in the cortical penumbra. FA was intravenously administered to rats at a dose of 100 mg/kg 24 h before ischemia (B-FA), 2 h before ischemia (P-FA), immediately after ischemic insult (I-FA), 2 h after reperfusion (R-FA), or 24 h after reperfusion (D-FA). Our study results indicated that P-FA, I-FA, and R-FA effectively reduced cerebral infarct areas and neurological deficits. P-FA, I-FA, and R-FA significantly downregulated glial fibrillary acidic protein (GFAP), mitochondrial Bax, cytochrome c, and cleaved caspase-3 expression, and effectively restored the phospho-p38 MAPK (p-p38 MAPK)/p38 MAPK ratio, phospho-90 kDa ribosomal S6 kinase (p-p90RSK) expression, phospho-Bad (p-Bad) expression, the phospho-cAMP response element-binding protein (p-CREB)/CREB ratio, the cytosolic and mitochondrial Bcl-2/Bax ratios, and the cytosolic Bcl-xL/Bax ratio in the cortical penumbra 7 d after reperfusion. SB203580, a specific inhibitor of p38 MAPK, administered 30 min prior to ischemia abrogated the downregulating effects of I-FA on cerebral infarction, and mitochondrial Bax and cleaved caspase-3 expression, and the upregulating effects of I-FA on the p-p38 MAPK/p38 MAPK ratio, p-p90RSK expression, p-Bad expression, and the p-CREB/CREB, and cytosolic and mitochondrial Bcl-2/Bax ratios. Our study results thus indicate that P-FA, I-FA, and R-FA effectively suppress reactive astrocytosis and exert neuroprotective effects against cerebral infarction by activating p38 MAPK signaling. The regulating effects of P-FA, I-FA, and R-FA on Bax-induced apoptosis result from activation of the p38 MAPK/p90RSK/CREB/Bcl-2 signaling pathway, and eventually contribute to inhibition of the cytochrome c-mediated caspase-3-dependent apoptotic pathway in the cortical penumbra 7 d after reperfusion.

Ferulic Acid Administered at Various Time Points Protects against Cerebral Infarction by Activating p38 MAPK/p90RSK/CREB/Bcl-2 Anti-Apoptotic Signaling in the Subacute Phase of Cerebral Ischemia-Reperfusion Injury in Rats

PubMed Central

Cheng, Chin-Yi; Tang, Nou-Ying; Kao, Shung-Te; Hsieh, Ching-Liang

2016-01-01

Objectives This study aimed to evaluate the effects of ferulic acid (FA) administered at various time points before or after 30 min of middle cerebral artery occlusion (MCAo) followed by 7 d of reperfusion and to examine the involvement of mitogen-activated protein kinase (MAPK) signaling pathways in the cortical penumbra. Methods FA was intravenously administered to rats at a dose of 100 mg/kg 24 h before ischemia (B-FA), 2 h before ischemia (P-FA), immediately after ischemic insult (I-FA), 2 h after reperfusion (R-FA), or 24 h after reperfusion (D-FA). Results Our study results indicated that P-FA, I-FA, and R-FA effectively reduced cerebral infarct areas and neurological deficits. P-FA, I-FA, and R-FA significantly downregulated glial fibrillary acidic protein (GFAP), mitochondrial Bax, cytochrome c, and cleaved caspase-3 expression, and effectively restored the phospho-p38 MAPK (p-p38 MAPK)/p38 MAPK ratio, phospho-90 kDa ribosomal S6 kinase (p-p90RSK) expression, phospho-Bad (p-Bad) expression, the phospho-cAMP response element-binding protein (p-CREB)/CREB ratio, the cytosolic and mitochondrial Bcl-2/Bax ratios, and the cytosolic Bcl-xL/Bax ratio in the cortical penumbra 7 d after reperfusion. SB203580, a specific inhibitor of p38 MAPK, administered 30 min prior to ischemia abrogated the downregulating effects of I-FA on cerebral infarction, and mitochondrial Bax and cleaved caspase-3 expression, and the upregulating effects of I-FA on the p-p38 MAPK/p38 MAPK ratio, p-p90RSK expression, p-Bad expression, and the p-CREB/CREB, and cytosolic and mitochondrial Bcl-2/Bax ratios. Conclusions Our study results thus indicate that P-FA, I-FA, and R-FA effectively suppress reactive astrocytosis and exert neuroprotective effects against cerebral infarction by activating p38 MAPK signaling. The regulating effects of P-FA, I-FA, and R-FA on Bax-induced apoptosis result from activation of the p38 MAPK/p90RSK/CREB/Bcl-2 signaling pathway, and eventually contribute to inhibition of the cytochrome c-mediated caspase-3-dependent apoptotic pathway in the cortical penumbra 7 d after reperfusion. PMID:27187745

Celastrol nanomicelles attenuate cytokine secretion in macrophages and inhibit macrophage-induced corneal neovascularization in rats.

PubMed

Li, Zhanrong; Li, Jingguo; Zhu, Lei; Zhang, Ying; Zhang, Junjie; Yao, Lin; Liang, Dan; Wang, Liya

The aim of the present study was to investigate the inhibitory effects of celastrol-loaded nanomicelles (CNMs) on activated macrophage-induced corneal neovascularization (CNV) in rats and cytokine secretion in macrophages. Using an angiogenesis assay in vitro, we detected the effects of CNMs on human umbilical vein endothelial cell (HUVEC) migration and invasion. In addition, the expression levels of cytokines secreted from hypoxia-induced macrophages were assessed through cytokine array analysis. The expression of hypoxia-inducible factors-1α (HIF-1α), nuclear factor-kappa B p65 (NF-κB p65), phospho-nuclear factor-kappa B p65 (phospho-NF-κB p65), p38 mitogen-activated protein kinase (p38 MAPK), phospho-p38 MAPK, extracellular signal-regulated kinase 1/2 (ERK1/2), and phospho-ERK1/2 was analyzed by western blotting. Activated macrophages were elicited through mineral oil lumbar injection, labeled with 1,19-dioctadecyl-3-3-39,39-tetramethylindocarbocyanine (DiI) and implanted into the corneal micro-pocket to induce CNV and to assess the antiangiogenic effect in rats. CNV was morphometrically analyzed using ImageJ software. Histopathological features were evaluated by immunofluorescence immunostaining for vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) on day 2 after surgery. In the present study, the results indicated that CNMs significantly inhibited the migration and invasion of HUVECs; remarkably attenuated the expression of VEGF, tumor necrosis factor-α, interleukin-1α, monocyte chemoattractant protein 1, cytokine-induced neutrophil chemoattractant 3, and MMP-9 protein; and downregulated ERK1/2, p38 MAPK, NF-κB activation, and HIF-1α expression in macrophages. The peritoneal cells elicited using mineral oil were highly purified macrophages, and the length and area of CNV were significantly decreased in the CNMs group compared with the control group. There was a significant reduction in the expression of VEGF and MMP-9 in activated macrophages and corneal tissue after pretreatment with CNMs in this model. In conclusion, CNMs potently suppressed macrophage-induced CNV via the inhibition of VEGF and MMP-9 expression. This effect might be mediated through attenuating macrophages via HIF-1α, MAPK, and NF-κB signaling pathways.

Involvement of the phosphoinositide 3-kinase/Akt pathway in apoptosis induced by capsaicin in the human pancreatic cancer cell line PANC-1.

PubMed

Zhang, Jian-Hong; Lai, Fu-Ji; Chen, Hui; Luo, Jiang; Zhang, Ri-Yuan; Bu, He-Qi; Wang, Zhao-Hong; Lin, Hong-Hai; Lin, Sheng-Zhang

2013-01-01

Capsaicin, one of the major pungent ingredients found in red peppers, has been recently demonstrated to induce apoptosis in various malignant cell lines through an unclear mechanism. In this study, the effect of capsaicin on proliferation and apoptosis in the human pancreatic cancer cell line PANC-1 and its possible mechanism(s) of action were investigated. The results of a Cell Counting Kit-8 (CCK-8) assay revealed that capsaicin significantly decreased the viability of PANC-1 cells in a dose-dependent manner. Capsaicin induced G0/G1 phase cell cycle arrest and apoptosis in PANC-1 cells as demonstrated by a flow cytometric assessment. Caspase-3 expression at both the protein and mRNA level was promoted following capsaicin treatment. Furthermore, we revealed that phospho-PI3 Kinase p85 (Tyr458) and phospho-Akt (Ser473) in PANC-1 cells were downregulated in response to capsaicin. Moreover, capsaicin gavage significantly inhibited the growth of pancreatic cancer PANC-1 cell xenografts in athymic nude mice. An increased number of TUNEL-positive cells and cleaved caspase-3 were observed in capsaicin-treated mice. In vivo, capsaicin downregulated the expression of phospho-PI3 Kinase p85 (Tyr458) and phospho-Akt (Ser473). In conclusion, we have demonstrated that capsaicin is an inhibitor of growth of PANC-1 cells, and downregulation of the phosphoinositide 3-kinase/Akt pathway may be involved in capsaicin-induced apoptosis in vitro and in vivo.

Phospho-Rb mediating cell cycle reentry induces early apoptosis following oxygen-glucose deprivation in rat cortical neurons.

PubMed

Yu, Ying; Ren, Qing-Guo; Zhang, Zhao-Hui; Zhou, Ke; Yu, Zhi-Yuan; Luo, Xiang; Wang, Wei

2012-03-01

The aim of this study was to investigate the relationship between cell cycle reentry and apoptosis in cultured cortical neurons following oxygen-glucose deprivation (OGD). We found that the percentage of neurons with BrdU uptake, TUNEL staining, and colocalized BrdU uptake and TUNEL staining was increased relative to control 6, 12 and 24 h after 1 h of OGD. The number of neurons with colocalized BrdU and TUNEL staining was decreased relative to the number of TUNEL-positive neurons at 24 h. The expression of phosphorylated retinoblastoma protein (phospho-Rb) was significantly increased 6, 12 and 24 h after OGD, parallel with the changes in BrdU uptake. Phospho-Rb and TUNEL staining were colocalized in neurons 6 and 12 h after OGD. This colocalization was strikingly decreased 24 h after OGD. Treatment with the cyclin-dependent kinase inhibitor roscovitine (100 μM) decreased the expression of phospho-Rb and reduced neuronal apoptosis in vitro. These results demonstrated that attempted cell cycle reentry with phosphorylation of Rb induce early apoptosis in neurons after OGD and there must be other mechanisms involved in the later stages of neuronal apoptosis besides cell cycle reentry. Phosphoralated Rb may be an important factor which closely associates aberrant cell cycle reentry with the early stages of neuronal apoptosis following ischemia/hypoxia in vitro, and pharmacological interventions for neuroprotection may be useful directed at this keypoint.

Synergistic effect of atorvastatin and cyanidin-3-glucoside against angiotensin II-mediated vascular smooth muscle cell proliferation and migration through MAPK and PI3K/Akt pathways.

