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Sample records for altered protein phosphorylation

  1. Altered protein phosphorylation as a resource for potential AD biomarkers.

    PubMed

    Henriques, Ana Gabriela; Müller, Thorsten; Oliveira, Joana Machado; Cova, Marta; da Cruz E Silva, Cristóvão B; da Cruz E Silva, Odete A B

    2016-01-01

    The amyloidogenic peptide, Aβ, provokes a series of events affecting distinct cellular pathways regulated by protein phosphorylation. Aβ inhibits protein phosphatases in a dose-dependent manner, thus it is expected that the phosphorylation state of specific proteins would be altered in response to Aβ. In fact several Alzheimer's disease related proteins, such as APP and TAU, exhibit pathology associated hyperphosphorylated states. A systems biology approach was adopted and the phosphoproteome, of primary cortical neuronal cells exposed to Aβ, was evaluated. Phosphorylated proteins were recovered and those whose recovery increased or decreased, upon Aβ exposure across experimental sets, were identified. Significant differences were evident for 141 proteins and investigation of their interactors revealed key protein clusters responsive to Aβ treatment. Of these, 73 phosphorylated proteins increased and 68 decreased upon Aβ addition. These phosphorylated proteins represent an important resource of potential AD phospho biomarkers that should be further pursued. PMID:27466139

  2. Altered protein phosphorylation as a resource for potential AD biomarkers

    PubMed Central

    Henriques, Ana Gabriela; Müller, Thorsten; Oliveira, Joana Machado; Cova, Marta; da Cruz e Silva, Cristóvão B.; da Cruz e Silva, Odete A. B.

    2016-01-01

    The amyloidogenic peptide, Aβ, provokes a series of events affecting distinct cellular pathways regulated by protein phosphorylation. Aβ inhibits protein phosphatases in a dose-dependent manner, thus it is expected that the phosphorylation state of specific proteins would be altered in response to Aβ. In fact several Alzheimer’s disease related proteins, such as APP and TAU, exhibit pathology associated hyperphosphorylated states. A systems biology approach was adopted and the phosphoproteome, of primary cortical neuronal cells exposed to Aβ, was evaluated. Phosphorylated proteins were recovered and those whose recovery increased or decreased, upon Aβ exposure across experimental sets, were identified. Significant differences were evident for 141 proteins and investigation of their interactors revealed key protein clusters responsive to Aβ treatment. Of these, 73 phosphorylated proteins increased and 68 decreased upon Aβ addition. These phosphorylated proteins represent an important resource of potential AD phospho biomarkers that should be further pursued. PMID:27466139

  3. Rapid alteration of protein phosphorylation during postmortem: implication in the study of protein phosphorylation.

    PubMed

    Wang, Yifan; Zhang, Yanchong; Hu, Wen; Xie, Shutao; Gong, Cheng-Xin; Iqbal, Khalid; Liu, Fei

    2015-01-01

    Protein phosphorylation is an important post-translational modification of proteins. Postmortem tissues are widely being utilized in the biomedical studies, but the effects of postmortem on protein phosphorylation have not been received enough attention. In the present study, we found here that most proteins in mouse brain, heart, liver, and kidney were rapidly dephosphorylated to various degrees during 20 sec to 10 min postmortem. Phosphorylation of tau at Thr212 and glycogen synthase kinase 3β (GSK-3β) at Ser9 was reduced by 50% in the brain with 40 sec postmortem, a regular time for tissue processing. During postmortem, phosphorylation of cAMP-dependent protein kinase (PKA) and AMP activated kinase (AMPK) was increased in the brain, but not in other organs. Perfusion of the brain with cold or room temperature phosphate-buffered saline (PBS) also caused significant alteration of protein phosphorylation. Cooling down and maintaining mouse brains in the ice-cold buffer prevented the alteration effectively. This study suggests that phosphorylation of proteins is rapidly changed during postmortem. Thus, immediate processing of tissues followed by cooling down in ice-cold buffer is vitally important and perfusion has to be avoided when protein phosphorylation is to be studied. PMID:26511732

  4. Rapid alteration of protein phosphorylation during postmortem: implication in the study of protein phosphorylation

    PubMed Central

    Wang, Yifan; Zhang, Yanchong; Hu, Wen; Xie, Shutao; Gong, Cheng-Xin; Iqbal, Khalid; Liu, Fei

    2015-01-01

    Protein phosphorylation is an important post-translational modification of proteins. Postmortem tissues are widely being utilized in the biomedical studies, but the effects of postmortem on protein phosphorylation have not been received enough attention. In the present study, we found here that most proteins in mouse brain, heart, liver, and kidney were rapidly dephosphorylated to various degrees during 20 sec to 10 min postmortem. Phosphorylation of tau at Thr212 and glycogen synthase kinase 3β (GSK-3β) at Ser9 was reduced by 50% in the brain with 40 sec postmortem, a regular time for tissue processing. During postmortem, phosphorylation of cAMP-dependent protein kinase (PKA) and AMP activated kinase (AMPK) was increased in the brain, but not in other organs. Perfusion of the brain with cold or room temperature phosphate-buffered saline (PBS) also caused significant alteration of protein phosphorylation. Cooling down and maintaining mouse brains in the ice-cold buffer prevented the alteration effectively. This study suggests that phosphorylation of proteins is rapidly changed during postmortem. Thus, immediate processing of tissues followed by cooling down in ice-cold buffer is vitally important and perfusion has to be avoided when protein phosphorylation is to be studied. PMID:26511732

  5. Microgravity alters protein phosphorylation changes during initiation of sea urchin sperm motility

    NASA Technical Reports Server (NTRS)

    Tash, J. S.; Bracho, G. E.

    1999-01-01

    European Space Agency (ESA) studies demonstrated that bull sperm swim with higher velocity in microgravity (microG) than at 1 G. Coupling between protein phosphorylation and sperm motility during activation in microG and at 1 G was examined in the ESA Biorack on two space shuttle missions. Immotile sperm were activated to swim (86-90% motility) at launch +20 h by dilution into artificial seawater (ASW). Parallel ground controls were performed 2 h after the flight experiment. Activation after 0, 30, and 60 s was terminated with electrophoresis sample buffer and samples analyzed for phosphoamino acids by Western blotting. Phosphorylation of a 130-kDa phosphothreonine-containing protein (FP130) occurred three to four times faster in microG than at 1 G. A 32-kDa phosphoserine-containing protein was significantly stimulated at 30 s but returned to 1 G control levels at 60 s. The rate of FP130 phosphorylation in microG was attenuated by D2O, suggesting that changes in water properties participate in altering signal transduction. Changes in FP130 phosphorylation triggered by the egg peptide speract were delayed in microG. These results demonstrate that previously observed effects of microG on sperm motility are coupled to changes in phosphorylation of specific flagellar proteins and that early events of sperm activation and fertilization are altered in microG.

  6. Tumor promoters alter gene expression and protein phosphorylation in avian cells in culture

    SciTech Connect

    Laszlo, A.; Radke, K.; Chin, S.; Bissell, M.J.

    1981-10-01

    We have investigated the effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the synthesis and modification of polypeptides in normal avian cells and cells infected by wild-type and temperature-sensitive Rous sarcoma virus (RSV). Using two-dimensional gel electrophoresis, we have detected alterations in both the abundance of cellular polypeptides and in their phosphorylation that seem unique to TPA treatment. However, the state of phosphorylation of the major putative substrate for the action of the src gene-associated protein kinase, the 34- to 36-kilodalton protein, was not altered. Moreover, examination of the phosphorylated amino acid content of total cellular phosphoproteins revealed that the response to TPA was not associated with detectable increases in their phosphotyrosine content. These results make it unlikely that TPA acts by the activation of the phosphorylating activity of the cellular proto-src gene or by the activation of other cellular phosphotyrosine-specific kinases. We have shown previously that temperature-sensitive RSV-infected cells at nonpermissive temperature demonstrate an increased sensitivity to TPA treatment (Bissell, M.J., Hatie, C. and Calfin, M. (1979) Proc. Natl. Acad. Sci. USA 76, 348-352). Our present results indicate that this is not due to reactivation of the phosphorylating activity of the defective src gene product or to its leakiness, and they lend support to the notion of multistep viral carcinogenesis.

  7. Altered phosphorylation of. tau. protein in heat-shocked rats and patients with Alzheimer disease

    SciTech Connect

    Papasozomenos, S.C.; Yuan Su Baylor College of Medicine, Houston, TX )

    1991-05-15

    Six hours after heat shocking 2- to 3-month-old male and female Sprague-Dawley rats at 42C for 15 min, the authors analyzed {tau} protein immunoreactivity in SDS extracts of cerebrums and peripheral nerves by using immunoblot analysis and immunohistochemistry with the anti-{tau} monoclonal antibody Tau-1, which recognizes a phosphate-dependent nonphosphorylated epitope, and with {sup 125}I-labeled protein A. In the cerebal extracts, the authors found altered phosphorylation of {tau} in heat-shocked females, characterized by a marked reduction in the amount of nonphosphorylated {tau}, a doubling of the ratio of total (phosphorylated plus nonphosphorylated) {tau} to nonphosphorylated {tau}, and the appearance of the slowest moving phosphorylated {tau} polypeptide (68 kDa). Similar, but milder, changes were observed in male rats. Quantitative immunoblot analysis of cortex and the underlying white matter with Tau-1 and {sup 125}I-labeled protein A showed that the amount of phosphorylated {tau} progressively increased in the Alzheimer disease-affected cerebral cortex, while concurrently a proportionally lesser amount of {tau} entered the white matter axons. The similar findings for the rat heat-shock model and Alzheimer disease suggest that life stressors may play a role in the etiopathogenesis of Alzheimer's disease.

  8. CA+/CALMODULIN-DEPENDENT PROTEIN PHOSPHORYLATION IS NOT ALTERED BY AMYGDALOID KINDLING

    EPA Science Inventory

    The effects of amygdaloid kindling on Ca2+/Calmodulin (CaM) dependent protein phosphorylation were assessed using one- and two-dimensional gel electrophoresis. n vitro phosphorylation of membrane and cytosol fractions in the presence or absence of Ca2+/CaM did not differentiate b...

  9. Acrylamide administration alters protein phosphorylation and phospholipid metabolism in rat sciatic nerve

    SciTech Connect

    Berti-Mattera, L.N.; Eichberg, J.; Schrama, L.; LoPachin, R.M. )

    1990-05-01

    The effects of ACR on protein phosphorylation and phospholipid metabolism were assessed in rat sciatic nerve. After 5 days of ACR administration (50 mg/kg/day) an increase in the incorporation of 32P into phosphatidylinositol-4,5-bisphosphate, phosphatidylinositol-4-phosphate, and phosphatidylcholine was detected in proximal sciatic nerve segments. In contrast, no changes in phospholipid metabolism were observed in distal segments. After 9 days of ACR treatment when neurotoxicological symptoms were clearly apparent, a generalized increase in radiolabel uptake into phospholipids was noted exclusively in proximal nerve regions. ACR-induced increases in phospholipid metabolism were toxicologically specific since comparable administration of MBA (108 mg/kg/day X 5 or 9 days) produced only minor changes. ACR intoxication was also associated with a rise in sciatic nerve protein phosphorylation. After 9 days of ACR treatment, phosphorylation of beta-tubulin, P0, and several unidentified proteins (38 and 180 kDa) was increased in distal segments. In contrast, chronic administration of MBA caused increases in phosphorylation of beta-tubulin and the major myelin proteins of proximal nerve segments. In cell free homogenates prepared from sciatic nerves of treated and control rats, MBA caused an increase in phosphorylation of major myelin proteins similar to its effect in intact proximal nerve segments. The most striking effect observed in nerve homogenates of ACR-treated rats was a marked decrease in phosphorylation of an 80-kDa protein. Addition of ACR (1 mM) to homogenates of normal nerve had no effect on protein phosphorylation. Our results indicate that changes in the phosphorylation of phospholipids and proteins in sciatic nerve might be a component of the neurotoxic mechanism of ACR.

  10. Mutations associated with retinopathies alter mitogen-activated protein kinase-induced phosphorylation of neural retina leucine-zipper

    PubMed Central

    Kumar, Sandeep; Patel, Dharmesh; Richong, Sushmita; Oberoi, Pranav; Ghosh, Madhumita; Swaroop, Anand

    2007-01-01

    Purpose Neural retina leucine-zipper (NRL), a member of the basic motif leucine zipper family of transcription factors, is preferentially expressed in rod photoreceptors of the mammalian retina. Mutations in NRL are associated with retinopathies; many of these are suggested to change phosphorylation status and alter NRL-mediated transactivation of rhodopsin promoter. The purpose of this study was to identify potential kinases responsible for the phosphorylation of NRL and determine if such kinase-dependent phosphorylation is altered in disease-associated NRL mutations. Methods Metabolic labeling with 33P-orthophosphate was used to study phosphorylation of NRL in transfected COS-1 cells. NRL or NRL mutants were expressed as glutathione S-transferase (GST)-fusion proteins and used as substrate to screen various kinases by in vitro phosphorylation assays. CV-1 cells were co-transfected with rhodopsin promoter-reporter construct and expression plasmids, with or without specific mitogen-activated protein kinase (MAPK) inhibitors, to examine their effect on NRL-mediated transactivation. Expression of activated MAPKs in postnatal mice retina was determined by immunoblot analysis. Results Metabolic labeling of NRL produces multiple phosphorylated protein bands in transfected COS-1 cells. Fewer but more intense radiolabeled bands are observed for NRL-S50T, -S50A, and -P51L mutants compared to wild-type NRL. We show that MAPK2 and p38 induce specific phosphorylation of NRL, but this pattern is altered in NRL mutants. Immunoblot analysis of extracts from developing mouse retina reveals enhanced expression of activated MAPK2 at postnatal day 0-3, concordant with the reported phosphorylation pattern of NRL in vivo. Inhibition of MAPK signaling pathways decreases NRL and CRX -mediated synergistic activation of rhodopsin promoter in transfected CV-1 cells. Conclusions Our results suggest that multiple MAPKs can phosphorylate NRL and this phosphorylation pattern is altered by

  11. Altered protein phosphorylation in sciatic nerve from rats with streptozocin-induced diabetes

    SciTech Connect

    Schrama, L.H.; Berti-Mattera, L.N.; Eichberg, J.

    1987-11-01

    The effect of experimental diabetes on the phosphorylation of proteins in the rat sciatic nerve was studied. Nerves from animals made diabetic with streptozocin were incubated in vitro with (/sup 32/P)orthophosphate and divided into segments from the proximal to the distal end, and proteins from each segment were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The principal labeled species were the major myelin proteins, P0, and the basic proteins. After 6 wk of diabetes, the incorporation of isotope into these proteins rose as a function of distance along the nerve in a proximal to distal direction and was significantly higher at the distal end compared with incorporation into nerves from age-matched controls. The overall level of isotope uptake was similar in nerves from diabetic animals and weight-matched controls. The distribution of /sup 32/P among proteins also differed in diabetic nerve compared with both control groups in that P0 and the small basic protein accounted for a greater proportion of total label incorporated along the entire length of nerve. In contrast to intact nerve, there was no significant difference in protein phosphorylation when homogenates from normal and diabetic nerve were incubated with (/sup 32/P)-gamma-ATP. The results suggest that abnormal protein phosphorylation, particularly of myelin proteins, is a feature of experimental diabetic neuropathy and that the changes are most pronounced in the distal portion of the nerve.

  12. Zinc ions and alkaline pH alter the phosphorylation state of human erythrocyte membrane proteins

    SciTech Connect

    Fennell, R.L. Jr.

    1988-01-01

    Since the phosphorylation state of the red cell membrane proteins in vitro is likely to be regulated by phosphorylation and dephosphorylation, this research was carried out to investigate the possible role of membrane-bound phosphatase activities. These studies were conducted with red blood cell ghosts and IOVs from normal individuals and from an individual with hereditary spherocytosis. In vitro phosphorylation with ({gamma}-{sup 32}P) ATP was conducted in the presence and the absence of Zn{sup ++}, or erythrocyte ghosts and IOVs were pretreated for 30 minutes at 37{degree}C and pH 7-11 in the presence and the absence of calf intestine alkaline phosphatase. The resulting phosphoproteins were analyzed by SDS-polyacrylamide gel electrophoresis, stained with Coomassie blue, and fluorographed. In the presence of Zn{sup ++}, the red blood ghosts, with or without pretreatment, demonstrated enhanced phosphorylation of membrane proteins, including band 4.2. Preincubation at pH 10 in the presence of absence of exogenous phosphatase further stimulates phosphorylation of these proteins. Under similar conditions, the erythrocyte membranes also demonstrated the ability to hydrolyze p-nitrophenyl phosphate and to remove {sup 32}P from red blood cell phosphoproteins.

  13. Mosquito Protein Kinase G Phosphorylates Flavivirus NS5 and Alters Flight Behavior in Aedes aegypti and Anopheles gambiae

    PubMed Central

    Keating, Julie A.; Bhattacharya, Dipankar; Rund, Samuel S.C.; Hoover, Spencer; Dasgupta, Ranjit; Lee, Samuel J.; Duffield, Giles E.

    2013-01-01

    Abstract Many arboviral proteins are phosphorylated in infected mammalian cells, but it is unknown if the same phosphorylation events occur when insects are similarly infected. One of the mammalian kinases responsible for phosphorylation, protein kinase G (PKG), has been implicated in the behavior of multiple nonvector insects, but is unstudied in mosquitoes. PKG from Aedes aegypti was cloned, and phosphorylation of specific viral sites was monitored by mass spectrometry from biochemical and cell culture experiments. PKG from Aedes mosquitoes is able to phosphorylate dengue nonstructural protein 5 (NS5) at specific sites in cell culture and cell-free systems and autophosphorylates its own regulatory domain in a cell-free system. Injecting Aedes aegypti and Anopheles gambiae mosquitoes with a pharmacological PKG activator resulted in increased Aedes wing activity during periods of their natural diurnal/crepuscular activity and increased Anopheles nocturnal locomotor/flight activity. Thus, perturbation of the PKG signaling pathway in mosquitoes alters flight behavior. The demonstrated effect of PKG alterations is consistent with a viral PKG substrate triggering increased PKG activity. This increased PKG activity could be the mechanism by which dengue virus increases flight behavior and possibly facilitates transmission. Whether or not PKG is part of the mechanism by which dengue increases flight behavior, this report is the first to show PKG can modulate behavior in hematophagous disease vectors. PMID:23930976

  14. Experimental manipulation of compaction of the mouse embryo alters patterns of protein phosphorylation

    SciTech Connect

    Bloom, T. )

    1991-03-01

    Compaction, occurring at the eight-cell stage of mouse development, is the process of cell flattening and polarisation by which cellular asymmetry is first established. Changes in the pattern of protein phosphorylation have been correlated with this early event of development. In the study reported here, groups of embryos were treated in ways known to affect particular features of compaction and were then labeled with ({sup 32}P)orthophosphate; the phosphoproteins obtained were examined following electrophoresis in one and two dimensions. Four-cell embryos were treated with protein synthesis inhibitors, which advance cell flattening. This treatment resulted in only minor differences from the phosphoprotein profile of untreated four-cell embryos. Inhibition of protein synthesis at the eight-cell stage has little effect on cell flattening or polarisation. However, some phosphoproteins that are observed normally in eight-cell but not in four-cell embryos were no longer detectable if labeling took place in the presence of protein synthesis inhibitors. Eight-cell embryos incubated in phorbol 12-myristate 13-acetate, which disrupts various features of compaction, showed a relative increase in the phosphorylation of a group of phosphoprotein spots associated with the eight-cell but not with the four-cell stage. Embryos incubated in Ca2(+)-free medium, which prevents intercellular flattening and delays polarisation, showed a relative decrease in the phosphorylation of the same group of phosphoprotein spots. The behaviour of these phosphoproteins may therefore be correlated with some of the features of compaction.

  15. The cyclic nucleotide cGMP is involved in plant hormone signalling and alters phosphorylation of Arabidopsis thaliana root proteins

    PubMed Central

    Isner, Jean Charles; Nühse, Thomas; Maathuis, Frans J. M.

    2012-01-01

    The cyclic nucleotide cGMP has been shown to play important roles in plant development and responses to abiotic and biotic stress. Yet much controversy remains regarding the exact role of this second messenger. Progress in unravelling cGMP function in plants was hampered by laborious and time-consuming methodology to measure changes in cellular [cGMP] but the development of fluorescence-based reporters has removed this disadvantage. This study used the FlincG cGMP reporter to investigate potential interactions between phytohormone and cGMP signalling and found a rapid and significant effect of the hormones abscisic acid (ABA), auxin (IAA), and jasmonic acid (JA) on cytoplasmic cGMP levels. In contrast, brassinosteroids and cytokinin did not evoke a cGMP signal. The effects of ABA, IAA, and JA were apparent at external concentrations in the nanomolar range with EC50 values of around 1000, 300, and 0.03 nmoles for ABA, IAA, and JA respectively. To examine potential mechanisms for how hormone-induced cGMP signals are propagated, the role of protein phosphorylation was tested. A phosphoproteomics analysis on Arabidopsis thaliana root microsomal proteins in the absence and presence of membrane-permeable cGMP showed 15 proteins that rapidly (within minutes) changed in phosphorylation status. Out of these, nine were previously shown to also alter phosphorylation status in response to plant hormones, pointing to protein phosphorylation as a target for hormone-induced cGMP signalling. PMID:22345640

  16. The C2 Domain and Altered ATP-Binding Loop Phosphorylation at Ser359 Mediate the Redox-Dependent Increase in Protein Kinase C-δ Activity

    PubMed Central

    Gong, Jianli; Yao, Yongneng; Zhang, Pingbo; Udayasuryan, Barath; Komissarova, Elena V.; Chen, Ju; Sivaramakrishnan, Sivaraj; Van Eyk, Jennifer E.

    2015-01-01

    The diverse roles of protein kinase C-δ (PKCδ) in cellular growth, survival, and injury have been attributed to stimulus-specific differences in PKCδ signaling responses. PKCδ exerts membrane-delimited actions in cells activated by agonists that stimulate phosphoinositide hydrolysis. PKCδ is released from membranes as a Tyr313-phosphorylated enzyme that displays a high level of lipid-independent activity and altered substrate specificity during oxidative stress. This study identifies an interaction between PKCδ's Tyr313-phosphorylated hinge region and its phosphotyrosine-binding C2 domain that controls PKCδ's enzymology indirectly by decreasing phosphorylation in the kinase domain ATP-positioning loop at Ser359. We show that wild-type (WT) PKCδ displays a strong preference for substrates with serine as the phosphoacceptor residue at the active site when it harbors phosphomimetic or bulky substitutions at Ser359. In contrast, PKCδ-S359A displays lipid-independent activity toward substrates with either a serine or threonine as the phosphoacceptor residue. Additional studies in cardiomyocytes show that oxidative stress decreases Ser359 phosphorylation on native PKCδ and that PKCδ-S359A overexpression increases basal levels of phosphorylation on substrates with both phosphoacceptor site serine and threonine residues. Collectively, these studies identify a C2 domain-pTyr313 docking interaction that controls ATP-positioning loop phosphorylation as a novel, dynamically regulated, and physiologically relevant structural determinant of PKCδ catalytic activity. PMID:25755284

  17. Cisplatin stimulates protein tyrosine phosphorylation in macrophages.

    PubMed

    Kumar, R; Shrivastava, A; Sodhi, A

    1995-03-01

    Cisplatin [cis-dichlorodiamine platinum (II)], a potent anti-tumor compound, stimulates immune responses by activating monocyte-macrophages and other cells of the immune system. The mechanism by which cisplatin activates these cells is poorly characterized. Since protein tyrosine phosphorylation appears to be a major intracellular signalling event that mediates cellular responses, we examined whether cisplatin alters tyrosine phosphorylation in macrophages. We found that cisplatin increased tyrosine phosphorylation of several proteins in peritoneal macrophages and in P388D1 and IC-21 macrophage cell lines. Treatment of macrophages with tyrosine kinase inhibitors, genestein and lavendustin A, inhibited cisplatin-stimulated protein tyrosine phosphorylation in macrophages. Macrophages treated with cisplatin also exhibit increased fluorescence with anti-phosphotyrosine-FITC antibody. These data indicate that protein tyrosine phosphorylation plays a role in cisplatin-induced activation of macrophages. PMID:7539662

  18. Protein phosphorylation in stomatal movement

    PubMed Central

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2014-01-01

    As research progresses on how guard cells perceive and transduce environmental cues to regulate stomatal movement, plant biologists are discovering key roles of protein phosphorylation. Early research efforts focused on characterization of ion channels and transporters in guard cell hormonal signaling. Subsequent genetic studies identified mutants of kinases and phosphatases that are defective in regulating guard cell ion channel activities, and recently proteins regulated by phosphorylation have been identified. Here we review the essential role of protein phosphorylation in ABA-induced stomatal closure and in blue light-induced stomatal opening. We also highlight evidence for the cross-talk between different pathways, which is mediated by protein phosphorylation. PMID:25482764

  19. Protein phosphorylation in stomatal movement.

    PubMed

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2014-01-01

    As research progresses on how guard cells perceive and transduce environmental cues to regulate stomatal movement, plant biologists are discovering key roles of protein phosphorylation. Early research efforts focused on characterization of ion channels and transporters in guard cell hormonal signaling. Subsequent genetic studies identified mutants of kinases and phosphatases that are defective in regulating guard cell ion channel activities, and recently proteins regulated by phosphorylation have been identified. Here we review the essential role of protein phosphorylation in ABA-induced stomatal closure and in blue light-induced stomatal opening. We also highlight evidence for the cross-talk between different pathways, which is mediated by protein phosphorylation. PMID:25482764

  20. Cryopreservation-induced alterations in protein tyrosine phosphorylation of spermatozoa from different portions of the boar ejaculate.

    PubMed

    Kumaresan, A; Siqueira, A P; Hossain, M S; Bergqvist, A S

    2011-12-01

    Previous studies have shown that boar sperm quality after cryopreservation differs depending on the ejaculate fraction used and that spermatozoa contained in the first 10mL (P1) of the sperm-rich fraction (SRF) show better cryosurvival than those in the SRF-P1. Since protein tyrosine phosphorylation (PTP) in spermatozoa is related with the tolerance of spermatozoa to frozen storage and cryocapacitation, we assessed the dynamics of cryopreservation-induced PTP and intracellular calcium ([Ca(2+)]i) in spermatozoa, using flow cytometry, from P1 and SRF-P1 of the boar ejaculate at different stages of cryopreservation. Sperm kinetics, assessed using a computer-assisted semen analyzer, did not differ between P1 and SRF-P1 during cryopreservation but the decrease in sperm velocity during cryopreservation was significant (P<0.05) in SRF-P1 compared to P1. There were no significant differences in percentages of spermatozoa with high [Ca(2+)]i between P1 and SRF-P1 in fresh as well as in frozen-thawed semen. A higher (P<0.001) proportion of spermatozoa displayed PTP during the course of cryopreservation indicating a definite effect of the cryopreservation process on sperm PTP. The proportion of spermatozoa with PTP did not differ significantly between portions of the boar ejaculate. However at any given step during cryopreservation the percentage of spermatozoa with PTP was comparatively higher in SRF-P1 than P1. A 32kDa tyrosine phosphorylated protein, associated with capacitation, appeared after cooling suggesting that cooling induces capacitation-like changes in boar spermatozoa. In conclusion, the study has shown that the cryopreservation process induced PTP in spermatozoa and their proportions were similar between portions of SRF. PMID:21893053

  1. SYMPOSIUM ON PLANT PROTEIN PHOSPHORYLATION

    SciTech Connect

    JOHN C WALKER

    2011-11-01

    Protein phosphorylation and dephosphorylation play key roles in many aspects of plant biology, including control of cell division, pathways of carbon and nitrogen metabolism, pattern formation, hormonal responses, and abiotic and biotic responses to environmental signals. A Symposium on Plant Protein Phosphorylation was hosted on the Columbia campus of the University of Missouri from May 26-28, 2010. The symposium provided an interdisciplinary venue at which scholars studying protein modification, as it relates to a broad range of biological questions and using a variety of plant species, presented their research. It also provided a forum where current international challenges in studies related to protein phosphorylation could be examined. The symposium also stimulated research collaborations through interactions and networking among those in the research community and engaged students and early career investigators in studying issues in plant biology from an interdisciplinary perspective. The proposed symposium, which drew 165 researchers from 13 countries and 21 States, facilitated a rapid dissemination of acquired knowledge and technical expertise regarding protein phosphorylation in plants to a broad range of plant biologists worldwide.

  2. Autophagy proteins regulate ERK phosphorylation

    PubMed Central

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

    2013-01-01

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

  3. Phosphorylation of human link proteins

    SciTech Connect

    Oester, D.A.; Caterson, B.; Schwartz, E.R.

    1986-06-13

    Three link proteins of 48, 44 and 40 kDa were purified from human articular cartilage and identified with monoclonal anti-link protein antibody 8-A-4. Two sets of lower molecular weight proteins of 30-31 kDa and 24-26 kDa also contained link protein epitopes recognized by the monoclonal antibody and were most likely degradative products of the intact link proteins. The link proteins of 48 and 40 kDa were identified as phosphoproteins while the 44 kDa link protein did not contain /sup 32/P. The phosphorylated 48 and 40 kDa link proteins contained approximately 2 moles PO/sub 4//mole link protein.

  4. Effect of epidermal growth factor (EGF) on the phosphorylation of mitogen-activated protein kinase (MAPK) in the bovine oviduct in vitro: Alteration by heat stress

    PubMed Central

    WIJAYAGUNAWARDANE, Missaka P. B.; HAMBRUCH, Nina; HAEGER, Jan-Dirk; PFARRER, Christiane

    2015-01-01

    Epidermal growth factor (EGF) has been shown to be involved in control of the oviductal microenvironment. To elucidate the potential mechanisms responsible for the detrimental effect of heat stress and to identify the relation with the endocrine status, the effects of EGF on the level of phosphorylated mitogen-activated-protein kinase (MAPK) and proliferation of bovine oviductal epithelial cells (OECs) exposed to different cyclic ovarian steroidal environments (luteal phase (LP), follicular phase (FP) and postovulatory phase (PO)) and temperatures (mild heat stress (40 C) and severe heat stress (43 C)) were investigated. Western blot was performed to evaluate phosphorylated MAPK, while proliferation was analyzed by MTT assay. Stimulation of OECs with EGF alone or with EGF in the PO and FP environments significantly increased the amount of phosphorylated MAPK, with MAPK 44 phosphorylation being highest during exposure to PO conditions. These effects were not observed in the LP. Heat treatment completely blocked effects of EGF on phosphorylated MAPK. Additionally, severe heat stress led to a significantly lower basal level of phosphorylated MAPK. PD98059 (MAPK inhibitor) completely abolished EGF-stimulated MAPK phosphorylation and OECs proliferation. Overall the results indicate that EGF has the potential to increase the amount of phosphorylated MAPK in OECs and therefore could be involved in regulation of the bovine oviductal microenvironment. However, these regulatory mechanisms may be compromised in the presence of heat stress (high ambient temperature), leading to low fertility rates and impaired embryo survival. PMID:26050642

  5. Myosin Binding Protein-C Slow Phosphorylation is Altered in Duchenne Dystrophy and Arthrogryposis Myopathy in Fast-Twitch Skeletal Muscles

    PubMed Central

    Ackermann, Maegen A.; Ward, Christopher W.; Gurnett, Christina; Kontrogianni-Konstantopoulos, Aikaterini

    2015-01-01

    Myosin Binding Protein-C slow (sMyBP-C), encoded by MYBPC1, comprises a family of regulatory proteins of skeletal muscles that are phosphorylated by PKA and PKC. MYBPC1 missense mutations are linked to the development of Distal Arthrogryposis-1 (DA-1). Although structure-function details for this myopathy are evolving, function is undoubtedly driven by sequence variations and post-translational modifications in sMyBP-C. Herein, we examined the phosphorylation profile of sMyBP-C in mouse and human fast-twitch skeletal muscles. We used Flexor Digitorum Brevis (FDB) isolated from young (~2-months old) and old (~14-months old) wild type and mdx mice, and human Abductor Hallucis (AH) and gastrocnemious muscles carrying the DA-1 mutations. Our results indicate both constitutive and differential phosphorylation of sMyBP-C in aged and diseased muscles. We report a 7–35% reduction in the phosphorylation levels of select sites in old wild type and young or old mdx FDB mouse muscles, compared to young wild type tissue. Similarly, we observe a 30–70% decrease in the phosphorylation levels of all PKA and PKC phospho-sites in the DA-1 AH, but not gastrocnemius, muscle. Overall, our studies show that the phosphorylation pattern of sMyBP-C is differentially regulated in response to age and disease, suggesting that phosphorylation plays important roles in these processes. PMID:26287277

  6. Cellular regulation by protein phosphorylation.

    PubMed

    Fischer, Edmond H

    2013-01-11

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

  7. Interfacing protein lysine acetylation and protein phosphorylation

    PubMed Central

    Tran, Hue T.; Uhrig, R. Glen; Nimick, Mhairi; Moorhead, Greg B.

    2012-01-01

    Recognition that different protein covalent modifications can operate in concert to regulate a single protein has forced us to re-think the relationship between amino acid side chain modifications and protein function. Results presented by Tran et al. 2012 demonstrate the association of a protein phosphatase (PP2A) with a histone/lysine deacetylase (HDA14) on plant microtubules along with a histone/lysine acetyltransferase (ELP3). This finding reveals a regulatory interface between two prevalent covalent protein modifications, protein phosphorylation and acetylation, emphasizing the integrated complexity of post-translational protein regulation found in nature. PMID:22827947

  8. Platelet activation via the collagen receptor GPVI is not altered in platelets from chronic myeloid leukaemia patients despite the presence of the constitutively phosphorylated adapter protein CrkL.

    PubMed

    Best, D; Pasquet, S; Littlewood, T J; Brunskill, S J; Pallister, C J; Watson, S P

    2001-03-01

    In this study, we show that the adapter proteins CrkL and Cbl undergo increases in tyrosine phosphorylation and form an intracellular complex in platelets stimulated with the snake venom toxin convulxin, a selective agonist at the collagen receptor glycoprotein VI (GPVI). Constitutive tyrosine phosphorylation of CrkL has previously been reported in platelets from chronic myeloid leukaemia (CML) patients. This was confirmed in the present study, and shown to result in a weak constitutive association of CrkL with Cbl and a number of other unidentified tyrosine-phosphorylated proteins. There was no further increase in phosphorylation of CrkL in CML platelets in response to GPVI activation, whereas phosphorylation of Cbl and its association with CrkL were potentiated. In addition, this was accompanied by a small increase in p42/ 44 mapkinase (MAPK) activity in CML platelets. The functional consequence of the presence of constitutively phosphorylated proteins in CML platelets was investigated by measurement of aminophospholipid exposure and alpha-granule secretion. This revealed little alteration in the concentration-response curves for either in CML platelets stimulated via GPVI, although maximal levels of P-selectin were depressed. Despite the minimal effect on platelet activation in CML patients, we cannot exclude a role for CrkL or Cbl in signal transduction pathways stimulated via GPVI. PMID:11260061

  9. Mycobacterium tuberculosis supports protein tyrosine phosphorylation

    PubMed Central

    Kusebauch, Ulrike; Ortega, Corrie; Ollodart, Anja; Rogers, Richard S.; Sherman, David R.; Moritz, Robert L.; Grundner, Christoph

    2014-01-01

    Reversible protein phosphorylation determines growth and adaptive decisions in Mycobacterium tuberculosis (Mtb). At least 11 two-component systems and 11 Ser/Thr protein kinases (STPKs) mediate phosphorylation on Asp, His, Ser, and Thr. In contrast, protein phosphorylation on Tyr has not been described previously in Mtb. Here, using a combination of phospho-enrichment and highly sensitive mass spectrometry, we show extensive protein Tyr phosphorylation of diverse Mtb proteins, including STPKs. Several STPKs function as dual-specificity kinases that phosphorylate Tyr in cis and in trans, suggesting that dual-specificity kinases have a major role in bacterial phospho-signaling. Mutation of a phosphotyrosine site of the essential STPK PknB reduces its activity in vitro and in live Mtb, indicating that Tyr phosphorylation has a functional role in bacterial growth. These data identify a previously unrecognized phosphorylation system in a human pathogen that claims ∼1.4 million lives every year. PMID:24927537

  10. Partial regeneration and long-term survival of rat retinal ganglion cells after optic nerve crush is accompanied by altered expression, phosphorylation and distribution of cytoskeletal proteins.