PubMed

Pantan, Rungusa; Tocharus, Jiraporn; Phatsara, Manussabhorn; Suksamrarn, Apichart; Tocharus, Chainarong

2016-09-13

This study aimed to investigate the mechanism of cyanidin-3-glucoside (C3G) in synergy with atorvastatin, even when it is used in low concentrations. Human aortic smooth muscle cells (HASMCs) were used to verify the synergistic mechanism of atorvastatin and C3G against angiotensin II-induced proliferation and migration. BrdU incorporation assay was used to evaluate cell proliferation. Wound healing and Boyden chamber assays were used to investigate cell migration. The cell cycle was examined using flow cytometry. The results revealed that atorvastatin and C3G exhibit a synergistic effect in ameliorating HASMC proliferation and migration by enhancing cell cycle arrest. In addition, these effects also decreased mitogen-activated protein kinase (MAPK) activity by attenuating the expression of phospho-p38, phospho-extracellular signaling-regulated kinase 1/2, and phospho-c-Jun N-terminal kinase. Furthermore, the combination of atorvastatin and C3G modulated the PI3K/Akt pathway and upregulated p21 Cip1 , which was associated with decreases in cyclin D 1 and phospho-retinoblastoma expressions. The synergistic effect of atorvastatin and C3G induced anti-proliferation and anti-migration through MAPK and PI3K/Akt pathways mediated by AT 1 R. These results suggest that the synergistic effect of atorvastatin and C3G may be an alternative therapy for atherosclerosis patients.

Angiotensin II receptor blocker telmisartan enhances running endurance of skeletal muscle through activation of the PPAR-δ/AMPK pathway.

PubMed

Feng, Xiaoli; Luo, Zhidan; Ma, Liqun; Ma, Shuangtao; Yang, Dachun; Zhao, Zhigang; Yan, Zhencheng; He, Hongbo; Cao, Tingbing; Liu, Daoyan; Zhu, Zhiming

2011-07-01

Clinical trials have shown that angiotensin II receptor blockers reduce the new onset of diabetes in hypertensives; however, the underlying mechanisms remain unknown. We investigated the effects of telmisartan on peroxisome proliferator activated receptor γ (PPAR-δ) and the adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway in cultured myotubes, as well as on the running endurance of wild-type and PPAR-δ-deficient mice. Administration of telmisartan up-regulated levels of PPAR-δ and phospho-AMPKα in cultured myotubes. However, PPAR-δ gene deficiency completely abolished the telmisartan effect on phospho-AMPKαin vitro. Chronic administration of telmisartan remarkably prevented weight gain, enhanced running endurance and post-exercise oxygen consumption, and increased slow-twitch skeletal muscle fibres in wild-type mice, but these effects were absent in PPAR-δ-deficient mice. The mechanism is involved in PPAR-δ-mediated stimulation of the AMPK pathway. Compared to the control mice, phospho-AMPKα level in skeletal muscle was up-regulated in mice treated with telmisartan. In contrast, phospho-AMPKα expression in skeletal muscle was unchanged in PPAR-δ-deficient mice treated with telmisartan. These findings highlight the ability of telmisartan to improve skeletal muscle function, and they implicate PPAR-δ as a potential therapeutic target for the prevention of type 2 diabetes. © 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2013-11-01

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

Regulation of ciliary motility: conserved protein kinases and phosphatases are targeted and anchored in the ciliary axoneme

PubMed Central

Wirschell, Maureen; Yamamoto, Ryosuke; Alford, Lea; Gokhale, Avanti; Gaillard, Anne; Sale, Winfield S.

2011-01-01

Recent evidence has revealed that the dynein motors and highly conserved signaling proteins are localized within the ciliary 9 + 2 axoneme. One key mechanism for regulation of motility is phosphorylation. Here, we review diverse evidence, from multiple experimental organisms, that ciliary motility is regulated by phosphorylation / dephosphorylation of the dynein arms through kinases and phosphatases that are anchored immediately adjacent to their axonemal substrates. PMID:21513695

Molecular characterization of the Salmonella typhi StpA protein that is related to both Yersinia YopE cytotoxin and YopH tyrosine phosphatase.

PubMed

Arricau, N; Hermant, D; Waxin, H; Popoff, M Y

1997-01-01

Analysis of the nucleotide sequence of a 4-kb DNA fragment located between the sip and iag loci on Salmonella typhi chromosome revealed three open reading frames, termed sipF, ctpA and stpA. The 82-amino-acid (aa) sipF product showed extensive similarity to the lacP protein from S. typhimurium. The StpA protein (535 aa) exhibited significant similarity to both Yersinia enterocolitica YopE cytotoxin and YopH tyrosine phosphatase. The CtpA polypeptide (130 aa) might be the molecular chaperone of the StpA protein.

Regulation of tumor cell migration by protein tyrosine phosphatase (PTP)-proline-, glutamate-, serine-, and threonine-rich sequence (PEST)

PubMed Central

Zheng, Yanhua; Lu, Zhimin

2013-01-01

Protein tyrosine phosphatase (PTP)–proline-, glutamate-, serine-, and threonine-rich sequence (PEST) is ubiquitously expressed and is a critical regulator of cell adhesion and migration. PTP-PEST activity can be regulated transcriptionally via gene deletion or mutation in several types of human cancers or via post-translational modifications, including phosphorylation, oxidation, and caspase-dependent cleavage. PTP-PEST interacts with and dephosphorylates cytoskeletal and focal adhesion-associated proteins. Dephosphorylation of PTP-PEST substrates regulates their enzymatic activities and/or their interaction with other proteins and plays an essential role in the tumor cell migration process. PMID:23237212

Structural Basis for the Catalytic Activity of Human Serine/Threonine Protein Phosphatase-5

NASA Technical Reports Server (NTRS)

Swingle, M. R.; Honkanen, R.; Ciszak, E. M.

2004-01-01

Serinehhreonine protein phosphatase-5 (PP5) affects many signaling networks that regulate cell growth and cellular responses to stress. Here we report the crystal structure of the PP5 catalytic domain (PP5c) at a resolution of 1.6 A. From this structure we resolved the mechanism for PP5-mediated hydrolysis of phosphoprotein substrates, which requires the precise positioning of two metal ions within a con served Aspn-271-M(sub 1):M(sub 2)-W(sup 1)-His-427-His-304-Asp-274 catalytic motif. The structure of PPSc provides a structural basis for explaining the exceptional catalytic proficiency of protein phosphatases, which are among the most powerful known catalysts. Resolution of the entire C-terminus revealed a novel subdomain, and the structure of the PP5c should also aid development of type-specific inhibitors.

cDNA isolated from a human T-cell library encodes a member of the protein-tyrosine-phosphatase family

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

Cool, D.E.; Tonks, N.K.; Charbonneau, H.

1989-07-01

A human peripheral T-cell cDNA library was screened with two labeled synthetic oligonucleotides encoding regions of a human placenta protein-tyrosine-phosphatase. One positive clone was isolated and the nucleotide sequence was determined. It contained 1,305 base pairs of open reading frame followed by a TAA stop codon and 978 base pairs of 3{prime} untranslated end, although a poly(A){sup +} tail was not found. An initiator methionine residue was predicted at position 61, which would result in a protein of 415 amino acid residues. This was supported by the synthesis of a M{sub r} 48,000 protein in an in vitro reticulocyte lysatemore » translation system using RNA transcribed from the cloned cDNA and T7 RNA polymerase. The deduced amino acid sequence was compared to other known proteins revealing 65% identity to the low M{sub r} PTPase 1B isolated from placenta. In view of the high degree of similarity, the T-cell cDNA likely encodes a newly discovered protein-tyrosine-phosphatase, thus expanding this family of genes.« less

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

Shen, Wen-jun; Dong, Rui; Chen, Gong, E-mail: chengongzlp@hotmail.com

Highlights: • The RRV infected group showed cholestasis, retardation and extrahepatic biliary atresia. • miR-222 was highly expressed, and PPP2R2A was inhibited in the murine biliary atresia model. • miR-222 profoundly modulated the process of fibrosis in the murine biliary atresia model. • miR-222 might represent a potential target for improving biliary atresia prognosis. - Abstract: microRNA-222 (miR-222) has been shown to initiate the activation of hepatic stellate cells, which plays an important role in the pathogenesis of liver fibrosis. The aim of our study was to evaluate the role of miR-22 in a mouse model of biliary atresia (BA)more » induced by Rhesus Rotavirus (RRV) infection. New-born Balb/c mice were randomized into control and RRV infected groups. The extrahepatic bile ducts were evaluated. The experimental group was divided into BA group and negative group based on histology. The expression of miR-222, protein phosphatase 2 regulatory subunit B alpha (PPP2R2A), proliferating cell nuclear antigen (PCNA) and phospho-Akt were detected. We found that the experimental group showed signs of cholestasis, retardation and extrahepatic biliary atresia. No abnormalities were found in the control group. In the BA group, miR-222, PCNA and Akt were highly expressed, and PPP2R2A expression was significantly inhibited. Our findings suggest that miR-222 profoundly modulated the process of fibrosis in the murine BA model, which might represent a potential target for improving BA prognosis.« less

Role of Plastid Protein Phosphatase TAP38 in LHCII Dephosphorylation and Thylakoid Electron Flow

PubMed Central

Pribil, Mathias; Pesaresi, Paolo; Hertle, Alexander; Barbato, Roberto; Leister, Dario

2010-01-01

Short-term changes in illumination elicit alterations in thylakoid protein phosphorylation and reorganization of the photosynthetic machinery. Phosphorylation of LHCII, the light-harvesting complex of photosystem II, facilitates its relocation to photosystem I and permits excitation energy redistribution between the photosystems (state transitions). The protein kinase STN7 is required for LHCII phosphorylation and state transitions in the flowering plant Arabidopsis thaliana. LHCII phosphorylation is reversible, but extensive efforts to identify the protein phosphatase(s) that dephosphorylate LHCII have been unsuccessful. Here, we show that the thylakoid-associated phosphatase TAP38 is required for LHCII dephosphorylation and for the transition from state 2 to state 1 in A. thaliana. In tap38 mutants, thylakoid electron flow is enhanced, resulting in more rapid growth under constant low-light regimes. TAP38 gene overexpression markedly decreases LHCII phosphorylation and inhibits state 1→2 transition, thus mimicking the stn7 phenotype. Furthermore, the recombinant TAP38 protein is able, in an in vitro assay, to directly dephosphorylate LHCII. The dependence of LHCII dephosphorylation upon TAP38 dosage, together with the in vitro TAP38-mediated dephosphorylation of LHCII, suggests that TAP38 directly acts on LHCII. Although reversible phosphorylation of LHCII and state transitions are crucial for plant fitness under natural light conditions, LHCII hyperphosphorylation associated with an arrest of photosynthesis in state 2 due to inactivation of TAP38 improves photosynthetic performance and plant growth under state 2-favoring light conditions. PMID:20126264

Immunohistochemical expression of the mammalian target of rapamycin pathway in penile squamous cell carcinomas: a tissue microarray study of 112 cases.

PubMed

Chaux, Alcides; Munari, Enrico; Cubilla, Antonio L; Hicks, Jessica; Lecksell, Kristen; Burnett, Arthur L; Netto, George J

2014-05-01

The aim of this study was to evaluate the immunohistochemical expression of mammalian target of rapamycin (mTOR) pathway-related biomarkers in penile carcinomas, and to assess associations with histological type, histological grade, and human papillomavirus (HPV) infection. We built four tissue microarrays from 112 invasive penile squamous cell carcinomas, and evaluated the immunohistochemical expression of PTEN, phospho-AKT, phospho-mTOR, and phospho-S6. We found decreased or loss of PTEN expression in 87% of cases. Warty and/or basaloid carcinomas had a higher proportion of PTEN loss (P = 0.02), whereas keratinizing tumours showed higher levels of phospho-S6 (P = 0.009); phospho-AKT and phospho-mTOR levels were not significantly different between warty/basaloid and keratinizing carcinomas (P = 0.75 and P = 0.77, respectively). PTEN was not associated with histological grade (P = 0.18). Expression levels of phospho-S6 were significantly higher in low-grade tumours (P = 0.001), whereas expression levels of phospho-AKT and phospho-mTOR were slightly higher in high-grade tumours (P = 0.01 and P = 0.35, respectively). We did not find any association between HPV infection and mTOR markers (P ≥ 0.2 in all cases). Our results provide evidence of dysregulation of the mTOR pathway in penile carcinomas independently of HPV infection. Future clinical studies should further evaluate the prognostic and predictive usefulness of these markers in patients with penile cancer. © 2013 John Wiley & Sons Ltd.

Effects of intravitreal insulin and insulin signaling cascade inhibitors on emmetropization in the chick

PubMed Central

Penha, Alexandra Marcha; Burkhardt, Eva; Schaeffel, Frank

2012-01-01

Purpose Intravitreal insulin has been shown to be a powerful stimulator of myopia in chickens, in particular if the retinal image is degraded or defocused. In most tissues, the insulin receptor activates two main signaling pathways: a) the mitogen-activated protein kinase (MAPK) cascade (e.g., mitogen-activated protein kinasem kinase [MEK] and extracellular regulated kinase [ERK]) and b) the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. In the current study, insulin was injected, and these pathways were separately inhibited to determine which is activated when the retinal image is defocused by spectacle lenses. Methods Chicks were treated with either +7 D, −7 D, or no lenses. They were intravitreally injected with insulin, the MEK inhibitor U0126, the PI3K inhibitor Ly294002, or a combination of insulin and one of the inhibitors. Refractions and ocular dimension were measured at the beginning and after four days of treatment. The retinal proteins of the chicks were measured with western blots after 2 h and four days of treatment. Incubation occurred with anti-Akt1, anti-Erk1/2, anti-phospho-AktThr308, and anti-phospho-Erk1/2(Thr202/Tyr204) antibodies, and the ratio between the relative intensity of the phospho-form and the total-form was calculated. Results Chicks wearing positive lenses and injected with saline and with PI3K inhibitor compensated for the imposed defocus and became hyperopic. Insulin injections and insulin plus PI3K inhibitor injections prevented lens-induced hyperopia, whereas the MEK inhibitor alone and insulin plus MEK inhibitor had no effect. Obviously, the MEK inhibitor suppressed the effect of insulin on eye growth in the plus lens–treated animals. Chicks treated with negative lenses and injected with insulin, or with insulin plus MEK inhibitor, overcompensated for the imposed defocus. This effect of insulin was not detected in eyes injected with PI3K inhibitor plus insulin, suggesting that the PI3K inhibitor suppressed the effects of insulin in minus lens–treated animals. Insulin increased the ratio of phospho-Akt/total-Akt in animals with normal visual exposure but even more so in chicks wearing plus or minus lenses. The increase was blocked by simultaneous PI3K inhibitor injections in control eyes but not in lens-treated eyes. Insulin also increased the ratio of phospho-ERK/total-ERK in animals with normal visual exposure and in animals wearing positive lenses, compared to U0126- and Ly294002-injected eyes. In contrast, no significant activation of the MEK/ERK pathway was observed in the negative lens–treated animals. Conclusions Intravitreal insulin promoted axial eye growth and stimulated both signaling pathways. The PI3K/Akt pathway was activated in control and plus and minus lens–treated eyes, but the MEK/ERK pathway was activated only with positive lenses or no lenses. With negative lenses, insulin did not stimulate the MEK/ERK signaling cascade. Independent of the pathway stimulated after insulin binding, the effect on insulin was always the same: an increase in eye growth. PMID:23112573

Effects of intravitreal insulin and insulin signaling cascade inhibitors on emmetropization in the chick.

PubMed

Penha, Alexandra Marcha; Burkhardt, Eva; Schaeffel, Frank; Feldkaemper, Marita P

2012-01-01

Intravitreal insulin has been shown to be a powerful stimulator of myopia in chickens, in particular if the retinal image is degraded or defocused. In most tissues, the insulin receptor activates two main signaling pathways: a) the mitogen-activated protein kinase (MAPK) cascade (e.g., mitogen-activated protein kinasem kinase [MEK] and extracellular regulated kinase [ERK]) and b) the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. In the current study, insulin was injected, and these pathways were separately inhibited to determine which is activated when the retinal image is defocused by spectacle lenses. Chicks were treated with either +7 D, -7 D, or no lenses. They were intravitreally injected with insulin, the MEK inhibitor U0126, the PI3K inhibitor Ly294002, or a combination of insulin and one of the inhibitors. Refractions and ocular dimension were measured at the beginning and after four days of treatment. The retinal proteins of the chicks were measured with western blots after 2 h and four days of treatment. Incubation occurred with anti-Akt1, anti-Erk1/2, anti-phospho-Akt(Thr308), and anti-phospho-Erk1/2((Thr202/Tyr204)) antibodies, and the ratio between the relative intensity of the phospho-form and the total-form was calculated. Chicks wearing positive lenses and injected with saline and with PI3K inhibitor compensated for the imposed defocus and became hyperopic. Insulin injections and insulin plus PI3K inhibitor injections prevented lens-induced hyperopia, whereas the MEK inhibitor alone and insulin plus MEK inhibitor had no effect. Obviously, the MEK inhibitor suppressed the effect of insulin on eye growth in the plus lens-treated animals. Chicks treated with negative lenses and injected with insulin, or with insulin plus MEK inhibitor, overcompensated for the imposed defocus. This effect of insulin was not detected in eyes injected with PI3K inhibitor plus insulin, suggesting that the PI3K inhibitor suppressed the effects of insulin in minus lens-treated animals. Insulin increased the ratio of phospho-Akt/total-Akt in animals with normal visual exposure but even more so in chicks wearing plus or minus lenses. The increase was blocked by simultaneous PI3K inhibitor injections in control eyes but not in lens-treated eyes. Insulin also increased the ratio of phospho-ERK/total-ERK in animals with normal visual exposure and in animals wearing positive lenses, compared to U0126- and Ly294002-injected eyes. In contrast, no significant activation of the MEK/ERK pathway was observed in the negative lens-treated animals. Intravitreal insulin promoted axial eye growth and stimulated both signaling pathways. The PI3K/Akt pathway was activated in control and plus and minus lens-treated eyes, but the MEK/ERK pathway was activated only with positive lenses or no lenses. With negative lenses, insulin did not stimulate the MEK/ERK signaling cascade. Independent of the pathway stimulated after insulin binding, the effect on insulin was always the same: an increase in eye growth.

Characterisation and expression of a PP1 serine/threonine protein phosphatase (PfPP1) from the malaria parasite, Plasmodium falciparum: demonstration of its essential role using RNA interference

PubMed Central

Kumar, Rajinder; Adams, Brian; Oldenburg, Anja; Musiyenko, Alla; Barik, Sailen

2002-01-01

Background Reversible protein phosphorylation is relatively unexplored in the intracellular protozoa of the Apicomplexa family that includes the genus Plasmodium, to which belong the causative agents of malaria. Members of the PP1 family represent the most highly conserved protein phosphatase sequences in phylogeny and play essential regulatory roles in various cellular pathways. Previous evidence suggested a PP1-like activity in Plasmodium falciparum, not yet identified at the molecular level. Results We have identified a PP1 catalytic subunit from P. falciparum and named it PfPP1. The predicted primary structure of the 304-amino acid long protein was highly similar to PP1 sequences of other species, and showed conservation of all the signature motifs. The purified recombinant protein exhibited potent phosphatase activity in vitro. Its sensitivity to specific phosphatase inhibitors was characteristic of the PP1 class. The authenticity of the PfPP1 cDNA was further confirmed by mutational analysis of strategic amino acid residues important in catalysis. The protein was expressed in all erythrocytic stages of the parasite. Abrogation of PP1 expression by synthetic short interfering RNA (siRNA) led to inhibition of parasite DNA synthesis. Conclusions The high sequence similarity of PfPP1 with other PP1 members suggests conservation of function. Phenotypic gene knockdown studies using siRNA confirmed its essential role in the parasite. Detailed studies of PfPP1 and its regulation may unravel the role of reversible protein phosphorylation in the signalling pathways of the parasite, including glucose metabolism and parasitic cell division. The use of siRNA could be an important tool in the functional analysis of Apicomplexan genes. PMID:12057017

The Ndc80 complex targets Bod1 to human mitotic kinetochores

PubMed Central

2017-01-01

Regulation of protein phosphatase activity by endogenous protein inhibitors is an important mechanism to control protein phosphorylation in cells. We recently identified Biorientation defective 1 (Bod1) as a small protein inhibitor of protein phosphatase 2A containing the B56 regulatory subunit (PP2A-B56). This phosphatase controls the amount of phosphorylation of several kinetochore proteins and thus the establishment of load-bearing chromosome-spindle attachments in time for accurate separation of sister chromatids in mitosis. Like PP2A-B56, Bod1 directly localizes to mitotic kinetochores and is required for correct segregation of mitotic chromosomes. In this report, we have probed the spatio-temporal regulation of Bod1 during mitotic progression. Kinetochore localization of Bod1 increases from nuclear envelope breakdown until metaphase. Phosphorylation of Bod1 at threonine 95 (T95), which increases Bod1's binding to and inhibition of PP2A-B56, peaks in prometaphase when PP2A-B56 localization to kinetochores is highest. We demonstrate here that kinetochore targeting of Bod1 depends on the outer kinetochore protein Ndc80 and not PP2A-B56. Crucially, Bod1 depletion functionally affects Ndc80 phosphorylation at the N-terminal serine 55 (S55), as well as a number of other phosphorylation sites within the outer kinetochore, including Knl1 at serine 24 and 60 (S24, S60), and threonine T943 and T1155 (T943, T1155). Therefore, Ndc80 recruits a phosphatase inhibitor to kinetochores which directly feeds forward to regulate Ndc80, and Knl1 phosphorylation, including sites that mediate the attachment of microtubules to kinetochores. PMID:29142109

Activation of the Jnk signaling pathway by a dual-specificity phosphatase, JSP-1.

PubMed

Shen, Y; Luche, R; Wei, B; Gordon, M L; Diltz, C D; Tonks, N K

2001-11-20

The mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli, such as growth factors, hormones, and cytokines, and to a wide variety of environmental stresses. The MAPKs, which are stimulated by phosphorylation of a TXY motif in their activation loop, are components of signal transduction cascades in which sequential activation of protein kinases culminates in their activation and their subsequent phosphorylation of various effector proteins that mediate the physiological response. MAPKs are also subject to dephosphorylation and inactivation, both by enzymes that recognize the residues of the TXY motif independently and by dual specificity phosphatases, which dephosphroylate both Tyr and Ser/Thr residues. We report the identification and characterization of a novel dual specificity phosphatase. Contrary to expectation, this broadly expressed enzyme did not inactivate MAPKs in transient cotransfection assays but instead displayed the capacity to function as a selective activator of the MAPK Jnk, hence the name, Jnk Stimulatory Phosphatase-1 (JSP-1). This study illustrates a new aspect of the regulation of MAPK-dependent signal transduction and raises the possibility that JSP-1 may offer a different perspective to the study of various inflammatory and proliferative disorders associated with dysfunctional Jnk signaling.

Phosphatase control of 4E-BP1 phosphorylation state is central for glycolytic regulation of retinal protein synthesis.