    PubMed

    Dieterich, Daniela C; Trivedi, Niraj; Engelmann, Ralf; Gundelfinger, Eckart D; Gordon-Weeks, Phillip R; Kreutz, Michael R

    2002-05-01

    In a screen to identify genes that are expressed differentially in the retina after partial optic nerve crush, we identified MAP1B as an up-regulated transcript. Western blot analysis of inner retina protein preparations confirmed changes in the protein composition of the microtubule-associated cytoskeleton of crushed vs. uncrushed nerve. MAP1B immunoreactivity and transcript levels were elevated for two weeks after crush. Immunostaining and Western blots with monoclonal antibodies directed against developmentally regulated phosphorylation sites on MAP1B revealed a gradient of MAP1B phosphorylation from the proximal optic nerve stump to the soma of retinal ganglion cells. Most interestingly, using antibodies directed against developmentally regulated phosphorylation sites on MAP1B, we observed that a significant number of crushed optic nerve axons develop MAP1B-immunopositive growth cones, which cross the crush site and migrate along the distal nerve fragment. In parallel, an abnormal distribution of highly phosphorylated neurofilament protein (pNF-H) in the cell soma and dendrites of presumably axotomized retinal ganglion cells was observed following partial nerve crush. This redistribution is present for the period between day 7 and 28 postcrush and is not seen in cells that stay connected to the superior colliculus. Axotomized ganglion cells, which contain pNF-H in soma and dendrites appear to have been disconnected from the colliculus at an early stage but survive axonal trauma for long periods. PMID:12028353

  11. Altered protein phosphatase 2A methylation and Tau phosphorylation in the young and aged brain of methylenetetrahydrofolate reductase (MTHFR) deficient mice

    PubMed Central

    Sontag, Jean-Marie; Wasek, Brandi; Taleski, Goce; Smith, Josephine; Arning, Erland; Sontag, Estelle; Bottiglieri, Teodoro

    2014-01-01

    Common functional polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene, a key enzyme in folate and homocysteine metabolism, influence risk for a variety of complex disorders, including developmental, vascular, and neurological diseases. MTHFR deficiency is associated with elevation of homocysteine levels and alterations in the methylation cycle. Here, using young and aged Mthfr knockout mouse models, we show that mild MTHFR deficiency can lead to brain-region specific impairment of the methylation of Ser/Thr protein phosphatase 2A (PP2A). Relative to wild-type controls, decreased expression levels of PP2A and leucine carboxyl methyltransferase (LCMT1) were primarily observed in the hippocampus and cerebellum, and to a lesser extent in the cortex of young null Mthfr−/− and aged heterozygous Mthfr+/− mice. A marked down regulation of LCMT1 correlated with the loss of PP2A/Bα holoenzymes. Dietary folate deficiency significantly decreased LCMT1, methylated PP2A and PP2A/Bα levels in all brain regions examined from aged Mthfr+/+ mice, and further exacerbated the regional effects of MTHFR deficiency in aged Mthfr+/− mice. In turn, the down regulation of PP2A/Bα was associated with enhanced phosphorylation of Tau, a neuropathological hallmark of Alzheimer’s disease (AD). Our findings identify hypomethylation of PP2A enzymes, which are major CNS phosphatases, as a novel mechanism by which MTHFR deficiency and Mthfr gene-diet interactions could lead to disruption of neuronal homeostasis, and increase the risk for a variety of neuropsychiatric disorders, including age-related diseases like sporadic AD. PMID:25202269

  12. Altered protein phosphatase 2A methylation and Tau phosphorylation in the young and aged brain of methylenetetrahydrofolate reductase (MTHFR) deficient mice.

    PubMed

    Sontag, Jean-Marie; Wasek, Brandi; Taleski, Goce; Smith, Josephine; Arning, Erland; Sontag, Estelle; Bottiglieri, Teodoro

    2014-01-01

    Common functional polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene, a key enzyme in folate and homocysteine metabolism, influence risk for a variety of complex disorders, including developmental, vascular, and neurological diseases. MTHFR deficiency is associated with elevation of homocysteine levels and alterations in the methylation cycle. Here, using young and aged Mthfr knockout mouse models, we show that mild MTHFR deficiency can lead to brain-region specific impairment of the methylation of Ser/Thr protein phosphatase 2A (PP2A). Relative to wild-type controls, decreased expression levels of PP2A and leucine carboxyl methyltransferase (LCMT1) were primarily observed in the hippocampus and cerebellum, and to a lesser extent in the cortex of young null Mthfr (-/-) and aged heterozygous Mthfr (+/-) mice. A marked down regulation of LCMT1 correlated with the loss of PP2A/Bα holoenzymes. Dietary folate deficiency significantly decreased LCMT1, methylated PP2A and PP2A/Bα levels in all brain regions examined from aged Mthfr (+/+) mice, and further exacerbated the regional effects of MTHFR deficiency in aged Mthfr (+/-) mice. In turn, the down regulation of PP2A/Bα was associated with enhanced phosphorylation of Tau, a neuropathological hallmark of Alzheimer's disease (AD). Our findings identify hypomethylation of PP2A enzymes, which are major CNS phosphatases, as a novel mechanism by which MTHFR deficiency and Mthfr gene-diet interactions could lead to disruption of neuronal homeostasis, and increase the risk for a variety of neuropsychiatric disorders, including age-related diseases like sporadic AD. PMID:25202269

  13. Protein phosphorylation in isolated hepatocytes of septic and endotoxemic rats

    SciTech Connect

    Deaciuc, I.V.; Spitzer, J.A. )

    1989-11-01

    The purpose of this study was to investigate possible alterations induced by sepsis and endotoxicosis in the late phase of Ca2+-dependent signaling in rat liver. Hepatocytes isolated from septic or chronically endotoxin (ET)-treated rats were labeled with (32P)H3PO4 and stimulated with various agents. Proteins were resolved by one-dimensional polyacrylamide gel electrophoresis and autoradiographed. Vasopressin (VP)- and phenylephrine (PE)-induced responses were attenuated in both septic and ET-treated rats for cytosolic and membrane proteins compared with their respective controls. Glucagon and 12-O-myristate phorbol-13-acetate (TPA) affected only the phosphorylation of membrane proteins. Glucagon-induced changes in the phosphorylation of membrane proteins were affected by both sepsis and endotoxicosis, whereas TPA-stimulated phosphorylation was lowered only in endotoxicosis. Response to the Ca2+ ionophore A23187 was depressed in septic rats for cytosolic proteins. The phosphorylation of two cytosolic proteins, i.e., 93 and 61 kDa (previously identified as glycogen phosphorylase and pyruvate kinase, respectively), in response to VP, PE, and A23187 was severely impaired by endotoxicosis and sepsis. TPA did not affect the phosphorylation state of these two proteins. The results show that sepsis and endotoxicosis produce perturbations of the phosphorylation step in Ca2+ transmembrane signaling. Such changes can explain alterations of glycogenolysis and gluconeogenesis associated with sepsis and endotoxicosis.

  14. Protein phosphorylation in chloroplasts - a survey of phosphorylation targets.

    PubMed

    Baginsky, Sacha

    2016-06-01

    The development of new software tools, improved mass spectrometry equipment, a suite of optimized scan types, and better-quality phosphopeptide affinity capture have paved the way for an explosion of mass spectrometry data on phosphopeptides. Because phosphoproteomics achieves good sensitivity, most studies use complete cell extracts for phosphopeptide enrichment and identification without prior enrichment of proteins or subcellular compartments. As a consequence, the phosphoproteome of cell organelles often comes as a by-product from large-scale studies and is commonly assembled from these in meta-analyses. This review aims at providing some guidance on the limitations of meta-analyses that combine data from analyses with different scopes, reports on the current status of knowledge on chloroplast phosphorylation targets, provides initial insights into phosphorylation site conservation in different plant species, and highlights emerging information on the integration of gene expression with metabolism and photosynthesis by means of protein phosphorylation. PMID:26969742

  15. In the Beginning, There Was Protein Phosphorylation

    PubMed Central

    Kyriakis, John M.

    2014-01-01

    The importance of reversible protein phosphorylation to cellular regulation cannot be overstated. In eukaryotic cells, protein kinase/phosphatase signaling pathways regulate a staggering number of cellular processes, including cell proliferation, cell death (apoptosis, necroptosis, necrosis), metabolism (at both the cellular and organismal levels), behavior and neurological function, development, and pathogen resistance. Although protein phosphorylation as a mode of eukaryotic cell regulation is familiar to most biochemists, many are less familiar with protein kinase/phosphatase signaling networks that function in prokaryotes. In this thematic minireview series, we present four minireviews that cover the important field of prokaryotic protein phosphorylation. PMID:24554697

  16. Fibronectin phosphorylation by ecto-protein kinase

    SciTech Connect

    Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru )

    1988-12-01

    The presence of membrane-associated, extracellular protein kinase (ecto-protein kinase) and its substrate proteins was examined with serum-free cultures of Swiss 3T3 fibroblast. When cells were incubated with ({gamma}-{sup 32})ATP for 10 min at 37{degree}C, four proteins with apparent molecular weights between 150 and 220 kDa were prominently phosphorylated. These proteins were also radiolabeled by lactoperoxidase catalyzed iodination and were sensitive to mild tryptic digestion, suggesting that they localized on the cell surface or in the extracellular matrix. Phosphorylation of extracellular proteins with ({gamma}-{sup 32}P)ATP in intact cell culture is consistent with the existence of ecto-protein kinase. Anti-fibronectin antibody immunoprecipitated one of the phosphoproteins which comigrated with a monomer and a dimer form of fibronectin under reducing and nonreducing conditions of electrophoresis, respectively. The protein had affinity for gelatin as demonstrated by retention with gelatin-conjugated agarose. This protein substrate of ecto-protein kinase was thus concluded to be fibronectin. The sites of phosphorylation by ecto-protein kinase were compared with those of intracellularly phosphorylated fibronectin by the analysis of radiolabeled amino acids and peptides. Ecto-protein kinase phosphorylated fibronectin at serine and threonine residues which were distinct from the sites of intracellular fibronectin phosphorylation.

  17. Protein phosphorylation: Localization in regenerating optic axons

    SciTech Connect

    Larrivee, D. )

    1990-09-01

    A number of axonal proteins display changes in phosphorylation during goldfish optic nerve regeneration. (1) To determine whether the phosphorylation of these proteins was closely linked to their synthesis in the retinal ganglion cell body, cycloheximide was injected intraocularly into goldfish whose optic nerves had been regenerating for 3 weeks. Cycloheximide reduced the incorporation of (3H)proline and 32P orthophosphate into total nerve protein by 84% and 46%, respectively. Of the 20 individual proteins examined, 17 contained less than 15% of the (3H)proline label measured in corresponding controls, whereas 18 proteins contained 50% or more of the 32P label, suggesting that phosphorylation was largely independent of synthesis. (2) To determine whether the proteins were phosphorylated in the ganglion cell axons, axonal transport of proteins was blocked by intraocular injection of vincristine. Vincristine reduced (3H)proline labeling of total protein by 88% and 32P labeling by 49%. Among the individual proteins (3H)proline labeling was reduced by 90% or more in 18 cases but 32P labeling was reduced only by 50% or less. (3) When 32P was injected into the cranial cavity near the ends of the optic axons, all of the phosphoproteins were labeled more intensely in the optic tract than in the optic nerve. These results suggest that most of the major phosphoproteins that undergo changes in phosphorylation in the course of regeneration are phosphorylated in the optic axons.

  18. Protein phosphorylation in neurodegeneration: friend or foe?

    PubMed Central

    Tenreiro, Sandra; Eckermann, Katrin; Outeiro, Tiago F.

    2014-01-01

    Protein misfolding and aggregation is a common hallmark in neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), and fronto-temporal dementia (FTD). In these disorders, the misfolding and aggregation of specific proteins occurs alongside neuronal degeneration in somewhat specific brain areas, depending on the disorder and the stage of the disease. However, we still do not fully understand the mechanisms governing protein aggregation, and whether this constitutes a protective or detrimental process. In PD, alpha-synuclein (aSyn) forms protein aggregates, known as Lewy bodies, and is phosphorylated at serine 129. Other residues have also been shown to be phosphorylated, but the significance of phosphorylation in the biology and pathophysiology of the protein is still controversial. In AD and in FTD, hyperphosphorylation of tau protein causes its misfolding and aggregation. Again, our understanding of the precise consequences of tau phosphorylation in the biology and pathophysiology of the protein is still limited. Through the use of a variety of model organisms and technical approaches, we are now gaining stronger insight into the effects of phosphorylation in the behavior of these proteins. In this review, we cover recent findings in the field and discuss how targeting phosphorylation events might be used for therapeutic intervention in these devastating diseases of the nervous system. PMID:24860424

  19. Protein phosphorylation during Plasmodium berghei gametogenesis.

    PubMed

    Alonso-Morales, Alberto; González-López, Lorena; Cázares-Raga, Febe Elena; Cortés-Martínez, Leticia; Torres-Monzón, Jorge Aurelio; Gallegos-Pérez, José Luis; Rodríguez, Mario Henry; James, Anthony A; Hernández-Hernández, Fidel de la Cruz

    2015-09-01

    Plasmodium gametogenesis within the mosquito midgut is a complex differentiation process involving signaling mediated by phosphorylation, which modulate metabolic routes and protein synthesis required to complete this development. However, the mechanisms leading to gametogenesis activation are poorly understood. We analyzed protein phosphorylation during Plasmodium berghei gametogenesis in vitro in serum-free medium using bidimensional electrophoresis (2-DE) combined with immunoblotting (IB) and antibodies specific to phosphorylated serine, threonine and tyrosine. Approximately 75 protein exhibited phosphorylation changes, of which 23 were identified by mass spectrometry. These included components of the cytoskeleton, heat shock proteins, and proteins involved in DNA synthesis and signaling pathways among others. Novel phosphorylation events support a role for these proteins during gametogenesis. The phosphorylation sites of six of the identified proteins, HSP70, WD40 repeat protein msi1, enolase, actin-1 and two isoforms of large subunit of ribonucleoside reductase were investigated using TiO2 phosphopeptides enrichment and tandem mass spectrometry. In addition, transient exposure to hydroxyurea, an inhibitor of ribonucleoside reductase, impaired male gametocytes exflagellation in a dose-dependent manner, and provides a resource for functional studies. PMID:26008612

  20. The Chemical Biology of Protein Phosphorylation

    PubMed Central

    Tarrant, Mary Katherine; Cole, Philip A.

    2011-01-01

    The explosion of scientific interest in protein kinase-mediated signaling networks has led to the infusion of new chemical methods and their applications related to the analysis of phosphorylation pathways. We highlight some of these chemical biology approaches across three areas. First, we discuss the development of chemical tools to modulate the activity of protein kinases to explore kinase mechanisms and their contributions to phosphorylation events and cellular processes. Second, we describe chemical techniques developed in the past few years to dissect the structural and functional effects of phosphate modifications at specific sites in proteins. Third, we cover newly developed molecular imaging approaches to elucidate the spatiotemporal aspects of phosphorylation cascades in live cells. Exciting advances in our understanding of protein phosphorylation have been obtained with these chemical biology approaches, but continuing opportunities for technological innovation remain. PMID:19489734

  1. Protein phosphorylation systems in postmortem human brain

    SciTech Connect

    Walaas, S.I.; Perdahl-Wallace, E.; Winblad, B.; Greengard, P. )

    1989-01-01

    Protein phosphorylation systems regulated by cyclic adenosine 3',5'-monophosphate (cyclic AMP), or calcium in conjunction with calmodulin or phospholipid/diacylglycerol, have been studied by phosphorylation in vitro of particulate and soluble fractions from human postmortem brain samples. One-dimensional or two-dimensional gel electrophoretic protein separations were used for analysis. Protein phosphorylation catalyzed by cyclic AMP-dependent protein kinase was found to be highly active in both particulate and soluble preparations throughout the human CNS, with groups of both widely distributed and region-specific substrates being observed in different brain nuclei. Dopamine-innervated parts of the basal ganglia and cerebral cortex contained the phosphoproteins previously observed in rodent basal ganglia. In contrast, calcium/phospholipid-dependent and calcium/calmodulin-dependent protein phosphorylation systems were less prominent in human postmortem brain than in rodent brain, and only a few widely distributed substrates for these protein kinases were found. Protein staining indicated that postmortem proteolysis, particularly of high-molecular-mass proteins, was prominent in deeply located, subcortical regions in the human brain. Our results indicate that it is feasible to use human postmortem brain samples, when obtained under carefully controlled conditions, for qualitative studies on brain protein phosphorylation. Such studies should be of value in studies on human neurological and/or psychiatric disorders.

  2. Hypertension alters phosphorylation of VASP in brain endothelial cells.

    PubMed

    Arlier, Zulfikar; Basar, Murat; Kocamaz, Erdogan; Kiraz, Kemal; Tanriover, Gamze; Kocer, Gunnur; Arlier, Sefa; Giray, Semih; Nasırcılar, Seher; Gunduz, Filiz; Senturk, Umit K; Demir, Necdet

    2015-04-01

    Hypertension impairs cerebral vascular function. Vasodilator-stimulated phosphoprotein (VASP) mediates active reorganization of the cytoskeleton via membrane ruffling, aggregation and tethering of actin filaments. VASP regulation of endothelial barrier function has been demonstrated by studies using VASP(-/-) animals under conditions associated with tissue hypoxia. We hypothesize that hypertension regulates VASP expression and/or phosphorylation in endothelial cells, thereby contributing to dysfunction in the cerebral vasculature. Because exercise has direct and indirect salutary effects on vascular systems that have been damaged by hypertension, we also investigated the effect of exercise on maintenance of VASP expression and/or phosphorylation. We used immunohistochemistry, Western blotting and immunocytochemistry to examine the effect of hypertension on VASP expression and phosphorylation in brain endothelial cells in normotensive [Wistar-Kyoto (WKY)] and spontaneously hypertensive (SH) rats under normal and exercise conditions. In addition, we analyzed VASP regulation in normoxia- and hypoxia-induced endothelial cells. Brain endothelial cells exhibited significantly lower VASP immunoreactivity and phosphorylation at the Ser157 residue in SHR versus WKY rats. Exercise reversed hypertension-induced alterations in VASP phosphorylation. Western blotting and immunocytochemistry indicated reduction in VASP phosphorylation in hypoxic versus normoxic endothelial cells. These results suggest that diminished VASP expression and/or Ser157 phosphorylation mediates endothelial changes associated with hypertension and exercise may normalize these changes, at least in part, by restoring VASP phosphorylation. PMID:24894047

  3. dimerization and DNA binding alter phosphorylation of Fos and Jun

    SciTech Connect

    Abate, C.; Baker, S.J.; Curran, T. ); Lees-Miller, S.P.; Anderson, C.W. ); Marshak, D.R. )

    1993-07-15

    Fos and Jun form dimeric complexes that bind to activator protein 1 (AP-1) DNA sequences and regulate gene expression. The levels of expression and activities of these proteins are regulated by a variety of extracellular stimuli. They are thought to function in nuclear signal transduction processes in many different cell types. The role of Fos and Jun in gene transcription is complex and may be regulated in several ways including association with different dimerization partners, interactions with other transcription factors, effects on DNA topology, and reduction/oxidation of a conserved cysteine residue in the DNA-binding domain. In addition, phosphorylation has been suggested to control the activity of Fos and Jun. Here the authors show that phosphorylation of Fos and Jun by several protein kinases is affected by dimerization and binding to DNA. Jun homodimers are phosphorylated efficiently by casein kinase II, whereas Fos-Jun heterodimers are not. DNA binding also reduces phosphorylation of Jun by casein kinase II, p34[sup cdc2] (cdc2) kinase, and protein kinase C. Phosphorylation of Fos by cAMP-dependent protein kinase and cdc2 is relatively insensitive to dimerization and DNA binding, whereas phosphorylation of Fos and Jun by DNA-dependent protein kinase is dramatically stimulated by binding to the AP-1 site. These results imply that different protein kinases can distinguish among Fos and Jun proteins in the form of monomers, homodimers, and heterodimers and between DNA-bound and non-DNA-bound proteins. Thus, potentially, these different states of Fos and Jun can be recognized and regulated independently by phosphorylation. 44 refs., 4 figs.

  4. Protein phosphorylation is involved in bacterial chemotaxis.

    PubMed Central

    Hess, J F; Oosawa, K; Matsumura, P; Simon, M I

    1987-01-01

    The nature of the biochemical signal that is involved in the excitation response in bacterial chemotaxis is not known. However, ATP is required for chemotaxis. We have purified all of the proteins involved in signal transduction and show that the product of the cheA gene is rapidly autophosphorylated, while some mutant CheA proteins cannot be phosphorylated. The presence of stoichiometric levels of two other purified components in the chemotaxis system, the CheY and CheZ proteins, induces dephosphorylation. We suggest that the phosphorylation of CheA by ATP plays a central role in signal transduction in chemotaxis. Images PMID:3313398

  5. Identification of extracellularly phosphorylated membrane proteins.

    PubMed

    Burghoff, Sandra; Willberg, Wibke; Schrader, Jürgen

    2015-10-01

    Ecto-protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC-MS/MS. A "false-positive" strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G-protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface-exposed peptides when extracellular adenosine-5'-triphosphate is elevated during purinergic signalling. PMID:26152529

  6. Metaphase protein phosphorylation in Xenopus laevis eggs.

    PubMed Central

    Lohka, M J; Kyes, J L; Maller, J L

    1987-01-01

    Cytoplasmic extracts of metaphase (M-phase)-arrested Xenopus laevis eggs support nuclear envelope breakdown and chromosome condensation in vitro. Induction of nuclear breakdown is inhibited by AMPP(NH)P, a nonhydrolyzable ATP analog, but not by ATP or gamma-S-ATP, a hydrolyzable ATP analog, suggesting that protein phosphorylation may be required for M-phase nuclear events in vitro. By addition of [gamma-32P]ATP, we have identified in cytoplasmic extracts and in intact eggs at least six phosphoproteins that are present during M-phase but absent in G1/S-phase. These phosphoproteins also appear in response to partially purified preparations of maturation-promoting factor. A subset of these proteins are thiophosphorylated by gamma-S-ATP under conditions that promote nuclear envelope breakdown and chromosome condensation. Each of these proteins is phosphorylated on serine and threonine, and one, a 42-kilodalton protein, is also phosphorylated on tyrosine both in extracts and in intact eggs. These results indicate that activation of protein kinases accounts for at least part of the increased phosphorylation in M-phase and that both protein-serine-threonine kinases and protein-tyrosine kinases may play a role in controlling M-phase nuclear behavior. Images PMID:3821728

  7. Ligand-induced alterations in the phosphorylation state of ethylene receptors in tomato fruit.

    PubMed

    Kamiyoshihara, Yusuke; Tieman, Denise M; Huber, Donald J; Klee, Harry J

    2012-09-01

    Perception of the plant hormone ethylene is essential to initiate and advance ripening of climacteric fruits. Since ethylene receptors negatively regulate signaling, the suppression is canceled upon ethylene binding, permitting responses including fruit ripening. Although receptors have autophosphorylation activity, the mechanism whereby signal transduction occurs has not been fully determined. Here we demonstrate that LeETR4, a critical receptor for tomato (Solanum lycopersicum) fruit ripening, is multiply phosphorylated in vivo and the phosphorylation level is dependent on ripening stage and ethylene action. Treatment of preclimacteric fruits with ethylene resulted in accumulation of LeETR4 with reduced phosphorylation whereas treatments of ripening fruits with ethylene antagonists, 1-methylcyclopropene and 2,5-norbornadiene, induced accumulation of the phosphorylated isotypes. A similar phosphorylation pattern was also observed for Never ripe, another ripening-related receptor. Alteration in the phosphorylation state of receptors is likely to be an initial response upon ethylene binding since treatments with ethylene and 1-methylcyclopropene rapidly influenced the LeETR4 phosphorylation state rather than protein abundance. The LeETR4 phosphorylation state closely paralleled ripening progress, suggesting that the phosphorylation state of receptors is implicated in ethylene signal output in tomato fruits. We provide insights into the nature of receptor on and off states. PMID:22797658

  8. Regulation of protein phosphorylation in oat mitochondria

    SciTech Connect

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

    1989-04-01

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

  9. Phosphorylation of plastoglobular proteins in Arabidopsis thaliana.

    PubMed

    Lohscheider, Jens N; Friso, Giulia; van Wijk, Klaas J

    2016-06-01

    Plastoglobules (PGs) are plastid lipid-protein particles with a small specialized proteome and metabolome. Among the 30 core PG proteins are six proteins of the ancient ABC1 atypical kinase (ABC1K) family and their locations in an Arabidopsis mRNA-based co-expression network suggested central regulatory roles. To identify candidate ABC1K targets and a possible ABC1K hierarchical phosphorylation network within the chloroplast PG proteome, we searched Arabidopsis phosphoproteomics data from publicly available sources. Evaluation of underlying spectra and/or associated information was challenging for a variety of reasons, but supported pSer sites and a few pThr sites in nine PG proteins, including five FIBRILLINS. PG phosphorylation motifs are discussed in the context of possible responsible kinases. The challenges of collection and evaluation of published Arabidopsis phosphorylation data are discussed, illustrating the importance of deposition of all mass spectrometry data in well-organized repositories such as PRIDE and ProteomeXchange. This study provides a starting point for experimental testing of phosho-sites in PG proteins and also suggests that phosphoproteomics studies specifically designed toward the PG proteome and its ABC1K are needed to understand phosphorylation networks in these specialized particles. PMID:26962209

  10. Phosphorylation of plastoglobular proteins in Arabidopsis thaliana

    PubMed Central

    Lohscheider, Jens N.; Friso, Giulia; van Wijk, Klaas J.

    2016-01-01

    Plastoglobules (PGs) are plastid lipid–protein particles with a small specialized proteome and metabolome. Among the 30 core PG proteins are six proteins of the ancient ABC1 atypical kinase (ABC1K) family and their locations in an Arabidopsis mRNA-based co-expression network suggested central regulatory roles. To identify candidate ABC1K targets and a possible ABC1K hierarchical phosphorylation network within the chloroplast PG proteome, we searched Arabidopsis phosphoproteomics data from publicly available sources. Evaluation of underlying spectra and/or associated information was challenging for a variety of reasons, but supported pSer sites and a few pThr sites in nine PG proteins, including five FIBRILLINS. PG phosphorylation motifs are discussed in the context of possible responsible kinases. The challenges of collection and evaluation of published Arabidopsis phosphorylation data are discussed, illustrating the importance of deposition of all mass spectrometry data in well-organized repositories such as PRIDE and ProteomeXchange. This study provides a starting point for experimental testing of phosho-sites in PG proteins and also suggests that phosphoproteomics studies specifically designed toward the PG proteome and its ABC1K are needed to understand phosphorylation networks in these specialized particles. PMID:26962209

  11. Phosphorylation of proteins in Clostridium thermohydrosulfuricum

    SciTech Connect

    Londesborough, J.

    1986-02-01

    Cell extracts of the thermophile Clostridium thermohydrosulfuricum catalyzed the phosphorylation by (..gamma..-/sup 32/P)ATP of several endogenous proteins with M/sub r/s between 13,000 and 100,000. Serine and tyrosine were the main acceptors. Distinct substrate proteins were found in the soluble (e.g., proteins p66, p63, and p53 of M/sub r/s 66,000, 63,000, and 53,000, respectively) and particulate (p76 and p30) fractions, both of which contained protein kinase and phosphatase activity. The soluble fraction suppressed the phosphorylation of particulate proteins and contained a protein kinase inhibitor. Phosphorylation of p53 was promoted by 10..mu..M fructose 1,6-bisphosphate or glucose 1,6-bisphosphate and suppressed by hexose monophosphates, whereas p30 and p13 were suppressed by 5 ..mu..M brain (but not spinach) calmodulin. Polyamines, including the odd polyamines characteristic of thermophiles, modulated the labeling of most of the phosphoproteins. Apart from p66, all the proteins labeled in vitro were also rapidly labeled in intact cells by /sub 32/P/sub i/. Several proteins strongly labeled in vivo were labeled slowly or not at all in vitro.

  12. Protein Synthesis Initiation Factors: Phosphorylation and Regulation

    SciTech Connect

    Karen S. Browning

    2009-06-15

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

  13. Regulation of cardiac C-protein phosphorylation

    SciTech Connect

    Titus, F.L.

    1985-01-01

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

  14. Alterations of Histone H1 Phosphorylation During Bladder Carcinogenesis

    PubMed Central

    Telu, Kelly H.; Abbaoui, Besma; Thomas-Ahner, Jennifer M.; Zynger, Debra L.; Clinton, Steven K.

    2013-01-01

    There is a crucial need for development of prognostic and predictive biomarkers in human bladder carcinogenesis in order to personalize preventive and therapeutic strategies and improve outcomes. Epigenetic alterations, such as histone modifications, are implicated in the genetic dysregulation that is fundamental to carcinogenesis. Here we focus on profiling the histone modifications during the progression of bladder cancer. Histones were extracted from normal human bladder epithelial cells, an immortalized human bladder epithelial cell line (hTERT), and four human bladder cancer cell lines (RT4, J82, T24, and UMUC3) ranging from superficial low-grade to invasive high-grade cancers. Liquid Chromatography-Mass Spectrometry (LC-MS) profiling revealed a statistically significant increase in phosphorylation of H1 linker histones from normal human bladder epithelial cells to low-grade superficial to high-grade invasive bladder cancer cells. This finding was further validated by immunohistochemical staining of the normal epithelium and transitional cell cancer from human bladders. Cell cycle analysis of histone H1 phosphorylation by western blotting showed an increase of phosphorylation from G0/G1 phase to M phase, again supporting this as a proliferative marker. Changes in histone H1 phosphorylation status may further clarify epigenetic changes during bladder carcinogenesis and provide diagnostic and prognostic biomarkers or targets for future therapeutic interventions. PMID:23675690

  15. Heat shock triggers rapid protein phosphorylation in soybean seedings

    SciTech Connect

    Krishnan, H.B.; Pueppke, S.G.

    1987-10-29

    Heat shock arrests the synthesis of many cellular proteins and simultaneously initiates expression of a unique set of proteins, termed heat shock proteins. We have found that heat shock rapidly triggers phosphorylation of a set of proteins in soybean seedlings. Although the kinetics of phosphorylation and the heat shock response are similar, the major identified phosphorylation products do not comigrate with heat shock proteins on polyacrylamide gels. Cadmium, which is known to induce the heat shock response, stimulates phosphorylation of the same set of proteins. The rapidity of phosphorylation suggests that it may play a pivotal role in sensing and transducing elevated temperature stress in plants.

  16. Induction of protein tyrosine phosphorylation in macrophages incubated with tumor cells.

    PubMed

    Sodhi, A; Shrivastava, A; Kumar, R

    1995-03-01

    The cellular and molecular interaction between monocyte/macrophage and tumor cells leading to macrophage activation is not clearly understood. Since protein tyrosine phosphorylation appears to be a major intracellular signalling event, we checked whether the tumor cells alter tyrosine phosphorylation of proteins in macrophages. We found that both L929 and Yac-1 tumor cells induced increased tyrosine phosphorylation of several polypeptides in peritoneal as well as P388D-1 and IC-21 macrophages. Macrophages co-cultured with tumor cells also showed increased fluorescence with anti-phosphotyrosine-FITC antibody. These observations suggest that increased tyrosine phosphorylation plays a role in tumor cell-induced activation of macrophages. PMID:7539664

  17. Survey of phosphorylation near drug binding sites in the Protein Data Bank (PDB) and their effects.

    PubMed

    Smith, Kyle P; Gifford, Kathleen M; Waitzman, Joshua S; Rice, Sarah E

    2015-01-01

    While it is currently estimated that 40 to 50% of eukaryotic proteins are phosphorylated, little is known about the frequency and local effects of phosphorylation near pharmaceutical inhibitor binding sites. In this study, we investigated how frequently phosphorylation may affect the binding of drug inhibitors to target proteins. We examined the 453 non-redundant structures of soluble mammalian drug target proteins bound to inhibitors currently available in the Protein Data Bank (PDB). We cross-referenced these structures with phosphorylation data available from the PhosphoSitePlus database. Three hundred twenty-two of 453 (71%) of drug targets have evidence of phosphorylation that has been validated by multiple methods or labs. For 132 of 453 (29%) of those, the phosphorylation site is within 12 Å of the small molecule-binding site, where it would likely alter small molecule binding affinity. We propose a framework for distinguishing between drug-phosphorylation site interactions that are likely to alter the efficacy of drugs versus those that are not. In addition we highlight examples of well-established drug targets, such as estrogen receptor alpha, for which phosphorylation may affect drug affinity and clinical efficacy. Our data suggest that phosphorylation may affect drug binding and efficacy for a significant fraction of drug target proteins. PMID:24833420

  18. Survey of phosphorylation near drug binding sites in the Protein Data Bank (PDB) and their effects

    PubMed Central

    Smith, Kyle P.; Gifford, Kathleen M.; Waitzman, Joshua S.; Rice, Sarah E.

    2014-01-01

    While it is currently estimated that 40–50% of eukaryotic proteins are phosphorylated, little is known about the frequency and local effects of phosphorylation near pharmaceutical inhibitor binding sites. In this study, we investigated how frequently phosphorylation may affect the binding of drug inhibitors to target proteins. We examined the 453 non-redundant structures of soluble mammalian drug target proteins bound to inhibitors currently available in the Protein Data Bank (PDB). We cross-referenced these structures with phosphorylation data available from the PhosphoSitePlus database. 322/453 (71%) of drug targets have evidence of phosphorylation that has been validated by multiple methods or labs. For 132/453 (29%) of those, the phosphorylation site is within 12Å of the small molecule-binding site, where it would likely alter small molecule binding affinity. We propose a framework for distinguishing between drug-phosphorylation site interactions that are likely to alter the efficacy of drugs vs. those that are not. In addition we highlight examples of well-established drug targets, such as estrogen receptor alpha, for which phosphorylation may affect drug affinity and clinical efficacy. Our data suggest that phosphorylation may affect drug binding and efficacy for a significant fraction of drug target proteins. PMID:24833420

  19. The Timing of Multiple Retrieval Events Can Alter GluR1 Phosphorylation and the Requirement for Protein Synthesis in Fear Memory Reconsolidation

    ERIC Educational Resources Information Center

    Jarome, Timothy J.; Kwapis, Janine L.; Werner, Craig T.; Parsons, Ryan G.; Gafford, Georgette M.; Helmstetter, Fred J.

    2012-01-01

    Numerous studies have indicated that maintaining a fear memory after retrieval requires de novo protein synthesis. However, no study to date has examined how the temporal dynamics of repeated retrieval events affect this protein synthesis requirement. The present study varied the timing of a second retrieval of an established auditory fear memory…

  20. Tonoplast-Bound Protein Kinase Phosphorylates Tonoplast Intrinsic Protein 1

    PubMed Central

    Johnson, Kenneth D.; Chrispeels, Maarten J.

    1992-01-01

    Tonoplast intrinsic protein (TIP) is a member of a family of putative membrane channels found in bacteria, animals, and plants. Plants have seed-specific, vegetative/reproductive organ-specific, and water-stress-induced forms of TIP. Here, we report that the seed-specific TIP is a phosphoprotein whose phosphorylation can be monitored in vivo by allowing bean cotyledons to take up [32P]orthophosphate and in vitro by incubating purified tonoplasts with γ-labeled [32P]ATP. Characterization of the in vitro phosphorylation of TIP indicates that a membrane-bound protein kinase phosphorylates TIP in a Ca2+-dependent manner. The capacity of the isolated tonoplast membranes to phosphorylate TIP declined markedly during seed germination, and this decline occurred well before the development-mediated decrease in TIP occurs. Phosphoamino acid analysis of purified, radiolabeled TIP showed that serine is the major, if not only, phosphorylated residue, and cyanogen bromide cleavage yielded a single radioactive peptide peak on a reverse-phase high-performance liquid chromatogram. Estimation of the molecular mass of the cyanogen bromide phosphopeptide by laser desorption mass spectroscopy led to its identification as the hydrophilic N-terminal domain of TIP. The putative phosphate-accepting serine residue occurs in a consensus phosphorylation site for serine/threonine protein kinases. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:16653198

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

    PubMed

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

    2014-06-01

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

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

    PubMed Central

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

    2014-01-01

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

  3. The importance of intrinsic disorder for protein phosphorylation.

    PubMed

    Iakoucheva, Lilia M; Radivojac, Predrag; Brown, Celeste J; O'Connor, Timothy R; Sikes, Jason G; Obradovic, Zoran; Dunker, A Keith

    2004-01-01

    Reversible protein phosphorylation provides a major regulatory mechanism in eukaryotic cells. Due to the high variability of amino acid residues flanking a relatively limited number of experimentally identified phosphorylation sites, reliable prediction of such sites still remains an important issue. Here we report the development of a new web-based tool for the prediction of protein phosphorylation sites, DISPHOS (DISorder-enhanced PHOSphorylation predictor, http://www.ist.temple. edu/DISPHOS). We observed that amino acid compositions, sequence complexity, hydrophobicity, charge and other sequence attributes of regions adjacent to phosphorylation sites are very similar to those of intrinsically disordered protein regions. Thus, DISPHOS uses position-specific amino acid frequencies and disorder information to improve the discrimination between phosphorylation and non-phosphorylation sites. Based on the estimates of phosphorylation rates in various protein categories, the outputs of DISPHOS are adjusted in order to reduce the total number of misclassified residues. When tested on an equal number of phosphorylated and non-phosphorylated residues, the accuracy of DISPHOS reaches 76% for serine, 81% for threonine and 83% for tyrosine. The significant enrichment in disorder-promoting residues surrounding phosphorylation sites together with the results obtained by applying DISPHOS to various protein functional classes and proteomes, provide strong support for the hypothesis that protein phosphorylation predominantly occurs within intrinsically disordered protein regions. PMID:14960716

  4. A strategy to quantitate global phosphorylation of bone matrix proteins.

    PubMed

    Sroga, Grażyna E; Vashishth, Deepak

    2016-04-15

    Current studies of protein phosphorylation focus primarily on the importance of specific phosphoproteins and their landscapes of phosphorylation in the regulation of different cellular functions. However, global changes in phosphorylation of extracellular matrix phosphoproteins measured "in bulk" are equally important. For example, correct global phosphorylation of different bone matrix proteins is critical to healthy tissue biomineralization. To study changes of bone matrix global phosphorylation, we developed a strategy that combines a procedure for in vitro phosphorylation/dephosphorylation of fully mineralized bone in addition to quantitation of the global phosphorylation levels of bone matrix proteins. For the first time, we show that it is possible to enzymatically phosphorylate/dephosphorylate fully mineralized bone originating from either cadaveric human donors or laboratory animals (mice). Using our strategy, we detected the difference in the global phosphorylation levels of matrix proteins isolated from wild-type and osteopontin knockout mice. We also observed that the global phosphorylation levels of matrix proteins isolated from human cortical bone were lower than those isolated from trabecular bone. The developed strategy has the potential to open new avenues for studies on the global phosphorylation of bone matrix proteins and their role in biomineralization as well for other tissues/cells and protein-based materials. PMID:26851341

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

    PubMed Central

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

    1996-01-01

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

  6. Phosphorylated tyrosine in the flagellum filament protein of Pseudomonas aeruginosa

    SciTech Connect

    Kelly-Wintenberg, K.; Anderson, T.; Montie, T.C. )

    1990-09-01

    Purified flagella from two strains of {sup 32}P-labeled Pseudomonas aeruginosa were shown to be phosphorylated. This was confirmed by autoradiography of flagellin protein in polyacrylamide gels. Thin-layer electrophoresis and autoradiography of flagellin partial hydrolysates indicated that phosphotyrosine was the major phosphorylated amino acid. High-pressure liquid chromatographic analysis confirmed the presence of phosphotyrosine in flagellum filament protein. Preliminary data indicated that less than one tyrosine per subunit was phosphorylated. No evidence was found for phosphorylation of serine or threonine. A function related to tyrosine phosphorylation has not been determined.