PubMed

Gardner, Thomas W; Abcouwer, Steven F; Losiewicz, Mandy K; Fort, Patrice E

2015-09-15

Control of protein synthesis in insulin-responsive tissues has been well characterized, but relatively little is known about how this process is regulated in nervous tissues. The retina exhibits a relatively high protein synthesis rate, coinciding with high basal Akt and metabolic activities, with the majority of retinal ATP being derived from aerobic glycolysis. We examined the dependency of retinal protein synthesis on the Akt-mTOR signaling and glycolysis using ex vivo rat retinas. Akt inhibitors significantly reduced retinal protein synthesis but did not affect glycolytic lactate production. Surprisingly, the glycolytic inhibitor 2-deoxyglucose (2-DG) markedly inhibited Akt1 and Akt3 activities, as well as protein synthesis. The effects of 2-DG, and 2-fluorodeoxyglucose (2-FDG) on retinal protein synthesis correlated with inhibition of lactate production and diminished ATP content, with all these effects reversed by provision of d-mannose. 2-DG treatment was not associated with increased AMPK, eEF2, or eIF2α phosphorylation; instead, it caused rapid dephosphorylation of 4E-BP1. 2-DG reduced total mTOR activity by 25%, but surprisingly, it did not reduce mTORC1 activity, as indicated by unaltered raptor-associated mTOR autophosphorylation and ribosomal protein S6 phosphorylation. Dephosphorylation of 4E-BP1 was largely prevented by inhibition of PP1/PP2A phosphatases with okadaic acid and calyculin A, and inhibition of PPM1 phosphatases with cadmium. Thus, inhibition of retinal glycolysis diminished Akt and protein synthesis coinciding with accelerated dephosphorylation of 4E-BP1 independently of mTORC1. These results demonstrate a novel mechanism regulating protein synthesis in the retina involving an mTORC1-independent and phosphatase-dependent regulation of 4E-BP1. Copyright © 2015 the American Physiological Society.

Immunocytochemical detection of the microsomal glucose-6-phosphatase in human brain astrocytes.

PubMed

Bell, J E; Hume, R; Busuttil, A; Burchell, A

1993-10-01

Using an antibody raised against the catalytic subunit of glucose-6-phosphatase, this enzyme was immunolocalized in many astrocytes in 20 normal human brains. Double immunofluorescence studies showed co-localization of glial fibrillary acidic protein (GFAP) with glucose-6-phosphatase in astrocytes. However, not all GFAP-positive cells were also glucose-6-phosphatase positive, indicating that some astrocytes do not contain demonstrable expression of this enzyme. Reactive astrocytes in a variety of abnormal brains were strongly glucose-6-phosphatase positive, but neoplastic astrocytes were often only weakly positive. Expression of the enzyme could not be demonstrated in radial glia, neurons or oligodendroglia. Astrocytes normally contain glycogen and the demonstration that some astrocytes also contain glucose-6-phosphatase indicates that they are competent for both glycogenolysis and gluconeogenesis, which may be critical for neuronal welfare.

Omega-3 polyunsaturated fatty acids augment the muscle protein anabolic response to hyperinsulinaemia-hyperaminoacidaemia in healthy young and middle-aged men and women.

PubMed

Smith, Gordon I; Atherton, Philip; Reeds, Dominic N; Mohammed, B Selma; Rankin, Debbie; Rennie, Michael J; Mittendorfer, Bettina

2011-09-01

Increased dietary LCn-3PUFA (long-chain n-3 polyunsaturated fatty acid) intake stimulates muscle protein anabolism in individuals who experience muscle loss due to aging or cancer cachexia. However, it is not known whether LCn-3PUFAs elicit similar anabolic effects in healthy individuals. To answer this question, we evaluated the effect of 8 weeks of LCn-3PUFA supplementation (4 g of Lovaza®/day) in nine 25-45-year-old healthy subjects on the rate of muscle protein synthesis (by using stable isotope-labelled tracer techniques) and the activation (phosphorylation) of elements of the mTOR (mammalian target of rapamycin)/p70S6K (p70 S6 kinase) signalling pathway during basal post-absorptive conditions and during a hyperinsulinaemic-hyperaminoacidaemic clamp. We also measured the concentrations of protein, RNA and DNA in muscle to obtain indices of the protein synthetic capacity, translational efficiency and cell size. Neither the basal muscle protein fractional synthesis rate nor basal signalling element phosphorylation changed in response to LCn-3PUFA supplementation, but the anabolic response to insulin and amino acid infusion was greater after LCn-3PUFA [i.e. the muscle protein fractional synthesis rate during insulin and amino acid infusion increased from 0.062±0.004 to 0.083±0.007%/h and the phospho-mTOR (Ser2448) and phospho-p70S6K (Thr389) levels increased by ∼50%; all P<0.05]. In addition, the muscle protein concentration and the protein/DNA ratio (i.e. muscle cell size) were both greater (P<0.05) after LCn-3PUFA supplementation. We conclude that LCn-3PUFAs have anabolic properties in healthy young and middle-aged adults.

Evaluating transition state structures of vanadium-phosphatase protein complexes using shape analysis.

PubMed

Sánchez-Lombardo, Irma; Alvarez, Santiago; McLauchlan, Craig C; Crans, Debbie C

2015-06-01

Shape analysis of coordination complexes is well-suited to evaluate the subtle distortions in the trigonal bipyramidal (TBPY-5) geometry of vanadium coordinated in the active site of phosphatases and characterized by X-ray crystallography. Recent studies using the tau (τ) analysis support the assertion that vanadium is best described as a trigonal bipyramid, because this geometry is the ideal transition state geometry of the phosphate ester substrate hydrolysis (C.C. McLauchlan, B.J. Peters, G.R. Willsky, D.C. Crans, Coord. Chem. Rev. http://dx.doi.org/10.1016/j.ccr.2014.12.012 ; D.C. Crans, M.L. Tarlton, C.C. McLauchlan, Eur. J. Inorg. Chem. 2014, 4450-4468). Here we use continuous shape measures (CShM) analysis to investigate the structural space of the five-coordinate vanadium-phosphatase complexes associated with mechanistic transformations between the tetrahedral geometry and the five-coordinate high energy TBPY-5 geometry was discussed focusing on the protein tyrosine phosphatase 1B (PTP1B) enzyme. No evidence for square pyramidal geometries was observed in any vanadium-protein complexes. The shape analysis positioned the metal ion and the ligands in the active site reflecting the mechanism of the cleavage of the organic phosphate in a phosphatase. We identified the umbrella distortions to be directly on the reaction path between tetrahedral phosphate and the TBPY-5-types of high-energy species. The umbrella distortions of the trigonal bipyramid are therefore identified as being the most relevant types of transition state structures for the phosphoryl group transfer reactions for phosphatases and this may be related to the possibility that vanadium is an inhibitor for enzymes that support both exploded and five-coordinate transition states. Copyright © 2015 Elsevier Inc. All rights reserved.

Study of Protein Phosphatase 2A (PP2A) Activity in LPS-Induced Tolerance Using Fluorescence-Based and Immunoprecipitation-Aided Methodology.

PubMed

Sun, Lei; Ii, Adlai L Pappy; Pham, Tiffany T; Shanley, Thomas P

2015-06-29

Protein phosphatase 2A (PP2A) is one of the most abundant intracellular serine/threonine (Ser/Thr) phosphatases accounting for 1% of the total cellular protein content. PP2A is comprised of a heterodimeric core enzyme and a substrate-specific regulatory subunit. Potentially, at least seventy different compositions of PP2A exist because of variable regulatory subunit binding that accounts for various activity modulating numerous cell functions. Due to the constitutive phosphatase activity present inside cells, a sensitive assay is required to detect the changes of PP2A activity under various experimental conditions. We optimized a fluorescence assay (DIFMU assay) by combining it with prior anti-PP2A immunoprecipitation to quantify PP2A-specific phosphatase activity. It is also known that prior exposure to lipopolysaccharides (LPS) induces "immune tolerance" of the cells to subsequent stimulation. Herein we report that PP2A activity is upregulated in tolerized peritoneal macrophages, corresponding to decreased TNF-α secretion upon second LPS stimulation. We further examined the role of PP2A in the tolerance effect by using PP2ACαl°xl°x;lyM-Cre conditional knockout macrophages. We found that PP2A phosphatase activity cannot be further increased by tolerance. TNF-α secretion from tolerized PP2ACαl°xl°x;lyM-Cre macrophages is higher than tolerized control macrophages. Furthermore, we showed that the increased TNF-α secretion may be due to an epigenetic transcriptionally active signature on the promoter of TNF-α gene rather than regulation of the NFκB/IκB signaling pathway. These results suggest a role for increased PP2A activity in the regulation of immune tolerance.

Alkaline phosphatase from the hyperthermophilic bacterium T. maritima requires cobalt for activity

PubMed Central

Wojciechowski, Cheryl L.; Cardia, James P.; Kantrowitz, Evan R.

2002-01-01

The hyperthermophilic bacterium Thermotoga maritima encodes a gene sharing sequence similarities with several known genes for alkaline phosphatase (AP). The putative gene was isolated and the corresponding protein expressed in Escherichia coli, with and without a predicted signal sequence. The recombinant protein showed phosphatase activity toward the substrate p-nitrophenyl-phosphate with a kcat of 16 s−1 and a Km of 175 μM at a pH optimum of 8.0 when assayed at 25°C. T. maritima phosphatase activity increased at high temperatures, reaching a maximum kcat of 100 s−1, with a Km of 93 μM at 65°C. Activity was stable at 65°C for >24 h and at 90°C for 5 h. Phosphatase activity was dependent on divalent metal ions, specifically Co(II) and Mg(II). Circular dichroism spectra showed that the enzyme gains secondary structure on addition of these metals. Zinc, the most common divalent metal ion required for activity in known APs, was shown to inhibit the T. maritima phosphatase enzyme at concentrations above 0.3 moles Zn: 1 mole monomer. All activity was abolished in the presence of 0.1 mM EDTA. The T. maritima AP primary sequence is 28% identical when compared with E. coli AP. Based on a structural model, the active sites are superimposable except for two residues near the E. coli AP Mg binding site, D153 and K328 (E. coli numbering) corresponding to histidine and tryptophan in T. maritima AP, respectively. Sucrose-density gradient sedimentation experiments showed that the protein exists in several quaternary forms predominated by an octamer. PMID:11910033

A Novel Arsenate Reductase from the Arsenic Hyperaccumulating Fern Pteris vittata1

PubMed Central

Ellis, Danielle R.; Gumaelius, Luke; Indriolo, Emily; Pickering, Ingrid J.; Banks, Jo Ann; Salt, David E.