  7. Protein phosphorylation in response to stress in Clostridium acetobutylicum

    SciTech Connect

    Balodimos, I.A.; Rapaport, E.; Kashket, E.R. )

    1990-07-01

    The possible involvement of protein phosphorylation in the clostridial stress response was investigated by radioactively labeling growing cells of Clostridium acetobutylicum with {sup 32}P{sub i} or cell extracts with ({gamma}-{sup 32}P)ATP. Several phosphoproteins were identified; these were not affected by the growth stage of the culture. Although the extent of protein phosphorylation was increased by heat stress, the phosphoproteins did not correspond to known stress proteins seen in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified clostridial DnaK, a stress protein, acted as a kinase catalyzing the phosphorylation of a 50-kilodalton protein. The phosphorylation of this protein was enhanced in extracts prepared from heat-stressed cells. Diadenosine-5{prime},5{double prime}{prime}-P{sup 1},P{sup 4}-tetraphosphate had no influence on protein phosphorylation.

  8. Diazinon alters sperm chromatin structure in mice by phosphorylating nuclear protamines

    SciTech Connect

    Pina-Guzman, B.; Solis-Heredia, M.J.; Quintanilla-Vega, B. . E-mail: mquintan@mail.cinvestav.mx

    2005-01-15

    Organophosphorus (OP) pesticides, widely used in agriculture and pest control, are associated with male reproductive effects, including sperm chromatin alterations, but the mechanisms underlying these effects are unknown. The main toxic action of OP is related to phosphorylation of proteins. Chemical alterations in sperm nuclear proteins (protamines), which pack DNA during the last steps of spermatogenesis, contribute to male reproductive toxicity. Therefore, in the present study, we tested the ability of diazinon (DZN), an OP compound, to alter sperm chromatin by phosphorylating nuclear protamines. Mice were injected with a single dose of DZN (8.12 mg/kg, i.p.), and killed 8 and 15 days after treatment. Quality of sperm from epididymis and vas deferens was evaluated through standard methods and chromatin condensation by flow cytometry (DNA Fragmented Index parameters: DFI and DFI%) and fluorescence microscopy using chromomycin-A{sub 3} (CMA{sub 3}). Increases in DFI (15%), DFI% (4.5-fold), and CMA{sub 3} (2-fold) were observed only at 8 days post-treatment, indicating an alteration in sperm chromatin condensation and DNA damage during late spermatid differentiation. In addition, an increase of phosphorous content (approximately 50%) in protamines, especially in the phosphoserine content (approximately 73%), was found at 8 days post-treatment. Sperm viability, motility, and morphology showed significant alterations at this time. These data strongly suggest that spermatozoa exposed during the late steps of maturation were the targets of DZN exposure. The correlation observed between the phosphorous content in nuclear protamines with DFI%, DFI, and CMA{sub 3} provides evidence that phosphorylation of nuclear protamines is involved in the OP effects on sperm chromatin.

  9. Methods for the Analysis of Protein Phosphorylation-Mediated Cellular Signaling Networks.

    PubMed

    White, Forest M; Wolf-Yadlin, Alejandro

    2016-06-12

    Protein phosphorylation-mediated cellular signaling networks regulate almost all aspects of cell biology, including the responses to cellular stimulation and environmental alterations. These networks are highly complex and comprise hundreds of proteins and potentially thousands of phosphorylation sites. Multiple analytical methods have been developed over the past several decades to identify proteins and protein phosphorylation sites regulating cellular signaling, and to quantify the dynamic response of these sites to different cellular stimulation. Here we provide an overview of these methods, including the fundamental principles governing each method, their relative strengths and weaknesses, and some examples of how each method has been applied to the analysis of complex signaling networks. When applied correctly, each of these techniques can provide insight into the topology, dynamics, and regulation of protein phosphorylation signaling networks. PMID:27049636

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

    SciTech Connect

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

    1988-03-01

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

  11. Phosphorylation of influenza A virus NS1 protein at threonine 49 suppresses its interferon antagonistic activity.

    PubMed

    Kathum, Omer Abid; Schräder, Tobias; Anhlan, Darisuren; Nordhoff, Carolin; Liedmann, Swantje; Pande, Amit; Mellmann, Alexander; Ehrhardt, Christina; Wixler, Viktor; Ludwig, Stephan

    2016-06-01

    Phosphorylation and dephosphorylation acts as a fundamental molecular switch that alters protein function and thereby regulates many cellular processes. The non-structural protein 1 (NS1) of influenza A virus is an important factor regulating virulence by counteracting cellular immune responses against viral infection. NS1 was shown to be phosphorylated at several sites; however, so far, no function has been conclusively assigned to these post-translational events yet. Here, we show that the newly identified phospho-site threonine 49 of NS1 is differentially phosphorylated in the viral replication cycle. Phosphorylation impairs binding of NS1 to double-stranded RNA and TRIM25 as well as complex formation with RIG-I, thereby switching off its interferon antagonistic activity. Because phosphorylation was shown to occur at later stages of infection, we hypothesize that at this stage other functions of the multifunctional NS1 beyond its interferon-antagonistic activity are needed. PMID:26687707

  12. Calcium-regulated in vivo protein phosphorylation in Zea mays L. root tips

    NASA Technical Reports Server (NTRS)

    Raghothama, K. G.; Reddy, A. S.; Friedmann, M.; Poovaiah, B. W.

    1987-01-01

    Calcium dependent protein phosphorylation was studied in corn (Zea mays L.) root tips. Prior to in vivo protein phosphorylation experiments, the effect of calcium, ethyleneglycol-bis-(beta-aminoethyl ether)-N-N' -tetraacetic acid (EGTA) and calcium ionophore (A-23187) on phosphorus uptake was studied. Calcium increased phosphorus uptake, whereas EGTA and A-23187 decreased it. Consequently, phosphorus concentration in the media was adjusted so as to attain similar uptake in different treatments. Phosphoproteins were analyzed by two-dimensional gel electrophoresis. Distinct changes in phosphorylation were observed following altered calcium levels. Calcium depletion in root tips with EGTA and A-23187 decreased protein phosphorylation. However, replenishment of calcium following EGTA and ionophore pretreatment enhanced phosphorylation of proteins. Preloading of the root tips with 32P in the presence of EGTA and A-23187 followed by a ten minute calcium treatment, resulted in increased phosphorylation indicating the involvement of calcium, calcium and calmodulin-dependent kinases. Calmodulin antagonist W-7 was effective in inhibiting calcium-promoted phosphorylation. These studies suggest a physiological role for calcium-dependent phosphorylation in calcium-mediated processes in plants.

  13. Cardiac mitochondrial matrix and respiratory complex protein phosphorylation

    PubMed Central

    Covian, Raul

    2012-01-01

    It has become appreciated over the last several years that protein phosphorylation within the cardiac mitochondrial matrix and respiratory complexes is extensive. Given the importance of oxidative phosphorylation and the balance of energy metabolism in the heart, the potential regulatory effect of these classical signaling events on mitochondrial function is of interest. However, the functional impact of protein phosphorylation and the kinase/phosphatase system responsible for it are relatively unknown. Exceptions include the well-characterized pyruvate dehydrogenase and branched chain α-ketoacid dehydrogenase regulatory system. The first task of this review is to update the current status of protein phosphorylation detection primarily in the matrix and evaluate evidence linking these events with enzymatic function or protein processing. To manage the scope of this effort, we have focused on the pathways involved in energy metabolism. The high sensitivity of modern methods of detecting protein phosphorylation and the low specificity of many kinases suggests that detection of protein phosphorylation sites without information on the mole fraction of phosphorylation is difficult to interpret, especially in metabolic enzymes, and is likely irrelevant to function. However, several systems including protein translocation, adenine nucleotide translocase, cytochrome c, and complex IV protein phosphorylation have been well correlated with enzymatic function along with the classical dehydrogenase systems. The second task is to review the current understanding of the kinase/phosphatase system within the matrix. Though it is clear that protein phosphorylation occurs within the matrix, based on 32P incorporation and quantitative mass spectrometry measures, the kinase/phosphatase system responsible for this process is ill-defined. An argument is presented that remnants of the much more labile bacterial protein phosphoryl transfer system may be present in the matrix and that the

  14. Phosphorylation and Ionic Strength Alter the LRAP-HAP Interface in the N-terminus

    SciTech Connect

    Lu, Junxia; Xu, Yimin; Shaw, Wendy J.

    2013-04-02

    The conditions present during enamel crystallite development change dramatically as a function of time, including the pH, protein concentration, surface type and ionic strength. In this work, we investigate the role that two of these changing conditions, pH and ionic strength, have in modulating the interaction of amelogenin, LRAP, with hydroxyapatite (HAP). Using solid state NMR dipolar recoupling and chemical shift data, we investigate the structure, orientation and dynamics of three regions in the N-terminus of the protein, L15 to V19, V19 to L23 and K24 to S28. These regions are also near the only phosphorylated residue in the protein, pS16, therefore, changes in the LRAP-HAP interaction as a function of phosphorylation (LRAP(-P) vs. LRAP(+P)) were also investigated. All of the regions and conditions studies for the surface immobilized proteins showed restricted motion, with more mobility under all conditions for L15(+P) and K24(-P). The structure and orientation of the LRAP-HAP interaction in the N-terminus of the phosphorylated protein is very stable to changing solution conditions. From REDOR dipolar recoupling data, the structure and orientation in the region L15V19(+P) did not change significantly as a function of pH or ionic strength. The structure and orientation of the region V19L23(+P) were also stable to changes in pH, with the only significant change observed at high ionic strength, where the region becomes extended, suggesting this may be an important region in regulating mineral development. Chemical shift studies also suggest minimal changes in all three regions studied for both LRAP(-P) and LRAP(+P) as a function of pH or ionic strength. Phosphorylation also alters the LRAP-HAP interface. All of the three residues investigated (L15, V19, and K24) are closer to the surface in LRAP(+P), but K24S28 also changes structure as a result of phosphorylation, from a random coil to a largely helical structure, and V19L23 becomes more extended at high ionic

  15. Conserved aspartate residues and phosphorylation in signal transduction by the chemotaxis protein CheY.

    PubMed Central

    Bourret, R B; Hess, J F; Simon, M I

    1990-01-01

    The CheY protein is phosphorylated by CheA and dephosphorylated by CheZ as part of the chemotactic signal transduction pathway in Escherichia coli. Phosphorylation of CheY has been proposed to occur on an aspartate residue. Each of the eight aspartate residues of CheY was replaced by using site-directed mutagenesis. Substitutions at Asp-12, Asp-13, or Asp-57 resulted in loss of chemotaxis. Most of the mutant CheY proteins were still phosphorylated by CheA but exhibited modified biochemical properties, including reduced ability to accept phosphate from CheA, altered phosphate group stability, and/or resistance to CheZ-mediated dephosphorylation. The properties of CheY proteins bearing a substitution at position 57 were most aberrant, consistent with the hypothesis that Asp-57 is the normal site of acyl phosphate formation. Evidence for an alternate site of phosphorylation in the Asp-57 mutants is presented. Phosphorylated CheY is believed to cause tumbling behavior. However, a dominant mutant CheY protein that was not phosphorylated in vitro caused tumbling in vivo in the absence of CheA. This phenotype suggests that the role of phosphorylation in the wild-type CheY protein is to stabilize a transient conformational change that can generate tumbling behavior. Images PMID:2404281

  16. Protein phosphorylation in isolated human adipocytes - Adrenergic control of the phosphorylation of hormone-sensitive lipase

    SciTech Connect

    Smiley, R.M. Columbia Univ College of Physicians and Surgeons, New York, NY ); Paul, S.; Browning, M.D.; Leibel, R.L.; Hirsch, J. )

    1990-01-01

    The effect of adrenergic agents on protein phosphorylation in human adipocytes was examined. Freshly isolated human fat cells were incubated with {sup 32}PO{sub 4} in order to label intracellular ATP, then treated with a variety of adrenergic and other pharmacologic agents. Treatment with the {beta}-adrenergic agonist isoproterenol led to a significant increase in phosphate content of at least five protein bands (M{sub r} 52, 53, 63, 67, 84 kDa). The increase in phosphorylation was partially inhibited by the {alpha}-2 agonist clonidine. Epinephrine, a combined {alpha} and {beta} agonist, was less effective at increasing phosphate content of the proteins than was isoproterenol. Neither insulin nor the {alpha}-1 agonist phenylephrine had any discernible effect on the pattern of protein phosphorylation. The 84 kDa phosphorylated peptide band appears to contain hormone-sensitive lipase, a key enzyme in the lipolytic pathway which is activated by phosphorylation. These results are somewhat different than previously reported results for rat adipocytes, and represent the first report of overall pattern and adrenergic modulation of protein phosphorylation in human adipocytes.

  17. Comprehensive analysis of phosphorylated proteins of Escherichia coli ribosomes.

    PubMed

    Soung, George Y; Miller, Jennifer L; Koc, Hasan; Koc, Emine C

    2009-07-01

    Phosphorylation of bacterial ribosomal proteins has been known for decades; however, there is still very limited information available on specific locations of the phosphorylation sites in ribosomal proteins and the role they might play in protein synthesis. In this study, we have mapped the specific phosphorylation sites in 24 Escherichia coli ribosomal proteins by tandem mass spectrometry. Detection of phosphorylation was achieved by either phosphorylation specific visualization techniques, ProQ staining, and antibodies for phospho-Ser, Thr, and Tyr; or by mass spectrometry equipped with a capability to detect addition and loss of the phosphate moiety. Enrichment by immobilized metal affinity and/or strong cation exchange chromatography was used to improve the success of detection of the low abundance phosphopeptides. We found the small subunit (30S) proteins S3, S4, S5, S7, S11, S12, S13, S18, and S21 and the large subunit (50S) proteins L1, L2, L3, L5, L6, L7/L12, L13, L14, L16, L18, L19, L21, L22, L28, and L31 to be phosphorylated at one or more residues. Potential roles for each specific site in ribosome function were deduced through careful evaluation of the given phosphorylation sites in 3D-crystal structure models of ribosomes and the previous mutational studies of E. coli ribosomal proteins. PMID:19469554

  18. Protein phosphorylation and its role in archaeal signal transduction.

    PubMed

    Esser, Dominik; Hoffmann, Lena; Pham, Trong Khoa; Bräsen, Christopher; Qiu, Wen; Wright, Phillip C; Albers, Sonja-Verena; Siebers, Bettina

    2016-09-01

    Reversible protein phosphorylation is the main mechanism of signal transduction that enables cells to rapidly respond to environmental changes by controlling the functional properties of proteins in response to external stimuli. However, whereas signal transduction is well studied in Eukaryotes and Bacteria, the knowledge in Archaea is still rather scarce. Archaea are special with regard to protein phosphorylation, due to the fact that the two best studied phyla, the Euryarchaeota and Crenarchaeaota, seem to exhibit fundamental differences in regulatory systems. Euryarchaeota (e.g. halophiles, methanogens, thermophiles), like Bacteria and Eukaryotes, rely on bacterial-type two-component signal transduction systems (phosphorylation on His and Asp), as well as on the protein phosphorylation on Ser, Thr and Tyr by Hanks-type protein kinases. Instead, Crenarchaeota (e.g. acidophiles and (hyper)thermophiles) only depend on Hanks-type protein phosphorylation. In this review, the current knowledge of reversible protein phosphorylation in Archaea is presented. It combines results from identified phosphoproteins, biochemical characterization of protein kinases and protein phosphatases as well as target enzymes and first insights into archaeal signal transduction by biochemical, genetic and polyomic studies. PMID:27476079

  19. Protein phosphorylation and its role in archaeal signal transduction

    PubMed Central

    Esser, Dominik; Hoffmann, Lena; Pham, Trong Khoa; Bräsen, Christopher; Qiu, Wen; Wright, Phillip C.; Albers, Sonja-Verena; Siebers, Bettina

    2016-01-01

    Reversible protein phosphorylation is the main mechanism of signal transduction that enables cells to rapidly respond to environmental changes by controlling the functional properties of proteins in response to external stimuli. However, whereas signal transduction is well studied in Eukaryotes and Bacteria, the knowledge in Archaea is still rather scarce. Archaea are special with regard to protein phosphorylation, due to the fact that the two best studied phyla, the Euryarchaeota and Crenarchaeaota, seem to exhibit fundamental differences in regulatory systems. Euryarchaeota (e.g. halophiles, methanogens, thermophiles), like Bacteria and Eukaryotes, rely on bacterial-type two-component signal transduction systems (phosphorylation on His and Asp), as well as on the protein phosphorylation on Ser, Thr and Tyr by Hanks-type protein kinases. Instead, Crenarchaeota (e.g. acidophiles and (hyper)thermophiles) only depend on Hanks-type protein phosphorylation. In this review, the current knowledge of reversible protein phosphorylation in Archaea is presented. It combines results from identified phosphoproteins, biochemical characterization of protein kinases and protein phosphatases as well as target enzymes and first insights into archaeal signal transduction by biochemical, genetic and polyomic studies. PMID:27476079

  20. Large-scale analysis of phosphorylated proteins in maize leaf.

    PubMed

    Bi, Ying-Dong; Wang, Hong-Xia; Lu, Tian-Cong; Li, Xiao-Hui; Shen, Zhuo; Chen, Yi-Bo; Wang, Bai-Chen

    2011-02-01

    Phosphorylation is an ubiquitous regulatory mechanism governing the activity, subcellular localization, and intermolecular interactions of proteins. To identify a broad range of phosphoproteins from Zea mays, we enriched phosphopeptides from Zea mays leaves using titanium dioxide microcolumns and then extensively fractionated and identified the phosphopeptides by mass spectrometry. A total of 165 unique phosphorylation sites with a putative role in biological processes were identified in 125 phosphoproteins. Most of these proteins are involved in metabolism, including carbohydrate and protein metabolism. We identified novel phosphorylation sites on translation initiation factors, splicing factors, nucleolar RNA helicases, and chromatin-remodeling proteins such as histone deacetylases. Intriguingly, we also identified phosphorylation sites on several proteins associated with photosynthesis, and we speculate that these sites may be involved in carbohydrate metabolism or electron transport. Among these phosphoproteins, phosphoenolpyruvate carboxylase and NADH: nitrate reductase (NR) which catalyzes the rate-limiting and regulated step in the pathway of inorganic nitrogen assimilation were identified. A conserved phosphorylation site was found in the cytochrome b5 heme-binding domain of NADH: nitrate reductase, suggesting that NADH: nitrate reductase is phosphorylated by the same protein kinase or highly related kinases. These data demonstrate that the pathways that regulate diverse processes in plants are major targets of phosphorylation. PMID:21053013

  1. A secretory kinase complex regulates extracellular protein phosphorylation.

    PubMed

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

    2015-01-01

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

  2. Enrichment of phosphorylated peptides and proteins by selective precipitation methods.

    PubMed

    Rainer, Matthias; Bonn, Günther K

    2015-01-01

    Protein phosphorylation is one of the most prominent post-translational modifications involved in the regulation of cellular processes. Fundamental understanding of biological processes requires appropriate bioanalytical methods for selectively enriching phosphorylated peptides and proteins. Most of the commonly applied enrichment approaches include chromatographic materials including Fe(3+)-immobilized metal-ion affinity chromatography or metal oxides. In the last years, the introduction of several non-chromatographic isolation technologies has increasingly attracted the interest of many scientists. Such approaches are based on the selective precipitation of phosphorylated peptides and proteins by applying various metal cations. The excellent performance of precipitation-based enrichment methods can be explained by the absence of any stationary phase, resin or sorbent, which usually leads to unspecific binding. This review provides an overview of recently published methods for the selective precipitation of phosphorylated peptides and proteins. PMID:25587840

  3. Rapid changes in plasma membrane protein phosphorylation during initiation of cell wall digestion

    SciTech Connect

    Blowers, D.P.; Boss, W.F.; Trewavas, A.J. )

    1988-02-01

    Plasma membrane vesicles from wild carrot cells grown in suspension culture were isolated by aqueous two-phase partitioning, and ATP-dependent phosphorylation was measured with ({gamma}-{sup 32}P)ATP in the presence and absence of calcium. Treatment of the carrot cells with the cell wall digestion enzymes, driselase, in a sorbitol osmoticum for 1.5 min altered the protein phosphorylation pattern compared to that of cells treated with sorbitol alone. Driselase treatment resulted in decreased phosphorylation of a band of M{sub r} 80,000 which showed almost complete calcium dependence in the osmoticum treated cells; decreased phosphorylation of a band of M{sub r} 15,000 which showed little calcium activation, and appearance of a new band of calcium-dependent phosphorylation at M{sub r} 22,000. However, protein phosphorylation was decreased. Adding driselase to the in vitro reaction mixture caused a general decrease in the membrane protein phosphorylation either in the presence or absence of calcium which did not mimic the in vivo response. Cells labeled in vivo with inorganic {sup 32}P also showed a response to the Driselase treatment. An enzymically active driselas preparation was required for the observed responses.

  4. Altered dopamine transporter function and phosphorylation following chronic cocaine self-administration and extinction in rats.

    PubMed

    Ramamoorthy, Sammanda; Samuvel, Devadoss J; Balasubramaniam, Annamalai; See, Ronald E; Jayanthi, Lankupalle D

    2010-01-15

    Cocaine binds with the dopamine transporter (DAT), an effect that has been extensively implicated in its reinforcing effects. However, persisting adaptations in DAT regulation after cocaine self-administration have not been extensively investigated. Here, we determined the changes in molecular mechanisms of DAT regulation in the caudate-putamen (CPu) and nucleus accumbens (NAcc) of rats with a history of cocaine self-administration, followed by 3weeks of withdrawal under extinction conditions (i.e., no cocaine available). DA uptake was significantly higher in the CPu of cocaine-experienced animals as compared to saline-yoked controls. DAT V(max) was elevated in the CPu without changes in apparent affinity for DA. In spite of elevated CPu DAT activity, total and surface DAT density and DAT-PP2Ac (protein phosphatase 2A catalytic subunit) interaction remained unaltered, although p-Ser- DAT phosphorylation was elevated. In contrast to the CPu, there were no differences between cocaine and saline rats in the levels of DA uptake, DAT V(max) and K(m) values, total and surface DAT, p-Ser-DAT phosphorylation, or DAT-PP2Ac interactions in the NAcc. These results show that chronic cocaine self-administration leads to lasting, regionally specific alterations in striatal DA uptake and DAT-Ser phosphorylation. Such changes may be related to habitual patterns of cocaine-seeking observed during relapse. PMID:20035724

  5. Inhibition by calmodulin of calcium/phospholipid-dependent protein phosphorylation.

    PubMed Central

    Albert, K A; Wu, W C; Nairn, A C; Greengard, P

    1984-01-01

    Calmodulin was previously found to inhibit the Ca2+/phospholipid-dependent phosphorylation of an endogenous substrate, called the 87-kilodalton protein, in a crude extract prepared from rat brain synaptosomal cytosol. We investigated the mechanism of this inhibition, using Ca2+/phospholipid-dependent protein kinase and the 87-kilodalton protein, both of which had been purified to homogeneity from bovine brain. Rabbit brain calmodulin and some other Ca2+-binding proteins inhibited the phosphorylation of the 87-kilodalton protein by this kinase in the purified system. Calmodulin also inhibited the Ca2+/phospholipid-dependent phosphorylation of H1 histone, synapsin I, and the delta subunit of the acetylcholine receptor, with use of purified components. These results suggest that calmodulin may be a physiological regulator of Ca2+/phospholipid-dependent protein kinase. Images PMID:6233611

  6. METHIONINE OXIDATION AND PROTEIN PHOSPHORYLATION: INTERACTIVE PARTNERS IN SIGNALING?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein phosphorylation can affect the activity, stability or localization of a protein and as result plays a broad role in regulation of processes ranging from metabolism to control of plant growth and development. One aspect of current interest in our lab is how protein kinases target their substr...

  7. A novel sphingosine-dependent protein kinase (SDK1) specifically phosphorylates certain isoforms of 14-3-3 protein.

    PubMed

    Megidish, T; Cooper, J; Zhang, L; Fu, H; Hakomori, S

    1998-08-21

    Protein kinases activated by sphingosine or N,N'-dimethylsphingosine, but not by other lipids, have been detected and are termed sphingosine-dependent protein kinases (SDKs). These SDKs were previously shown to phosphorylate endogenous 14-3-3 proteins (Megidish, T., White, T., Takio, K., Titani, K., Igarashi, Y., and Hakomori, S. (1995) Biochem. Biophys. Res. Commun. 216, 739-747). We have now partially purified one SDK, termed SDK1, from cytosol of mouse Balb/c 3T3(A31) fibroblasts. SDK1 is a serine kinase with molecular mass 50-60 kDa that is strongly activated by N, N'-dimethylsphingosine and sphingosine, but not by ceramide, sphingosine 1-phosphate, or other sphingo-, phospho-, or glycerolipids tested. Its activity is inhibited by the protein kinase C activator phosphatidylserine. Activity of SDK1 is clearly distinct from other types of serine kinases tested, including casein kinase II, the alpha and zeta isoforms of protein kinase C, extracellular signal-regulated mitogene-activated protein kinase 1 (Erk-1), Erk-2, and Raf-1. SDK1 specifically phosphorylates certain isoforms of 14-3-3 (eta, beta, zeta) but not others (sigma, tau). The phosphorylation site was identified as Ser* in the sequence Arg-Arg-Ser-Ser*-Trp-Arg in 14-3-3 beta. The sigma and tau isoforms of 14-3-3 lack serine at this position, potentially explaining their lack of phosphorylation by SDK1. Interestingly, the phosphorylation site is located on the dimer interface of 14-3-3. Phosphorylation of this site by SDK1 was studied in 14-3-3 mutants. Mutation of a lysine residue, located 9 amino acids N-terminal to the phosphorylation site, abolished 14-3-3 phosphorylation. Furthermore, co-immunoprecipitation experiments demonstrate an association between an SDK and 14-3-3 in situ. Exogenous N, N'-dimethylsphingosine stimulates 14-3-3 phosphorylation in Balb/c 3T3 fibroblasts, suggesting that SDK1 may phosphorylate 14-3-3 in situ. These data support a biological role of SDK1 activation and consequent

  8. Protein Phosphorylation during Coconut Zygotic Embryo Development1

    PubMed Central

    Islas-Flores, Ignacio; Oropeza, Carlos; Hernández-Sotomayor, S.M. Teresa

    1998-01-01

    Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L.) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [γ-32P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [32P]phosphoserine, [32P]phosphothreonine, and [32P]phosphotyrosine in [32P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60src (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species. PMID:9733545

  9. Aberrant protein phosphorylation in Alzheimer disease brain disturbs pro-survival and cell death pathways.

    PubMed

    Perluigi, M; Barone, E; Di Domenico, F; Butterfield, D A

    2016-10-01

    Protein phosphorylation of serine, threonine, and tyrosine residues is one of the most prevalent post-translational modifications fundamental in mediating diverse cellular functions in living cells. Aberrant protein phosphorylation is currently recognized as a critical step in the pathogenesis and progression of Alzheimer disease (AD). Changes in the pattern of protein phosphorylation of different brain regions are suggested to promote AD transition from a presymptomatic to a symptomatic state in response to accumulating amyloid β-peptide (Aβ). Several experimental approaches have been utilized to profile alteration of protein phosphorylation in the brain, including proteomics. Among central pathways regulated by kinases/phosphatases those involved in the activation/inhibition of both pro survival and cell death pathways play a central role in AD pathology. We discuss in detail how aberrant phosphorylation could contribute to dysregulate p53 activity and insulin-mediated signaling. Taken together these results highlight that targeted therapeutic intervention, which can restore phosphorylation homeostasis, either acting on kinases and phosphatases, conceivably may prove to be beneficial to prevent or slow the development and progression of AD. PMID:27425034

  10. A Rapid Screening Assay to Search for Phosphorylated Proteins in Tissue Extracts

    PubMed Central

    Garaguso, Ignazio; Borlak, Juergen

    2012-01-01

    Reversible protein phosphorylation is an essential mechanism in the regulation of diverse biological processes, nonetheless is frequently altered in disease. As most phosphoproteome studies are based on optimized in-vitro cell culture studies new methods are in need to improve de novo identification and characterization of phosphoproteins in extracts from tissues. Here, we describe a rapid and reliable method for the detection of phosphoproteins in tissue extract based on an experimental strategy that employs 1D and 2D SDS PAGE, Western immunoblotting of phosphoproteins, in-gel protease digestion and enrichment of phosphorpeptides using metal oxide affinity chromatography (MOAC). Subsequently, phosphoproteins are identified by MALDI-TOF-MS/MS with the CHCA-TL or DHB ML sample matrix preparation method and further characterized by various bioinformatic software tools to search for candidate kinases and phosphorylation-dependent binding motifs. The method was applied to mouse lung tissue extracts and resulted in an identification of 160 unique phosphoproteins. Notably, TiO2 enrichment of pulmonary protein extracts resulted in an identification of additional 17 phosphoproteins and 20 phosphorylation sites. By use of MOAC, new phosphorylation sites were identified as evidenced for the advanced glycosylation end product-specific receptor. So far this protein was unknown to be phosphorylated in lung tissue of mice. Overall the developed methodology allowed efficient and rapid screening of phosphorylated proteins and can be employed as a general experimental strategy for an identification of phosphoproteins in tissue extracts. PMID:23166814

  11. Global Analysis of Protein Expression and Phosphorylation Levels in Nicotine-Treated Pancreatic Stellate Cells.

    PubMed

    Paulo, Joao A; Gaun, Aleksandr; Gygi, Steven P

    2015-10-01

    Smoking is a risk factor in pancreatic disease; however, the biochemical mechanisms correlating smoking with pancreatic dysfunction remain poorly understood. Strategies using multiplexed isobaric tag-based mass spectrometry facilitate the study of drug-induced perturbations on biological systems. Here, we present the first large-scale analysis of the proteomic and phosphoproteomic alterations in pancreatic stellate cells following treatment with two nicotinic acetylcholine receptor (nAChR) ligands: nicotine and α-bungarotoxin. We treated cells with nicotine or α-bungarotoxin for 12 h in triplicate and compared alterations in protein expression and phosphorylation levels to mock-treated cells using a tandem mass tag (TMT9plex)-based approach. Over 8100 proteins were quantified across all nine samples, of which 46 were altered in abundance upon treatment with nicotine. Proteins with increased abundance included those associated with neurons, defense mechanisms, indicators of pancreatic disease, and lysosomal proteins. In addition, we measured differences for ∼16 000 phosphorylation sites across all nine samples using a titanium dioxide-based strategy, of which 132 sites were altered with nicotine and 451 with α-bungarotoxin treatment. Many altered phosphorylation sites were involved in nuclear function and transcriptional events. This study supports the development of future targeted investigations to establish a better understanding for the role of nicotine and associated receptors in pancreatic disease. PMID:26265067

  12. Global analysis of protein expression and phosphorylation levels in nicotine-treated pancreatic stellate cells

    PubMed Central

    Paulo, Joao A.; Gaun, Aleksandr; Gygi, Steven P.

    2016-01-01

    Smoking is a risk factor in pancreatic disease, however, the biochemical mechanisms correlating smoking with pancreatic dysfunction remain poorly understood. Strategies using multiplexed isobaric tag-based mass spectrometry facilitate the study of drug-induced perturbations on biological systems. Here, we present the first large scale analysis of the proteomic and phosphoproteomic alterations in pancreatic stellate cells following treatment with two nicotinic acetylcholine receptor (nAChR) ligands: nicotine and α-bungarotoxin. We treated cells with nicotine or α-bungarotoxin for 12hr in triplicate and compared alterations in protein expression and phosphorylation levels to mock treated cells using a tandem mass tag (TMT9plex)-based approach. Over 8,100 proteins were quantified across all nine samples of which 46 were altered in abundance upon treatment with nicotine. Proteins with increased abundance included those associated with neurons, defense mechanisms, indicators of pancreatic disease and lysosomal proteins. In addition, we measured differences for ∼16,000 phosphorylation sites across all nine samples using a titanium dioxide-based strategy, of which 132 sites were altered with nicotine and 451 with α-bungarotoxin treatment. Many altered phosphorylation sites were involved in nuclear function and transcriptional events. This study supports the development of future targeted investigations to establish a better understanding for the role of nicotine and associated receptors in pancreatic disease. PMID:26265067

  13. Abundant protein phosphorylation potentially regulates Arabidopsis anther development

    PubMed Central

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

    2016-01-01

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

  14. Abundant protein phosphorylation potentially regulates Arabidopsis anther development.

    PubMed

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

    2016-09-01

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

  15. Phosphorylation of Intrinsically Disordered Regions in Remorin Proteins

    PubMed Central

    Marín, Macarena; Ott, Thomas

    2012-01-01

    Plant-specific remorin proteins reside in subdomains of plasma membranes, originally termed membrane rafts. They probably facilitate cellular signal transduction by direct interaction with signaling proteins such as receptor-like kinases and may dynamically modulate their lateral segregation within plasma membranes. Recent evidence suggests such functions of remorins during plant–microbe interactions and innate immune responses, where differential phosphorylation of some of these proteins has been described to be dependent on the perception of the microbe-associated molecular pattern (MAMP) flg22 and the presence of the NBS–LRR resistance protein RPM1. A number of specifically phosphorylated residues in their highly variable and intrinsically disordered N-terminal regions have been identified. Sequence diversity of these evolutionary distinct domains suggests that remorins may serve a wide range of biological functions. Here, we describe patterns and features of intrinsic disorder in remorin protein and discuss possible functional implications of phosphorylation within these rapidly evolving domains. PMID:22639670

  16. Predicting and analyzing protein phosphorylation sites in plants using musite.

    PubMed

    Yao, Qiuming; Gao, Jianjiong; Bollinger, Curtis; Thelen, Jay J; Xu, Dong

    2012-01-01

    Although protein phosphorylation sites can be reliably identified with high-resolution mass spectrometry, the experimental approach is time-consuming and resource-dependent. Furthermore, it is unlikely that an experimental approach could catalog an entire phosphoproteome. Computational prediction of phosphorylation sites provides an efficient and flexible way to reveal potential phosphorylation sites and provide hypotheses in experimental design. Musite is a tool that we previously developed to predict phosphorylation sites based solely on protein sequence. However, it was not comprehensively applied to plants. In this study, the phosphorylation data from Arabidopsis thaliana, B. napus, G. max, M. truncatula, O. sativa, and Z. mays were collected for cross-species testing and the overall plant-specific prediction as well. The results show that the model for A. thaliana can be extended to other organisms, and the overall plant model from Musite outperforms the current plant-specific prediction tools, Plantphos, and PhosphAt, in prediction accuracy. Furthermore, a comparative study of predicted phosphorylation sites across orthologs among different plants was conducted to reveal potential evolutionary features. A bipolar distribution of isolated, non-conserved phosphorylation sites, and highly conserved ones in terms of the amino acid type was observed. It also shows that predicted phosphorylation sites conserved within orthologs do not necessarily share more sequence similarity in the flanking regions than the background, but they often inherit protein disorder, a property that does not necessitate high sequence conservation. Our analysis also suggests that the phosphorylation frequencies among serine, threonine, and tyrosine correlate with their relative proportion in disordered regions. Musite can be used as a web server (http://musite.net) or downloaded as an open-source standalone tool (http://musite.sourceforge.net/). PMID:22934099

  17. Protein kinase activators alter glial cholesterol esterification

    SciTech Connect

    Jeng, I.; Dills, C.; Klemm, N.; Wu, C.