2006-01-01

Pteris vittata sporophytes hyperaccumulate arsenic to 1% to 2% of their dry weight. Like the sporophyte, the gametophyte was found to reduce arsenate [As(V)] to arsenite [As(III)] and store arsenic as free As(III). Here, we report the isolation of an arsenate reductase gene (PvACR2) from gametophytes that can suppress the arsenate sensitivity and arsenic hyperaccumulation phenotypes of yeast (Saccharomyces cerevisiae) lacking the arsenate reductase gene ScACR2. Recombinant PvACR2 protein has in vitro arsenate reductase activity similar to ScACR2. While PvACR2 and ScACR2 have sequence similarities to the CDC25 protein tyrosine phosphatases, they lack phosphatase activity. In contrast, Arath;CDC25, an Arabidopsis (Arabidopsis thaliana) homolog of PvACR2 was found to have both arsenate reductase and phosphatase activities. To our knowledge, PvACR2 is the first reported plant arsenate reductase that lacks phosphatase activity. CDC25 protein tyrosine phosphatases and arsenate reductases have a conserved HCX5R motif that defines the active site. PvACR2 is unique in that the arginine of this motif, previously shown to be essential for phosphatase and reductase activity, is replaced with a serine. Steady-state levels of PvACR2 expression in gametophytes were found to be similar in the absence and presence of arsenate, while total arsenate reductase activity in P. vittata gametophytes was found to be constitutive and unaffected by arsenate, consistent with other known metal hyperaccumulation mechanisms in plants. The unusual active site of PvACR2 and the arsenate reductase activities of cell-free extracts correlate with the ability of P. vittata to hyperaccumulate arsenite, suggesting that PvACR2 may play an important role in this process. PMID:16766666

High-resolution Structures of Protein-Membrane Complexes by Neutron Reflection and MD Simulation: Membrane Association of the PTEN Tumor Suppressor

NASA Astrophysics Data System (ADS)

Lösche, Matthias

2012-02-01

The lipid matrix of biomembranes is an in-plane fluid, thermally and compositionally disordered leaflet of 5 nm thickness and notoriously difficult to characterize in structural terms. Yet, biomembranes are ubiquitous in the cell, and membrane-bound proteins are implicated in a variety of signaling pathways and intra-cellular transport. We developed methodology to study proteins associated with model membranes using neutron reflection measurements and showed recently that this approach can resolve the penetration depth and orientation of membrane proteins with ångstrom resolution if their crystal or NMR structure is known. Here we apply this technology to determine the membrane bindung and unravel functional details of the PTEN phosphatase, a key player in the PI3K apoptosis pathway. PTEN is an important regulatory protein and tumor suppressor that performs its phosphatase activity as an interfacial enzyme at the plasma membrane-cytoplasm boundary. Acting as an antagonist to phosphoinositide-3-kinase (PI3K) in cell signaling, it is deleted in many human cancers. Despite its importance in regulating the levels of the phosphoinositoltriphosphate PI(3,4,5)P3, there is little understanding of how PTEN binds to membranes, is activated and then acts as a phosphatase. We investigated the structure and function of PTEN by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P2) act synergetically in attracting the enzyme to the membrane surface. Membrane affinities depend strongly on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P2. Neutron reflection measurements show that the PTEN phosphatase ``scoots'' along the membrane surface (penetration < 5 å) but binds the membrane tightly with its two major domains, the C2 and phosphatase domains. In the bound state, PTEN's regulatory C-terminal tail is displaced from the membrane and organized on the far side of the protein, ˜ 60 å away from the bilayer surface, in a rather compact structure. The combination of binding studies and neutron reflection allows us to distinguish between PTEN mutant proteins and ultimately may identify the structural features required for membrane binding and activation of PTEN. Molecular dynamics simulations, currently in progress, refine this structural picture further.

Regulation of cortical contractility and spindle positioning by the protein phosphatase 6 PPH-6 in one-cell stage C. elegans embryos

PubMed Central

Afshar, Katayoun; Werner, Michael E.; Tse, Yu Chung; Glotzer, Michael; Gönczy, Pierre

2010-01-01

Modulation of the microtubule and the actin cytoskeleton is crucial for proper cell division. Protein phosphorylation is known to be an important regulatory mechanism modulating these cytoskeletal networks. By contrast, there is a relative paucity of information regarding how protein phosphatases contribute to such modulation. Here, we characterize the requirements for protein phosphatase PPH-6 and its associated subunit SAPS-1 in one-cell stage C. elegans embryos. We establish that the complex of PPH-6 and SAPS-1 (PPH-6/SAPS-1) is required for contractility of the actomyosin network and proper spindle positioning. Our analysis demonstrates that PPH-6/SAPS-1 regulates the organization of cortical non-muscle myosin II (NMY-2). Accordingly, we uncover that PPH-6/SAPS-1 contributes to cytokinesis by stimulating actomyosin contractility. Furthermore, we demonstrate that PPH-6/SAPS-1 is required for the proper generation of pulling forces on spindle poles during anaphase. Our results indicate that this requirement is distinct from the role in organizing the cortical actomyosin network. Instead, we uncover that PPH-6/SAPS-1 contributes to the cortical localization of two positive regulators of pulling forces, GPR-1/2 and LIN-5. Our findings provide the first insights into the role of a member of the PP6 family of phosphatases in metazoan development. PMID:20040490

Effects of alkaloids from Sophora flavescens on osteoblasts infected with Staphylococcus aureus and osteoclasts.

PubMed

Wang, Xuping; Zheng, Rongzong; Huang, Xiaowen; Mao, Zhujun; Wang, Nani; Li, Hongyu; Wen, Chengping; Shou, Dan

2018-03-25

Chronic osteomyelitis is primarily caused by infection with Staphylococcus aureus (S. aureus). Antibiotics are commonly administered; however, it is a challenge to promote bone healing. The aim of this study was to investigate the in vitro effects of alkaloids from the herbal remedy Sophora flavescens (ASF) on rat calvarial osteoblasts (ROBs) infected with S. aureus and healthy osteoclasts. Cell proliferation and alkaline phosphatase, interleukin-6, and tumour necrosis factor-α activity was measured in infected ROBs; tartrate-resistant acid phosphatase was evaluated in osteoclasts via enzyme-linked immunosorbent assay. The mRNA and protein expression levels of bone morphogenetic protein 2, runt-related transcription factor 2, osteoprotegerin, and receptor activator of nuclear factor kappa-B ligand were assessed in infected ROBs through reverse transcription-polymerase chain reaction and western blotting analysis, respectively. Results indicated that ASF increased the viability of uninfected ROBs and infected ROBs treated with vancomycin via regulation of bone morphogenetic protein 2, runt-related transcription factor, osteoprotegerin, and receptor activator of nuclear factor kappa-B ligand mRNA and protein expression levels. In addition, the secretion of the inflammatory factor tumour necrosis factor-α was decreased and alkaline phosphatase activity was increased, inhibiting the viability of osteoclasts and tartrate-resistant acid phosphatase activity. Therefore, the herbal remedy ASF has potential as a new treatment for chronic osteomyelitis. Copyright © 2018 John Wiley & Sons, Ltd.

New Treatment Options for Osteosarcoma - Inactivation of Osteosarcoma Cells by Cold Atmospheric Plasma.

PubMed

Gümbel, Denis; Gelbrich, Nadine; Weiss, Martin; Napp, Matthias; Daeschlein, Georg; Sckell, Axel; Ender, Stephan A; Kramer, Axel; Burchardt, Martin; Ekkernkamp, Axel; Stope, Matthias B

2016-11-01

Cold atmospheric plasma has been shown to inhibit tumor cell growth and induce tumor cell death. The aim of the study was to investigate the effects of cold atmospheric plasma treatment on proliferation of human osteosarcoma cells and to characterize the underlying cellular mechanisms. Human osteosarcoma cells (U2-OS and MNNG/HOS) were treated with cold atmospheric plasma and seeded in culture plates. Cell proliferation, p53 and phospho-p53 protein expression and nuclear morphology were assessed. The treated human osteosarcoma cell lines exhibited attenuated proliferation rates by up to 66%. The cells revealed an induction of p53, as well as phospho-p53 expression, by 2.3-fold and 4.5-fold, respectively, compared to controls. 4',6-diamidino-2-phenylindole staining demonstrated apoptotic nuclear condensation following cold atmospheric plasma treatment. Cold atmospheric plasma treatment significantly attenuated cell proliferation in a preclinical in vitro osteosarcoma model. The resulting increase in p53 expression and phospho-activation in combination with characteristic nuclear changes indicate this was through induction of apoptosis. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Chromatin recruitment of activated AMPK drives fasting response genes co-controlled by GR and PPARα.

PubMed

Ratman, Dariusz; Mylka, Viacheslav; Bougarne, Nadia; Pawlak, Michal; Caron, Sandrine; Hennuyer, Nathalie; Paumelle, Réjane; De Cauwer, Lode; Thommis, Jonathan; Rider, Mark H; Libert, Claude; Lievens, Sam; Tavernier, Jan; Staels, Bart; De Bosscher, Karolien

2016-12-15

Adaptation to fasting involves both Glucocorticoid Receptor (GRα) and Peroxisome Proliferator-Activated Receptor α (PPARα) activation. Given both receptors can physically interact we investigated the possibility of a genome-wide cross-talk between activated GR and PPARα, using ChIP- and RNA-seq in primary hepatocytes. Our data reveal extensive chromatin co-localization of both factors with cooperative induction of genes controlling lipid/glucose metabolism. Key GR/PPAR co-controlled genes switched from transcriptional antagonism to cooperativity when moving from short to prolonged hepatocyte fasting, a phenomenon coinciding with gene promoter recruitment of phosphorylated AMP-activated protein kinase (AMPK) and blocked by its pharmacological inhibition. In vitro interaction studies support trimeric complex formation between GR, PPARα and phospho-AMPK. Long-term fasting in mice showed enhanced phosphorylation of liver AMPK and GRα Ser211. Phospho-AMPK chromatin recruitment at liver target genes, observed upon prolonged fasting in mice, is dampened by refeeding. Taken together, our results identify phospho-AMPK as a molecular switch able to cooperate with nuclear receptors at the chromatin level and reveal a novel adaptation mechanism to prolonged fasting. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Molecular structure of EmbR, a response element of Ser/Thr kinase signaling in Mycobacterium tuberculosis

PubMed Central

Alderwick, Luke J.; Molle, Virginie; Kremer, Laurent; Cozzone, Alain J.; Dafforn, Timothy R.; Besra, Gurdyal S.; Fütterer, Klaus

2006-01-01

Ser/Thr phosphorylation has emerged as a critical regulatory mechanism in a number of bacteria, including Mycobacterium tuberculosis. This problematic pathogen encodes 11 eukaryotic-like Ser/Thr kinases, yet few substrates or signaling targets have been characterized. Here, we report the structure of EmbR (2.0 Å), a putative transcriptional regulator of key arabinosyltransferases (EmbC, -A, and -B), and an endogenous substrate of the Ser/Thr-kinase PknH. EmbR presents a unique domain architecture: the N-terminal winged-helix DNA-binding domain forms an extensive interface with the all-helical central bacterial transcriptional activation domain and is positioned adjacent to the regulatory C-terminal forkhead-associated (FHA) domain, which mediates binding to a Thr-phosphorylated site in PknH. The structure in complex with a phospho-peptide (1.9 Å) reveals a conserved mode of phospho-threonine recognition by the FHA domain and evidence for specific recognition of the cognate kinase. The present structures suggest hypotheses as to how EmbR might propagate the phospho-relay signal from its cognate kinase, while serving as a template for the structurally uncharacterized Streptomyces antibiotic regulatory protein family of transcription factors. PMID:16477027

mTOR Pathway in Papillary Thyroid Carcinoma: Different Contributions of mTORC1 and mTORC2 Complexes for Tumor Behavior and SLC5A5 mRNA Expression

PubMed Central

Tavares, Catarina; Eloy, Catarina; Melo, Miguel; Gaspar da Rocha, Adriana; Pestana, Ana; Batista, Rui; Rios, Elisabete; Sobrinho Simões, Manuel

2018-01-01

The mammalian target of rapamycin (mTOR) pathway is overactivated in thyroid cancer (TC). We previously demonstrated that phospho-mTOR expression is associated with tumor aggressiveness, therapy resistance, and lower mRNA expression of SLC5A5 in papillary thyroid carcinoma (PTC), while phospho-S6 (mTORC1 effector) expression was associated with less aggressive clinicopathological features. The distinct behavior of the two markers led us to hypothesize that mTOR activation may be contributing to a preferential activation of the mTORC2 complex. To approach this question, we performed immunohistochemistry for phospho-AKT Ser473 (mTORC2 effector) in a series of 182 PTCs previously characterized for phospho-mTOR and phospho-S6 expression. We evaluated the impact of each mTOR complex on SLC5A5 mRNA expression by treating cell lines with RAD001 (mTORC1 blocker) and Torin2 (mTORC1 and mTORC2 blocker). Phospho-AKT Ser473 expression was positively correlated with phospho-mTOR expression. Nuclear expression of phospho-AKT Ser473 was significantly associated with the presence of distant metastases. Treatment of cell lines with RAD001 did not increase SLC5A5 mRNA levels, whereas Torin2 caused a ~6 fold increase in SLC5A5 mRNA expression in the TPC1 cell line. In PTC, phospho-mTOR activation may lead to the activation of the mTORC2 complex. Its downstream effector, phospho-AKT Ser473, may be implicated in distant metastization, therapy resistance, and downregulation of SLC5A5 mRNA expression. PMID:29757257

mTOR Pathway in Papillary Thyroid Carcinoma: Different Contributions of mTORC1 and mTORC2 Complexes for Tumor Behavior and SLC5A5 mRNA Expression.

PubMed

Tavares, Catarina; Eloy, Catarina; Melo, Miguel; Gaspar da Rocha, Adriana; Pestana, Ana; Batista, Rui; Bueno Ferreira, Luciana; Rios, Elisabete; Sobrinho Simões, Manuel; Soares, Paula

2018-05-13

The mammalian target of rapamycin (mTOR) pathway is overactivated in thyroid cancer (TC). We previously demonstrated that phospho-mTOR expression is associated with tumor aggressiveness, therapy resistance, and lower mRNA expression of SLC5A5 in papillary thyroid carcinoma (PTC), while phospho-S6 (mTORC1 effector) expression was associated with less aggressive clinicopathological features. The distinct behavior of the two markers led us to hypothesize that mTOR activation may be contributing to a preferential activation of the mTORC2 complex. To approach this question, we performed immunohistochemistry for phospho-AKT Ser473 (mTORC2 effector) in a series of 182 PTCs previously characterized for phospho-mTOR and phospho-S6 expression. We evaluated the impact of each mTOR complex on SLC5A5 mRNA expression by treating cell lines with RAD001 (mTORC1 blocker) and Torin2 (mTORC1 and mTORC2 blocker). Phospho-AKT Ser473 expression was positively correlated with phospho-mTOR expression. Nuclear expression of phospho-AKT Ser473 was significantly associated with the presence of distant metastases. Treatment of cell lines with RAD001 did not increase SLC5A5 mRNA levels, whereas Torin2 caused a ~6 fold increase in SLC5A5 mRNA expression in the TPC1 cell line. In PTC, phospho-mTOR activation may lead to the activation of the mTORC2 complex. Its downstream effector, phospho-AKT Ser473, may be implicated in distant metastization, therapy resistance, and downregulation of SLC5A5 mRNA expression.

Relation of placental alkaline phosphatase expression in human term placenta with maternal and offspring fat mass.

PubMed

Hirschmugl, Birgit; Crozier, Sarah; Matthews, Nina; Kitzinger, Eva; Klymiuk, Ingeborg; Inskip, Hazel M; Harvey, Nicholas C; Cooper, Cyrus; Sibley, Colin P; Glazier, Jocelyn; Wadsack, Christian; Godfrey, Keith M; Desoye, Gernot; Lewis, Rohan M

2018-06-13

Alkaline phosphatase is implicated in intestinal lipid transport and in the development of obesity. Placental alkaline phosphatase is localised to the microvillous plasma membrane of the placental syncytiotrophoblast at the maternal-fetal interface, but its role is unclear. We investigated the relations of placental alkaline phosphatase activity and mRNA expression with maternal body composition and offspring fat mass in humans. Term human placentas from the UK Birthright cohort (n = 52) and the Southampton Women's Survey (SWS) (n = 95) were studied. In the Birthright cohort, alkaline phosphatase activity was measured in placental microvillous plasma membrane vesicles. In the SWS, alkaline phosphatase mRNA was measured using Nanostring. Alkaline phosphatase gene expression was compared to other lipid-related genes. In Birthright samples placental microvillous plasma membrane alkaline phosphatase activity was positively associated with maternal triceps skinfold thickness and BMI (β = 0.04 (95% CI: 0.01-0.06) and β = 0.02 (0.00-0.03) µmol/mg protein/min per SD, P = 0.002 and P = 0.05, respectively) after adjusting for potential confounders. In SWS samples placental alkaline phosphatase mRNA expression in term placenta was positively associated with maternal triceps skinfold (β = 0.24 (0.04, 0.44) SD/SD, P = 0.02), had no association with neonatal %fat mass (β = 0.01 (-0.20 to 0.21) SD/SD, P = 0.93) and was negatively correlated with %fat mass at ages 4 (β = -0.28 (-0.52 to -0.04) SD/SD, P = 0.02), 6-7 (β = -0.25 (-0.49 to -0.02) SD/SD, P = 0.03) years. When compared with placental expression of other genes, alkaline phosphatase expression was positively related to genes including the lysophosphatidylcholine transporter MFSD2A (major facilitator superfamily domain containing 2A, P < 0.001) and negatively related to genes including the fatty acid transport proteins 2 and 3 (P = 0.001, P < 0.001). Our findings suggest relationships between placental alkaline phosphatase and both maternal and childhood adiposity. The inverse relationship between placental alkaline phosphatase gene expression and childhood %fat mass suggests that placental alkaline phosphatase may help to protect the foetus from the adverse effects of maternal obesity.

Hydrostatic pressure and muscarinic receptors are involved in the release of inflammatory cytokines in human bladder smooth muscle cells.

PubMed

Liang, Zhou; Xin, Wei; Qiang, Liu; Xiang, Cai; Bang-Hua, Liao; Jin, Yang; De-Yi, Luo; Hong, Li; Kun-Jie, Wang

2017-06-01

Abnormal intravesical pressure results in a series of pathological changes. We investigated the effects of hydrostatic pressure and muscarinic receptors on the release of inflammatory cytokines in rat and human bladder smooth muscle cells (HBSMCs). Animal model of bladder outlet obstruction was induced by urethra ligation. HBSMCs were subjected to elevated hydrostatic pressure and/or acetylcholine (Ach). Macrophage infiltration in the bladder wall was determined by immunohistochemical staining. The expression of inflammatory genes was measured by RT-PCR, ELISA and immunofluorescence. In obstructed bladder, inflammatory genes and macrophage infiltration were remarkably induced. When HBSMCs were subjected to 200-300 cm H 2 O pressure for 2-24 h in vitro, the expressions of IL-6 and RANTES were significantly increased. Hydrostatic pressure promoted the protein levels of phospho-NFκB p65 and phospho-ERK1/2 as well as muscarinic receptors. Moreover, NFκB or ERK1/2 inhibitors suppressed pressure-induced inflammatory genes mRNA. When cells were treated with 1 μM acetylcholine for 6 h, a significant increase in IL-6 mRNA expression was detected. Acetylcholine also enhanced pressure-induced phospho-NFκB p65 and IL-6 protein expression. Additionally, pressure-induced IL-6 was partially suppressed by muscarinic receptors antagonists. Hydrostatic pressure and muscarinic receptors were involved in the secretion of inflammatory cytokines in HBSMCs, indicating a pro-inflammatory effect of the two factors in the pathological process of BOO. © 2016 Wiley Periodicals, Inc.

Docosahexaenoic acid attenuates Western diet-induced hepatic fibrosis in Ldlr−/− mice by targeting the TGFβ-Smad3 pathway[S

PubMed Central

Lytle, Kelli A.; Depner, Christopher M.; Wong, Carmen P.; Jump, Donald B.

2015-01-01

DHA (22:6,ω3), but not EPA (20:5,ω3), attenuates Western diet (WD)-induced hepatic fibrosis in a Ldlr−/− mouse model of nonalcoholic steatohepatitis. We examined the molecular basis for the differential effect of dietary EPA and DHA on WD-induced hepatic fibrosis. DHA was more effective than EPA at preventing WD-induced effects on hepatic transcripts linked to fibrosis, including collagen 1A1 (Col1A1), transforming growth factor-β (TGFβ) signaling and proteins involved in remodeling the extracellular matrix, including metalloproteases, tissue inhibitors of metalloproteases, and lysyl oxidase subtypes. Examination of the TGFβ pathway showed that mice fed the WD supplemented with either olive oil or EPA had a significant (≥2.5-fold) increase in hepatic nuclear abundance of phospho-mothers against decapentaplegic homolog (Smad)3 when compared with mice fed the reference diet (RD); Smad3 is a key regulator of Col1A1 expression in stellate cells. In contrast, mice fed the WD supplemented with DHA had no increase in phospho-Smad3 when compared with mice fed the RD. Changes in hepatic phospho-Smad3 nuclear content correlated with proCol1A1 mRNA and protein abundance. Pretreatment of human LX2 stellate cells with DHA, but not other unsaturated fatty acids, blocked TGFβ1-mediated induction of Col1A1. In conclusion, DHA attenuates WD-induced fibrosis by targeting the TGFβ-Smad3-Col1A1 pathway in stellate cells. PMID:26315048

Structure-Function Correlation of G6, a Novel Small Molecule Inhibitor of Jak2

PubMed Central

Majumder, Anurima; Govindasamy, Lakshmanan; Magis, Andrew; Kiss, Róbert; Polgár, Tímea; Baskin, Rebekah; Allan, Robert W.; Agbandje-McKenna, Mavis; Reuther, Gary W.; Keserű, György M.; Bisht, Kirpal S.; Sayeski, Peter P.

2010-01-01

Somatic mutations in the Jak2 protein, such as V617F, cause aberrant Jak/STAT signaling and can lead to the development of myeloproliferative neoplasms. This discovery has led to the search for small molecule inhibitors that target Jak2. Using structure-based virtual screening, our group recently identified a novel small molecule inhibitor of Jak2 named G6. Here, we identified a structure-function correlation of this compound. Specifically, five derivative compounds of G6 having structural similarity to the original lead compound were obtained and analyzed for their ability to (i) inhibit Jak2-V617F-mediated cell growth, (ii) inhibit the levels of phospho-Jak2, phospho-STAT3, and phospho-STAT5; (iii) induce apoptosis in human erythroleukemia cells; and (iv) suppress pathologic cell growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Additionally, we computationally examined the interactions of these compounds with the ATP-binding pocket of the Jak2 kinase domain. We found that the stilbenoid core-containing derivatives of G6 significantly inhibited Jak2-V617F-mediated cell proliferation in a time- and dose-dependent manner. They also inhibited phosphorylation of Jak2, STAT3, and STAT5 proteins within cells, resulting in higher levels of apoptosis via the intrinsic apoptotic pathway. Finally, the stilbenoid derivatives inhibited the pathologic growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Collectively, our data demonstrate that G6 has a stilbenoid core that is indispensable for maintaining its Jak2 inhibitory potential. PMID:20667821

Phosphorylation of Rpt6 regulates synaptic strength in hippocampal neurons.