    1986-05-01

    Similar to nonneural tissues, the activity of glial acyl-CoA cholesterol acyltransferase is controlled by a phosphorylation and dephosphorylation mechanism. Manipulation of cyclic AMP content did not alter the cellular cholesterol esterification, suggesting that cyclic AMP is not a bioregulator in this case. Therefore, the authors tested the effect of phorbol-12-myristate 13-acetate (PMA) on cellular cholesterol esterification to determine the involvement of protein kinase C. PMA has a potent effect on cellular cholesterol esterification. PMA depresses cholesterol esterification initially, but cells recover from inhibition and the result was higher cholesterol esterification, suggesting dual effects of protein kinase C. Studies of other phorbol analogues and other protein kinase C activators such as merezein indicate the involvement of protein kinase C. Oleoyl-acetyl glycerol duplicates the effect of PMA. This observation is consistent with a diacyl-glycerol-protein kinase-dependent reaction. Calcium ionophore A23187 was ineffective in promoting the effect of PMA. They concluded that a calcium-independent and protein C-dependent pathway regulated glial cholesterol esterification.

  18. Microfluidic IEF technique for sequential phosphorylation analysis of protein kinases

    NASA Astrophysics Data System (ADS)

    Choi, Nakchul; Song, Simon; Choi, Hoseok; Lim, Bu-Taek; Kim, Young-Pil

    2015-11-01

    Sequential phosphorylation of protein kinases play the important role in signal transduction, protein regulation, and metabolism in living cells. The analysis of these phosphorylation cascades will provide new insights into their physiological functions in many biological functions. Unfortunately, the existing methods are limited to analyze the cascade activity. Therefore, we suggest a microfluidic isoelectric focusing technique (μIEF) for the analysis of the cascade activity. Using the technique, we show that the sequential phosphorylation of a peptide by two different kinases can be successfully detected on a microfluidic chip. In addition, the inhibition assay for kinase activity and the analysis on a real sample have also been conducted. The results indicate that μIEF is an excellent means for studies on phosphorylation cascade activity.

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

    PubMed Central

    Biever, Anne; Valjent, Emmanuel; Puighermanal, Emma

    2015-01-01

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

  20. Systematic Analysis of Protein Phosphorylation Networks From Phosphoproteomic Data*

    PubMed Central

    Song, Chunxia; Ye, Mingliang; Liu, Zexian; Cheng, Han; Jiang, Xinning; Han, Guanghui; Songyang, Zhou; Tan, Yexiong; Wang, Hongyang; Ren, Jian; Xue, Yu; Zou, Hanfa

    2012-01-01

    In eukaryotes, hundreds of protein kinases (PKs) specifically and precisely modify thousands of substrates at specific amino acid residues to faithfully orchestrate numerous biological processes, and reversibly determine the cellular dynamics and plasticity. Although over 100,000 phosphorylation sites (p-sites) have been experimentally identified from phosphoproteomic studies, the regulatory PKs for most of these sites still remain to be characterized. Here, we present a novel software package of iGPS for the prediction of in vivo site-specific kinase-substrate relations mainly from the phosphoproteomic data. By critical evaluations and comparisons, the performance of iGPS is satisfying and better than other existed tools. Based on the prediction results, we modeled protein phosphorylation networks and observed that the eukaryotic phospho-regulation is poorly conserved at the site and substrate levels. With an integrative procedure, we conducted a large-scale phosphorylation analysis of human liver and experimentally identified 9719 p-sites in 2998 proteins. Using iGPS, we predicted a human liver protein phosphorylation networks containing 12,819 potential site-specific kinase-substrate relations among 350 PKs and 962 substrates for 2633 p-sites. Further statistical analysis and comparison revealed that 127 PKs significantly modify more or fewer p-sites in the liver protein phosphorylation networks against the whole human protein phosphorylation network. The largest data set of the human liver phosphoproteome together with computational analyses can be useful for further experimental consideration. This work contributes to the understanding of phosphorylation mechanisms at the systemic level, and provides a powerful methodology for the general analysis of in vivo post-translational modifications regulating sub-proteomes. PMID:22798277

  1. Phosphorylation of vaccinia virus core proteins during transcription in vitro.

    PubMed Central

    Moussatche, N; Keller, S J

    1991-01-01

    The phosphorylation of vaccinia virus core proteins has been studied in vitro during viral transcription. The incorporation of [gamma-32P]ATP into protein is linear for the first 2 min of the reaction, whereas incorporation of [3H]UTP into RNA lags for 1 to 2 min before linear synthesis. At least 12 different proteins are phosphorylated on autoradiograms of acrylamide gels, and the majority of label is associated with low-molecular-weight proteins. If the transcription reaction is reduced by dropping the pH to 7 from its optimal of 8.5, two proteins (70 and 80 kDa) are no longer phosphorylated. RNA isolated from the pH 7 transcription reaction hybridized primarily to the vaccinia virus HindIII DNA fragments D to F, whereas the transcripts synthesized at pH 8.5 hybridized to almost all of the HindIII-digested vaccinia virus DNA fragments. The differences between the pH 7.0 and 8.5 transcription reactions in phosphorylation and transcription could be eliminated by preincubating the viral cores with 2 mM ATP. In sum, the results suggest that the phosphorylation of the 70- and 80-kDa peptides may contribute to the regulation of early transcription. Images PMID:2016772

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

    PubMed

    Cao, Muqing; Li, Guihua; Pan, Junmin

    2009-01-01

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

  3. An evolutionary view on thylakoid protein phosphorylation uncovers novel phosphorylation hotspots with potential functional implications.

    PubMed

    Grieco, Michele; Jain, Arpit; Ebersberger, Ingo; Teige, Markus

    2016-06-01

    The regulation of photosynthetic light reactions by reversible protein phosphorylation is well established today, but functional studies have so far mostly been restricted to processes affecting light-harvesting complex II and the core proteins of photosystem II. Virtually no functional data are available on regulatory effects at the other photosynthetic complexes despite the identification of multiple phosphorylation sites. Therefore we summarize the available data from 50 published phospho-proteomics studies covering the main complexes involved in photosynthetic light reactions in the 'green lineage' (i.e. green algae and land plants) as well as its cyanobacterial counterparts. In addition, we performed an extensive orthologue search for the major photosynthetic thylakoid proteins in 41 sequenced genomes and generated sequence alignments to survey the phylogenetic distribution of phosphorylation sites and their evolutionary conservation from green algae to higher plants. We observed a number of uncharacterized phosphorylation hotspots at photosystem I and the ATP synthase with potential functional relevance as well as an unexpected divergence of phosphosites. Although technical limitations might account for a number of those differences, we think that many of these phosphosites have important functions. This is particularly important for mono- and dicot plants, where these sites might be involved in regulatory processes such as stress acclimation. PMID:27117338

  4. Phosphorylation of alfalfa mosaic virus movement protein in vivo.

    PubMed

    Kim, Bong-Suk; Halk, Edward L; Merlo, Donald J; Nelson, Steven E; Loesch-Fries, L Sue

    2014-07-01

    The 32-kDa movement protein, P3, of alfalfa mosaic virus (AMV) is essential for cell-to-cell spread of the virus in plants. P3 shares many properties with other virus movement proteins (MPs); however, it is not known if P3 is posttranslationally modified by phosphorylation, which is important for the function of other MPs. When expressed in Nicotiana tabacum, P3 accumulated primarily in the cell walls of older leaves or in the cytosol of younger leaves. When expressed in Pischia pastoris, P3 accumulated primarily in a soluble form. Metabolic labeling indicated that a portion of P3 was phosphorylated in both tobacco and yeast, suggesting that phosphorylation regulates the function of this protein as it does for other virus MPs. PMID:24435161

  5. Phosphorylation of proteins in Dictyostelium discoideum during development

    SciTech Connect

    Coffman, D.S.

    1982-01-01

    The phosphoproteins in D. discoideum were studied with respect to their formation, metabolic stability, cellular and subcellular distribution. Special emphasis was on the role of cAMP on the pattern of phosphorylation. Amoebae were metabolically labeled with /sup 32/P/sub i/; subsequently proteins of the total lysate, nuclei and membranes were resolved by SDS-polyacrylamide gel electrophoresis and subjected to autoradiography. Numerous changes in the profile of phosphoproteins were observed during development. Functions were assigned to four membranal phosphoproteins; only one protein, the heavy chain of myosin, was susceptible to phosphorylation in vitro when purified membranes and /sup 32/P-ATP were used. A comparison between the time of protein synthesis and phosphorylation, as examined in vivo using /sup 35/S-methionine and /sup 32/P/sub i/ labeling of amoebae and two-dimensional gel electrophoresis, indicated that phosphorylation is concurrent with synthesis. It appears then that there are two classes of membranal phosphoproteins in D. discoideum which differ with respect to the stability of the phosphate moiety. It is evident that the turnover of the phosphate moiety in myosin heavy chain plays a crucial role in the function of myosin; a role for the metabolically inert phosphate of other membranal proteins remains to be established. The G protein which couples occupancy of hormone receptor to stimulation of adenylate cyclase in higher multicellular eukaryotes was detected in D. discoideum. The G protein is present in approximately equal amounts in vegetative and in developing amoebae.

  6. Phosphorylation of tau protein over time in rats subjected to transient brain ischemia.

    PubMed

    Song, Bo; Ao, Qiang; Wang, Zhen; Liu, Weiqiang; Niu, Ying; Shen, Qin; Zuo, Huancong; Zhang, Xiufang; Gong, Yandao

    2013-12-01

    Transient brain ischemia has been shown to induce hyperphosphorylation of the microtubule-associated protein tau. To further determine the mechanisms underlying these processes, we investigated the interaction between tau, glycogen synthase kinase (GSK)-3β and protein phos-phatase 2A. The results confirmed that tau protein was dephosphorylated during brain ischemia; in addition, the activity of GSK-3β was increased and the activity of protein phosphatase 2A was decreased. After reperfusion, tau protein was hyperphosphorylated, the activity of GSK-3β was decreased and the activity of protein phosphatase 2A remained low. Importantly, the interaction of tau with GSK-3β and protein phosphatase 2A was altered during ischemia and reperfusion. Lithium chloride could affect tau phosphorylation by regulating the interaction of tau with GSK-3β and protein phosphatase 2A, and improve learning and memory ability of rats after transient brain ischemia. The present study demonstrated that it was the interaction of tau with GSK-3β and protein phosphatase 2A, rather than their individual activities, that dominates the phosphorylation of tau in transient brain ischemia. Hyperphosphorylated tau protein may play an important role in the evolution of brain injury in ischemic stroke. The neuroprotective effects of lithium chloride partly depend on the inhibition of tau phosphorylation during transient brain ischemia. PMID:25206638

  7. Phosphorylation of Cysteine String Protein Triggers a Major Conformational Switch.

    PubMed

    Patel, Pryank; Prescott, Gerald R; Burgoyne, Robert D; Lian, Lu-Yun; Morgan, Alan

    2016-08-01

    Cysteine string protein (CSP) is a member of the DnaJ/Hsp40 chaperone family that localizes to neuronal synaptic vesicles. Impaired CSP function leads to neurodegeneration in humans and model organisms as a result of misfolding of client proteins involved in neurotransmission. Mammalian CSP is phosphorylated in vivo on Ser10, and this modulates its protein interactions and effects on neurotransmitter release. However, there are no data on the structural consequences of CSP phosphorylation to explain these functional effects. We show that Ser10 phosphorylation causes an order-to-disorder transition that disrupts CSP's extreme N-terminal α helix. This triggers the concomitant formation of a hairpin loop stabilized by ionic interactions between phosphoSer10 and the highly conserved J-domain residue, Lys58. These phosphorylation-induced effects result in significant changes to CSP conformation and surface charge distribution. The phospho-switch revealed here provides structural insight into how Ser10 phosphorylation modulates CSP function and also has potential implications for other DnaJ phosphoproteins. PMID:27452402

  8. Altering the antigenicity of proteins.

    PubMed Central

    Alexander, H; Alexander, S; Getzoff, E D; Tainer, J A; Geysen, H M; Lerner, R A

    1992-01-01

    To better understand the binding interaction between antigen and antibody we need to distinguish protein residues critical to the binding energy and mechanism from residues merely localized in the interface. By analyzing the binding of monoclonal antibodies to recombinant wild-type and mutant myohemerythrin (MHr) proteins, we were able to test the role of individual critical residues at the highly antigenic site MHr-(79-84), within the context of the folded protein. The results directly show the existence of antigenically critical residues, whose mutations significantly reduce antibody binding to the folded protein, thus verifying peptide-based assignments of these critical residues and demonstrating the ability of buried side chains to influence antigenicity. Taken together, these results (i) distinguish the antigenic surface from the solvent-exposed protein surface before binding, (ii) support a two-stage interaction mechanism allowing inducible changes in protein antigens by antibody binding, and (iii) show that protein antigenicity can be significantly reduced by alteration of single critical residues without destroying biological activity. Images PMID:1373498

  9. Screening for protein phosphorylation using nanoscale reactions on microdroplet arrays.

    PubMed

    Küster, Simon K; Pabst, Martin; Zenobi, Renato; Dittrich, Petra S

    2015-01-26

    We present a novel and straightforward screening method to detect protein phosphorylations in complex protein mixtures. A proteolytic digest is separated by a conventional nanoscale liquid chromatography (nano-LC) separation and the eluate is immediately compartmentalized into microdroplets, which are spotted on a microarray MALDI plate. Subsequently, the enzyme alkaline phosphatase is applied to every second microarray spot to remove the phosphate groups from phosphorylated peptides, which results in a mass shift of n×-80 Da. The MALDI-MS scan of the microarray is then evaluated by a software algorithm to automatically identify the phosphorylated peptides by exploiting the characteristic chromatographic peak profile induced by the phosphatase treatment. This screening method does not require extensive MS/MS experiments or peak list evaluation and can be easily extended to other enzymatic or chemical reactions. PMID:25504774

  10. Phosphorylation of mouse melanopsin by protein kinase A.

    PubMed

    Blasic, Joseph R; Brown, R Lane; Robinson, Phyllis R

    2012-01-01

    The visual pigment melanopsin is expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs) in the mammalian retina, where it is involved in non-image forming light responses including circadian photoentrainment, pupil constriction, suppression of pineal melatonin synthesis, and direct photic regulation of sleep. It has recently been shown that the melanopsin-based light response in ipRGCs is attenuated by the neurotransmitter dopamine. Here, we use a heterologous expression system to demonstrate that mouse melanopsin can be phosphorylated by protein kinase A, and that phosphorylation can inhibit melanopsin signaling in HEK cells. Site-directed mutagenesis experiments revealed that this inhibitory effect is primarily mediated by phosphorylation of sites T186 and S287 located in the second and third intracellular loops of melanopsin, respectively. Furthermore, we show that this phosphorylation can occur in vivo using an in situ proximity-dependent ligation assay (PLA). Based on these data, we suggest that the attenuation of the melanopsin-based light response by dopamine is mediated by direct PKA phosphorylation of melanopsin, rather than phosphorylation of a downstream component of the signaling cascade. PMID:23049792

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

    PubMed Central

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

    2009-01-01

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

  12. Phosphorylation in protein-protein binding: effect on stability and function

    PubMed Central

    Nishi, Hafumi; Hashimoto, Kosuke; Panchenko, Anna R.

    2011-01-01

    Summary Post-translational modifications offer a dynamic way to regulate protein activity, subcellular localization and stability. Here we estimate the effect of phosphorylation on protein binding and function for different types of complexes from human proteome. We find that phosphorylation sites have a tendency to be located on binding interfaces in heterooligomeric and weak transient homooligomeric complexes. The analysis of molecular mechanisms of phosphorylation shows that phosphorylation may modulate the strength of interactions directly on interfaces and binding hotspots have a tendency to be phosphorylated in heterooligomers. Although majority of phosphosites do not show significant estimated stability differences upon attaching the phosphate groups, for about one third of all complexes it causes relatively large changes in binding energy. We discuss the cases where phosphorylation mediates the complex formation and regulates the function. We show that phosphorylation sites are not only more likely to be evolutionary conserved than surface residues but even more so than other interfacial residues. PMID:22153503

  13. A secretory kinase complex regulates extracellular protein phosphorylation

    PubMed Central

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

    2015-01-01

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

  14. HALOACETIC ACIDS PERTURB PROTEIN PHOSPHORYLATION IN MOUSE EMBRYOS IN VITRO

    EPA Science Inventory

    HALOACETIC ACIDS PERTURB PROTEIN PHOSPHORYLATION IN MOUSE EMBRYOS IN VITRO. MR Blanton and ES Hunter. Reproductive Toxicology Division, NHEERL, ORD, US EPA, RTP, NC, USA.
    Sponsor: JM Rogers.
    Haloacetic Acids (HAAs) formed during the disinfection process are present in drin...

  15. In planta changes in protein phosphorylation induced by the plant hormone abscisic acid

    PubMed Central

    Kline, Kelli G.; Barrett-Wilt, Gregory A.; Sussman, Michael R.

    2010-01-01

    Abscisic acid (ABA) is a hormone that controls seed dormancy and germination as well as the overall plant response to important environmental stresses such as drought. Recent studies have demonstrated that the ABA-bound receptor binds to and inhibits a class of protein phosphatases. To identify more broadly the phosphoproteins affected by this hormone in vivo, we used 14N/15N metabolic labeling to perform a quantitative untargeted mass spectrometric analysis of the Arabidopsis thaliana phosphoproteome following ABA treatment. We found that 50 different phosphopeptides had their phosphorylation state significantly altered by ABA over a treatment period lasting 5–30 min. Among these changes were increases in phosphorylation of subfamily 2 SNF1-related kinases and ABA-responsive basic leucine zipper transcription factors implicated in ABA signaling by previous in vitro studies. Furthermore, four members of the aquaporin family showed decreased phosphorylation at a carboxy-terminal serine which is predicted to cause closure of the water-transporting aquaporin gate, consistent with ABA's role in ameliorating the effect of drought. Finally, more than 20 proteins not previously known to be involved with ABA were found to have significantly altered phosphorylation levels. Many of these changes are phosphorylation decreases, indicating that an expanded model of ABA signaling, beyond simple phosphatase inhibition, may be necessary. This quantitative proteomics dataset provides a more comprehensive, albeit incomplete, view both of the protein targets whose biochemical activities are likely to be controlled by ABA and of the nature of the emerging phosphorylation and dephosphorylation cascades triggered by this hormone. PMID:20733066

  16. Protein tyrosine phosphorylation during meiotic divisions of starfish oocytes

    SciTech Connect

    Peaucellier, G.; Andersen, A.C.; Kinsey, W.H. )

    1990-04-01

    We have used an antibody specific for phosphotyrosine to investigate protein phosphorylation on tyrosine during hormone-induced maturation of starfish oocytes. Analysis of immunoprecipitates from cortices of in vivo labeled Marthasterias glacialis oocytes revealed the presence of labeled phosphotyrosine-containing proteins only after hormone addition. Six major phosphoproteins of 195, 155, 100, 85, 45, and 35 kDa were detected. Total activity in immunoprecipitates increased until first polar body emission and was greatly reduced upon completion of meiosis but some proteins exhibited different kinetics. The labeling of the 155-kDa protein reached a maximum at germinal vesicle breakdown, while the 35-kDa appeared later and disappeared after polar body emission. Similar results were obtained with Asterias rubens oocytes. In vitro phosphorylation of cortices showed that tyrosine kinase activity is a major protein kinase activity in this fraction, the main endogenous substrate being a 68-kDa protein. The proteins phosphorylated on tyrosine in vitro were almost similar in extracts from oocytes treated or not with the hormone.

  17. Phosphorylation of ornithine decarboxylase by a polyamine-dependent protein kinase.

    PubMed Central

    Atmar, V J; Kuehn, G D

    1981-01-01

    This paper presents evidence that a polyamine-dependent protein kinase (EC 2.7.1.37) purified from nuclei of the slime mold Physarum polycephalum catalyzes phosphorylation of ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17). The protein kinase had properties similar to OrnDCase antizyme. Phosphocellulose chromatography of nuclear preparations from P. polycephalum yielded the polyamine-dependent protein kinase of subunit Mr 26,000 that was resolved from a second fraction in which the protein kinase copurified with a phosphate-acceptor protein of subunit Mr 70,000. At Na+ concentrations less than approximately 150 mM, a complex formed between the protein kinase and the phosphate-acceptor protein. The complex did not demonstrate protein kinase or OrnDCase activity. The complex was dissociated by greater than 150 mM Na+ into its constituent proteins. The dissociated complex catalyzed phosphorylation of the Mr 70,000 component in the presence of spermidine and spermine, and it also demonstrated OrnDCase activity. The purified Mr 70,000 component from the complex and authentic OrnDCase, purified by procedures previously reported, were virtually identical with respect to OrnDCase activity, capacity to be phosphorylated by the polyamine-dependent protein kinase, amino acid composition, and immunological crossreactivity. Phosphorylation of OrnDCase by the polyamine-dependent protein kinase sharply inhibited OrnDCase activity. Thus, this is an example of posttranslational covalent modification of OrnDCase with concurrent alteration of its catalytic function. It is also an unusual example of control of the first enzyme in a biosynthetic pathway by a protein kinase that is, in turn, modulated by the immediate end products of the pathway. Images PMID:6946489

  18. IGF-1-induced phosphorylation and altered distribution of TSC1/TSC2 in C2C12 myotubes

    PubMed Central

    Miyazaki, Mitsunori; McCarthy, John J; Esser, Karyn A

    2010-01-01

    Insulin like growth factor-1 (IGF-1) is established as an anabolic factor that can induce skeletal muscle growth through activating the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. While this signaling pathway has been heavily studied, the molecular mechanisms linking IGF-1 binding to mTOR activation are still poorly defined in muscle. The purpose of this study was to test the hypothesis that IGF-1 activation of mTOR in C2C12 myotubes requires a phosphorylation dependent, altered distribution of the tuberous sclerosis complex (TSC)1/TSC2 complex from the membrane to the cytosol. We found that IGF-1 treatment does not affect complex formation between TSC1 and TSC2, but rather IGF-1 induces an altered distribution of the TSC1/TSC2 complex in C2C12 myotubes. In response to IGF-1 treatment, there was a relative re-distribution of the TSC1/TSC2 complex, composed of TSC1 and phosphorylated TSC2, from the membrane to the cytosol. IGF-1 stimulated TSC1/TSC2 phosphorylation and re-distribution were completely prevented by the PI3K inhibitor wortmannin, but were not with the downstream mTOR inhibitor, rapamycin. When a non-phosphorylatable form of TSC2 (S939A) was overexpressed, phosphorylation-dependent binding of the scaffold protein 14-3-3 to TSC2 was diminished and no re-distribution of the TSC1/TSC2 complex was observed following IGF-1 stimulation. These results indicate that TSC2 phosphorylation in response to IGF-1 treatment is necessary for the altered distribution of the TSC1/TSC2 complex to the cytosol and we suggest that this translocation is likely critical for mTOR activation by dissociating the interaction between the GAP activity of the TSC1/TSC2 complex and its downstream target Rheb. PMID:20412061

  19. Phosphorylation of Cardiac Myosin Binding Protein-C is a Critical Mediator of Diastolic Function

    PubMed Central

    Rosas, Paola C.; Liu, Yang; Abdalla, Mohamed I.; Thomas, Candice M.; Kidwell, David T.; Dusio, Giuseppina F.; Mukhopadhyay, Dhriti; Kumar, Rajesh; Baker, Kenneth M.; Mitchell, Brett M.; Powers, Patricia A.; Fitzsimons, Daniel P.; Patel, Bindiya G.; Warren, Chad M.; Solaro, R. John; Moss, Richard L.; Tong, Carl W.

    2015-01-01

    Background Heart failure with preserved ejection fraction (HFpEF) accounts for approximately 50% of all cases of heart failure and currently has no effective treatment. Diastolic dysfunction underlies HFpEF; therefore, elucidation of the mechanisms that mediate relaxation can provide new potential targets for treatment. Cardiac myosin binding protein-C (cMyBP-C) is a thick filament protein that modulates cross-bridge cycling rates via alterations in its phosphorylation status. Thus, we hypothesize that phosphorylated cMyBP-C accelerates rate of cross-bridge detachment, thereby enhancing relaxation to mediate diastolic function. Methods and Results We compared mouse models expressing phosphorylation deficient cMyBP-C(S273A/S282A/S302A)-cMyBP-C(t3SA), phosphomimetic cMyBP-C(S273D/S282D/S302D)-cMyBP-C(t3SD), and WT-control cMyBP-C(tWT) to elucidate the functional effects of cMyBP-C phosphorylation. Decreased voluntary running distances, increased lung/body weight ratios, and increased brain natriuretic peptide (BNP) levels in cMyBP-C(t3SA) mice demonstrate that phosphorylation deficiency is associated with signs of heart failure. Echocardiography (ejection fraction, myocardial relaxation velocity) and pressure/volume measurements (−dP/dtmin, pressure decay time constant Tau-Glantz, passive filling stiffness) show that cMyBP-C phosphorylation enhances myocardial relaxation in cMyBP-C(t3SD) mice while deficient cMyBP-C phosphorylation causes diastolic dysfunction with preserved ejection fraction in cMyBP-C(t3SA) mice. Simultaneous force and [Ca2+]i measurements on intact papillary muscles show that enhancement of relaxation in cMyBP-C(t3SD) mice and impairment of relaxation in cMyBP-C(t3SA) mice are not due to altered [Ca2+]i handling, implicating that altered cross-bridge detachment rates mediate these changes in relaxation rates. Conclusions cMyBP-C phosphorylation enhances relaxation while deficient phosphorylation causes diastolic dysfunction and phenotypes

  20. Ceramide-mediated depression in cardiomyocyte contractility through PKC activation and modulation of myofilament protein phosphorylation

    PubMed Central

    Simon, Jillian N.; Chowdhury, Shamim A.K.; Warren, Chad M.; Sadayappan, Sakthivel; Wieczorek, David F.; Solaro, R. John; Wolska, Beata M.

    2015-01-01

    Although ceramide accumulation in the heart is considered a major factor in promoting apoptosis and cardiac disorders, including heart failure, lipotoxicity and ischemia-reperfusion injury, little is known about ceramide’s role in mediating changes in contractility. In the present study, we measured the functional consequences of acute exposure of isolated field stimulated adult rat cardiomyocytes to C6-ceramide. Exogenous ceramide treatment depressed the peak amplitude and the maximal velocity of shortening without altering intracellular calcium levels or kinetics. The inactive ceramide analog C6-dihydroceramide had no effect on myocyte shortening or [Ca2+]i transients. Experiments testing a potential role for C6-ceramide-mediated effects on activation of protein kinase C (PKC) demonstrated evidence for signaling through the calcium-independent isoform, PKCε. We employed 2 dimensional electrophoresis and anti-phospho-peptide antibodies to test whether treatment of the cardiomyocytes with C6-ceramide altered myocyte shortening via PKC dependent phosphorylation of myofilament proteins. Compared to controls, myocytes treated with ceramide exhibited increased phosphorylation of myosin binding protein-C (cMyBP-C), specifically at Ser273 and Ser302, and troponin I (cTnI) at sites apart from Ser23/24, which could be attenuated with PKC inhibition. We conclude that the altered myofilament response to calcium resulting from multiple sites of PKC-dependent phosphorylation contributes to contractile dysfunction that is associated with cardiac diseases in which elevations in ceramides are present. PMID:25280528

  1. Protein phosphorylation in Bradyrhizobium japonicum bacteroids and cultures.

    PubMed Central

    Karr, D B; Emerich, D W

    1989-01-01

    Protein phosphorylation was demonstrated in Bradyrhizobium japonicum bacteroids in vivo and in cultures in vivo and in vitro. Comparison of in vivo-labeled phosphoproteins of bacteroids and of cultured cells showed differences in both the pattern and intensity of labeling. In cultured cells, comparison of the labeling patterns and intensities of in vivo- and in vitro-labeled phosphoproteins showed a number of similarities; however, several phosphoproteins were found only after one of the two labeling conditions. The labeling intensity was time dependent in both in vivo and in vitro assays and was dependent on the presence of magnesium in in vitro assays. Differences in the rates of phosphorylation and dephosphorylation were noted for a number of proteins. The level of incorporation of 32P into protein was only 2% or less of the total phosphate accumulated during the in vivo labeling period. Several isolation and sample preparation procedures resulted in differences in labeling patterns. Phosphatase inhibitors and several potential metabolic effectors had negligible effects on the phosphorylation pattern. There were no significant changes in the phosphorylation patterns of cells cultured on mannitol, acetate, and succinate, although the intensity of the labeling did vary with the carbon source. Images PMID:2498290

  2. Protein phosphorylation in Bradyrhizobium japonicum bacteroids and cultures

    SciTech Connect

    Karr, D.B.; Emerich, D.W. )

    1989-06-01

    Protein phosphorylation was demonstrated in Bradyrhizobium japonicum bacteroids in vivo and in cultures in vivo and in vitro. Comparison of in vivo-labeled phosphoproteins of bacteroids and of cultured cells showed differences in both the pattern and intensity of labeling. In cultured cells, comparison of the labeling patterns and intensities of in vivo- and in vitro-labeled phosphoproteins showed a number of similarities; however, several phosphoproteins were found only after one of the two labeling conditions. The labeling intensity was time dependent in both in vivo and in vitro assays and was dependent on the presence of magnesium in in vitro assays. Differences in the rates of phosphorylation and dephosphorylation were noted for a number of proteins. The level of incorporation of {sup 32}P into protein was only 2% or less of the total phosphate accumulated during the in vivo labeling period. Several isolation and sample preparation procedures resulted in differences in labeling patterns. Phosphatase inhibitors and several potential metabolic effectors had negligible effects on the phosphorylation pattern. There were no significant changes in the phosphorylation patterns of cells cultured on mannitol, acetate, and succinate, although the intensity of the labeling did vary with the carbon source.

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

    PubMed

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

    2007-04-01

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

  4. Protein kinase C coordinates histone H3 phosphorylation and acetylation

    PubMed Central

    Darieva, Zoulfia; Webber, Aaron; Warwood, Stacey; Sharrocks, Andrew D

    2015-01-01

    The re-assembly of chromatin following DNA replication is a critical event in the maintenance of genome integrity. Histone H3 acetylation at K56 and phosphorylation at T45 are two important chromatin modifications that accompany chromatin assembly. Here we have identified the protein kinase Pkc1 as a key regulator that coordinates the deposition of these modifications in S. cerevisiae under conditions of replicative stress. Pkc1 phosphorylates the histone acetyl transferase Rtt109 and promotes its ability to acetylate H3K56. Our data also reveal novel cross-talk between two different histone modifications as Pkc1 also enhances H3T45 phosphorylation and this modification is required for H3K56 acetylation. Our data therefore uncover an important role for Pkc1 in coordinating the deposition of two different histone modifications that are important for chromatin assembly. DOI: http://dx.doi.org/10.7554/eLife.09886.001 PMID:26468616

  5. Calcium-phospholipid enhanced protein phosphorylation in human placenta

    SciTech Connect

    Moore, J.J.; Moore, R.; Cardaman, R.C.

    1986-07-01

    Calcium-activated, phospholipid-dependent protein phosphorylation has not been studied in placenta. Human placental cytosol was subjected to an endogenous protein phosphorylation assay using (..gamma..-/sup 32/P)ATP in the presence of calcium and phosphatidylserine. Protein phosphorylation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. When compared to basal levels, calcium (10/sup -6/ M) in combination with phosphatidylserine (50 ..mu..g/ml) significantly enhanced (P < 100) /sup 32/P incorporation into phosphoproteins having mol wt 47,000, 43,000, and 37,000. Half-maximal /sup 22/P incorporation was observed with 3.5 x 10/sup -7/ M Ca/sup 2 +/ in the presence of phosphatidylserine (50 ..mu..g/ml). The effect of phosphatidylserine was biphasic. In the presence of Ca 10/sup -6/ M, /sup 32/P incorporation increased to a maximum at 70 /sup +/g/ml of phosphatidylserine. The increase was suppressed at 150 ..mu..g/ml. Tetracaine caused a dose-dependent inhibition of calcium-activated, phospholipid-dependent enhancement of the three phosphoproteins. Calcium in the absence of phospholipid enhanced the phosphorylation of a protein of 98,000 mol wt. Phosphatidylserine suppressed this enhancement. Calmodulin (10/sup -6/ M) had no detectable effect upon phosphorylation beyond that of calcium alone, but the calmodulin inhibitor R-24571 specifically inhibited the calcium-stimulated 98,000 mol wt phosphoprotein. Calcium-activated, phospholipid-dependent phospholipid-dependent phosphoproteins are present in human placental cytosol; whether calcium-activated, calmodulin-dependent phosphoproteins also are present remains a question.

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

    PubMed Central

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

    2011-01-01

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

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

    SciTech Connect

    Coughlan, S.J.

    1990-01-01

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

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

    PubMed

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

    2015-12-01

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

  9. Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure

    PubMed Central

    North, Justin A.; Šimon, Marek; Ferdinand, Michelle B.; Shoffner, Matthew A.; Picking, Jonathan W.; Howard, Cecil J.; Mooney, Alex M.; van Noort, John; Poirier, Michael G.; Ottesen, Jennifer J.

    2014-01-01

    Nucleosomes contain ∼146 bp of DNA wrapped around a histone protein octamer that controls DNA accessibility to transcription and repair complexes. Posttranslational modification (PTM) of histone proteins regulates nucleosome function. To date, only modest changes in nucleosome structure have been directly attributed to histone PTMs. Histone residue H3(T118) is located near the nucleosome dyad and can be phosphorylated. This PTM destabilizes nucleosomes and is implicated in the regulation of transcription and repair. Here, we report gel electrophoretic mobility, sucrose gradient sedimentation, thermal disassembly, micrococcal nuclease digestion and atomic force microscopy measurements of two DNA–histone complexes that are structurally distinct from nucleosomes. We find that H3(T118ph) facilitates the formation of a nucleosome duplex with two DNA molecules wrapped around two histone octamers, and an altosome complex that contains one DNA molecule wrapped around two histone octamers. The nucleosome duplex complex forms within short ∼150 bp DNA molecules, whereas altosomes require at least ∼250 bp of DNA and form repeatedly along 3000 bp DNA molecules. These results are the first report of a histone PTM significantly altering the nucleosome structure. PMID:24561803

  10. Interleukin 2 signaling involves the phosphorylation of Stat proteins.

    PubMed

    Frank, D A; Robertson, M J; Bonni, A; Ritz, J; Greenberg, M E

    1995-08-15

    One of the most important cytokines involved in immune response regulation is interleukin 2 (IL-2), a potent activator of the proliferation and function of T lymphocytes and natural killer cells. The mechanisms by which the effects of IL-2 are propagated within cells are not understood. While the binding of IL-2 to its receptor was recently shown to lead to the activation of two kinases, Jak-1 and Jak-3, subsequent steps in the signaling pathway to the nucleus that lead to the activation of specific genes had not been characterized. Since many cytokines that activate Jak kinases also lead to the tyrosine phosphorylation and activation of members of the Stat family of transcription factors, the ability of IL-2 to trigger Stat phosphorylation was examined. Exposure of activated human T lymphocytes or of a natural killer cell line (NKL) to IL-2 leads to the phosphorylation of Stat1 alpha, Stat1 beta, and Stat3, as well as of two Stat-related proteins, p94 and p95. p94 and p95 share homology with Stat1 at the phosphorylation site and in the Src homology 2 (SH2) domain, but otherwise are immunologically distinct from Stat1. These Stat proteins were found to translocate to the nucleus and to bind to a specific DNA sequence. These findings suggest a mechanism by which IL-2 binding to its receptor may activate specific genes involved in immune cell function. PMID:7544001

  11. Changes of testicular phosphorylated proteins in response to restraint stress in male rats*

    PubMed Central

    Arun, Supatcharee; Burawat, Jaturon; Sukhorum, Wannisa; Sampannang, Apichakan; Uabundit, Nongnut; Iamsaard, Sitthichai

    2016-01-01

    Objective: To investigate male reproductive parameters via changes of potential testicular protein markers in restraint-stress rats. Methods: Male Sprague-Dawley rats were divided into two groups (non-immobilized control and restraint-immobilized/stress groups, n=8 each group). The stress animals were immobilized (12 h/d) by a restraint cage for 7 consecutive days. All reproductive parameters, morphology and histology were observed and compared between groups. In addition, the expression of steroidogenic acute regulatory (StAR) and phosphotyrosine proteins (previously localized in Sertoli and late spermatid cells) in testicular lysate was assayed by immuno-Western blotting. Results: Testosterone level, sperm concentration and sperm head normality of stress rats were significantly decreased while the corticosterone level was increased as compared with the control (P<0.05). Histologically, stress rats showed low sperm mass in epididymal lumen and some atrophy of seminiferous tubules. Although the expression of testicular StAR protein was not significantly different between groups, changed patterns of the 131, 95, and 75 kDa testicular phosphorylated proteins were observed in the stress group compared with the control group. The intensity of a testicular 95-kDa phosphorylated protein was significantly decreased in stress rats. Conclusions: This study has demonstrated the alteration of testicular phosphorylated protein patterns, associated with adverse male reproductive parameters in stress rats. It could be an explanation of some infertility in stress males. PMID:26739523

  12. Functional phosphorylation sites in cardiac myofilament proteins are evolutionarily conserved in skeletal myofilament proteins.