PubMed

Djakovic, Stevan N; Marquez-Lona, Esther M; Jakawich, Sonya K; Wright, Rebecca; Chu, Carissa; Sutton, Michael A; Patrick, Gentry N

2012-04-11

It has become increasingly evident that protein degradation via the ubiquitin proteasome system plays a fundamental role in the development, maintenance and remodeling of synaptic connections in the CNS. We and others have recently described the activity-dependent regulation of proteasome activity and recruitment of proteasomes into spine compartments involving the phosphorylation of the 19S ATPase subunit, Rpt6, by the plasticity kinase Ca(2+)/calmodulin-dependent protein kinase II α (CaMKIIα) (Bingol and Schuman, 2006; Djakovic et al., 2009; Bingol et al, 2010). Here, we investigated the role of Rpt6 phosphorylation on proteasome function and synaptic strength. Utilizing a phospho-specific antibody we verified that Rpt6 is phosphorylated at Serine 120 (S120) by CaMKIIα. In addition, we found that Rpt6 is phosphorylated by CaMKIIα in an activity-dependent manner. Furthermore, we showed that a serine 120 to aspartic acid phospho-mimetic mutant of Rpt6 (S120D) increases its resistance to detergent extraction in rat hippocampal dendrites, indicating phosphorylated Rpt6 may promote the tethering of proteasomes to scaffolds and cytoskeletal components. Expression of Rpt6 S120D decreased miniature EPSC (mEPSC) amplitude, while expression of a phospho-dead mutant (S120A) increased mEPSC amplitude. Surprisingly, homeostatic scaling of mEPSC amplitude produced by chronic application of bicuculline or tetrodotoxin is both mimicked and occluded by altered Rpt6 phosphorylation. Together, these data suggest that CaMKII-dependent phosphorylation of Rpt6 at S120 may be an important regulatory mechanism for proteasome-dependent control of synaptic remodeling in slow homeostatic plasticity.

Expression, purification and preliminary crystallographic analysis of Mycobacterium tuberculosis CysQ, a phosphatase involved in sulfur metabolism

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

Erickson, Anna I.; Sarsam, Reta D.; Fisher, Andrew J., E-mail: ajfisher@ucdavis.edu

The cysQ gene from Mycobacterium tuberculosis was cloned and the expressed protein, a 3′-phosphoadenosine-5′’-phosphatase, was purified and crystallized. X-ray diffraction data were collected to 1.7 Å resolution.

Structure-Based Virtual Screening of Protein Tyrosine Phosphatase Inhibitors: Significance, Challenges, and Solutions.

PubMed

Reddy, Rallabandi Harikrishna; Kim, Hackyoung; Cha, Seungbin; Lee, Bongsoo; Kim, Young Jun

2017-05-28

Phosphorylation, a critical mechanism in biological systems, is estimated to be indispensable for about 30% of key biological activities, such as cell cycle progression, migration, and division. It is synergistically balanced by kinases and phosphatases, and any deviation from this balance leads to disease conditions. Pathway or biological activity-based abnormalities in phosphorylation and the type of involved phosphatase influence the outcome, and cause diverse diseases ranging from diabetes, rheumatoid arthritis, and numerous cancers. Protein tyrosine phosphatases (PTPs) are of prime importance in the process of dephosphorylation and catalyze several biological functions. Abnormal PTP activities are reported to result in several human diseases. Consequently, there is an increased demand for potential PTP inhibitory small molecules. Several strategies in structure-based drug designing techniques for potential inhibitory small molecules of PTPs have been explored along with traditional drug designing methods in order to overcome the hurdles in PTP inhibitor discovery. In this review, we discuss druggable PTPs and structure-based virtual screening efforts for successful PTP inhibitor design.

Identification and partial characterization of a latent ATP, Mg-dependent protein phosphatase in rabbit skeletal muscle cytosol.

PubMed

Vandenheede, J R; Staquet, S; Merlevede, W

1989-05-04

Fractionation of rabbit skeletal muscle cytosol on Aminohexyl-Sepharose has resulted in the identification of a latent ATP, Mg-dependent protein phosphatase whose catalytic subunit is in the active conformation, but is inhibited by the presence of more than one modulator unit. The partially purified enzyme is converted to an inactive, kinase FA-dependent form upon incubation at 30 degrees C unless modulator-specific polyclonal antibodies are added to the preparation. The immunoglobulins also relieve the inhibition which is responsible for the low basal phosphatase activity of the enzyme, and they counteract all of the heat-stable inhibitor activity present in the preparation. Addition of free catalytic subunit abolishes the inhibition of the latent enzyme in a dose-dependent way, but cannot prevent the inactivation process. The inactivated phosphatase and the original latent enzyme exhibit the same apparent Mr in sucrose density-gradient centrifugation (70,000) and in gel filtration (110,000).

Ozone (O{sub 3}) elicits neurotoxicity in spinal cord neurons (SCNs) by inducing ER Ca{sup 2+} release and activating the CaMKII/MAPK signaling pathway

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

Li, Yun; Lin, Xiaowen; Zhao, XueJun

Ozone (O{sub 3}) is widely used in the treatment of spinal cord related diseases. Excess or accumulation of this photochemical air can however be neurotoxic. In this study, in vitro cultured Wister rat spinal cord neurons (SCNs) were used to investigate the detrimental effects and underlying mechanisms of O{sub 3}. Ozone in a dose-dependent manner inhibited cell viability at a range of 20 to 500 μg/ml, with the dose at 40 μg/ml resulting in a decrease of cell viability to 75%. The cell death after O{sub 3} exposure was related to endoplasmic reticulum (ER) calcium (Ca{sup 2+}) release. Intracellular Ca{supmore » 2+} chelator, ER stabilizer (inositol 1,4,5-trisphosphate receptor (IP3R) antagonist and ryanodine receptor (RyR) antagonist) and calcium/calmodulin-dependent protein kinase II (CaMKII) antagonist could effectively block Ca{sup 2+} mobilization and inhibit cell death following 40 μg/ml O{sub 3} exposure. In addition, ER Ca{sup 2+} release due to O{sub 3} exposure enhanced phospho-p38 and phospho-JNK levels and apoptosis of SCNs through activating CaMKII. Based on these results, we confirm that ozone elicits neurotoxicity in SCNs via inducing ER Ca{sup 2+} release and activating CaMKII/MAPK signaling pathway. Therefore, physicians should get attention to the selection of treatment concentrations of oxygen/ozone. And, approaches, such as chelating intracellular Ca{sup 2+} and stabilizing neuronal Ca{sup 2+} homeostasis could effectively ameliorate the neurotoxicity of O{sub 3}. - Highlights: • Exposure to O{sub 3} can reduce the viability of SCNs and cause the cell death. • Exposure to O{sub 3} can trigger RyR and IP3R dependent intracellular Ca{sup 2+} release. • Exposure to O{sub 3} can enhance the phospho-CaMKII, phospho-JNK and phospho-p38 levels.« less

Arabidopsis PHOSPHOTYROSYL PHOSPHATASE ACTIVATOR Is Essential for PROTEIN PHOSPHATASE 2A Holoenzyme Assembly and Plays Important Roles in Hormone Signaling, Salt Stress Response, and Plant Development1[W][OPEN

PubMed Central

Chen, Jian; Hu, Rongbin; Zhu, Yinfeng; Shen, Guoxin; Zhang, Hong

2014-01-01

PROTEIN PHOSPHATASE 2A (PP2A) is a major group of serine/threonine protein phosphatases in eukaryotes. It is composed of three subunits: scaffolding subunit A, regulatory subunit B, and catalytic subunit C. Assembly of the PP2A holoenzyme in Arabidopsis (Arabidopsis thaliana) depends on Arabidopsis PHOSPHOTYROSYL PHOSPHATASE ACTIVATOR (AtPTPA). Reduced expression of AtPTPA leads to severe defects in plant development, altered responses to abscisic acid, ethylene, and sodium chloride, and decreased PP2A activity. In particular, AtPTPA deficiency leads to decreased methylation in PP2A-C subunits (PP2Ac). Complete loss of PP2Ac methylation in the suppressor of brassinosteroid insensitive1 mutant leads to 30% reduction of PP2A activity, suggesting that PP2A with a methylated C subunit is more active than PP2A with an unmethylated C subunit. Like AtPTPA, PP2A-A subunits are also required for PP2Ac methylation. The interaction between AtPTPA and PP2Ac is A subunit dependent. In addition, AtPTPA deficiency leads to reduced interactions of B subunits with C subunits, resulting in reduced functional PP2A holoenzyme formation. Thus, AtPTPA is a critical factor for committing the subunit A/subunit C dimer toward PP2A heterotrimer formation. PMID:25281708

AMA0428, A Potent Rock Inhibitor, Attenuates Early and Late Experimental Diabetic Retinopathy.

PubMed

Hollanders, Karolien; Hove, Inge Van; Sergeys, Jurgen; Bergen, Tine Van; Lefevere, Evy; Kindt, Nele; Castermans, Karolien; Vandewalle, Evelien; van Pelt, Jos; Moons, Lieve; Stalmans, Ingeborg

2017-02-01

Diabetic retinopathy (DR) is characterized by an early stage of inflammation and vessel leakage, and an advanced vasoproliferative stage. Also, neurodegeneration might play an important role in disease pathogenesis. The aim of this study was to investigate the effect of the Rho kinase (ROCK) inhibitor, AMA0428, on these processes. The response to ROCK inhibition by AMA0428 (1 µg) was studied in vivo using the murine model for streptozotocin (STZ)-induced diabetes, focusing on early non-proliferative DR features and the oxygen-induced retinopathy (OIR) model to investigate proliferative DR. Intravitreal (IVT) administration of AMA0428 was compared with murine anti-VEGF-R2 antibody (DC101, 6.2 µg) and placebo (H 2 O/PEG; 1C8). Outcome was assessed by analyzing leukostasis using fluorescein isothiocyanate coupled concanavalin A (FITC-ConA) and vessel leakage (bovine serum albumin conjugated with fluorescein isothiocyanate; FITC-BSA)/neovascularization and neurodegeneration by immunohistological approaches (hematoxylin and eosin (H&E), terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL), Brn3a). ELISA and Western blotting were employed to unravel the consequences of ROCK inhibition (1 µM AMA0428) on myosin phosphatase target protein (MYPT)-1 phosphorylation, endothelial nitric oxide synthase (eNOS) phosphorylation, and vascular endothelial growth factor (VEGF) levels in retinas of diabetic mice, on NF-κβ activity and ICAM-1 expression in endothelial cells (ECs). In vivo, AMA0428 significantly reduced vessel leakage and neovascularization, respectively, in the STZ and OIR model, comparable to DC101 therapy. Additionally, the ROCK inhibitor decreased neurodegeneration in both models and inhibited leukostasis by 30% (p < 0.05) in the STZ model (p < 0.05), while DC101 had no positive effect on the outcome of these latter processes. ROCK activity was upregulated in the diabetic retina and AMA0428 administration resulted in decreased phospho-MYPT-1, enhanced phospho-eNOS, and reduced VEGF levels. In vitro, AMA0428 interfered with NF-κβ activity, thereby inhibiting ICAM-1 expression in ECs. Targeting ROCK with AMA0428 effectively attenuated outcome in an early DR model (STZ) and a late vasoproliferative retinopathy model (OIR). These findings make AMA0428 a promising candidate with an additional anti-inflammatory and neuroprotective benefit for DR patients, as compared with anti-VEGF treatment.