    PubMed

    Gross, Sean M; Lehman, Steven L

    2016-06-01

    Protein phosphorylation plays an important role in regulating cardiac contractile function, but phosphorylation is not thought to play a regulatory role in skeletal muscle. To examine how myofilament phosphorylation arose in the human heart, we analyzed the amino acid sequences of 25 cardiac phosphorylation sites in animals ranging from fruit flies to humans. These analyses indicated that of the 25 human phosphorylation sites examined, 11 have been conserved across vertebrates and four have been sporadically present in vertebrates. Furthermore, all 11 of the cardiac sites found across vertebrates were present in skeletal muscle isoforms, along with three sites that were sporadically present. Based on the conservation of amino acid sequences between cardiac and skeletal contractile proteins, we tested for phosphorylation in mammalian skeletal muscle using several biochemical techniques and found evidence that multiple myofilament proteins were phosphorylated. Several of these phosphorylation sites were validated using mass spectrometry, including one site that is present in slow- and fast-twitch troponin I (TnI), but was lost in cardiac TnI. Thus, several myofilament phosphorylation sites present in the human heart likely arose in invertebrate muscle, have been evolutionarily conserved in skeletal muscle, and potentially have functional effects in both skeletal and cardiac muscle. PMID:26993364

  13. Phosphorylation Modulates the Mechanical Stability of the Cardiac Myosin-Binding Protein C Motif

    PubMed Central

    Michalek, Arthur J.; Howarth, Jack W.; Gulick, James; Previs, Michael J.; Robbins, Jeffrey; Rosevear, Paul R.; Warshaw, David M.

    2013-01-01

    Cardiac myosin-binding protein C (cMyBP-C) is a thick-filament-associated protein that modulates cardiac contractility through interactions of its N-terminal immunoglobulin (Ig)-like C0-C2 domains with actin and/or myosin. These interactions are modified by the phosphorylation of at least four serines located within the motif linker between domains C1 and C2. We investigated whether motif phosphorylation alters its mechanical properties by characterizing force-extension relations using atomic force spectroscopy of expressed mouse N-terminal cMyBP-C fragments (i.e., C0-C3). Protein kinase A phosphorylation or serine replacement with aspartic acids did not affect persistence length (0.43 ± 0.04 nm), individual Ig-like domain unfolding forces (118 ± 3 pN), or Ig extension due to unfolding (30 ± 0.38 nm). However, phosphorylation did significantly decrease the C0-C3 mean contour length by 24 ± 2 nm. These results suggest that upon phosphorylation, the motif, which is freely extensible in the nonphosphorylated state, adopts a more stable and/or different structure. Circular dichroism and dynamic light scattering data for shorter expressed C1-C2 fragments with all four serines replaced by aspartic acids confirmed that the motif did adopt a more stable structure that was not apparent in the nonphosphorylated motif. These biophysical data provide both a mechanical and structural basis for cMyBP-C regulation by motif phosphorylation. PMID:23442866

  14. The eFIP system for text mining of protein interaction networks of phosphorylated proteins

    PubMed Central

    Wu, Cathy H.; Vijay-Shanker, K.

    2012-01-01

    Protein phosphorylation is a central regulatory mechanism in signal transduction involved in most biological processes. Phosphorylation of a protein may lead to activation or repression of its activity, alternative subcellular location and interaction with different binding partners. Extracting this type of information from scientific literature is critical for connecting phosphorylated proteins with kinases and interaction partners, along with their functional outcomes, for knowledge discovery from phosphorylation protein networks. We have developed the Extracting Functional Impact of Phosphorylation (eFIP) text mining system, which combines several natural language processing techniques to find relevant abstracts mentioning phosphorylation of a given protein together with indications of protein–protein interactions (PPIs) and potential evidences for impact of phosphorylation on the PPIs. eFIP integrates our previously developed tools, Extracting Gene Related ABstracts (eGRAB) for document retrieval and name disambiguation, Rule-based LIterature Mining System (RLIMS-P) for Protein Phosphorylation for extraction of phosphorylation information, a PPI module to detect PPIs involving phosphorylated proteins and an impact module for relation extraction. The text mining system has been integrated into the curation workflow of the Protein Ontology (PRO) to capture knowledge about phosphorylated proteins. The eFIP web interface accepts gene/protein names or identifiers, or PubMed identifiers as input, and displays results as a ranked list of abstracts with sentence evidence and summary table, which can be exported in a spreadsheet upon result validation. As a participant in the BioCreative-2012 Interactive Text Mining track, the performance of eFIP was evaluated on document retrieval (F-measures of 78–100%), sentence-level information extraction (F-measures of 70–80%) and document ranking (normalized discounted cumulative gain measures of 93–100% and mean average

  15. Rapid Oligo-Galacturonide Induced Changes in Protein Phosphorylation in Arabidopsis.

    PubMed

    Kohorn, Bruce D; Hoon, Divya; Minkoff, Benjamin B; Sussman, Michael R; Kohorn, Susan L

    2016-04-01

    The wall-associated kinases (WAKs)(1)are receptor protein kinases that bind to long polymers of cross-linked pectin in the cell wall. These plasma-membrane-associated protein kinases also bind soluble pectin fragments called oligo-galacturonides (OGs) released from the wall after pathogen attack and damage. WAKs are required for cell expansion during development but bind water soluble OGs generated from walls with a higher affinity than the wall-associated polysaccharides. OGs activate a WAK-dependent, distinct stress-like response pathway to help plants resist pathogen attack. In this report, a quantitative mass-spectrometric-based phosphoproteomic analysis was used to identify Arabidopsis cellular events rapidly induced by OGsin planta Using N(14/)N(15)isotopicin vivometabolic labeling, we screened 1,000 phosphoproteins for rapid OG-induced changes and found 50 proteins with increased phosphorylation, while there were none that decreased significantly. Seven of the phosphosites within these proteins overlap with those altered by another signaling molecule plants use to indicate the presence of pathogens (the bacterial "elicitor" peptide Flg22), indicating distinct but overlapping pathways activated by these two types of chemicals. Genetic analysis of genes encoding 10 OG-specific and two Flg22/OG-induced phosphoproteins reveals that null mutations in eight proteins compromise the OG response. These phosphorylated proteins with genetic evidence supporting their role in the OG response include two cytoplasmic kinases, two membrane-associated scaffold proteins, a phospholipase C, a CDPK, an unknown cadmium response protein, and a motor protein. Null mutants in two proteins, the putative scaffold protein REM1.3, and a cytoplasmic receptor like kinase ROG2, enhance and suppress, respectively, a dominantWAKallele. Altogether, the results of these chemical and genetic experiments reveal the identity of several phosphorylated proteins involved in the kinase

  16. Structural Mechanism for Regulation of Bcl-2 protein Noxa by phosphorylation

    NASA Astrophysics Data System (ADS)

    Karim, Christine B.; Michel Espinoza-Fonseca, L.; James, Zachary M.; Hanse, Eric A.; Gaynes, Jeffrey S.; Thomas, David D.; Kelekar, Ameeta

    2015-09-01

    We showed previously that phosphorylation of Noxa, a 54-residue Bcl-2 protein, at serine 13 (Ser13) inhibited its ability to promote apoptosis through interactions with canonical binding partner, Mcl-1. Using EPR spectroscopy, molecular dynamics (MD) simulations and binding assays, we offer evidence that a structural alteration caused by phosphorylation partially masks Noxa’s BH3 domain, inhibiting the Noxa-Mcl-1 interaction. EPR of unphosphorylated Noxa, with spin-labeled amino acid TOAC incorporated within the BH3 domain, revealed equilibrium between ordered and dynamically disordered states. Mcl-1 further restricted the ordered component for non-phosphorylated Noxa, but left the pSer13 Noxa profile unchanged. Microsecond MD simulations indicated that the BH3 domain of unphosphorylated Noxa is housed within a flexible loop connecting two antiparallel β-sheets, flanked by disordered N- and C-termini and Ser13 phosphorylation creates a network of salt-bridges that facilitate the interaction between the N-terminus and the BH3 domain. EPR showed that a spin label inserted near the N-terminus was weakly immobilized in unphosphorylated Noxa, consistent with a solvent-exposed helix/loop, but strongly constrained in pSer13 Noxa, indicating a more ordered peptide backbone, as predicted by MD simulations. Together these studies reveal a novel mechanism by which phosphorylation of a distal serine inhibits a pro-apoptotic BH3 domain and promotes cell survival.

  17. Enhanced prediction of conformational flexibility and phosphorylation in proteins.

    PubMed

    Swaminathan, Karthikeyan; Adamczak, Rafal; Porollo, Aleksey; Meller, Jarosław

    2010-01-01

    Many sequence-based predictors of structural and functional properties of proteins have been developed in the past. In this study, we developed new methods for predicting measures of conformational flexibility in proteins, including X-ray structure-derived temperature (B-) factors and the variance within NMR structural ensemble, as effectively measured by the solvent accessibility standard deviations (SASDs). We further tested whether these predicted measures of conformational flexibility in crystal lattices and solution, respectively, can be used to improve the prediction of phosphorylation in proteins. The latter is an example of a common post-translational modification that modulates protein function, e.g., by affecting interactions and conformational flexibility of phosphorylated sites. Using robust epsilon-insensitive support vector regression (ε-SVR) models, we assessed two specific representations of protein sequences: one based on the position-specific scoring matrices (PSSMs) derived from multiple sequence alignments, and an augmented representation that incorporates real-valued solvent accessibility and secondary structure predictions (RSA/SS) as additional measures of local structural propensities. We showed that a combination of PSSMs and real-valued SS/RSA predictions provides systematic improvements in the accuracy of both B-factors and SASD prediction. These intermediate predictions were subsequently combined into an enhanced predictor of phosphorylation that was shown to significantly outperform methods based on PSSM alone. We would like to stress that to the best of our knowledge, this is the first example of using predicted from sequence NMR structure-based measures of conformational flexibility in solution for the prediction of other properties of proteins. Phosphorylation prediction methods typically employ a two-class classification approach with the limitation that the set of negative examples used for training may include some sites that are

  18. Synthesis and phosphorylation of the glial fibrillary acidic protein during brain development: A tissue slice study

    SciTech Connect

    Noetzel, M.J. )

    1990-01-01

    Brain slices were incubated with either (3H) amino acids or (32P) orthophosphate in order to characterize the synthesis and phosphorylation of the glial fibrillary acidic protein (GFAP) in the rat nervous system. The incorporation of (3H) amino acids into GFAP was found to increase significantly during early postnatal development, reaching a peak of activity on day 5 of life and then declining over the next 2 weeks. Concomitant with this peak of synthetic activity the content of GFAP in rat brain was also observed to increase dramatically. GFAP continued to accumulate in brain through postnatal day 30 despite a decrease in the synthesis of the protein. These results indicate that the increase in GFAP during the first month of life cannot be ascribed solely to the rate of GFAP synthesis. The findings are consistent with the hypothesis that during later stages of astrocytic development the accumulation of GFAP may be primarily dependent upon a low rate of protein degradation. The pattern of GFAP phosphorylation in the developing rat brain differed from that observed for the incorporation of (3H) amino acids. The peak incorporation of 32P into GFAP occurred on postnatal day 10 at a time when synthesis of the protein had declined by 43%. These findings suggest that during development phosphorylation of GFAP is mediated by factors different from those directing its synthesis. In addition, phosphorylation of GFAP did not alter its solubility in cytoskeletal preparations indicating that GFAP phosphorylation is probably not a major regulatory mechanism in disassembly of the astroglial filaments.

  19. Solution structures of Mengovirus Leader protein, its phosphorylated derivatives, and in complex with nuclear transport regulatory protein, RanGTPase

    PubMed Central

    Bacot-Davis, Valjean R.; Ciomperlik, Jessica J.; Basta, Holly A.; Cornilescu, Claudia C.; Palmenberg, Ann C.

    2014-01-01

    Cardiovirus Leader (L) proteins induce potent antihost inhibition of active cellular nucleocytoplasmic trafficking by triggering aberrant hyperphosphorylation of nuclear pore proteins (Nup). To achieve this, L binds protein RanGTPase (Ran), a key trafficking regulator, and diverts it into tertiary or quaternary complexes with required kinases. The activity of L is regulated by two phosphorylation events not required for Ran binding. Matched NMR studies on the unphosphorylated, singly, and doubly phosphorylated variants of Mengovirus L (LM) show both modifications act together to partially stabilize a short internal α-helix comprising LM residues 43–46. This motif implies that ionic and Van der Waals forces contributed by phosphorylation help organize downstream residues 48–67 into a new interface. The full structure of LM as bound to Ran (unlabeled) and Ran (216 aa) as bound by LM (unlabeled) places LM into the BP1 binding site of Ran, wrapped by the conformational flexible COOH tail. The arrangement explains the tight KD for this complex and places the LM zinc finger and phosphorylation interface as surface exposed and available for subsequent reactions. The core structure of Ran, outside the COOH tail, is not altered by LM binding and remains accessible for canonical RanGTP partner interactions. Pull-down assays identify at least one putative Ran:LM partner as an exportin, Crm1, or CAS. A model of Ran:LM:Crm1, based on the new structures suggests LM phosphorylation status may mediate Ran’s selection of exportin(s) and cargo(s), perverting these native trafficking elements into the lethal antihost Nup phosphorylation pathways. PMID:25331866

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

    PubMed Central

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

    2013-01-01

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

  1. Rapid changes in protein phosphorylation associated with light-induced gravity perception in corn roots

    NASA Technical Reports Server (NTRS)

    McFadden, J. J.; Poovaiah, B. W.

    1988-01-01

    The effect of light and calcium depletion on in vivo protein phosphorylation was tested using dark-grown roots of Merit corn. Light caused rapid and specific promotion of phosphorylation of three polypeptides. Pretreatment of roots with ethylene glycol bis N,N,N',N' tetraacetic acid and A23187 prevented light-induced changes in protein phosphorylation. We postulate that these changes in protein phosphorylation are involved in the light-induced gravity response.

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

    PubMed Central

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

    2016-01-01

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

  3. Regulation of the autophagy protein LC3 by phosphorylation

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

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

    1989-01-01

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

  5. Phosphorylation of synthetic peptides by a tyrosine protein kinase from the particulate fraction of a lymphoma cell line.

    PubMed Central

    Casnellie, J E; Harrison, M L; Pike, L J; Hellström, K E; Krebs, E G

    1982-01-01

    The particulate fraction from a lymphoma cell line, LSTRA, was found to contain an apparent high level of tyrosine protein kinase activity. When this fraction was incubated with [gamma-32P]ATP in the presence of 10 mM MnCl2, hydrolyzed, and assayed, 70--80% of the radioactivity recovered in phosphoamino acids was in phosphotyrosine. Gel electrophoresis of the proteins showed that a large portion of the 32P was in a single protein with a molecular weight of approximately 58,000. The phosphorylated residue in this protein was identified as phosphotyrosine. Detergent extracts of the particulate fraction from LSTRA cells contained both the Mr 58,000 protein and the enzyme responsible for its phosphorylation. These extracts were found to catalyze the phosphorylation of the tyrosine residue in the synthetic peptide, Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gln-Gly, corresponding to the sequence around the tyrosine that is phosphorylated in pp60src; the Km for the peptide in this reaction was 5 mM. High-performance liquid chromatography was used to assay for this phosphorylation. A second peptide was synthesized that contained two additional arginine residues whose presence permitted the phosphorylation of the peptide to be measured by a simple assay using phosphocellulose paper. The Km for this peptide was 3--4 mM, indicating that the presence of the additional arginine residues did not alter the apparent affinity of the kinase for the peptide. Images PMID:6804939

  6. Evidence that tyrphostins AG10 and AG18 are mitochondrial uncouplers that alter phosphorylation-dependent cell signaling.

    PubMed

    Soltoff, Stephen P

    2004-03-19

    Receptor agonists that initiate fluid secretion in salivary gland epithelial cells also increase protein phosphorylation. To assess contributions of tyrosine phosphorylation to secretion, changes in muscarinic receptor-initiated secretion (estimated from sodium pump-dependent increases in oxygen consumption) were measured in parotid acinar cells exposed to tyrosine kinase inhibitors. However, like the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, tyrphostins AG10 and AG18 increased the rate of oxygen consumption and reduced cellular ATP by approximately 90% in the absence of the muscarinic agonist carbachol, indicating that these tyrphostins uncouple mitochondria. Exposure of isolated mitochondria to five structurally related tyrphostins demonstrated that their relative potencies as uncouplers differed from their in vitro kinase-inhibitory potencies due to different molecular requirements for the two effects. AG10 and AG18 blocked parotid phosphorylation events only at concentrations that reduced ATP content. The tyrosine kinase inhibitor genistein reduced ATP content by 15-20% and weakly uncoupled isolated mitochondria, but its inhibition of carbachol-mediated protein kinase Cdelta tyrosine phosphorylation and ERK1/2 activation appeared attributable to blocking tyrosine kinases directly. Carbachol itself rapidly reduced ATP content by 15-20%. Carbachol, 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (P2X(7) receptor agonist), AG10, AG18, and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone rapidly activated the fuel sensor AMP-activated protein kinase (AMPK); however, only AMPK activation by carbachol and BzATP was due to sodium pump stimulation. AG10 and AG18 also activated AMPK and/or uncoupled mitochondria in PC12, HeLa, and HEK293 cells. These studies demonstrate that some tyrosine kinase inhibitors produce cellular effects that are mechanistically different from their primary in vitro characterizations and, as do salivary

  7. Protein kinase C mediated phosphorylation blocks juvenile hormone action.

    PubMed

    Kethidi, Damu R; Li, Yiping; Palli, Subba R

    2006-03-01

    Juvenile hormones (JH) regulate a wide variety of developmental and physiological processes in insects. Although the biological actions of JH are well documented, the molecular mechanisms underlying JH action are poorly understood. We studied the molecular basis of JH action using a JH response element (JHRE) identified in the promoter region of JH esterase gene cloned from Choristoneura fumiferana, which is responsive to JH and 20-hydroxyecdysone (20E). In Drosophila melanogaster L57 cells, the JHRE-regulated reporter gene was induced by JH I, JH III, methoprene, and hydroprene. Nuclear proteins isolated from L57 cells bound to the JHRE and exposure of these proteins to ATP resulted in a reduction in their DNA binding. Either JH III or calf intestinal alkaline phosphatase (CIAP) was able to restore the binding of nuclear proteins to the DNA. In addition, protein kinase C inhibitors increased and protein kinase C activators reduced the binding of nuclear proteins to the JHRE. In transactivation assays, protein kinase C inhibitors induced the luciferase gene placed under the control of a minimal promoter and the JHRE. These data suggest that protein kinase C mediated phosphorylation prevents binding of nuclear proteins to juvenile hormone responsive promoters resulting in suppression of JH action. PMID:16448742

  8. Protein kinase A dependent membrane protein phosphorylation and chloride conductance in endosomal vesicles from kidney cortex

    SciTech Connect

    Reenstra, W.W.; Bae, H.R.; Verkman, A.S. Univ. of California, San Francisco ); Sabolic, I. Harvard Medical School, Charlestown, MA )

    1992-01-14

    Regulation of Cl conductance by protein kinase A action, cell-free measurements of Cl transport and membrane protein phosphorylation were carried out in apical endocytic vesicles from rabbit kidney proximal tubule. Cl transport was measured by a stopped-flow quenching assay in endosomes labeled in vivo with the fluorescent Cl indicator 6-methoxy-N-(3-sulfopropyl)quinolinium. Phosphorylation was studied in a purified endosomal preparation by SDS-PAGE and autoradiography of membrane proteins labeled by ({gamma}-{sup 32}P)ATP. These results suggest that, in a cell-free system, protein kinase A increases Cl conductance in endosomes from kidney proximal tubule by a phosphorylation mechanism. The labeled protein has a size similar to that of the 64-kDa putative kidney Cl channel reported by Landry et al. but is much smaller than the {approximately}170-kDa cystic fibrosis transmembrane conductance regulatory protein.

  9. Spatial distributions of phosphorylated membrane proteins aquaporin 0 and MP20 across young and aged human lenses.

    PubMed

    Gutierrez, Danielle B; Garland, Donita L; Schwacke, John H; Hachey, David L; Schey, Kevin L

    2016-08-01

    In the human ocular lens it is now realized that post-translational modifications can alter protein function and/or localization in fiber cells that no longer synthesize proteins. The specific sites of post-translational modification to the abundant ocular lens membrane proteins AQP0 and MP20 have been previously identified and their functional effects are emerging. To further understand how changes in protein function and/or localization induced by these modifications alter lens homeostasis, it is necessary to determine the spatial distributions of these modifications across the lens. In this study, a quantitative LC-MS approach was used to determine the spatial distributions of phosphorylated AQP0 and MP20 peptides from manually dissected, concentric layers of fiber cells from young and aged human lenses. The absolute amounts of phosphorylation were determined for AQP0 Ser235 and Ser229 and for MP20 Ser170 in fiber cells from the lens periphery to the lens center. Phosphorylation of AQP0 Ser229 represented a minor portion of the total phosphorylated AQP0. Changes in spatial distributions of phosphorylated APQ0 Ser235 and MP20 Ser170 correlated with regions of physiological interest in aged lenses, specifically, where barriers to water transport and extracellular diffusion form. PMID:27339748

  10. SOLID-PHASE ASSAY FOR THE PHOSPHORYLATION OF PROTEINS BLOTTED ON NITROCELLULOSE MEMBRANE FILTERS

    EPA Science Inventory

    A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters and the blotted polypeptides are phosphorylated with ...

  11. Xenobiotics that affect oxidative phosphorylation alter differentiation of human adipose-derived stem cells at concentrations that are found in human blood

    PubMed Central

    Llobet, Laura; Toivonen, Janne M.; Montoya, Julio; Ruiz-Pesini, Eduardo; López-Gallardo, Ester

    2015-01-01

    ABSTRACT Adipogenesis is accompanied by differentiation of adipose tissue-derived stem cells to adipocytes. As part of this differentiation, biogenesis of the oxidative phosphorylation system occurs. Many chemical compounds used in medicine, agriculture or other human activities affect oxidative phosphorylation function. Therefore, these xenobiotics could alter adipogenesis. We have analyzed the effects on adipocyte differentiation of some xenobiotics that act on the oxidative phosphorylation system. The tested concentrations have been previously reported in human blood. Our results show that pharmaceutical drugs that decrease mitochondrial DNA replication, such as nucleoside reverse transcriptase inhibitors, or inhibitors of mitochondrial protein synthesis, such as ribosomal antibiotics, diminish adipocyte differentiation and leptin secretion. By contrast, the environmental chemical pollutant tributyltin chloride, which inhibits the ATP synthase of the oxidative phosphorylation system, can promote adipocyte differentiation and leptin secretion, leading to obesity and metabolic syndrome as postulated by the obesogen hypothesis. PMID:26398948

  12. Calcium and protein phosphorylation in the transduction of gravity signal in corn roots

    NASA Technical Reports Server (NTRS)

    Friedmann, M.; Poovaiah, B. W.

    1991-01-01

    The involvement of calcium and protein phosphorylation in the transduction of gravity signal was studied using corn roots of a light-insensitive variety (Zea mays L., cv. Patriot). The gravitropic response was calcium-dependent. Horizontal placement of roots preloaded with 32P for three minutes resulted in changes in protein phosphorylation of polypeptides of 32 and 35 kD. Calcium depletion resulted in decreased phosphorylation of these phosphoproteins and replenishment of calcium restored the phosphorylation.

  13. Phosphorylation of spore coat proteins by a family of atypical protein kinases.

    PubMed

    Nguyen, Kim B; Sreelatha, Anju; Durrant, Eric S; Lopez-Garrido, Javier; Muszewska, Anna; Dudkiewicz, Małgorzata; Grynberg, Marcin; Yee, Samantha; Pogliano, Kit; Tomchick, Diana R; Pawłowski, Krzysztof; Dixon, Jack E; Tagliabracci, Vincent S

    2016-06-21

    The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. Here, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology. PMID:27185916

  14. Phosphorylation of Transcription Factor Specificity Protein 4 Is Increased in Peripheral Blood Mononuclear Cells of First-Episode Psychosis

    PubMed Central

    Fusté, Montserrat; Meléndez-Pérez, Iria; Villalta-Gil, Victoria; Haro, Josep Maria; Gill, Grace; Ramos, Belén

    2015-01-01

    Background Altered expression of transcription factor specificity protein 4 (SP4) has been found in the postmortem brain of patients with psychiatric disorders including schizophrenia and bipolar disorder. Reduced levels of SP4 protein have recently been reported in peripheral blood mononuclear cells in first-episode psychosis. Also, SP4 levels are modulated by lithium treatment in cultured neurons. Phosphorylation of SP4 at S770 is increased in the cerebellum of bipolar disorder subjects and upon inhibition of NMDA receptor signaling in cultured neurons. The aim of this study was to investigate whether SP4 S770 phosphorylation is increased in lymphocytes of first-episode psychosis patients and the effect of lithium treatment on this phosphorylation. Methods A cross-sectional study of S770 phosphorylation relative to total SP4 immunoreactivity using specific antibodies in peripheral blood mononuclear cells in first-episode psychosis patients (n = 14, treated with lithium or not) and matched healthy controls (n = 14) by immunoblot was designed. We also determined the effects of the prescribed drugs lithium, olanzapine or valproic acid on SP4 phosphorylation in rat primary cultured cerebellar granule neurons. Results We found that SP4 S770 phosphorylation was significantly increased in lymphocytes in first-episode psychosis compared to controls and decreased in patients treated with lithium compared to patients who did not receive lithium. Moreover, incubation with lithium but not olanzapine or valproic acid reduced SP4 phosphorylation in rat cultured cerebellar granule neurons. Conclusions The findings presented here indicate that SP4 S770 phosphorylation is increased in lymphocytes in first-episode psychosis which may be reduced by lithium treatment in patients. Moreover, our study shows lithium treatment prevents this phosphorylation in vitro in neurons. This pilot study suggests that S770 SP4 phosphorylation could be a peripheral biomarker of psychosis, and may

  15. Alterations in connexin 43 during diabetic cardiomyopathy: competition of tyrosine nitration versus phosphorylation

    PubMed Central

    COOK, Angela C.; SCHANBACHER, Brandon L.; BAUER, John Anthony

    2014-01-01

    Objective Cardiac conduction abnormalities are observed early in the progression of Type I diabetes, but the mechanism(s) involved are undefined. Connexin 43, a critical component of ventricular gap junctions, depends on tyrosine phosphorylation status to modulate channel conductance - alterations in connexin 43 content, distributions, and/or phosphorylation status may be involved in cardiac rhythm disturbances. We tested the hypothesis that cardiac content/distribution of connexin 43 are altered in a rat model of Type I diabetic cardiomyopathy, investigating a mechanistic role for tyrosine. Methods We conducted electrocardiographic analyses during the progression of diabetic cardiomyopathy in rats dosed with streptozotocin (65mg/kg), at 3, 7, and 35 days post-induction of diabetes. Following functional analyses, we conducted immunohistochemical and immunoprecipitation studies to assess alterations in connexin 43. Results We observed significant evidence of ventricular conduction abnormalities (QRS complex, Q-T interval) as early as 7 days post-streptozotocin, persisting throughout the study. Connexin 43 levels were increased 7d post- streptozotocin and remained elevated throughout the study. Connexin 40 content was unchanged relative to controls throughout the study. Changes in Connexin 43 distribution were also observed; connexin 43 staining was dispersed from myocyte short axis junctions. Connexin 43 tyrosine phosphorylation declined during the progression of diabetes, with concurrent increases in tyrosine nitration. Conclusions These data suggest that alterations in connexin 43 content and distribution occur during experimental diabetes and likely contribute to alterations in cardiac function, and that oxidative modification of tyrosine-mediated signaling may play a mechanistic role. PMID:24796789

  16. 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. PMID:27037847

  17. Heat-shock protein-25/27 phosphorylation by the delta isoform of protein kinase C.

    PubMed Central

    Maizels, E T; Peters, C A; Kline, M; Cutler, R E; Shanmugam, M; Hunzicker-Dunn, M

    1998-01-01

    Small heat-shock proteins (sHSPs) are widely expressed 25-28 kDa proteins whose functions are dynamically regulated by phosphorylation. While recent efforts have clearly delineated a stress-responsive p38 mitogen-activated protein-kinase (MAPK)-dependent kinase pathway culminating in activation of the heat-shock (HSP)-kinases, mitogen-activated protein-kinase-activated protein kinase-2 and -3, not all sHSP phosphorylation events can be explained by the p38 MAPK-dependent pathway. The contribution of protein kinase C (PKC) to sHSP phosphorylation was suggested by early studies but later questioned on the basis of the reported poor ability of purified PKC to phosphorylate sHSP in vitro. The current study re-evaluates the role of PKC in sHSP phosphorylation in the light of the isoform complexity of the PKC family. We evaluated the sHSP phosphorylation status in rat corpora lutea obtained from two stages of pregnancy, mid-pregnancy and late-pregnancy, which express different levels of the novel PKC isoform, PKC-delta. Two-dimensional Western blot analysis showed that HSP-27 was more highly phosphorylated in vivo in corpora lutea of late pregnancy, corresponding to the developmental stage in which PKC-delta is abundant and active. Late-pregnant luteal extracts contained a lipid-sensitive HSP-kinase activity which exactly co-purified with PKC-delta using hydroxyapatite and S-Sepharose column chromatography. To determine whether there might be preferential phosphorylation of sHSP by a particular PKC isoform, purified recombinant PKC isoforms corresponding to those PKC isoforms detected in rat corpora lutea were evaluated for HSP-kinase activity in vitro. Recombinant PKC-delta effectively catalysed the phosphorylation of sHSP in vitro, and PKC-alpha was 30-50% as effective as an HSP-kinase; other PKCs tested (beta1, beta2, epsilon and zeta) were poor HSP-kinases. These results show that select PKC family members can function as direct HSP-kinases in vitro. Moreover, the

  18. Protein phosphorylation and intermolecular electron transfer: a joint experimental and computational study of a hormone biosynthesis pathway.

    PubMed

    Zöllner, Andy; Pasquinelli, Melissa A; Bernhardt, Rita; Beratan, David N

    2007-04-11

    Protein phosphorylation is a common regulator of enzyme activity. Chemical modification of a protein surface, including phosphorylation, could alter the function of biological electron-transfer reactions. However, the sensitivity of intermolecular electron-transfer kinetics to post-translational protein modifications has not been widely investigated. We have therefore combined experimental and computational studies to assess the potential role of phosphorylation in electron-transfer reactions. We investigated the steroid hydroxylating system from bovine adrenal glands, which consists of adrenodoxin (Adx), adrenodoxin reductase (AdR), and a cytochrome P450, CYP11A1. We focused on the phosphorylation of Adx at Thr-71, since this residue is located in the acidic interaction domain of Adx, and a recent study has demonstrated that this residue is phosphorylated by casein kinase 2 (CK2) in vitro.1 Optical biosensor experiments indicate that the presence of this phosphorylation slightly increases the binding affinity of oxidized Adx with CYP11A1ox but not AdRox. This tendency was confirmed by KA values extracted from Adx concentration-dependent stopped-flow experiments that characterize the interaction between AdRred and Adxox or between Adxred and CYP11A1ox. In addition, acceleration of the electron-transfer kinetics measured with stopped-flow is seen only for the phosphorylated Adx-CYP11A1 reaction. Biphasic reaction kinetics are observed only when Adx is phosphorylated at Thr-71, and the Brownian dynamics (BD) simulations suggest that this phosphorylation may enhance the formation of a secondary Adx-CYP11A1 binding complex that provides an additional electron-transfer pathway with enhanced coupling. PMID:17358057

  19. Muscarinic agonists and phorbol esters increase tyrosine phosphorylation of a 40-kilodalton protein in hippocampal slices

    SciTech Connect

    Stratton, K.R.; Worley, P.F.; Huganir, R.L.; Baraban, J.M. )

    1989-04-01

    The authors have used the hippocampal slice preparation to investigate the regulation of protein tyrosine phosphorylation in brain. After pharmacological treatment of intact slices, proteins were separated by electrophoresis, and levels of protein tyrosine phosphorylation were assessed by immunoblotting with specific anti-phosphotyrosine antibodies. Phorbol esters, activators of the serine- and threonine-phosphorylating enzyme protein kinase C, selectively increase tyrosine phosphorylation of a soluble protein with an apparent molecular mass of approximately 40 kilodaltons. Muscarinic agonists such as carbachol and oxotremorine M that strongly activate the inositol phospholipid system also increase tyrosine phosphorylation of this protein. Neurotransmitter activation of the inositol phospholipid system and protein kinase C appears to trigger a cascade leading to increased tyrosine phosphorylation.

  20. Identifying Human Kinase-Specific Protein Phosphorylation Sites by Integrating Heterogeneous Information from Various Sources

    PubMed Central

    Li, Tingting; Du, Pufeng; Xu, Nanfang

    2010-01-01

    Phosphorylation is an important type of protein post-translational modification. Identification of possible phosphorylation sites of a protein is important for understanding its functions. Unbiased screening for phosphorylation sites by in vitro or in vivo experiments is time consuming and expensive; in silico prediction can provide functional candidates and help narrow down the experimental efforts. Most of the existing prediction algorithms take only the polypeptide sequence around the phosphorylation sites into consideration. However, protein phosphorylation is a very complex biological process in vivo. The polypeptide sequences around the potential sites are not sufficient to determine the phosphorylation status of those residues. In the current work, we integrated various data sources such as protein functional domains, protein subcellular location and protein-protein interactions, along with the polypeptide sequences to predict protein phosphorylation sites. The heterogeneous information significantly boosted the prediction accuracy for some kinase families. To demonstrate potential application of our method, we scanned a set of human proteins and predicted putative phosphorylation sites for Cyclin-dependent kinases, Casein kinase 2, Glycogen synthase kinase 3, Mitogen-activated protein kinases, protein kinase A, and protein kinase C families (avaiable at http://cmbi.bjmu.edu.cn/huphospho). The predicted phosphorylation sites can serve as candidates for further experimental validation. Our strategy may also be applicable for the in silico identification of other post-translational modification substrates. PMID:21085571

  1. Phosphorylation of C-protein, troponin I and phospholamban in isolated rabbit hearts.

    PubMed Central

    Garvey, J L; Kranias, E G; Solaro, R J

    1988-01-01

    Phosphorylation of myofibrillar and sacroplasmic-reticulum (SR) proteins was studied in Langendorff-perfused rabbit hearts subjected to various inotropic interventions. Stimulation of hearts with isoprenaline resulted in the phosphorylation of both troponin I (TnI) and C-protein in myofibrils and phospholamban in SR. Phosphorylation of phospholamban could be reversed by a 15 min perfusion with drug-free buffer, after a 1 minute pulse perfusion with isoprenaline, at which time the mechanical effects of isoprenaline stimulation had also been reversed. However, both TnI and C-protein remained phosphorylated at this time. Moreover, the inhibition of Ca2+ activation of the Mg2+-dependent ATPase (Mg-ATPase) activity associated with myofibrillar phosphorylation persisted in myofibrils prepared from hearts frozen after 15 min of washout of isoprenaline. To assess the contribution of C-protein phosphorylation in the decrease of Ca2+ activation of the myofibrillar Mg-ATPase activity, we reconstituted a regulated actomyosin system in which only C-protein was phosphorylated. In this system, C-protein phosphorylation did not contribute to the decrease in Ca2+ activation of Mg-ATPase activity, indicating that TnI phosphorylation is responsible for the diminished sensitivity of the myofibrils to Ca2+. These observations support the hypothesis that phospholamban phosphorylation plays a more dominant role than TnI or C-protein phosphorylation in the mechanical response of the mammalian heart to beta-adrenergic stimulation. Images Fig. 1. Fig. 3. PMID:2895634

  2. Effects of PKA phosphorylation on the conformation of the Na,K-ATPase regulatory protein FXYD1

    PubMed Central

    Teriete, Peter; Thai, Khang; Choi, Jungyuen; Marassi, Francesca M.