Enterococcus faecalis utilizes maltose by connecting two incompatible metabolic routes via a novel maltose-6’-phosphate phosphatase (MapP)

PubMed Central

Mokhtari, Abdelhamid; Blancato, Víctor S.; Repizo, Guillermo; Henry, Céline; Pikis, Andreas; Bourand, Alexa; de Fátima Álvarez, María; Immel, Stefan; Mechakra-Maza, Aicha; Hartke, Axel; Thompson, John; Magni, Christian; Deutscher, Josef

2013-01-01

Summary Similar to Bacillus subtilis, Enterococcus faecalis transports and phosphorylates maltose via a phosphoenolpyruvate (PEP):maltose phosphotransferase system (PTS). The maltose-specific PTS permease is encoded by the malT gene. However, E. faecalis lacks a malA gene encoding a 6-phospho-α-glucosidase which in B. subtilis hydrolyses maltose-6’-P into glucose and glucose-6-P. Instead, an operon encoding a maltose phosphorylase (MalP), a phosphoglucomutase and a mutarotase starts upstream from malT. MalP was suggested to split maltose-6-P into glucose-1-P and glucose-6-P. However, purified MalP phosphorolyses maltose but not maltose-6’-P. We discovered that the gene downstream from malT encodes a novel enzyme (MapP) that dephosphorylates maltose-6’-P formed by the PTS. The resulting intracellular maltose is cleaved by MalP into glucose and glucose-1-P. Slow uptake of maltose probably via a maltodextrin ABC transporter allows poor growth for the mapP but not the malP mutant. Synthesis of MapP in a B. subtilis mutant accumulating maltose-6’-P restored growth on maltose. MapP catalyzes the dephosphorylation of intracellular maltose-6’-P, and the resulting maltose is converted by the B. subtilis maltose phosphorylase into glucose and glucose-1-P. MapP therefore connects PTS-mediated maltose uptake to maltose phosphorylase-catalyzed metabolism. Dephosphorylation assays with a wide variety of phospho-substrates revealed that MapP preferably dephosphorylates disaccharides containing an O-α-glycosyl linkage. PMID:23490043

ACCA phosphopeptide recognition by the BRCT repeats of BRCA1.

PubMed

Ray, Hind; Moreau, Karen; Dizin, Eva; Callebaut, Isabelle; Venezia, Nicole Dalla

2006-06-16

The tumour suppressor gene BRCA1 encodes a 220 kDa protein that participates in multiple cellular processes. The BRCA1 protein contains a tandem of two BRCT repeats at its carboxy-terminal region. The majority of disease-associated BRCA1 mutations affect this region and provide to the BRCT repeats a central role in the BRCA1 tumour suppressor function. The BRCT repeats have been shown to mediate phospho-dependant protein-protein interactions. They recognize phosphorylated peptides using a recognition groove that spans both BRCT repeats. We previously identified an interaction between the tandem of BRCA1 BRCT repeats and ACCA, which was disrupted by germ line BRCA1 mutations that affect the BRCT repeats. We recently showed that BRCA1 modulates ACCA activity through its phospho-dependent binding to ACCA. To delineate the region of ACCA that is crucial for the regulation of its activity by BRCA1, we searched for potential phosphorylation sites in the ACCA sequence that might be recognized by the BRCA1 BRCT repeats. Using sequence analysis and structure modelling, we proposed the Ser1263 residue as the most favourable candidate among six residues, for recognition by the BRCA1 BRCT repeats. Using experimental approaches, such as GST pull-down assay with Bosc cells, we clearly showed that phosphorylation of only Ser1263 was essential for the interaction of ACCA with the BRCT repeats. We finally demonstrated by immunoprecipitation of ACCA in cells, that the whole BRCA1 protein interacts with ACCA when phosphorylated on Ser1263.

Cloning and characterization of rat density-enhanced phosphatase-1, a protein tyrosine phosphatase expressed by vascular cells.

PubMed

Borges, L G; Seifert, R A; Grant, F J; Hart, C E; Disteche, C M; Edelhoff, S; Solca, F F; Lieberman, M A; Lindner, V; Fischer, E H; Lok, S; Bowen-Pope, D F

1996-09-01

We have cloned from cultured vascular smooth muscle cells a protein tyrosine phosphatase, rat density-enhanced phosphatase-1 (rDEP-1), which is a probable rat homologue of DEP-1/HPTP eta. rDEP-1 is encoded by an 8.7-kb transcript and is expressed as a 180- to 220-kD protein. The rDEP-1 gene is located on human chromosome 11 (region p11.2) and on mouse chromosome 2 (region 2E). The cDNA sequence predicts a transmembrane protein consisting of a single phosphatase catalytic domain in the intracellular region, a single transmembrane domain, and eight fibronectin type III repeats in the extracellular region (GenBank accession number U40790). In situ hybridization analysis demonstrates that rDEP-1 is widely expressed in vivo but that expression is highest in cells that form epithelioid monolayers. In cultured cells with epitheliod morphology, including endothelial cells and newborn smooth muscle cells, but not in fibroblast-like cells, rDEP-1 transcript levels are dramatically upregulated as population density increases. In vivo, quiescent endothelial cells in normal arteries express relatively high levels of rDEP-1. During repair of vascular injury, expression of rDEP-1 is downregulated in migrating and proliferating endothelial cells. In vivo, rDEP-1 transcript levels are present in very high levels in megakaryocytes, and circulating plates have high levels of the rDEP-1 protein. In vitro, initiation of differentiation of the human megakaryoblastic cell line CHRF-288-11 with phorbol 12-myristate 13-acetate leads to a very strong upregulation of rDEP-1 transcripts. The deduced structure and the regulation of expression of rDEP-1 suggest that it may play a role in adhesion and/or signaling events involving cell-cell and cell-matrix contact.

Pharmacodynamic Assay Panel for Monitoring Phospho-Signaling Networks | Office of Cancer Clinical Proteomics Research

Cancer.gov

The DNA damage response (DDR) is a highly regulated signal transduction network that orchestrates the temporal and spatial organization of protein complexes required to repair (or tolerate) DNA damage (e.g., nucleotide excision repair, base excision repair, homologous recombination, non-homologous end joining, post-replication repair).

Increases in cAMP, MAPK Activity and CREB Phosphorylation during REM Sleep: Implications for REM Sleep and Memory Consolidation

PubMed Central

Luo, Jie; Phan, Trongha X.; Yang, Yimei; Garelick, Michael G.; Storm, Daniel R.

2013-01-01

The cyclic adenosine monophosphate (cAMP), mitogen-activated protein kinase (MAPK) and cAMP response element-binding protein (CREB) transcriptional pathway is required for consolidation of hippocampus-dependent memory. In mice, this pathway undergoes a circadian oscillation required for memory persistence that reaches a peak during the daytime. Since mice exhibit polyphasic sleep patterns during the day, this suggested the interesting possibility that cAMP, MAPK activity and CREB phosphorylation may be elevated during sleep. Here, we report that cAMP, phospho-p44/42 MAPK and phospho-CREB are higher in rapid eye movement (REM) sleep compared to awake mice but are not elevated in non-rapid eye movement (NREM) sleep. This peak of activity during REM sleep does not occur in mice lacking calmodulin-stimulated adenylyl cyclases, a mouse strain that learns but cannot consolidate hippocampus-dependent memory. We conclude that a preferential increase in cAMP, MAPK activity and CREB phosphorylation during REM sleep may contribute to hippocampus-dependent memory consolidation. PMID:23575844

Endoplasmic reticulum stress preconditioning attenuates methylmercury-induced cellular damage by inducing favorable stress responses

PubMed Central

Usuki, Fusako; Fujimura, Masatake; Yamashita, Akio

2013-01-01

We demonstrate that methylmercury (MeHg)-susceptible cells preconditioned with an inhibitor of endoplasmic reticulum (ER) Ca2+-ATPase, thapsigargin, showed resistance to MeHg cytotoxicity through favorable stress responses, which included phosphorylation of eukaryotic initiation factor 2 alpha (Eif2α), accumulation of activating transcription factor 4 (Atf4), upregulation of stress-related proteins, and activation of extracellular signal regulated kinase pathway. In addition, ER stress preconditioning induced suppression of nonsense-mediated mRNA decay (NMD) mainly through the phospho-Eif2α-mediated general suppression of translation initiation and possible combined effects of decreased several NMD components expression. Atf4 accumulation was not mediated by NMD inhibition but translation inhibition of its upstream open reading frame (uORF) and translation facilitation of its protein-coding ORF by the phospho-Eif2α. These results suggested that ER stress plays an important role in MeHg cytotoxicity and that the modulation of ER stress has therapeutic potential to attenuate MeHg cytotoxicity, the underlying mechanism being the induction of integrated stress responses. PMID:23907635

A widespread family of serine/threonine protein phosphatases shares a common regulatory switch with proteasomal proteases

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

Bradshaw, Niels; Levdikov, Vladimir M.; Zimanyi, Christina M.

PP2C phosphatases control biological processes including stress responses, development, and cell division in all kingdoms of life. Diverse regulatory domains adapt PP2C phosphatases to specific functions, but how these domains control phosphatase activity was unknown. We present structures representing active and inactive states of the PP2C phosphatase SpoIIE from Bacillus subtilis. Based on structural analyses and genetic and biochemical experiments, we identify an α-helical switch that shifts a carbonyl oxygen into the active site to coordinate a metal cofactor. Our analysis indicates that this switch is widely conserved among PP2C family members, serving as a platform to control phosphatase activitymore » in response to diverse inputs. Remarkably, the switch is shared with proteasomal proteases, which we identify as evolutionary and structural relatives of PP2C phosphatases. Although these proteases use an unrelated catalytic mechanism, rotation of equivalent helices controls protease activity by movement of the equivalent carbonyl oxygen into the active site.« less

E2/ER β Enhances Calcineurin Protein Degradation and PI3K/Akt/MDM2 Signal Transduction to Inhibit ISO-Induced Myocardial Cell Apoptosis.

PubMed

Lin, Kuan-Ho; Kuo, Wei-Wen; Shibu, Marthandam Asokan; Day, Cecilia-Hsuan; Hsieh, You-Liang; Chung, Li-Chin; Chen, Ray-Jade; Wen, Su-Ying; Viswanadha, Vijaya Padma; Huang, Chih-Yang

2017-04-24

Secretion of multifunctional estrogen and its receptor has been widely considered as the reason for markedly higher frequency of heart disease in men than in women. 17β-Estradiol (E2), for instance, has been reported to prevent development of cardiac apoptosis via activation of estrogen receptors (ERs). In addition, protein phosphatase such as protein phosphatase 1 (PP1) and calcineurin (PP2B) are also involved in cardiac hypertrophy and cell apoptosis signaling. However, the mechanism by which E2/ERβ suppresses apoptosis is not fully understood, and the role of protein phosphatase in E2/ERβ action also needs further investigation. In this study, we observed that E2/ERβ inhibited isoproterenol (ISO)-induced myocardial cell apoptosis, cytochrome c release and downstream apoptotic markers. Moreover, we found that E2/ERβ blocks ISO-induced apoptosis in H9c2 cells through the enhancement of calcineurin protein degradation through PI3K/Akt/MDM2 signaling pathway. Our results suggest that supplementation with estrogen and/or overexpression of estrogen receptor β gene may prove to be effective means to treat stress-induced myocardial damage.