    2009-01-01

    FXYD1 (phospholemman) is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the Na,K-ATPase enzyme complex in specific tissues and specific physiological states. In heart and skeletal muscle sarcolemma, FXYD1 is also the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinase A and by protein kinase C, which phosphorylate the protein at conserved Ser residues in its cytoplasmic domain, altering its Na,K-ATPase regulatory activity. FXYD1 adopts an L-shaped α-helical structure with the transmembrane helix loosely connected to a cytoplasmic amphipathic helix that rests on the membrane surface. In this paper we describe NMR experiments showing that neither PKA phosphorylation at Ser68 nor the physiologically relevant phosphorylation mimicking mutation Ser68Asp induces major changes in the protein conformation. The results, viewed in light of a model of FXYD1 associated with the Na,K-ATPase α and β subunits, indicate that the effects of phosphorylation on the Na,K-ATPase regulatory activity of FXYD1 could be due primarily to changes in electrostatic potential near the membrane surface and near the Na+/K+ ion binding site of the Na,K-ATPase α subunit. PMID:19761758

  3. Identification of phosphorylation sites in the nucleocapsid protein (N protein) of SARS-coronavirus

    NASA Astrophysics Data System (ADS)

    Lin, Liang; Shao, Jianmin; Sun, Maomao; Liu, Jinxiu; Xu, Gongjin; Zhang, Xumin; Xu, Ningzhi; Wang, Rong; Liu, Siqi

    2007-12-01

    After decoding the genome of SARS-coronavirus (SARS-CoV), next challenge is to understand how this virus causes the illness at molecular bases. Of the viral structural proteins, the N protein plays a pivot role in assembly process of viral particles as well as viral replication and transcription. The SARS-CoV N proteins expressed in the eukaryotes, such as yeast and HEK293 cells, appeared in the multiple spots on two-dimensional electrophoresis (2DE), whereas the proteins expressed in E. coli showed a single 2DE spotE These 2DE spots were further examined by Western blot and MALDI-TOF/TOF MS, and identified as the N proteins with differently apparent pI values and similar molecular mass of 50 kDa. In the light of the observations and other evidences, a hypothesis was postulated that the SARS-CoV N protein could be phosphorylated in eukaryotes. To locate the plausible regions of phosphorylation in the N protein, two truncated N proteins were generated in E. coli and treated with PKC[alpha]. The two truncated N proteins after incubation of PKC[alpha] exhibited the differently electrophoretic behaviors on 2DE, suggesting that the region of 1-256 aa in the N protein was the possible target for PKC[alpha] phosphorylation. Moreover, the SARS-CoV N protein expressed in yeast were partially digested with trypsin and carefully analyzed by MALDI-TOF/TOF MS. In contrast to the completely tryptic digestion, these partially digested fragments generated two new peptide mass signals with neutral loss, and MS/MS analysis revealed two phosphorylated peptides located at the "dense serine" island in the N protein with amino acid sequences, GFYAEGSRGGSQASSRSSSR and GNSGNSTPGSSRGNSPARMASGGGK. With the PKC[alpha] phosphorylation treatment and the partially tryptic digestion, the N protein expressed in E. coli released the same peptides as observed in yeast cells. Thus, this investigation provided the preliminary data to determine the phosphorylation sites in the SARS-CoV N protein, and

  4. Protein kinase CK2 phosphorylates Hsp105 alpha at Ser509 and modulates its function.

    PubMed Central

    Ishihara, Keiichi; Yamagishi, Nobuyuki; Hatayama, Takumi

    2003-01-01

    The 105 kDa heat-shock protein (Hsp) Hsp105 alpha is a mammalian stress protein that belongs to the HSP105/HSP110 family. We have shown previously that Hsp105 alpha exists as non-phosphorylated and phosphorylated forms in vivo, and is phosphorylated by protein kinase CK2 (CK2) in vitro. In this study, to elucidate the role of phosphorylation of Hsp105 alpha, we first analysed the site of phosphorylation of Hsp105 alpha by CK2. Peptide mapping analysis of Hsp105 alpha phosphorylated by CK2 and in vitro phosphorylation experiments using various deletion and substitution mutants of Hsp105 alpha revealed that Hsp105 alpha is phosphorylated at Ser(509) in the beta-sheet domain. Furthermore, Ser(509) in Hsp105 alpha was also phosphorylated in mammalian COS-7 cells, although other sites were phosphorylated as well. Next, we examined the effects of phosphorylation of Hsp105 alpha on its functions using CK2-phosphorylated Hsp105 alpha. Interestingly, Hsp105 alpha suppressed 70 kDa heat-shock cognate protein (Hsc70)-mediated protein folding, whereas the phosphorylation of Hsp105 alpha at Ser(509) abolished the inhibitory activity of Hsp105 alpha in vitro. In accordance with these findings, wild-type Hsp105 alpha, which was thought to be phosphorylated in vivo, had no effect on Hsp70-mediated refolding of heat-denatured luciferase, whereas a non-phosphorylatable mutant of Hsp105 alpha suppressed the Hsp70-mediated refolding of heat-denatured luciferase in mammalian cells. Thus it was suggested that CK2 phosphorylates Hsp105 alpha at Ser(509) and modulates the function of Hsp105 alpha. The regulation of Hsp105 alpha function by phosphorylation may play an important role in a variety of cellular events. PMID:12558502

  5. Global impact of protein arginine phosphorylation on the physiology of Bacillus subtilis

    PubMed Central

    Elsholz, Alexander K. W.; Turgay, Kürşad; Michalik, Stephan; Hessling, Bernd; Gronau, Katrin; Oertel, Dan; Mäder, Ulrike; Bernhardt, Jörg; Becher, Dörte; Hecker, Michael; Gerth, Ulf

    2012-01-01

    Reversible protein phosphorylation is an important and ubiquitous protein modification in all living cells. Here we report that protein phosphorylation on arginine residues plays a physiologically significant role. We detected 121 arginine phosphorylation sites in 87 proteins in the Gram-positive model organism Bacillus subtilis in vivo. Moreover, we provide evidence that protein arginine phosphorylation has a functional role and is involved in the regulation of many critical cellular processes, such as protein degradation, motility, competence, and stringent and stress responses. Our results suggest that in B. subtilis the combined activity of a protein arginine kinase and phosphatase allows a rapid and reversible regulation of protein activity and that protein arginine phosphorylation can play a physiologically important and regulatory role in bacteria. PMID:22517742

  6. In vitro phosphorylation of the N-terminal half of hordeivirus movement protein.

    PubMed

    Makarov, V V; Iconnikova, A Y; Guseinov, M A; Vishnichenko, V K; Kalinina, N O

    2012-09-01

    The N-terminal half of TGB1 movement protein of poa semilatent hordeivirus, which forms a ribonucleoprotein complex involved in movement of the viral genome in the plant, and its two domains, NTD and ID, are phosphorylated in vitro by a fraction enriched in cell walls from Nicotiana benthamiana. Using a set of protein kinase inhibitors with different specificities, it was found that enzymes possessing activities of casein kinase 1, protein kinase A, and protein kinase C are involved in phosphorylation. Commercial preparations of protein kinases A and C are able to phosphorylate in vitro recombinant proteins corresponding to the N-terminal half of the protein and its domains NTD and ID. Phosphorylation of the NTD has no effect on the efficiency and character of its binding to RNA. However, phosphorylation of the ID leads to a decrease in its RNA-binding activity and in the ability for homological protein-protein interactions. PMID:23157268

  7. Differentiation of HL60 cells: involvement of protein phosphorylation

    SciTech Connect

    Spearman, T.N.; Fontana, J.A.; Butcher, F.R.; Durham, J.P.

    1986-05-01

    The addition of retinoic acid (RA) to the human promyelocytic leukemic cell line HL60 in culture results in the cessation of growth and the acquisition of a more mature phenotype. Previous work in these laboratories has demonstrated a concomitant increase in the activity of calcium-dependent, phospholipid-sensitive protein kinase (PK-C). HL60 cells were incubated with /sup 32/P-P/sub i/ in the absence and presence of RA, homogenized, and aliquots subjected to two-dimensional electrophoresis. A comparison of autoradiograms made from these gels revealed several phosphoproteins whose radiolabeling was affected by RA. The radiolabeling of one particular phosphoprotein (49kd, pI 4.8) was found to be increased prior to phenotypic evidence of differentiation. It was demonstrated via incubating HL60 cytosol with /sup 32/P -ATP and Ca/sup 2 +/ in the absence and presence of phosphatidylserine and resolving the labeled proteins as above that this protein is phosphorylated by PK-C. The labeling of this protein was also increased by RA in other leukemic cell lines which showed phenotypic evidence of differentiation while no effect was seen in HL60 sublines resistant to RA or in mature neutrophils (the end product of myeloid differentiation). These results suggest that this protein may be an important intermediate in myeloid differentiation.

  8. Development of the affinity materials for phosphorylated proteins/peptides enrichment in phosphoproteomics analysis.

    PubMed

    Wang, Zhi-Gang; Lv, Nan; Bi, Wen-Zhi; Zhang, Ji-Lin; Ni, Jia-Zuan

    2015-04-29

    Reversible protein phosphorylation is a key event in numerous biological processes. Mass spectrometry (MS) is the most powerful analysis tool in modern phosphoproteomics. However, the direct MS analysis of phosphorylated proteins/peptides is still a big challenge because of the low abundance and insufficient ionization of phosphorylated proteins/peptides as well as the suppression effects of nontargets. Enrichment of phosphorylated proteins/peptides by affinity materials from complex biosamples is the most widely used strategy to enhance the MS detection. The demand of efficiently enriching phosphorylated proteins/peptides has spawned diverse affinity materials based on different enrichment principles (e.g., electronic attraction, chelating). In this review, we summarize the recent development of various affinity materials for phosphorylated proteins/peptides enrichment. We will highlight the design and fabrication of these affinity materials, discuss the enrichment mechanisms involved in different affinity materials, and suggest the future challenges and research directions in this field. PMID:25845677

  9. The effect of oviductal fluid on protein tyrosine phosphorylation in cryopreserved boar spermatozoa differs with the freezing method.

    PubMed

    Kumaresan, A; Johannisson, A; Saravia, F; Bergqvist, A S

    2012-02-01

    Sperm capacitation takes place in the oviduct and protein tyrosine phosphorylation of sperm proteins is a crucial step in capacitation and acquisition of fertilizing potential. Cryopreserved spermatozoa show altered expression of protein tyrosine phosphorylation in the oviduct. The present study compared two freezing methods (conventional-conventional freezing (CF) and simplified-simplified freezing (SF) methods) for their effect on the ability of boar spermatozoa to undergo protein tyrosine phosphorylation in response to oviductal fluid (ODF). Cryopreserved boar-spermatozoa were incubated with pre- and post-ovulatory ODF for 6 h at 38 °C under 5% CO(2). Aliquots of sperm samples were taken at hourly intervals and analyzed for kinematics and protein tyrosine phosphorylation. Global protein tyrosine phosphorylation in spermatozoa was measured using flow cytometry and different patterns of phosphorylation were assessed using confocal microscopy. Immediately after thawing, no significant difference was observed in post-thaw sperm motility, velocity and global tyrosine phosphorylation between the two methods of freezing although the freezing method significantly (P < 0.05) influenced the effect of oviductal fluid on these parameters during incubation. While spermatozoa frozen by the CF method showed a significantly higher (P < 0.001) proportion of phosphorylation in response to preovulatory ODF during incubation, spermatozoa frozen by the SF method did not elicit such significant response as there was no significant difference in the proportion of tyrosine phosphorylated spermatozoa between treatments at any given time during incubation. If the CF method was used, the proportion of spermatozoa displaying either tail or full sperm phosphorylation increased in response to both preovulatory (EODF) and postovulatory oviductal fluid. However, if the SF method was used, a significant increase in these patterns was noticed only in the EODF treated group. The present study

  10. Synaptic Activation of Ribosomal Protein S6 Phosphorylation Occurs Locally in Activated Dendritic Domains

    ERIC Educational Resources Information Center

    Pirbhoy, Patricia Salgado; Farris, Shannon; Steward, Oswald

    2016-01-01

    Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6)…

  11. Asymmetric Dimethylarginine Stimulates Akt1 Phosphorylation via Heat Shock Protein 70-Facilitated Carboxyl-Terminal Modulator Protein Degradation in Pulmonary Arterial Endothelial Cells.

    PubMed

    Sun, Xutong; Kellner, Manuela; Desai, Ankit A; Wang, Ting; Lu, Qing; Kangath, Archana; Qu, Ning; Klinger, Christina; Fratz, Sohrab; Yuan, Jason X-J; Jacobson, Jeffrey R; Garcia, Joe G N; Rafikov, Ruslan; Fineman, Jeffrey R; Black, Stephen M

    2016-08-01

    Asymmetric dimethylarginine (ADMA) induces the mitochondrial translocation of endothelial nitric oxide synthase (eNOS) through the nitration-mediated activation of Akt1. However, it is recognized that the activation of Akt1 requires phosphorylation events at threonine (T) 308 and serine (S) 473. Thus, the current study was performed to elucidate the potential effect of ADMA on Akt1 phosphorylation and the mechanisms that are involved. Exposure of pulmonary arterial endothelial cells to ADMA enhanced Akt1 phosphorylation at both threonine 308 and Ser473 without altering Akt1 protein levels, phosphatase and tensin homolog activity, or membrane Akt1 levels. Heat shock protein (Hsp) 90 plays a pivotal role in maintaining Akt1 activity, and our results demonstrate that ADMA decreased Hsp90-Akt1 interactions, but, surprisingly, overexpression of a dominant-negative Hsp90 mutant increased Akt1 phosphorylation. ADMA exposure or overexpression of dominant-negative Hsp90 increased Hsp70 levels, and depletion of Hsp70 abolished ADMA-induced Akt1 phosphorylation. ADMA decreased the interaction of Akt1 with its endogenous inhibitor, carboxyl-terminal modulator protein (CTMP). This was mediated by the proteasomal-dependent degradation of CTMP. The overexpression of CTMP attenuated ADMA-induced Akt1 phosphorylation at Ser473, eNOS phosphorylation at Ser617, and eNOS mitochondrial translocation. Finally, we found that the mitochondrial translocation of eNOS in our lamb model of pulmonary hypertension is associated with increased Akt1 and eNOS phosphorylation and reduced Akt1-CTMP protein interactions. In conclusion, our data suggest that CTMP is directly involved in ADMA-induced Akt1 phosphorylation in vitro and in vivo, and that increasing CTMP levels may be an avenue to treat pulmonary hypertension. PMID:26959555

  12. Phosphorylation of serine residues affects the conformation of the calmodulin binding domain of human protein 4.1.

    PubMed

    Vetter, S W; Leclerc, E

    2001-08-01

    We have previously characterized the calcium-dependent calmodulin (CaM)-binding domain (Ser76-Ser92) of the 135-kDa human protein 4.1 isoform using fluorescence spectroscopy and chemically synthesized nonphosphorylated or serine phosphorylated peptides [Leclerc, E. & Vetter, S. (1998) Eur. J. Biochem. 258, 567-671]. Here we demonstrate that phosphorylation of two serine residues within the 17-residue peptide alters their ability to adopt alpha helical conformation in a position-dependent manner. The helical content of the peptides was determined by CD-spectroscopy and found to increase from 36 to 45% for the Ser80 phosphorylated peptide and reduce to 28% for the Ser84 phosphorylated peptide; the di-phosphorylated peptide showed 32% helical content. Based on secondary structure prediction methods we propose that initial helix formation involves the central residues Leu82-Phe86. The ability of the peptides to adopt alpha helical conformations did not correlate with the observed binding affinities to CaM. We suggest that the reduced CaM-binding affinities observed for the phosphorylated peptides are more likely to be the result of unfavorable sterical and electrostatic interactions introduced into the CaM peptide-binding interface by the phosphate groups, rather than being due to the effect of phosphorylation on the secondary structure of the peptides. PMID:11488924

  13. Protein kinase C catalyses the phosphorylation and activation of rat liver phospholipid methyltransferase.

    PubMed Central

    Villalba, M; Pajares, M A; Renart, M F; Mato, J M

    1987-01-01

    When a partially purified rat liver phospholipid methyltransferase is incubated with [gamma-32P]ATP and rat brain protein kinase C, phospholipid methyltransferase (Mr 50,000, pI 4.75) becomes phosphorylated. Phosphorylation of the enzyme showed Ca2+/lipid-dependency. Protein kinase C-dependent phosphorylation of phospholipid methyltransferase was accompanied by an approx. 2-fold activation of the enzyme activity. Activity changes and enzyme phosphorylation showed the same time course. Activation of the enzyme also showed Ca2+/lipid-dependency. Protein kinase C mediates phosphorylation of predominantly serine residues of the methyltransferase. One major peak of phosphorylation was identified by analysis of tryptic phosphopeptides by isoelectrofocusing. This peak (pI 5.2) differs from that phosphorylated by the cyclic AMP-dependent protein kinase (pI 7.2), demonstrating the specificity of phosphorylation of protein kinase C. Tryptic-peptide mapping by h.p.l.c. of the methyltransferase phosphorylated by protein kinase C revealed one major peak of radioactivity, which could be resolved into two labelled phosphopeptides by t.l.c. The significance of protein kinase C-mediated phosphorylation of phospholipid methyltransferase is discussed. Images Fig. 1. Fig. 4. PMID:3593229

  14. Phosphorylation of TCF proteins by homeodomain-interacting protein kinase 2.

    PubMed

    Hikasa, Hiroki; Sokol, Sergei Y

    2011-04-01

    Wnt pathways play essential roles in cell proliferation, morphogenesis, and cell fate specification during embryonic development. According to the consensus view, the Wnt pathway prevents the degradation of the key signaling component β-catenin by the protein complex containing the negative regulators Axin and glycogen synthase kinase 3 (GSK3). Stabilized β-catenin associates with TCF proteins and enters the nucleus to promote target gene expression. This study examines the involvement of HIPK2 (homeodomain-interacting protein kinase 2) in the regulation of different TCF proteins in Xenopus embryos in vivo. We show that the TCF family members LEF1, TCF4, and TCF3 are phosphorylated in embryonic ectoderm after Wnt8 stimulation and HIPK2 overexpression. We also find that TCF3 phosphorylation is triggered by canonical Wnt ligands, LRP6, and dominant negative mutants for Axin and GSK3, indicating that this process shares the same upstream regulators with β-catenin stabilization. HIPK2-dependent phosphorylation caused the dissociation of LEF1, TCF4, and TCF3 from a target promoter in vivo. This result provides a mechanistic explanation for the context-dependent function of HIPK2 in Wnt signaling; HIPK2 up-regulates transcription by phosphorylating TCF3, a transcriptional repressor, but inhibits transcription by phosphorylating LEF1, a transcriptional activator. Finally, we show that upon HIPK2-mediated phosphorylation, TCF3 is replaced with positively acting TCF1 at a target promoter. These observations emphasize a critical role for Wnt/HIPK2-dependent TCF phosphorylation and suggest that TCF switching is an important mechanism of Wnt target gene activation in vertebrate embryos. PMID:21285352

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

    PubMed Central

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

    2012-01-01

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

  16. Extracellular Ser/Thr/Tyr phosphorylated proteins of Pseudomonas aeruginosa PA14 strain.

    PubMed

    Ouidir, Tassadit; Jarnier, Frédérique; Cosette, Pascal; Jouenne, Thierry; Hardouin, Julie

    2014-09-01

    Protein phosphorylation on serine, threonine, and tyrosine is known to be involved in a wide variety of cellular processes, signal transduction, and bacterial virulence. We characterized, for the first time, the extracellular phosphoproteins of the Pseudomonas aeruginosa PA14 strain. We identified 28 phosphoproteins (59 phosphosites) including enzymes, with various phosphorylation sites, known as potent secreted virulence factors in P. aeruginosa. The high phosphorylation level of these virulence factors might reflect a relationship between Ser/Thr/Tyr phosphorylation and virulence. PMID:24965220

  17. Protein Phosphorylation and Redox Modification in Stomatal Guard Cells.

    PubMed

    Balmant, Kelly M; Zhang, Tong; Chen, Sixue

    2016-01-01

    Post-translational modification (PTM) is recognized as a major process accounting for protein structural variation, functional diversity, and the dynamics and complexity of the proteome. Since PTMs can change the structure and function of proteins, they are essential to coordinate signaling networks and to regulate important physiological processes in eukaryotes. Plants are constantly challenged by both biotic and abiotic stresses that reduce productivity, causing economic losses in crops. The plant responses involve complex physiological, cellular, and molecular processes, with stomatal movement as one of the earliest responses. In order to activate such a rapid response, stomatal guard cells employ cellular PTMs of key protein players in the signaling pathways to regulate the opening and closure of the stomatal pores. Here we discuss two major types of PTMs, protein phosphorylation and redox modification that play essential roles in stomatal movement under stress conditions. We present an overview of PTMs that occur in stomatal guard cells, especially the methods and technologies, and their applications in PTM identification and quantification. Our focus is on PTMs that modify molecular components in guard cell signaling at the stages of signal perception, second messenger production, as well as downstream signaling events and output. Improved understanding of guard cell signaling will enable generation of crops with enhanced stress tolerance, and increased yield and bioenergy through biotechnology and molecular breeding. PMID:26903877

  18. Protein Phosphorylation and Redox Modification in Stomatal Guard Cells

    PubMed Central

    Balmant, Kelly M.; Zhang, Tong; Chen, Sixue

    2016-01-01

    Post-translational modification (PTM) is recognized as a major process accounting for protein structural variation, functional diversity, and the dynamics and complexity of the proteome. Since PTMs can change the structure and function of proteins, they are essential to coordinate signaling networks and to regulate important physiological processes in eukaryotes. Plants are constantly challenged by both biotic and abiotic stresses that reduce productivity, causing economic losses in crops. The plant responses involve complex physiological, cellular, and molecular processes, with stomatal movement as one of the earliest responses. In order to activate such a rapid response, stomatal guard cells employ cellular PTMs of key protein players in the signaling pathways to regulate the opening and closure of the stomatal pores. Here we discuss two major types of PTMs, protein phosphorylation and redox modification that play essential roles in stomatal movement under stress conditions. We present an overview of PTMs that occur in stomatal guard cells, especially the methods and technologies, and their applications in PTM identification and quantification. Our focus is on PTMs that modify molecular components in guard cell signaling at the stages of signal perception, second messenger production, as well as downstream signaling events and output. Improved understanding of guard cell signaling will enable generation of crops with enhanced stress tolerance, and increased yield and bioenergy through biotechnology and molecular breeding. PMID:26903877

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

    PubMed

    Morishita, Yuji; Tsutsumi, Koji; Ohta, Yasutaka

    2015-10-23

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

  20. Photosystem II antenna phosphorylation-dependent protein diffusion determined by fluorescence correlation spectroscopy.

    PubMed

    Iwai, Masakazu; Pack, Chan-Gi; Takenaka, Yoshiko; Sako, Yasushi; Nakano, Akihiko

    2013-01-01

    Flexibility of chloroplast thylakoid membrane proteins is essential for plant fitness and survival under fluctuating light environments. Phosphorylation of light-harvesting antenna complex II (LHCII) is known to induce dynamic protein reorganization that fine-tunes the rate of energy conversion in each photosystem. However, molecular details of how LHCII phosphorylation causes light energy redistribution throughout thylakoid membranes still remain unclear. By using fluorescence correlation spectroscopy, we here determined the LHCII phosphorylation-dependent protein diffusion in thylakoid membranes isolated from the green alga Chlamydomonas reinhardtii. As compared to the LHCII dephosphorylation-induced condition, the diffusion coefficient of LHCII increased nearly twofold under the LHCII phosphorylation-induced condition. We also verified the results by using the LHCII phosphorylation-deficient mutant. Our observation suggests that LHCII phosphorylation-dependent protein reorganization occurs along with the changes in the rate of protein diffusion, which would have an important role in mediating light energy redistribution throughout thylakoid membranes. PMID:24088948

  1. Solid-phase assay for the phosphorylation of proteins blotted on nitrocellulose membrane filters

    SciTech Connect

    Valtorta, F.; Schiebler, W.; Jahn, R.; Ceccarelli, B.; Greengard, P.

    1986-10-01

    A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters, and the blotted polypeptides are phyosphorylated with the catalytic subunit of cyclic AMP (adenosine 3':5'-monophosphate)-dependent protein kinase. The method was developed for the assay of dephosphosynapsin I, but it has also proven suitable for the phosphorylation of other proteins. The patterns of phosphorylation of tissue samples phosphorylated using the new method are similar to those obtained using the conventional test tube assay. Once phosphorylated, the adsorbed proteins can be digested with proteases and subjected to phosphopeptide mapping. The phosphorylated blotted proteins can also be analyzed by overlay techniques for the immunological detection of polypeptides.

  2. An isotope labeling strategy for quantifying the degree of phosphorylation at multiple sites in proteins.

    PubMed

    Hegeman, Adrian D; Harms, Amy C; Sussman, Michael R; Bunner, Anne E; Harper, Jeffrey F

    2004-05-01

    A procedure for determining the extent of phosphorylation at individual sites of multiply phosphorylated proteins was developed and applied to two polyphosphorylated proteins. The protocol, using simple chemical (Fischer methyl-esterification) and enzymatic (phosphatase) modification steps and an accessible isotopic labeling reagent (methyl alcohol-d(4)), is described in detail. Site-specific phosphorylation stoichiometries are derived from the comparison of chemically identical but isotopically distinct peptide species analyzed by microspray liquid chromatography-mass spectrometry (microLC-MS) using a Micromass Q-TOF2 mass spectrometer. Ten phosphorylation sites were unambiguously identified in tryptic digests of both proteins, and phosphorylation stoichiometries were determined for eight of the ten sites using the isotope-coded strategy. The extent of phosphorylation was also estimated from the mass spectral peak areas for the phosphorylated and unmodified peptides, and these estimates, when compared with stoichiometries determined using the isotope-coded technique, differed only marginally (within approximately 20%). PMID:15121193

  3. Extensive differential protein phosphorylation as intraerythrocytic Plasmodium falciparum schizonts develop into extracellular invasive merozoites.

    PubMed

    Lasonder, Edwin; Green, Judith L; Grainger, Munira; Langsley, Gordon; Holder, Anthony A

    2015-08-01

    Pathology of the most lethal form of malaria is caused by Plasmodium falciparum asexual blood stages and initiated by merozoite invasion of erythrocytes. We present a phosphoproteome analysis of extracellular merozoites revealing 1765 unique phosphorylation sites including 785 sites not previously detected in schizonts. All MS data have been deposited in the ProteomeXchange with identifier PXD001684 (http://proteomecentral.proteomexchange.org/dataset/PXD001684). The observed differential phosphorylation between extra and intraerythrocytic life-cycle stages was confirmed using both phospho-site and phospho-motif specific antibodies and is consistent with the core motif [K/R]xx[pS/pT] being highly represented in merozoite phosphoproteins. Comparative bioinformatic analyses highlighted protein sets and pathways with established roles in invasion. Within the merozoite phosphoprotein interaction network a subnetwork of 119 proteins with potential roles in cellular movement and invasion was identified and suggested that it is coregulated by a further small subnetwork of protein kinase A (PKA), two calcium-dependent protein kinases (CDPKs), a phosphatidyl inositol kinase (PI3K), and a GCN2-like elF2-kinase with a predicted role in translational arrest and associated changes in the ubquitinome. To test this notion experimentally, we examined the overall ubiquitination level in intracellular schizonts versus extracellular merozoites and found it highly upregulated in merozoites. We propose that alterations in the phosphoproteome and ubiquitinome reflect a starvation-induced translational arrest as intracellular schizonts transform into extracellular merozoites. PMID:25886026

  4. Rho kinase II phosphorylation of the lipoprotein receptor LR11/SORLA alters amyloid-beta production.

    PubMed

    Herskowitz, Jeremy H; Seyfried, Nicholas T; Gearing, Marla; Kahn, Richard A; Peng, Junmin; Levey, Allan I; Lah, James J

    2011-02-25

    LR11, also known as SorLA, is a mosaic low-density lipoprotein receptor that exerts multiple influences on Alzheimer disease susceptibility. LR11 interacts with the amyloid-β precursor protein (APP) and regulates APP traffic and processing to amyloid-β peptide (Aβ). The functional domains of LR11 suggest that it can act as a cell surface receptor and as an intracellular sorting receptor for trans-Golgi network to endosome traffic. We show that LR11 over-expressed in HEK293 cells is radiolabeled following incubation of cells with [(32)P(i)]orthophosphate. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to discover putative LR11 interacting kinases. Rho-associated coiled-coil containing protein kinase (ROCK) 2 was identified as a binding partner and a candidate kinase acting on LR11. LR11 and ROCK2 co-immunoprecipitate from post-mortem human brain tissue and drug inhibition of ROCK activity reduces LR11 phosphorylation in vivo. Targeted knockdown of ROCK2 with siRNA decreased LR11 ectodomain shedding while simultaneously increasing intracellular LR11 protein level. Site-directed mutagenesis of serine 2206 in the LR11 cytoplasmic tail reduced LR11 shedding, decreased LR11 phosphorylation in vitro, and abrogated LR11 mediated Aβ reduction. These findings provide direct evidence that LR11 is phosphorylated in vivo and indicate that ROCK2 phosphorylation of LR11 may enhance LR11 mediated processing of APP and amyloid production. PMID:21147781

  5. Phosphorylation at serine 52 and 635 does not alter the transport properties of glucosinolate transporter AtGTR1

    PubMed Central

    Jørgensen, Morten Egevang; Olsen, Carl Erik; Halkier, Barbara Ann; Nour-Eldin, Hussam Hassan

    2016-01-01

    Little is known about how plants regulate transporters of defense compounds. In A. thaliana, glucosinolates are transported between tissues by NPF2.10 (AtGTR1) and NPF2.11 (AtGTR2). Mining of the PhosPhat4.0 database showed two cytosol exposed phosphorylation sites for AtGTR1 and one membrane-buried phosphorylation site for AtGTR2. In this study, we investigate whether mutation of the two potential regulatory sites of AtGTR1 affected transport of glucosinolates in Xenopus oocytes. Characterization of AtGTR1 phosphorylation mutants showed that phosphorylation of AtGTR1 - at the two reported phosphorylation sites - is not directly involved in regulating AtGTR1 transport activity. We hypothesize a role for AtGTR1-phosphorylation in regulating protein-protein interactions. PMID:26340317

  6. STAT1:DNA sequence-dependent binding modulation by phosphorylation, protein:protein interactions and small-molecule inhibition

    PubMed Central

    Bonham, Andrew J.; Wenta, Nikola; Osslund, Leah M.; Prussin, Aaron J.; Vinkemeier, Uwe; Reich, Norbert O.

    2013-01-01

    The DNA-binding specificity and affinity of the dimeric human transcription factor (TF) STAT1, were assessed by total internal reflectance fluorescence protein-binding microarrays (TIRF-PBM) to evaluate the effects of protein phosphorylation, higher-order polymerization and small-molecule inhibition. Active, phosphorylated STAT1 showed binding preferences consistent with prior characterization, whereas unphosphorylated STAT1 showed a weak-binding preference for one-half of the GAS consensus site, consistent with recent models of STAT1 structure and function in response to phosphorylation. This altered-binding preference was further tested by use of the inhibitor LLL3, which we show to disrupt STAT1 binding in a sequence-dependent fashion. To determine if this sequence-dependence is specific to STAT1 and not a general feature of human TF biology, the TF Myc/Max was analysed and tested with the inhibitor Mycro3. Myc/Max inhibition by Mycro3 is sequence independent, suggesting that the sequence-dependent inhibition of STAT1 may be specific to this system and a useful target for future inhibitor design. PMID:23180800

  7. Deciphering the Interplay among Multisite Phosphorylation, Interaction Dynamics, and Conformational Transitions in a Tripartite Protein System

    PubMed Central

    2016-01-01

    Multisite phosphorylation is a common pathway to regulate protein function, activity, and interaction pattern in vivo, but routine biochemical analysis is often insufficient to identify the number and order of individual phosphorylation reactions and their mechanistic impact on the protein behavior. Here, we integrate complementary mass spectrometry (MS)-based approaches to characterize a multisite phosphorylation-regulated protein system comprising Polo-like kinase 1 (Plk1) and its coactivators Aurora kinase A (Aur-A) and Bora, the interplay of which is essential for mitotic entry after DNA damage-induced cell cycle arrest. Native MS and cross-linking–MS revealed that Aur-A/Bora-mediated Plk1 activation is accompanied by the formation of Aur-A/Bora and Plk1/Bora heterodimers. We found that the Aur-A/Bora interaction is independent of the Bora phosphorylation state, whereas the Plk1/Bora interaction is dependent on extensive Bora multisite phosphorylation. Bottom-up and top-down proteomics analyses showed that Bora multisite phosphorylation proceeds via a well-ordered sequence of site-specific phosphorylation reactions, whereby we could reveal the involvement of up to 16 phosphorylated Bora residues. Ion mobility spectrometry–MS demonstrated that this multisite phosphorylation primes a substantial structural rearrangement of Bora, explaining the interdependence between extensive Bora multisite phosphorylation and Plk1/Bora complex formation. These results represent a first benchmark of our multipronged MS strategy, highlighting its potential to elucidate the mechanistic and structural implications of multisite protein phosphorylation. PMID:27504491

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

    PubMed

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

    2016-04-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    1987-04-01

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

  11. The Emerging Role of Protein Phosphorylation as a Critical Regulatory Mechanism Controlling Cellulose Biosynthesis

    PubMed Central

    Jones, Danielle M.; Murray, Christian M.; Ketelaar, KassaDee J.; Thomas, Joseph J.; Villalobos, Jose A.; Wallace, Ian S.

    2016-01-01

    Plant cell walls are extracellular matrices that surround plant cells and critically influence basic cellular processes, such as cell division and expansion. Cellulose is a major constituent of plant cell walls, and this paracrystalline polysaccharide is synthesized at the plasma membrane by a large protein complex known as the cellulose synthase complex (CSC). Recent efforts have identified numerous protein components of the CSC, but relatively little is known about regulation of cellulose biosynthesis. Numerous phosphoproteomic surveys have identified phosphorylation events in CSC associated proteins, suggesting that protein phosphorylation may represent an important regulatory control of CSC activity. In this review, we discuss the composition and dynamics of the CSC in vivo, the catalog of CSC phosphorylation sites that have been identified, the function of experimentally examined phosphorylation events, and potential kinases responsible for these phosphorylation events. Additionally, we discuss future directions in cellulose synthase kinase identification and functional analyses of CSC phosphorylation sites. PMID:27252710

  12. The Emerging Role of Protein Phosphorylation as a Critical Regulatory Mechanism Controlling Cellulose Biosynthesis.

    PubMed

    Jones, Danielle M; Murray, Christian M; Ketelaar, KassaDee J; Thomas, Joseph J; Villalobos, Jose A; Wallace, Ian S

    2016-01-01

    Plant cell walls are extracellular matrices that surround plant cells and critically influence basic cellular processes, such as cell division and expansion. Cellulose is a major constituent of plant cell walls, and this paracrystalline polysaccharide is synthesized at the plasma membrane by a large protein complex known as the cellulose synthase complex (CSC). Recent efforts have identified numerous protein components of the CSC, but relatively little is known about regulation of cellulose biosynthesis. Numerous phosphoproteomic surveys have identified phosphorylation events in CSC associated proteins, suggesting that protein phosphorylation may represent an important regulatory control of CSC activity. In this review, we discuss the composition and dynamics of the CSC in vivo, the catalog of CSC phosphorylation sites that have been identified, the function of experimentally examined phosphorylation events, and potential kinases responsible for these phosphorylation events. Additionally, we discuss future directions in cellulose synthase kinase identification and functional analyses of CSC phosphorylation sites. PMID:27252710

  13. Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52

    NASA Astrophysics Data System (ADS)

    Jo, Chulman; Gundemir, Soner; Pritchard, Susanne; Jin, Youngnam N.; Rahman, Irfan; Johnson, Gail V. W.

    2014-03-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor in the defence against oxidative stress. Here we provide evidence that activation of the Nrf2 pathway reduces the levels of phosphorylated tau by induction of an autophagy adaptor protein NDP52 (also known as CALCOCO2) in neurons. The expression of NDP52, which we show has three antioxidant response elements (AREs) in its promoter region, is strongly induced by Nrf2, and its overexpression facilitates clearance of phosphorylated tau in the presence of an autophagy stimulator. In Nrf2-knockout mice, phosphorylated and sarkosyl-insoluble tau accumulates in the brains concurrent with decreased levels of NDP52. Moreover, NDP52 associates with phosphorylated tau from brain cortical samples of Alzheimer disease cases, and the amount of phosphorylated tau in sarkosyl-insoluble fractions is inversely proportional to that of NDP52. These results suggest that NDP52 plays a key role in autophagy-mediated degradation of phosphorylated tau in vivo.

  14. Phosphorylation of the mRNA cap binding protein and eIF-4A by different protein kinases

    SciTech Connect

    Hagedorn, C.H.

    1987-05-01

    These studies were done to determine the identity of a protein kinase that phosphorylates the mRNA cap binding protein (CBP). Two chromatographic steps (dye and ligand and ion exchange HPLC) produced a 500x purification of an enzyme activity in rabbit reticulocytes that phosphorylated CBP at serine residues. Isoelectric focusing analysis of kinase treated CBP demonstrated 5 isoelectric species of which the 2 most anodic species were phosphorylated (contained /sup 32/P). This kinase activity phosphorylated CBP when it was isolated or in the eIF-4F complex. Purified protein kinase C, cAMP or cGMP dependent protein kinase, casein kinase I or II, myosin light chain kinase or insulin receptor kinase did not significantly phosphorylate isolated CBP or CBP in the eIF-4F complex. However, cAMP and cGMP dependent protein kinases and casein kinase II phosphorylated eIF-4A but did not phosphorylate the 46 kDa component of eIF-4F. cAMP dependent protein kinase phosphorylated a approx. 220 kDa protein doublet in eIF-4F preparations. These studies indicate that CBP kinase activity probably represents a previously unidentified protein kinase. In addition, eIF-4A appears to be phosphorylated by several protein kinases whereas the 46 kDa component of the eIF-4F complex was not.

  15. Chronic hyperammonemia reduces the activity of neuronal nitric oxide synthase in cerebellum by altering its localization and increasing its phosphorylation by calcium-calmodulin kinase II.

    PubMed

    El-Mlili, Nisrin; Rodrigo, Regina; Naghizadeh, Bahareh; Cauli, Omar; Felipo, Vicente

    2008-08-01

    Impaired function of the glutamate-nitric oxide-cGMP pathway contributes to cognitive impairment in hyperammonemia and hepatic encephalopathy. The mechanisms by which hyperammonemia impairs this pathway remain unclear. Understanding these mechanisms would allow designing clinical treatments for cognitive deficits in hepatic encephalopathy. The aims of this work were: (i) to assess whether chronic hyperammonemia in vivo alters basal activity of neuronal nitric oxide synthase (nNOS) in cerebellum and/or its activation in response to NMDA receptor activation and (ii) to analyse the molecular mechanisms by which hyperammonemia induces these alterations. It is shown that hyperammonemia reduces both basal activity of nNOS and its activation following NMDA receptor activation. Reduced basal activity is because of increased phosphorylation in Ser847 (by 69%) which reduces basal activity of nNOS by about 40%. Increased phosphorylation of nNOS in Ser847 is because of increased activity of calcium-calmodulin-dependent protein kinases (CaMKII) which in turn is because of increased phosphorylation at Thr286. Inhibiting CaMKII with KN-62 normalizes phosphorylation of Ser847 and basal NOS activity in hyperammonemic rats, returning to values similar to controls. Reduced activation of nNOS in response to NMDA receptor activation in hyperammonemia is because of altered subcellular localization of nNOS, with reduced amount in post-synaptic membranes and increased amount in the cytosol. PMID:18498443

  16. Phosphorylation of Human CTP Synthetase 1 by Protein Kinase A: IDENTIFICATION OF Thr455 AS A MAJOR SITE OF PHOSPHORYLATION*

    PubMed Central

    Choi, Mal-Gi; Carman, George M.

    2007-01-01

    CTP synthetase is an essential enzyme that generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work, we examined the phosphorylation of the human CTPS1-encoded CTP synthetase 1 by protein kinase A. CTP synthetase 1 was expressed and purified from a Saccharomyces cerevisiae ura7Δ ura8Δ double mutant that lacks CTP synthetase activity. Using purified CTP synthetase 1 as a substrate, protein kinase A activity was time- and dose-dependent. The phosphorylation, which primarily occurred on a threonine residue, was accompanied by a 50% decrease in CTP synthetase 1 activity. The synthetic peptide LGKRRTLFQT that contains the protein kinase A motif for Thr455 was a substrate for protein kinase A. A Thr455 to Ala (T455A) mutation in CTP synthetase 1 was constructed by site-directed mutagenesis and was expressed and purified from the S. cerevisiae ura7Δ ura8Δ mutant. The T455A mutation caused a 78% decrease in protein kinase A phosphorylation, and the loss of the phosphothreonine residue and a major phosphopeptide that were present in the purified wild type enzyme phosphorylated by protein kinase A. The CTP synthetase 1 activity of the T455A mutant enzyme was 2-fold higher than the wild type enzyme. In addition, the T455A mutation caused a 44% decrease in the amount of human CTP synthetase 1 that was phosphorylated in S. cerevisiae cells, and this was accompanied by a 2.5-fold increase in the cellular concentration of CTP and a 1.5-fold increase in the choline-dependent synthesis of phosphatidylcholine. PMID:17189248

  17. G protein beta gamma subunits stimulate phosphorylation of Shc adapter protein.

    PubMed Central

    Touhara, K; Hawes, B E; van Biesen, T; Lefkowitz, R J

    1995-01-01

    The mechanism of mitogen-activated protein (MAP) kinase activation by pertussis toxin-sensitive Gi-coupled receptors is known to involve the beta gamma subunits of heterotrimeric G proteins (G beta gamma), p21ras activation, and an as-yet-unidentified tyrosine kinase. To investigate the mechanism of G beta gamma-stimulated p21ras activation, G beta gamma-mediated tyrosine phosphorylation was examined by overexpressing G beta gamma or alpha 2-C10 adrenergic receptors (ARs) that couple to Gi in COS-7 cells. Immunoprecipitation of phosphotyrosine-containing proteins revealed a 2- to 3-fold increase in the phosphorylation of two proteins of approximately 50 kDa (designated as p52) in G beta gamma-transfected cells or in alpha 2-C10 AR-transfected cells stimulated with the agonist UK-14304. The latter response was pertussis toxin sensitive. These proteins (p52) were also specifically immunoprecipitated with anti-Shc antibodies and comigrated with two Shc proteins, 46 and 52 kDa. The G beta gamma- or alpha 2-C10 AR-stimulated p52 (Shc) phosphorylation was inhibited by coexpression of the carboxyl terminus of beta-adrenergic receptor kinase (a G beta gamma-binding pleckstrin homology domain peptide) or by the tyrosine kinase inhibitors genistein and herbimycin A, but not by a dominant negative mutant of p21ras. Worthmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) inhibited phosphorylation of p52 (Shc), implying involvement of PI3K. These results suggest that G beta gamma-stimulated Shc phosphorylation represents an early step in the pathway leading to p21ras activation, similar to the mechanism utilized by growth factor tyrosine kinase receptors. Images Fig. 1 Fig. 3 PMID:7568118

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

    PubMed Central

    2015-01-01

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

  19. Spatial proximity statistics suggest a regulatory role of protein phosphorylation on compound binding.

    PubMed

    Korkuć, Paula; Walther, Dirk

    2016-05-01

    Phosphorylation is an important post-translational modification that regulates protein function by the attachment of negatively charged phosphate groups to phosphorylatable amino acid residues. As a mode of action, an influence of phosphorylation on the binding of compounds to proteins has been discussed and described for a number of proteins in the literature. However, a systematic statistical survey probing for enriched phosphorylation sites close to compound binding sites in support of this notion and with properly chosen random reference distributions has not been presented yet. Using high-resolution protein structures from the Protein Data Bank including their co-crystallized non-covalently bound compounds and experimentally determined phosphorylation sites, we analyzed the pairwise distance distributions of phosphorylation and compound binding sites on protein surfaces. We found that phosphorylation sites are indeed located at significantly closer distances to compounds than expected by chance holding true specifically also for the subset of compound binding sites serving as catalytic sites of metabolic reactions. This tendency was particularly evident when treating phosphorylation sites as collective sets supporting the relevance of phosphorylation hotspots. Interestingly, phosphorylation sites were found to be closer to negatively charged than to positively charged compounds suggesting a stronger modulation of the binding of negatively charged compounds in dependence on phosphorylation status than on positively charged compounds. The enrichment of phosphorylation sites near compound binding sites confirms a regulatory role of phosphorylation in compound binding and provides a solid statistical basis for the literature-reported selected events. Proteins 2016; 84:565-579. © 2016 Wiley Periodicals, Inc. PMID:26817627

  20. Histidine to aspartate phosphotransferase activity of nm23 proteins: phosphorylation of aldolase C on Asp-319.

    PubMed Central

    Wagner, P D; Vu, N D

    2000-01-01

    nm23 genes have been implicated in the suppression of tumour metastasis and cell motility; however, the biochemical mechanisms for these suppressions are not known. We have previously described the transfer of phosphate from the catalytic histidine residues of nm23 proteins to an aspartic or a glutamic residue on one or more 43 kDa proteins in detergent extracts of bovine brain membranes. To gain a better understanding of this transferase activity, we partly purified this 43 kDa protein and identified aldolases A and C as the major 43 kDa proteins present in the preparation. Aldolase was purified from brain cytosol; its phosphorylation by rat liver nm23 proteins and by recombinant human nm23-H1 was examined. The site of phosphorylation was identified as Asp-319 on aldolase C. The equivalent residue on aldolase A, a glutamic residue, was not phosphorylated. Aldolase C was rapidly phosphorylated by wild-type nm23-H1 but was not phosphorylated, or was phosphorylated very slowly, by either nm23-H1(P96S) or nm23-H1(S120G), mutants of nm23-H1 that do not suppress cell motility. This is the first identification of a protein that is phosphorylated on an aspartic residue by nm23 proteins. The sequence around Asp-319 of aldolase C has some similarities to those around the histidine residues on ATP-citrate lyase and succinic thiokinase that are phosphorylated by nm23 proteins. PMID:10698688

  1. Beryllium Alters Lipopolysaccharide-Mediated Intracellular Phosphorylation and Cytokine Release in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Silva, Shannon; Ganguly, Kumkum; Fresquez, Theresa M.; Gupta, Goutam; McCleskey, T. Mark; Chaudhary, Anu

    2013-01-01

    Beryllium exposure in susceptible individuals leads to the development of chronic beryllium disease, a lung disorder marked by release of inflammatory cytokine and granuloma formation. We have previously reported that beryllium induces an immune response even in blood mononuclear cells from healthy individuals. In this study, we investigate the effects of beryllium on lipopolysaccharide - mediated cytokine release in blood mononuclear and dendritic cells from healthy individuals. We find that in vitro treatment of beryllium sulfate inhibits the secretion of lipopolysaccharide-mediated interleukin 10, while the release of interleukin 1β is enhanced. Additionally, not all lipopolysaccharide - mediated responses are altered, as interleukin 6 release in unaffected upon beryllium treatment. Beryllium sulfate treated cells show altered phosphotyrosine levels upon lipopolysaccharide stimulation. Significantly, beryllium inhibits the phosphorylation of signal transducer and activator of transducer 3, induced by lipopolysaccharide. Finally, inhibitors of phosphoinositide-3 kinase mimic the effects of beryllium in inhibition of interleukin 10 release, while they have no effect on interleukin 1β secretion. This study strongly suggests that prior exposures to beryllium could alter host immune responses to bacterial infections in healthy individuals, by altering intracellular signaling. PMID:19894180

  2. Serine 574 phosphorylation alters transcriptional programming of FOXO3 by selectively enhancing apoptotic gene expression.

    PubMed

    Li, Z; Zhao, J; Tikhanovich, I; Kuravi, S; Helzberg, J; Dorko, K; Roberts, B; Kumer, S; Weinman, S A

    2016-04-01

    Forkhead box O3 (FOXO3) is a multispecific transcription factor that is responsible for multiple and conflicting transcriptional programs such as cell survival and apoptosis. The protein is heavily post-translationally modified and there is considerable evidence that post-transcriptional modifications (PTMs) regulate protein stability and nuclear-cytosolic translocation. Much less is known about how FOXO3 PTMs determine the specificity of its transcriptional program. In this study we demonstrate that exposure of hepatocytes to ethanol or exposure of macrophages to lipopolysaccharide (LPS) induces the c-Jun N-terminal kinase (JNK)-dependent phosphorylation of FOXO3 at serine-574. Chromatin immunoprecipitation (ChIP), mRNA and protein measurements demonstrate that p-574-FOXO3 selectively binds to promoters of pro-apoptotic genes but not to other well-described FOXO3 targets. Both unphosphorylated and p-574-FOXO3 bound to the B-cell lymphoma 2 (Bcl-2) promoter, but the unphosphorylated form was a transcriptional activator, whereas p-574-FOXO3 was a transcriptional repressor. The combination of increased TRAIL (TNF-related apoptosis-inducing ligand) and decreased Bcl-2 was both necessary and sufficient to induce apoptosis. LPS treatment of a human monocyte cell line (THP-1) induced FOXO3 S-574 phosphorylation and apoptosis. LPS-induced apoptosis was prevented by knockdown of FOXO3. It was restored by overexpressing wild-type FOXO3 but not by overexpressing a nonphosphorylatable S-574A FOXO3. Expression of an S-574D phosphomimetic form of FOXO3 induced apoptosis even in the absence of LPS. A similar result was obtained with mouse peritoneal macrophages where LPS treatment increased TRAIL, decreased Bcl-2 and induced apoptosis in wild-type but not FOXO3(-/-) cells. This work thus demonstrates that S-574 phosphorylation generates a specifically apoptotic form of FOXO3 with decreased transcriptional activity for other well-described FOXO3 functions. PMID:26470730

  3. Related proteins are phosphorylated at tyrosine in response to mitogenic stimuli and at meiosis.

    PubMed Central

    Cooper, J A

    1989-01-01

    Forty-two-kilodalton proteins that contain phosphotyrosine in metaphase-arrested Xenopus laevis eggs are closely related to p42, a protein that is phosphorylated at tyrosine when somatic cells are exposed to mitogenic stimuli. Images PMID:2779558

  4. Identification of in vivo protein phosphorylation sites in human pathogen Schistosoma japonicum by a phosphoproteomic approach.

    PubMed

    Luo, Rong; Zhou, Chunjing; Lin, Jiaojiao; Yang, Dehao; Shi, Yaojun; Cheng, Guofeng

    2012-01-01

    Schistosome is the causative agent of human schistosomiasis and related animal disease. Reversible protein phosphorylation plays a key role in signaling processing that are vital for a cell and organism. However, it remains to be undercharacterized in schistosomes. In the present study, we characterized in vivo protein phosphorylation events in different developmental stages (schistosomula and adult worms) of Schistosoma japonicum by using microvolume immobilized metal-ion affinity chromatography (IMAC) pipette tips coupled to nanoLC-ESI-MS/MS. In total, 127 distinct phosphorylation sites were identified in 92 proteins in S. japonicum. A comparison of the phosphopeptides identified between the schistosomula and the adult worms revealed 30 phosphoproteins co-detected in both of the two worms. These proteins included several signal molecules and enzymes such as 14-3-3 protein, cysteine string protein, heat shock protein 90, epidermal growth factor receptor pathway substrate 8, proliferation-associated protein 2G4, peptidyl-prolyl isomerase G, phosphofructokinase and thymidylate kinase. Additionally, the phosphorylation sites were examined for phosphorylation specific motif and evolutionarily conservation. The study represents the first attempt to determine in vivo protein phosphorylation in S. japonicum by using a phosphoproteomic approach. The results by providing an inventory of phosphorylated proteins may facilitate to further understand the mechanisms involved in schistosome development and growth, and then may result in the development of novel vaccine candidates and drug targets for schistosomiasis control. PMID:22036931

  5. Differential effects of vasopressin and phenylephrine on protein kinase C-mediated protein phosphorylations in isolated hepatocytes

    SciTech Connect

    Cooper, R.H.; Johanson, R.A.; Wiliamson, J.R.

    1986-05-01

    Receptor-mediated breakdown of inositol lipids produces two intracellular signals, diacylglycerol, which activates protein kinase C, and inositol trisphosphate, which causes release of intracellular vesicular Ca/sup 2 +/. This study examined the effects of Ca/sup 2 +/-ionophores, vasopressin, phenylephrine, and phorbol ester (PMA) on hepatocyte protein phosphorylations. (/sup 32/P) Phosphoproteins from hepatocytes prelabeled with /sup 32/P were resolved by 2-dimensional SDS-PAGE and corresponding autoradiographs were quantitated by densitometric analysis. The phosphorylation of five proteins, a plasma membrane bound 16 kDa protein with pI 6.4, a cytosolic 16 kDa protein with pI 5.8, and proteins with Mr's of 36 kDa, 52 kDa, and 68 kDa, could be attributed to phosphorylation by protein kinase C since the phosphorylation was stimulated by PMA. When the vasopressin concentration was varied, low vasopressin stimulated the phosphorylation of only the membrane bound 16 kDa protein of the above set of proteins, while higher vasopressin concentrations were required to stimulate the phosphorylation of all five proteins. Phenylephrine, even at supramaximal concentrations, stimulated the phosphorylation of only the membrane bound 16 kDa protein. These results suggest that phenylephrine is a less potent activator of protein kinase C than vasopressin by virtue of limited or localized diacylglycerol production.

  6. Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis.

    PubMed

    Kohansal-Nodehi, Mahdokht; Chua, John Je; Urlaub, Henning; Jahn, Reinhard; Czernik, Dominika

    2016-01-01

    Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity. PMID:27115346

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

    PubMed Central

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

    2006-01-01

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

  8. Auxin effects on in vitro and in vivo protein phosphorylation in pea. [Pisum sativum

    SciTech Connect

    Gallagher, S.R.; Ray, P.M.

    1987-04-01

    Terminal 8mm sections from the third internode of dark grown 7 day old Pisum sativum cv Alaska seedlings were separated into membrane and soluble fractions. SDS gradient PAGE identified approximately 50 in vivo phosphorylated proteins and proved superior to 2-D SDS PAGE in terms of resolution and repeatability. Addition of indoleacetic acid (IAA), fusicoccin, or 2,4 dichlorophenoxyacetic acid to membranes resulted in no detectable change in the number or phosphorylation level of the labeled proteins during in vitro phosphorylation in the presence of submicromolar concentrations of calcium. Similar results were obtained with soluble proteins. In the absence of calcium, the level of in vitro protein phosphorylation was much less, but not auxin effects could be identified. Furthermore, treatment of the sections with IAA in vivo followed by cell fractionation and in vitro phosphorylation failed to identify auxin responsive proteins. Lastly, when sections were labeled with /sup 32/P inorganic phosphate in the presence of 17 uM IAA, no auxin specific changes were found in the level of phosphorylation or in the number of phosphorylated proteins. Auxin effects on phosphorylation are thus slight or below their detection limit.

  9. Phosphorylation of ribosomal protein S6 in the aquatic fungus Blastocladiella emersonii.

    PubMed

    Bonato, M C; da Silva, A M; Maia, J C; Juliani, M H

    1984-11-01

    The changes in the degree of phosphorylation of ribosomal protein S6 during the life cycle of the aquatic fungus Blastocladiella emersonii were analyzed by two-dimensional gel electrophoresis. Three phosphorylated derivatives of S6 are present throughout the entire life cycle. However, under certain germination conditions, more highly phosphorylated derivatives of S6 appear. Nonetheless, the resumption of protein synthesis that occurs during germination is not dependent on those highly phosphorylated derivatives of S6. The pattern and sites of phosphorylation of S6 labelled in vivo with [32P]orthophosphate have been compared with those of 40S ribosomal subunit labelled in vitro by partially purified protein kinases. Three major phosphopeptides were found in S6 isolated from the zoospore, while six phosphopeptides were found after zoospore germination (in germling cells). The phosphopeptide patterns of S6 phosphorylated by the cAMP-dependent protein kinase and by casein kinases I and II were completely distinct. Only the cAMP-dependent protein kinase gives rise to a phosphopeptide found in 32P-labelled cells, indicating that one of sites phosphorylated in vivo is also phosphorylated in vitro by the cAMP-dependent protein kinase. PMID:6092077

  10. Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus

    SciTech Connect

    Yang, Feng; Camp, David G.; Gritsenko, Marina A.; Luo, Quanzhou; Kelly, Ryan T.; Clauss, Therese RW; Brinkley, William R.; Smith, Richard D.; Stenoien, David L.

    2007-11-16

    The chromosomal passenger complex (CPC) is a critical regulator of chromosome, cytoskeleton and membrane dynamics during mitosis. Here, we identified phosphopeptides and phosphoprotein complexes recognized by a phosphorylation specific antibody that labels the CPC using liquid chromatography coupled to mass spectrometry. A mitotic phosphorylation motif (PX{G/T/S}{L/M}[pS]P or WGL[pS]P) was identified in 11 proteins including Fzr/Cdh1 and RIC-8, two proteins with potential links to the CPC. Phosphoprotein complexes contained known CPC components INCENP, Aurora-B and TD-60, as well as SMAD2, 14-3-3 proteins, PP2A, and Cdk1, a likely kinase for this motif. Protein sequence analysis identified phosphorylation motifs in additional proteins including SMAD2, Plk3 and INCENP. Mitotic SMAD2 and Plk3 phosphorylation was confirmed using phosphorylation specific antibodies, and in the case of Plk3, phosphorylation correlates with its localization to the mitotic apparatus. A mutagenesis approach was used to show INCENP phosphorylation is required for midbody localization. These results provide evidence for a shared phosphorylation event that regulates localization of critical proteins during mitosis.

  11. Crosstalk between signaling pathways provided by single and multiple protein phosphorylation sites

    PubMed Central

    Nishi, Hafumi; Demir, Emek; Panchenko, Anna R.

    2014-01-01

    Cellular fate depends on the spatio-temporal separation and integration of signaling processes which can be provided by phosphorylation events. In this study we identify the crucial points in signaling crosstalk which can be triggered by discrete phosphorylation events on a single target protein. We integrated the data on individual human phosphosites with the evidence on their corresponding kinases, the functional consequences on phosphorylation on activity of the target protein and corresponding pathways. Our results show that there is a substantial fraction of phosphosites that can play critical roles in crosstalk between alternative or redundant pathways and regulatory outcome of phosphorylation can be linked to a type of phosphorylated residue. These regulatory phosphosites can serve as hubs in the signal flow and their functional roles are directly connected to their specific properties. Namely, phosphosites with similar regulatory functions are phosphorylated by the same kinases and participate in regulation of similar biochemical pathways. Such sites are more likely to cluster in sequence and space unlike sites with antagonistic outcomes of their phosphorylation on a target protein. In addition we found that in silico phosphorylation of sites with similar functional consequences have comparable outcomes on a target protein stability. An important role of phosphorylation sites in biological crosstalk is evident from the analysis of their evolutionary conservation. PMID:25451034

  12. Chronic restraint stress induces sperm acrosome reaction and changes in testicular tyrosine phosphorylated proteins in rats

    PubMed Central

    Arun, Supatcharee; Burawat, Jaturon; Sukhorum, Wannisa; Sampannang, Apichakan; Maneenin, Chanwit; Iamsaard, Sitthichai

    2016-01-01

    Background: Stress is a cause of male infertility. Although sex hormones and sperm quality have been shown to be low in stress, sperm physiology and testicular functional proteins, such as phosphotyrosine proteins, have not been documented. Objective: To investigate the acrosome status and alterations of testicular proteins involved in spermatogenesis and testosterone synthesis in chronic stress in rats. Materials and Methods: In this experimental study, male rats were divided into 2 groups (control and chronic stress (CS), n=7). CS rats were immobilized (4 hr/day) for 42 consecutive days. The blood glucose level (BGL), corticosterone, testosterone, acrosome status, and histopathology were examined. The expressions of testicular steroidogenic acute regulatory (StAR), cytochrome P450 side chain cleavage (CYP11A1), and phosphorylated proteins were analyzed. Results: Results showed that BGL (71.25±2.22 vs. 95.60±3.36 mg/dl), corticosterone level (24.33±4.23 vs. 36.9±2.01 ng/ml), acrosome reacted sperm (3.25±1.55 vs. 17.71±5.03%), and sperm head abnormality (3.29±0.71 vs. 6.21±1.18%) were significantly higher in CS group in comparison with control. In contrast, seminal vesicle (0.41±0.05 vs. 0.24±0.07 g/100g), testosterone level (3.37±0.79 vs. 0.61±0.29 ng/ml), and sperm concentration (115.33±7.70 vs. 79.13±3.65×106 cells/ml) of CS were significantly lower (p<0.05) than controls. Some atrophic seminiferous tubules and low sperm mass were apparent in CS rats. The expression of CYP11A1 except StAR protein was markedly decreased in CS rats. In contrast, a 55 kDa phosphorylated protein was higher in CS testes. Conclusion: CS decreased the expression of CYP11A, resulting in decreased testosterone, and increased acrosome-reacted sperm, assumed to be the result of an increase of 55 kDa phosphorylated protein. PMID:27525328

  13. Combinations of Physiologic Estrogens with Xenoestrogens Alter ERK Phosphorylation Profiles in Rat Pituitary Cells

    PubMed Central

    Jeng, Yow-Jiun; Watson, Cheryl S.

    2011-01-01

    Background Estrogens are potent nongenomic phospho-activators of extracellular-signal–regulated kinases (ERKs). A major concern about the toxicity of xenoestrogens (XEs) is potential alteration of responses to physiologic estrogens when XEs are present simultaneously. Objectives We examined estrogen-induced ERK activation, comparing the abilities of structurally related XEs (alkylphenols and bisphenol A) to alter ERK responses induced by physiologic concentrations (1 nM) of estradiol (E2), estrone (E1), and estriol (E3). Methods We quantified hormone/mimetic-induced ERK phosphorylations in the GH3/B6/F10 rat pituitary cell line using a plate immunoassay, comparing effects with those on cell proliferation and by estrogen receptor subtype-selective ligands. Results Alone, these structurally related XEs activate ERKs in an oscillating temporal pattern similar (but not identical) to that with physiologic estrogens. The potency of all estrogens was similar (active between femtomolar and nanomolar concentrations). XEs potently disrupted physiologic estrogen signaling at low, environmentally relevant concentrations. Generally, XEs potentiated (at the lowest, subpicomolar concentrations) and attenuated (at the highest, picomolar to 100 nM concentrations) the actions of the physiologic estrogens. Some XEs showed pronounced nonmonotonic responses/inhibitions. The phosphorylated ERK and proliferative responses to receptor-selective ligands were only partially correlated. Conclusions XEs are both imperfect potent estrogens and endocrine disruptors; the more efficacious an XE, the more it disrupts actions of physiologic estrogens. This ability to disrupt physiologic estrogen signaling suggests that XEs may disturb normal functioning at life stages where actions of particular estrogens are important (e.g., development, reproductive cycling, pregnancy, menopause). PMID:20870566

  14. Phosphorylation of five aminoacyl-tRNA synthetases in reticulocytes and identification of the protein kinases phosphorylating threonyl-tRNA synthetase from rat liver

    SciTech Connect

    Pendergast, A.M.; Traugh, J.A.

    1986-05-01

    Five aminoacyl-tRNA synthetases in the high molecular weight complex were phosphorylated in rabbit reticulocytes following labeling with /sup 32/P. The five synthetases phosphorylated were the glutamyl-, glutaminyl-, lysyl-, aspartyl- and methionyl-tRNA synthetases. In addition, a 37,000 dalton protein, associated with the synthetase complex and tentatively identified as casein kinase I, was also phosphorylated in intact cells. Phosphoamino acid analysis of the proteins indicated all of the phosphate was on seryl residues. Incubation of reticulocytes with /sup 32/P in the presence of 8-bromo-cAMP and o, the 3-isobutyl-1-methylxanthine resulted in a six-fold increase in phosphorylation of the glutaminyl-tRNA synthetase, a two-fold increase in phosphorylation of the aspartyl-tRNA synthetase, and a 50 to 60% decrease in phosphorylation of the glutamyl-, methionyl- and lysyl-tRNA synthetases and the M/sub r/ 37,000 protein. When the site(s) on the glutaminyl-tRNA synthetase phosphorylated in response to 8-bromo-cAMP was analyzed by two-dimensional tryptic phosphopeptide mapping, a single phosphopeptide was observed which was identical to that obtained in vitro upon phosphorylation with the cAMP-dependent protein kinase. Also, the authors identify here, the protein kinases phosphorylating threonyl-tRNA synthetase from rat liver. They are protease activated kinase I, the cAMP-dependent protein kinase and protein kinase C.

  15. Analysis of phosphorylation-dependent protein-protein interactions of histone h3.

    PubMed

    Klingberg, Rebecca; Jost, Jan Oliver; Schümann, Michael; Gelato, Kathy Ann; Fischle, Wolfgang; Krause, Eberhard; Schwarzer, Dirk

    2015-01-16

    Multiple posttranslational modifications (PTMs) of histone proteins including site-specific phosphorylation of serine and threonine residues govern the accessibility of chromatin. According to the histone code theory, PTMs recruit regulatory proteins or block their access to chromatin. Here, we report a general strategy for simultaneous analysis of both of these effects based on a SILAC MS scheme. We applied this approach for studying the biochemical role of phosphorylated S10 of histone H3. Differential pull-down experiments with H3-tails synthesized from l- and d-amino acids uncovered that histone acetyltransferase 1 (HAT1) and retinoblastoma-binding protein 7 (RBBP7) are part of the protein network, which interacts with the unmodified H3-tail. An additional H3-derived bait containing the nonhydrolyzable phospho-serine mimic phosphonomethylen-alanine (Pma) at S10 recruited several isoforms of the 14-3-3 family and blocked the recruitment of HAT1 and RBBP7 to the unmodified H3-tail. Our observations provide new insights into the many functions of H3S10 phosphorylation. In addition, the outlined methodology is generally applicable for studying specific binding partners of unmodified histone tails. PMID:25330109

  16. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    PubMed Central

    Shi, Lei; Pigeonneau, Nathalie; Ravikumar, Vaishnavi; Dobrinic, Paula; Macek, Boris; Franjevic, Damjan; Noirot-Gros, Marie-Francoise; Mijakovic, Ivan

    2014-01-01

    Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD, and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells. PMID:25278935

  17. Phosphorylation of ribosomal proteins induced by auxins in maize embryonic tissues. [Zea mays

    SciTech Connect

    Perez, L.; Aguilar, R.; Mendez, A.P.; de Jimenez, E.S.

    1990-11-01

    The effect of auxin on ribosomal protein phosphorylation of germinating maize (Zea mays) tissues was investigated. Two-dimensional gel electrophoresis and autoradiography of ({sup 32}P) ribosomal protein patterns for natural and synthetic auxin-treated tissues were performed. Both the rate of {sup 32}P incorporation and the electrophoretic patterns were dependent on {sup 32}P pulse length, suggesting that active protein phosphorylation-dephosphorylation occurred in small and large subunit proteins, in control as well as in auxin-treated tissues. The effect of ribosomal protein phosphorylation on in vitro translation was tested. Measurements of poly(U) translation rates as a function of ribosome concentration provided apparent K{sub m} values significantly different for auxin-treated and nontreated tissues. These findings suggest that auxin might exert some kind of translational control by regulating the phosphorylated status of ribosomal proteins.

  18. Effects of platelet inhibitors on propyl gallate-induced platelet aggregation, protein tyrosine phosphorylation, and platelet factor 3 activation.

    PubMed

    Xiao, Hongyan; Kovics, Richard; Jackson, Van; Remick, Daniel G

    2004-04-01

    Propyl gallate (PG) is a platelet agonist characterized by inducing platelet aggregation, protein tyrosine phosphorylation, and platelet factor 3 activity. The mechanisms of platelet activation following PG stimulation were examined by pre-incubating platelets with well-defined platelet inhibitors using platelet aggregation, protein tyrosine phosphorylation, activated plasma clotting time, and annexin V binding by flow cytometry. PG-induced platelet aggregation and tyrosine phosphorylation of multiple proteins were substantially abolished by aspirin, apyrase, and abciximab (c7E3), suggesting that PG is associated with activation of platelet cyclooxygenase 1, adenosine phosphate receptors, and glycoprotein IIb/IIIa, respectively. The phosphorylation of the cytoskeletal enzyme pp60(c-src) increased following PG stimulation, but was blunted by pre-incubation of platelets with aspirin, apyrase, and c7E3, suggesting that tyrosine kinase is important for the signal transduction of platelet aggregation. Propyl gallate also activates platelet factor 3 by decreasing the platelet coagulation time and increasing platelet annexin V binding. Platelet incubation with aspirin, apyrase, and c7E3 did not alter PG-induced platelet coagulation and annexin V binding. The results suggest that platelet factor 3 activation and membrane phosphotidylserine expression were not involved with activation of platelet cyclooxygenase, adenosine phosphate receptors, and glycoprotein IIb/IIIa. PG is unique in its ability to stimulate platelet aggregation and coagulation simultaneously, and platelet inhibitors in this study affect only platelet aggregation but not platelet coagulation. PMID:15060414

  19. Eukaryotic-Type Ser/Thr Protein Kinase Mediated Phosphorylation of Mycobacterial Phosphodiesterase Affects its Localization to the Cell Wall

    PubMed Central

    Malhotra, Neha; Chakraborti, Pradip K.

    2016-01-01

    Phosphodiesterase enzymes, involved in cAMP hydrolysis reaction, are present throughout phylogeny and their phosphorylation mediated regulation remains elusive in prokaryotes. In this context, we focused on this enzyme from Mycobacterium tuberculosis. The gene encoded by Rv0805 was PCR amplified and expressed as a histidine-tagged protein (mPDE) utilizing Escherichia coli based expression system. In kinase assays, upon incubation with mycobacterial Clade I eukaryotic-type Ser/Thr kinases (PknA, PknB, and PknL), Ni-NTA purified mPDE protein exhibited transphosphorylation ability albeit with varying degree. When mPDE was co-expressed one at a time with these kinases in E. coli, it was also recognized by an anti-phosphothreonine antibody, which further indicates its phosphorylating ability. Mass spectrometric analysis identified Thr-309 of mPDE as a phosphosite. In concordance with this observation, anti-phosphothreonine antibody marginally recognized mPDE-T309A mutant protein; however, such alteration did not affect the enzymatic activity. Interestingly, mPDE expressed in Mycobacterium smegmatis yielded a phosphorylated protein that preferentially localized to cell wall. In contrast, mPDE-T309A, the phosphoablative variant of mPDE, did not show such behavior. On the other hand, phosphomimics of mPDE (T309D or T309E), exhibited similar cell wall anchorage as was observed with the wild-type. Thus, our results provide credence to the fact that eukaryotic-type Ser/Thr kinase mediated phosphorylation of mPDE renders negative charge to the protein, promoting its localization on cell wall. Furthermore, multiple sequence alignment revealed that Thr-309 is conserved among mPDE orthologs of M. tuberculosis complex, which presumably emphasizes evolutionary significance of phosphorylation at this residue. PMID:26904001

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  2. Lead induced changes in phosphorylation of PSII proteins in low light grown pea plants.

    PubMed

    Wioleta, Wasilewska; Anna, Drożak; Ilona, Bacławska; Kamila, Kąkol; Elżbieta, Romanowska

    2015-02-01

    Light-intensity and redox-state induced thylakoid proteins phosphorylation involved in structural changes and in regulation of protein turnover. The presence of heavy metal ions triggers a wide range of cellular responses including changes in plant growth and photosynthesis. Plants have evolved a number of mechanisms to protect photosynthetic apparatus. We have characterized the effect of lead on PSII protein phosphorylation in pea (Pisum sativum L.) plants grown in low light conditions. Pb ions affected only slightly photochemical efficiency of PSII and had no effect on organization of thylakoid complexes. Lead activated strongly phosphorylation of PSII core D1 protein and dephosphorylation of this protein did not proceed in far red light. D1 protein was also not degraded in this conditions. However, phosphorylation of LHCII proteins was not affected by lead. These results indicate that Pb(2+) stimulate the phosphorylation of PSII core proteins and by disturbing the disassembly of supercomplexes play a role in PSII repair mechanism. LHCII phosphorylation could control the distribution of energy between the photosystems in low light conditions. This demonstrates that plants may respond to heavy metals by induction different pathways responsible for protein protection under stress conditions. PMID:25491575

  3. Responses of cyclic phosphorylation of MAPK-like proteins in intertidal macroalgae after environmental stress.

    PubMed

    Parages, María L; Capasso, Juan M; Niell, F Xavier; Jiménez, Carlos

    2014-02-15

    The presence and activation of MAPK-like proteins in intertidal macroalgae is described in the current study. Two MAPK-like proteins of 40 and 42 kDa in size similar to p38 and JNK, of mammalian cells have been identified in six representative species of intertidal macroalgae from the Strait of Gibraltar (Southern Spain), namely in the chlorophytes Ulva rigida and Chaetomorpha aerea, the rhodophytes Corallina elongata and Jania rubens, and the phaeophytes Dictyota dichotoma and Dilophus spiralis. Phosphorylation of MAPK-like proteins was studied during semi-tidal cycles. Analysis of p38-like and JNK-like MAPKs in macroalgae protein extracts was carried out by using specific antibodies against the phosphorylated forms of both MAPKs. Protein blot analysis of samples collected from 2009 to 2011 in natural growing sites on days when either low or high tide occurred at midday, indicated that MAPK-like proteins in all species were highly phosphorylated in response to desiccation imposed by low tide or high irradiance. Phosphorylation of p38-like MAPK always preceded that of JNK-like MAPK. In addition, phosphorylation of MAPKs was fastest in rhodophytes, followed by chlorophytes and then finally phaeophytes. In the first group, phosphorylation was mostly dependent on desiccation, whereas both high irradiance and desiccation were responsible for p38-like and JNK-like phosphorylation in chlorophytes. In phaeophytes, high irradiance was mostly responsible for MAPK-like activation. PMID:24120533

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

    PubMed Central

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

    2012-01-01

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

  5. Glutamate-induced protein phosphorylation in cerebellar granule cells: role of protein kinase C.

    PubMed

    Eboli, M L; Mercanti, D; Ciotti, M T; Aquino, A; Castellani, L

    1994-10-01

    Protein phosphorylation in response to toxic doses of glutamate has been investigated in cerebellar granule cells. 32P-labelled cells have been stimulated with 100 microM glutamate for up to 20 min and analysed by one and two dimensional gel electrophoresis. A progressive incorporation of label is observed in two molecular species of about 80 and 43 kDa (PP80 and PP43) and acidic isoelectric point. Glutamate-stimulated phosphorylation is greatly reduced by antagonists of NMDA and non-NMDA glutamate receptors. The effect of glutamate is mimicked by phorbol esters and is markedly reduced by inhibitors of protein kinase C (PKC) such as staurosporine and calphostin C. PP80 has been identified by Western blot analysis as the PKC substrate MARCKS (myristoylated alanine-rich C kinase substrate), while antibody to GAP-43 (growth associated protein-43), the nervous tissue-specific substrate of PKC, failed to recognize PP43. Our results suggest that PKC is responsible for the early phosphorylative events induced by toxic doses of glutamate in cerebellar granule cells. PMID:7891841

  6. Secreted beta-amyloid precursor protein stimulates mitogen-activated protein kinase and enhances tau phosphorylation.

    PubMed Central

    Greenberg, S M; Koo, E H; Selkoe, D J; Qiu, W Q; Kosik, K S

    1994-01-01

    Biological effects related to cell growth, as well as a role in the pathogenesis of Alzheimer disease, have been ascribed to the beta-amyloid precursor protein (beta-APP). Little is known, however, about the intracellular cascades that mediate these effects. We report that the secreted form of beta-APP potently stimulates mitogen-activated protein kinases (MAPKs). Brief exposure of PC-12 pheochromocytoma cells to beta-APP secreted by transfected Chinese hamster ovary cells stimulated the 43-kDa form of MAPK by > 10-fold. Induction of a dominant inhibitory form of ras in a PC12-derived cell line prevented the stimulation of MAPK by secreted beta-APP, demonstrating the dependence of the effect upon p21ras. Because the microtubule-associated protein tau is hyperphosphorylated in Alzheimer disease, we sought and found a 2-fold enhancement in tau phosphorylation associated with the beta-APP-induced MAPK stimulation. In the ras dominant inhibitory cell line, beta-APP failed to enhance phosphorylation of tau. The data presented here provide a link between secreted beta-APP and the phosphorylation state of tau. Images PMID:8041753

  7. The Roles of Phosphorylation and SHAGGY-Like Protein Kinases in Geminivirus C4 Protein Induced Hyperplasia

    PubMed Central

    Mills-Lujan, Katherine; Andrews, David L.; Chou, Chau-wen; Deom, C. Michael

    2015-01-01

    Even though plant cells are highly plastic, plants only develop hyperplasia under very specific abiotic and biotic stresses, such as when exposed to pathogens like Beet curly top virus (BCTV). The C4 protein of BCTV is sufficient to induce hyperplasia and alter Arabidopsis development. It was previously shown that C4 interacts with two Arabidopsis Shaggy-like protein kinases, AtSK21 and 23, which are negative regulators of brassinosteroid (BR) hormone signaling. Here we show that the C4 protein interacts with five additional AtSK family members. Bikinin, a competitive inhibitor of the seven AtSK family members that interact with C4, induced hyperplasia similar to that induced by the C4 protein. The Ser49 residue of C4 was found to be critical for C4 function, since: 1) mutagenesis of Ser49 to Ala abolished the C4-induced phenotype, abolished C4/AtSK interactions, and resulted in a mutant protein that failed to induce changes in the BR signaling pathway; 2) Ser49 is phosphorylated in planta; and 3) plant-encoded AtSKs must be catalytically active to interact with C4. A C4 N-myristoylation site mutant that does not localize to the plasma membrane and does not induce a phenotype, retained the ability to bind AtSKs. Taken together, these results suggest that plasma membrane associated C4 interacts with and co-opts multiple AtSKs to promote its own phosphorylation and activation to subsequently compromise cell cycle control. PMID:25815729

  8. The roles of phosphorylation and SHAGGY-like protein kinases in geminivirus C4 protein induced hyperplasia.

    PubMed

    Mills-Lujan, Katherine; Andrews, David L; Chou, Chau-Wen; Deom, C Michael

    2015-01-01

    Even though plant cells are highly plastic, plants only develop hyperplasia under very specific abiotic and biotic stresses, such as when exposed to pathogens like Beet curly top virus (BCTV). The C4 protein of BCTV is sufficient to induce hyperplasia and alter Arabidopsis development. It was previously shown that C4 interacts with two Arabidopsis Shaggy-like protein kinases, AtSK21 and 23, which are negative regulators of brassinosteroid (BR) hormone signaling. Here we show that the C4 protein interacts with five additional AtSK family members. Bikinin, a competitive inhibitor of the seven AtSK family members that interact with C4, induced hyperplasia similar to that induced by the C4 protein. The Ser49 residue of C4 was found to be critical for C4 function, since: 1) mutagenesis of Ser49 to Ala abolished the C4-induced phenotype, abolished C4/AtSK interactions, and resulted in a mutant protein that failed to induce changes in the BR signaling pathway; 2) Ser49 is phosphorylated in planta; and 3) plant-encoded AtSKs must be catalytically active to interact with C4. A C4 N-myristoylation site mutant that does not localize to the plasma membrane and does not induce a phenotype, retained the ability to bind AtSKs. Taken together, these results suggest that plasma membrane associated C4 interacts with and co-opts multiple AtSKs to promote its own phosphorylation and activation to subsequently compromise cell cycle control. PMID:25815729

  9. Protein kinase A alterations in adrenocortical tumors.

    PubMed

    Espiard, S; Ragazzon, B; Bertherat, J

    2014-11-01

    Stimulation of the cAMP pathway by adrenocorticotropin (ACTH) is essential for adrenal cortex maintenance, glucocorticoid and adrenal androgens synthesis, and secretion. Various molecular and cellular alterations of the cAMP pathway have been observed in endocrine tumors. Protein kinase A (PKA) is a central key component of the cAMP pathway. Molecular alterations of PKA subunits have been observed in adrenocortical tumors. PKA molecular defects can be germline in hereditary disorders or somatic in sporadic tumors. Heterozygous germline inactivating mutations of the PKA regulatory subunit RIα gene (PRKAR1A) can be observed in patients with ACTH-independent Cushing's syndrome (CS) due to primary pigmented nodular adrenocortical disease (PPNAD). PRKAR1A is considered as a tumor suppressor gene. Interestingly, these mutations can also be observed as somatic alterations in sporadic cortisol-secreting adrenocortical adenomas. Germline gene duplication of the catalytic subunits Cα (PRKACA) has been observed in patients with PPNAD. Furthermore, exome sequencing revealed recently activating somatic mutations of PRKACA in about 40% of cortisol-secreting adrenocortical adenomas. In vitro and in vivo functional studies help in the progress to understand the mechanisms of adrenocortical tumors development due to PKA regulatory subunits alterations. All these alterations are observed in benign oversecreting tumors and are mimicking in some way cAMP pathway constitutive activation. On the long term, unraveling these alterations will open new strategies of pharmacological treatment targeting the cAMP pathway in adrenal tumors and cortisol-secretion disorders. PMID:25105543

  10. Proteomic analysis of ERK1/2-mediated human sickle red blood cell membrane protein phosphorylation

    PubMed Central

    2013-01-01

    Background In sickle cell disease (SCD), the mitogen-activated protein kinase (MAPK) ERK1/2 is constitutively active and can be inducible by agonist-stimulation only in sickle but not in normal human red blood cells (RBCs). ERK1/2 is involved in activation of ICAM-4-mediated sickle RBC adhesion to the endothelium. However, other effects of the ERK1/2 activation in sickle RBCs leading to the complex SCD pathophysiology, such as alteration of RBC hemorheology are unknown. Results To further characterize global ERK1/2-induced changes in membrane protein phosphorylation within human RBCs, a label-free quantitative phosphoproteomic analysis was applied to sickle and normal RBC membrane ghosts pre-treated with U0126, a specific inhibitor of MEK1/2, the upstream kinase of ERK1/2, in the presence or absence of recombinant active ERK2. Across eight unique treatment groups, 375 phosphopeptides from 155 phosphoproteins were quantified with an average technical coefficient of variation in peak intensity of 19.8%. Sickle RBC treatment with U0126 decreased thirty-six phosphopeptides from twenty-one phosphoproteins involved in regulation of not only RBC shape, flexibility, cell morphology maintenance and adhesion, but also glucose and glutamate transport, cAMP production, degradation of misfolded proteins and receptor ubiquitination. Glycophorin A was the most affected protein in sickle RBCs by this ERK1/2 pathway, which contained 12 unique phosphorylated peptides, suggesting that in addition to its effect on sickle RBC adhesion, increased glycophorin A phosphorylation via the ERK1/2 pathway may also affect glycophorin A interactions with band 3, which could result in decreases in both anion transport by band 3 and band 3 trafficking. The abundance of twelve of the thirty-six phosphopeptides were subsequently increased in normal RBCs co-incubated with recombinant ERK2 and therefore represent specific MEK1/2 phospho-inhibitory targets mediated via ERK2. Conclusions These findings

  11. Altered Proteins in the Aging Brain

    PubMed Central

    Elobeid, Adila; Libard, Sylwia; Leino, Marina; Popova, Svetlana N.

    2016-01-01

    We assessed the prevalence of common altered brain proteins in 296 cognitively unimpaired subjects ranging from age 50 to 102 years. The incidence and the stage of hyperphosphorylated-τ (HPτ), β-amyloid, α-synuclein (αS), and transactive response DNA (TDP) binding protein 43 (TDP43)-immunoreactivity (-IR) increased with age. HPτ-IR was observed in 98% of the subjects; the locus coeruleus was solely affected in 46%, and 79% of the subjects were in Braak stages a to II. β-Amyloid was seen in 47% of subjects and the Thal phase correlated with the HPτ Braak stage and age. Intermediate Alzheimer disease-related pathology (ADRP) was seen in 12%; 52% of the subjects with HPτ-IR fulfilled criteria for definite primary age-related tauopathy (PART). The incidence of concomitant pathology (αS, TDP43) did not differ between those with PART and those with ADRP but the former were younger. TDP43-IR was observed in 36%; the most frequently affected region was the medulla; αS-IR was observed in 19% of subjects. In 41% of the subjects from 80 to 89 years at death, 3 altered proteins were seen in the brain. Thus, altered proteins are common in the brains of cognitively unimpaired aged subjects; this should be considered while developing diagnostic biomarkers, particularly for identifying subjects at early stages of neurodegenerative diseases. PMID:26979082

  12. Microgravity alters the expression of salivary proteins.

    PubMed

    Mednieks, Maija; Khatri, Aditi; Rubenstein, Renee; Burleson, Joseph A; Hand, Arthur R

    2014-06-01

    Spaceflight provides a unique opportunity to study how physiologic responses are influenced by the external environment. Microgravity has been shown to alter the function of a number of tissues and organ systems. Very little, however, is known about how microgravity affects the oral cavity. The rodent model is useful for study in that their salivary gland morphology and physiology is similar to that of humans. Useful also is the fact that saliva, a product of the salivary glands with a major role in maintaining oral health, can be easily collected in humans whereas the glands can be studied in experimental animals. Our working hypothesis is that expression of secretory proteins in saliva will respond to microgravity and will be indicative of the nature of physiologic reactions to travel in space. This study was designed to determine which components of the salivary proteome are altered in mice flown on the US space shuttle missions and to determine if a subset with predictive value can be identified using microscopy and biochemistry methods. The results showed that the expression of secretory proteins associated with beta-adrenergic hormone regulated responses and mediated via the cyclic AMP pathway was significantly altered, whereas that of a number of unrelated proteins was not. The findings are potentially applicable to designing a biochemical test system whereby specific salivary proteins can be biomarkers for stress associated with travel in space and eventually for monitoring responses to conditions on earth. PMID:24984624

  13. Altered Proteins in the Aging Brain.

    PubMed

    Elobeid, Adila; Libard, Sylwia; Leino, Marina; Popova, Svetlana N; Alafuzoff, Irina

    2016-04-01

    We assessed the prevalence of common altered brain proteins in 296 cognitively unimpaired subjects ranging from age 50 to 102 years. The incidence and the stage of hyperphosphorylated-τ (HPτ), β-amyloid, α-synuclein (αS), and transactive response DNA (TDP) binding protein 43 (TDP43)-immunoreactivity (-IR) increased with age. HPτ-IR was observed in 98% of the subjects; the locus coeruleus was solely affected in 46%, and 79% of the subjects were in Braak stages a to II. β-Amyloid was seen in 47% of subjects and the Thal phase correlated with the HPτ Braak stage and age. Intermediate Alzheimer disease-related pathology (ADRP) was seen in 12%; 52% of the subjects with HPτ-IR fulfilled criteria for definite primary age-related tauopathy (PART). The incidence of concomitant pathology (αS, TDP43) did not differ between those with PART and those with ADRP but the former were younger. TDP43-IR was observed in 36%; the most frequently affected region was the medulla; αS-IR was observed in 19% of subjects. In 41% of the subjects from 80 to 89 years at death, 3 altered proteins were seen in the brain. Thus, altered proteins are common in the brains of cognitively unimpaired aged subjects; this should be considered while developing diagnostic biomarkers, particularly for identifying subjects at early stages of neurodegenerative diseases. PMID:26979082

  14. Mitosis-specific phosphorylation of nucleolin by p34cdc2 protein kinase.

    PubMed Central

    Belenguer, P; Caizergues-Ferrer, M; Labbé, J C; Dorée, M; Amalric, F

    1990-01-01

    Nucleolin is a ubiquitous multifunctional protein involved in preribosome assembly and associated with both nucleolar chromatin in interphase and nucleolar organizer regions on metaphasic chromosomes in mitosis. Extensive nucleolin phosphorylation by a casein kinase (CKII) occurs on serine in growing cells. Here we report that while CKII phosphorylation is achieved in interphase, threonine phosphorylation occurs during mitosis. We provide evidence that this type of in vivo phosphorylation involves a mammalian homolog of the cell cycle control Cdc2 kinase. In vitro M-phase H1 kinase from starfish oocytes phosphorylated threonines in a TPXK motif present nine times in the amino-terminal part of the protein. The same sites which matched the p34cdc2 consensus phosphorylation sequence were used in vivo during mitosis. We propose that successive Cdc2 and CKII phosphorylation could modulate nucleolin function in controlling cell cycle-dependent nucleolar function and organization. Our results, along with previous studies, suggest that while serine phosphorylation is related to nucleolin function in the control of rDNA transcription, threonine phosphorylation is linked to mitotic reorganization of nucleolar chromatin. Images PMID:2192260

  15. Phosphorylation of Human Tristetraprolin in Response to Its Interaction with the Cbl Interacting Protein CIN85

    PubMed Central

    Kedar, Vishram P.; Darby, Martyn K.; Williams, Jason G.; Blackshear, Perry J.

    2010-01-01

    Background Tristetraprolin (TTP) is the prototype member of a family of CCCH tandem zinc finger proteins and is considered to be an anti-inflammatory protein in mammals. TTP plays a critical role in the decay of tumor necrosis factor alpha (TNF) mRNA, among others, by binding AU-rich RNA elements in the 3′-untranslated regions of this transcript and promoting its deadenylation and degradation. Methodology/Principal Findings We used yeast two-hybrid analysis to identify potential protein binding partners for human TTP (hTTP). Various regions of hTTP recovered 31 proteins that fell into 12 categories based on sequence similarities. Among these, the interactions between hTTP and CIN85, cytoplasmic poly (A) binding protein (PABP), nucleolin and heat shock protein 70 were confirmed by co-immunoprecipitation experiments. CIN85 and hTTP co-localized in the cytoplasm of cells as determined by confocal microscopy. CIN85 contains three SH3 domains that specifically bind a unique proline-arginine motif (PXXXPR) found in several CIN85 effectors. We found that the SH3 domains of CIN85 bound to a PXXXPR motif located near the C-terminus of hTTP. Co-expression of CIN85 with hTTP resulted in the increased phosphorylation of hTTP at serine residues in positions 66 and 93, possibly due in part to the demonstrated association of mitogen-activated protein kinase kinase kinase 4 (MEKK4) to both proteins. The presence of CIN85 did not appear to alter hTTP's binding to RNA probes or its stimulated breakdown of TNF mRNA. Conclusions/Significance These studies describe interactions between hTTP and nucleolin, cytoplasmic PABP, heat shock protein 70 and CIN85; these interactions were initially discovered by two-hybrid analysis, and confirmed by co-immunoprecipitation. We found that CIN85 binding to a C-terminal motif within hTTP led to the increased phosphorylation of hTTP, possibly through enhanced association with MEKK4. The functional consequences to each of the members of this

  16. Cellular progesterone receptor phosphorylation in response to ligands activating protein kinases

    SciTech Connect

    Rao, K.V.; Peralta, W.D.; Greene, G.L.; Fox, C.F.

    1987-08-14

    Progesterone receptors were immunoprecipitated with monoclonal antibodies KD68 from lysates of human breast carcinoma T47D cells labelled to steady state specific activity with /sup 32/Pi. The 120 kDa /sup 32/P-labelled progesterone receptor band was resolved by polyacrylamide gel electrophoresis and identified by autoradiography. Phosphoamino acid analysis revealed serine phosphorylation, but no threonine or tyrosine phosphorylation. Treatment of the /sup 32/Pi-labelled cells with EGF, TPA or dibutyryl cAMP had no significant quantitative effect on progesterone receptor phosphorylation, though the EGF receptor and the cAMP-dependent protein kinases have been reported to catalyze phosphorylation of purified avian progesterone receptor preparations in cell free systems. Progesterone receptor phosphorylation on serine residues was increased by 2-fold in cells treated with 10 nM progesterone; EGF had no effect on progesterone-mediated progesterone receptor phosphorylation.

  17. [Suppressive effect of protein kinase C inhibitors on tumor cell function via phosphorylation of p53 protein in mice].

    PubMed

    Nakamura, K; Shinozuka, K; Kunitomo, M

    2000-12-01

    We examined the role of protein kinase C (PKC) in the phosphorylation of a p53 protein. Exposure to a protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7), increased the phosphorylation of the wild type p53 protein, whereas exposure to a tumor promoter phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), decreased it in vivo after incubation with mouse epidermal JB6 cells for 3 h. Exposure to a cAMP dependent protein kinase (PKA) activator, forskolin, did not decrease the phosphorylation of p53 protein. In the transient transfection/luciferase reporter transactivation assay, H7 slightly increased the mouse double minute (MDM) 2 reporter transactivation activity of the p53 protein after treatment for 24 h, whereas TPA completely blocked it. Exposure to H7 and a specific PKC inhibitor, bisindolylmaleimide (bis), dose-dependently reduced the lung-colonizing potential of highly metastatic B16-F10 mouse melanoma cells in syngeneic mice. These results suggest that the phosphorylation of the wild type p53 protein is inversely related to PKC activation, and also suggest that the phosphorylation of the p53 protein is involved in the function of its transcription factor. The PKC inhibitor may exhibit a potent anti-metastatic effect through the phosphorylation of wild type p53 protein and the activation of its function. PMID:11193387

  18. Phosphorylated protein phosphatase 2A determines poor outcome in patients with metastatic colorectal cancer

    PubMed Central

    Cristóbal, I; Manso, R; Rincón, R; Caramés, C; Zazo, S; del Pulgar, T G; Cebrián, A; Madoz-Gúrpide, J; Rojo, F; García-Foncillas, J

    2014-01-01

    Background: Protein phosphatase 2A (PP2A) is a tumour suppressor frequently inactivated in human cancer and its tyrosine-307 phosphorylation has been reported as a molecular inhibitory mechanism. Methods: Expression of phosphorylated PP2A (p-PP2A) was evaluated in 250 metastatic colorectal cancer (CRC) patients. Chi-square, Kaplan–Meier and Cox analyses were used to determine correlations with clinical and molecular parameters and impact on clinical outcomes. Results: High p-PP2A levels were found in 17.2% cases and were associated with ECOG performance status (P=0.001) and presence of synchronous metastasis at diagnosis (P=0.035). This subgroup showed substantially worse overall survival (OS) (median OS, 6.0 vs 26.2 months, P<0.001) and progression-free survival (PFS) (median PFS, 3.8 vs 13.3 months, P<0.001). The prognostic impact of p-PP2A was particularly evident in patients aged <70 years (P<0.001). Multivariate analysis revealed that p-PP2A retained its prognostic impact for OS (hazard ratio 2.7; 95% confidence interval, 1.8–4.1; P<0.001) and PFS (hazard ratio 3.0; 95% confidence interval, 1.8–5.0; P<0.001). Conclusions: Phosphorylated PP2A is an alteration that determines poor outcome in metastatic CRC and represents a novel potential therapeutic target in this disease, thus enabling to define a subgroup of patients who could benefit from future treatments based on PP2A activators. PMID:25003662

  19. In vivo phosphorylation of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP): CNP in brain myelin is phosphorylated by forskolin- and phorbol ester-sensitive protein kinases.

    PubMed

    Agrawal, H C; Sprinkle, T J; Agrawal, D

    1994-06-01

    2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) was phosphorylated in vivo, in brain slices and in a cell free system. Phosphoamino acid analysis of immunoprecipitated CNP labeled in vivo and in brain slices revealed phosphorylation of phosphoserine (94%) and phosphothreonine (5%) residues. Phosphorylation of CNP increased by 3-fold after brain slices were incubated with forskolin. Similarly, incubation of isolated myelin with [gamma-32]ATP with cAMP (5 microM) and cAMP (5 microM)+catalytic unit of cAMP dependent protein kinase dramatically increased CNP2 phosphorylation by 4- and 6-fold, respectively. It is feasible that CNP2 was predominantly phosphorylated on serine and/or threonine residues of the amino terminal peptide of CNP2, and this phosphorylation was catalyzed by protein kinase A. Phosphorylation of CNP1 and CNP2 increased 2-fold by incubating brain slices with phorbol ester. Forskolin and phorbol ester increased the phosphorylation of single, but distinct, CNP peptides. We present the first biochemical evidence that CNP2, on a protein mass basis, is far more heavily phosphorylated than CNP1, suggesting there are more phosphorylation sites on CNP2 than CNP1 and that at least one site is located on the 20-amino acid terminus of CNP2 and that it is likely a PKA site. PMID:8065530

  20. CK2 Phosphorylation Inactivates DNA Binding by the Papillomavirus E1 and E2 Proteins

    PubMed Central

    Schuck, Stephen; Ruse, Cristian

    2013-01-01

    Papillomaviruses have complex life cycles that are understood only superficially. Although it is well established that the viral E1 and E2 proteins play key roles in controlling viral transcription and DNA replication, how these factors are regulated is not well understood. Here, we demonstrate that phosphorylation by the protein kinase CK2 controls the biochemical activities of the bovine papillomavirus E1 and E2 proteins by modifying their DNA binding activity. Phosphorylation at multiple sites in the N-terminal domain in E1 results in the loss of sequence-specific DNA binding activity, a feature that is also conserved in human papillomavirus (HPV) E1 proteins. The bovine papillomavirus (BPV) E2 protein, when phosphorylated by CK2 on two specific sites in the hinge, also loses its site-specific DNA binding activity. Mutation of these sites in E2 results in greatly increased levels of latent viral DNA replication, indicating that CK2 phosphorylation of E2 is a negative regulator of viral DNA replication during latent viral replication. In contrast, mutation of the N-terminal phosphorylation sites in E1 has no effect on latent viral DNA replication. We propose that the phosphorylation of the N terminus of E1 plays a role only in vegetative viral DNA replication, and consistent with such a role, caspase 3 cleavage of E1, which has been shown to be necessary for vegetative viral DNA replication, restores the DNA binding activity to phosphorylated E1. PMID:23637413

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

    PubMed Central

    Rankin, Michele L.; Sibley, David R.

    2010-01-01

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

  2. Computational Study of Pseudo-phosphorylation of the Microtubule associated Protein Tau

    NASA Astrophysics Data System (ADS)

    Prokopovich, Dmitriy; Larini, Luca

    This computational study focuses on the effect of pseudo-phosphorylation on the aggregation of the microtubule associated protein tau. In the axon of the neuron, tau regulates the assembly of microtubules in the cytoskeleton. This is important for both stabilization of and transport across the microtubules. One of the hallmarks of the Alzheimer's disease is that tau is hyper-phosphorylated and aggregates into neurofibrillary tangles that lay waste to the neurons. It is not known if hyper-phosphorylation directly causes the aggregation of tau into tangles. Experimentally, pseudo-phosphorylation mimics the effects of phosphorylation by mutating certain residues of the protein chain into charged residues. In this study, we will consider the fragment called PHF43 that belongs to the microtubule binding region and has been shown to readily aggregate.

  3. Identification and analysis of phosphorylation status of proteins in dormant terminal buds of poplar

    PubMed Central

    2011-01-01

    Background Although there has been considerable progress made towards understanding the molecular mechanisms of bud dormancy, the roles of protein phosphorylation in the process of dormancy regulation in woody plants remain unclear. Results We used mass spectrometry combined with TiO2 phosphopeptide-enrichment strategies to investigate the phosphoproteome of dormant terminal buds (DTBs) in poplar (Populus simonii × P. nigra). There were 161 unique phosphorylated sites in 161 phosphopeptides from 151 proteins; 141 proteins have orthologs in Arabidopsis, and 10 proteins are unique to poplar. Only 34 sites in proteins in poplar did not match well with the equivalent phosphorylation sites of their orthologs in Arabidopsis, indicating that regulatory mechanisms are well conserved between poplar and Arabidopsis. Further functional classifications showed that most of these phosphoproteins were involved in binding and catalytic activity. Extraction of the phosphorylation motif using Motif-X indicated that proline-directed kinases are a major kinase group involved in protein phosphorylation in dormant poplar tissues. Conclusions This study provides evidence about the significance of protein phosphorylation during dormancy, and will be useful for similar studies on other woody plants. PMID:22074553

  4. Cadmium inhibits mouse sperm motility through inducing tyrosine phosphorylation in a specific subset of proteins.

    PubMed

    Wang, Lirui; Li, Yuhua; Fu, Jieli; Zhen, Linqing; Zhao, Na; Yang, Qiangzhen; Li, Sisi; Li, Xinhong

    2016-08-01

    Cadmium (Cd) has been reported to impair male fertility, primarily by disrupting sperm motility, but the underlying molecular mechanism remains unclear. Here we investigated the effects of Cd on sperm motility, tyrosine phosphorylation, AMP-activated protein kinase (AMPK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, and ATP levels in vitro. Our results demonstrated that Cd inhibited sperm motility, GAPDH activity, AMPK activity and ATP production, and induced tyrosine phosphorylation of 55-57KDa proteins. Importantly, all the parameters affected by Cd were restored to normal levels when incubated with 10μM Cd in the presence of 30μM ethylene diamine tetraacetic acid (EDTA). Interestingly, changes of tyrosine phosphorylation levels of 55-57KDa proteins are completely contrary to that of other parameters. These results suggest that Cd-induced tyrosine phosphorylation of 55-57KDa proteins might act as an engine to block intracellular energy metabolism and thus decrease sperm motility. PMID:27233480

  5. Phosphorylation of Fe65 amyloid precursor protein-binding protein in response to neuronal differentiation.

    PubMed

    Koistinen, Niina A; Bacanu, Smaranda; Iverfeldt, Kerstin

    2016-02-01

    Fe65 is a brain enriched multi domain adaptor protein involved in diverse cellular functions. One of its binding partners is the amyloid-β (Aβ) precursor protein (APP), which after sequential proteolytic processing by secretases gives rise to the Alzheimer's Aβ peptide. Fe65 binds to the APP intracellular domain (AICD). Several studies have indicated that Fe65 binding promotes the amyloidogenic processing of APP. It has previously been shown that expression of APP increases concomitantly with a shift of its processing to the non-amyloidogenic pathway during neuronal differentiation. In this study we wanted to investigate the effects of neuronal differentiation on Fe65 expression. We observed that differentiation of SH-SY5Y human neuroblastoma cells induced by retinoic acid (RA), the phorbol ester PMA, or the γ-secretase inhibitor DAPT resulted in an electrophoretic mobility shift of Fe65. Similar effects were observed in rat PC6.3 cells treated with nerve growth factor. The electrophoretic mobility shift was shown to be due to phosphorylation. Previous studies have shown that Fe65 phosphorylation can prevent the APP-Fe65 interaction. We propose that phosphorylation is a way to modify the functions of Fe65 and to promote the non-amyloidogenic processing of APP during neuronal differentiation. PMID:26742640

  6. Altered sodium channel-protein associations in critical illness myopathy

    PubMed Central

    2012-01-01

    Background During the acute phase of critical illness myopathy (CIM) there is inexcitability of skeletal muscle. In a rat model of CIM, muscle inexcitability is due to inactivation of sodium channels. A major contributor to this sodium channel inactivation is a hyperpolarized shift in the voltage dependence of sodium channel inactivation. The goal of the current study was to find a biochemical correlate of the hyperpolarized shift in sodium channel inactivation. Methods The rat model of CIM was generated by cutting the sciatic nerve and subsequent injections of dexamethasone for 7 days. Skeletal muscle membranes were prepared from gastrocnemius muscles, and purification and biochemical analyses carried out. Immunoprecipitations were performed with a pan-sodium channel antibody, and the resulting complexes probed in Western blots with various antibodies. Results We carried out analyses of sodium channel glycosylation, phosphorylation, and association with other proteins. Although there was some loss of channel glycosylation in the disease, as assessed by size analysis of glycosylated and de-glycosylated protein in control and CIM samples, previous work by other investigators suggest that such loss would most likely shift channel inactivation gating in a depolarizing direction; thus such loss was viewed as compensatory rather than causative of the disease. A phosphorylation site at serine 487 was identified on the NaV 1.4 sodium channel α subunit, but there was no clear evidence of altered phosphorylation in the disease. Co-immunoprecipitation experiments carried out with a pan-sodium channel antibody confirmed that the sodium channel was associated with proteins of the dystrophin associated protein complex (DAPC). This complex differed between control and CIM samples. Syntrophin, dystrophin, and plectin associated strongly with sodium channels in both control and disease conditions, while β-dystroglycan and neuronal nitric oxide synthase (nNOS) associated

  7. Phosphorylation of Single Stranded RNA Virus Proteins and Potential for Novel Therapeutic Strategies

    PubMed Central

    Keck, Forrest; Ataey, Pouya; Amaya, Moushimi; Bailey, Charles; Narayanan, Aarthi

    2015-01-01

    Post translational modification of proteins is a critical requirement that regulates function. Among the diverse kinds of protein post translational modifications, phosphorylation plays essential roles in protein folding, protein:protein interactions, signal transduction, intracellular localization, transcription regulation, cell cycle progression, survival and apoptosis. Protein phosphorylation is also essential for many intracellular pathogens to establish a productive infection cycle. Preservation of protein phosphorylation moieties in pathogens in a manner that mirrors the host components underscores the co-evolutionary trajectory of pathogens and hosts, and sheds light on how successful pathogens have usurped, either in part or as a whole, the host enzymatic machinery. Phosphorylation of viral proteins for many acute RNA viruses including Flaviviruses and Alphaviruses has been demonstrated to be critical for protein functionality. This review focuses on phosphorylation modifications that have been documented to occur on viral proteins with emphasis on acutely infectious, single stranded RNA viruses. The review additionally explores the possibility of repurposing Food and Drug Administration (FDA) approved inhibitors as antivirals for the treatment of acute RNA viral infections. PMID:26473910

  8. Geminivirus C4 protein alters Arabidopsis development.

    PubMed

    Mills-Lujan, Katherine; Deom, Carl Michael

    2010-03-01

    The C4 protein of beet curly top virus [BCTV-B (US:Log:76)] induces hyperplasia in infected phloem tissue and tumorigenic growths in transgenic plants. The protein offers an excellent model for studying cell cycle control, cell differentiation, and plant development. To investigate the role of the C4 protein in plant development, transgenic Arabidopsis thaliana plants were generated in which the C4 transgene was expressed under the control of an inducible promoter. A detailed analysis of the developmental changes that occur in cotyledons and hypocotyls of seedlings expressing the C4 transgene showed extensive cell division in all tissues types examined, radically altered tissue layer organization, and the absence of a clearly defined vascular system. Induced seedlings failed to develop true leaves, lateral roots, and shoot and root apical meristems, as well as vascular tissue. Specialized epidermis structures, such as stomata and root hairs, were either absent or developmentally impaired in seedlings that expressed C4 protein. Exogenous application of brassinosteroid and abscisic acid weakly rescued the C4-induced phenotype, while induced seedlings were hypersensitive to gibberellic acid and kinetin. These results indicate that ectopic expression of the BCTV C4 protein in A. thaliana drastically alters plant development, possibly through the disruption of multiple hormonal pathways. PMID:20091067

  9. Partial purification of a spinach thylakoid protein kinase that can phosphorylate light-harvesting chlorophyll a/b proteins

    SciTech Connect

    Clark, R.D.; Hind, G.; Bennett, J.

    1985-01-01

    Protein phosphorylation in plant tissues is particularly marked in chloroplasts, protein kinase activity being associated with the outer envelope, the soluble stromal fraction, and the thylakoid membrane. Furthermore, thylakoid-bound activity probably includes several distinct kinases, as suggested by studies of divalent cation specificity and thermal lability carried out with intact thylakoids and by subfractionation of solubilized membranes. Illumination of thylakoids, particularly with red light, promotes the rapid and extensive phosphorylation of the light-harvesting chlorophyll a/b complex (LHCII) on a threonine residue near the amino terminus of the protein. This phosphorylation is thought to be involved in regulating the distribution of absorbed quanta between photosystems II and I and is modulated by the redox state of the thylakoid plastoquinone pool. Neither of the thylakoid kinases reported to date was capable of phosphorylating purified LHCII in vitro or of incorporating phosphate into threonyl residues of exogenous substrates, that some LHCII phosphorylation was catalyzed by a preliminary fraction led workers to suggest that at least one other kinase remained to be isolated. Here, the authors report the solubilization and partial purification of a protein kinase from spinach thylakoids that is capable of phosphorylating LHCII in vitro, and they show that the specific site of phosphorylation is very nearly the same as, if not identical with, the site phosphorylated in organello.

  10. Ca(2+)-calmodulin-dependent phosphorylation of islet secretory granule proteins

    SciTech Connect

    Watkins, D.T. )

    1991-08-01

    The effect of Ca2+ and calmodulin on phosphorylation of islet secretory granule proteins was studied. Secretory granules were incubated in a phosphorylation reaction mixture containing (32P)ATP and test reagents. The 32P-labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 32P content was visualized by autoradiography, and the relative intensities of specific bands were quantitated. When the reaction mixture contained EGTA and no added Ca2+, 32P was incorporated into two proteins with molecular weights of 45,000 and 13,000. When 10(-4) M Ca2+ was added without EGTA, two additional proteins (58,000 and 48,000 Mr) were phosphorylated, and the 13,000-Mr protein was absent. The addition of 2.4 microM calmodulin markedly enhanced the phosphorylation of the 58,000- and 48,000-Mr proteins and resulted in the phosphorylation of a major protein whose molecular weight (64,000 Mr) is identical to that of one of the calmodulin binding proteins located on the granule surface. Calmodulin had no effect on phosphorylation in the absence of Ca2+ but was effective in the presence of calcium between 10 nM and 50 microM. Trifluoperazine and calmidazolium, calmodulin antagonists, produced a dose-dependent inhibition of the calmodulin effect. 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester that activates protein kinase C, produced no increase in phosphorylation, and 1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride, an inhibitor of protein kinase C, had no effect. These results indicate that Ca(2+)-calmodulin-dependent protein kinases and endogenous substrates are present in islet secretory granules.

  11. Preventing Phosphorylation of Sterol Regulatory Element-Binding Protein 1a by MAP-Kinases Protects Mice from Fatty Liver and Visceral Obesity

    PubMed Central

    Haas, Jutta; Kremer, Lorena; Jacob, Sylvia; Hartwig, Sonja; Nitzgen, Ulrike; Muller–Wieland, Dirk

    2012-01-01

    The transcription factor sterol regulatory element binding protein (SREBP)-1a plays a pivotal role in lipid metabolism. Using the SREBP-1a expressing human hepatoma cell line HepG2 we have shown previously that human SREBP-1a is phosphorylated at serine 117 by ERK-mitogen-activated protein kinases (MAPK). Using a combination of cell biology and protein chemistry approach we show that SREBP-1a is also target of other MAPK-families, i.e. c-JUN N-terminal protein kinases (JNK) or p38 stress activated MAP kinases. Serine 117 is also the major phosphorylation site in SREBP-1a for JNK. In contrast to that the major phosphorylation sites of p38 MAPK family are serine 63 and threonine 426. Functional analyses reveal that phosphorylation of SREBP-1a does not alter protein/DNA interaction. The identified phosphorylation sites are specific for both kinase families also in cellular context. To provide direct evidence that phosphorylation of SREBP-1a is a regulatory principle of biological and clinical relevance, we generated transgenic mice expressing mature transcriptionally active N-terminal domain of human SREBP–1a variant lacking all identified phosphorylaton sites designed as alb-SREBP-1aΔP and wild type SREBP-1a designed as alb-SREBP-1a liver specific under control of the albumin promoter and a liver specific enhancer. In contrast to alb-SREBP–1a mice the phosphorylation–deficient mice develop no enlarged fatty livers under normocaloric conditions. Phenotypical examination reveales a massive accumulation of adipose tissue in alb-SREBP-1a but not in the phosphorylation deficient alb-SREBP-1aΔP mice. Moreover, preventing phosphorylation of SREBP-1a protects mice also from dyslipidemia. In conclusion, phosphorylation of SREBP-1a by ERK, JNK and p38 MAPK-families resembles a biological principle and plays a significant role, in vivo. PMID:22384276

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

    PubMed Central

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

    2016-01-01

    We previously reported that the levels of α-syn oligomers, which play pivotal pathogenic roles in age-related Parkinson's disease (PD) and dementia with Lewy bodies, increase heterogeneously in the aging brain. Here, we show that exogenous α-syn incubated with brain extracts from older cynomolgus monkeys and in Lewy body pathology (LBP)-susceptible brain regions (striatum and hippocampus) forms higher amounts of phosphorylated and oligomeric α-syn than that in extracts from younger monkeys and LBP-insusceptible brain regions (cerebellum and occipital cortex). The increased α-syn phosphorylation and oligomerization in the brain extracts from older monkeys and in LBP-susceptible brain regions were associated with higher levels of polo-like kinase 2 (PLK2), an enzyme promoting α-syn phosphorylation, and lower activity of protein phosphatase 2A (PP2A), an enzyme inhibiting α-syn phosphorylation, in these brain extracts. Further, the extent of the age- and brain-dependent increase in α-syn phosphorylation and oligomerization was reduced by inhibition of PLK2 and activation of PP2A. Inversely, phosphorylated α-syn oligomers reduced the activity of PP2A and showed potent cytotoxicity. In addition, the activity of GCase and the levels of ceramide, a product of GCase shown to activate PP2A, were lower in brain extracts from older monkeys and in LBP-susceptible brain regions. Our results suggest a role for altered intrinsic metabolic enzymes in age- and brain region-dependent α-syn oligomerization in aging brains. PMID:27032368

  13. Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants

    PubMed Central

    Bekešová, Slávka; Komis, George; Křenek, Pavel; Vyplelová, Petra; Ovečka, Miroslav; Luptovčiak, Ivan; Illés, Peter; Kuchařová, Anna; Šamaj, Jozef

    2015-01-01

    The aim of the present study is to rationalize acrylamide pendant Phos-Tag™ in-gel discrimination of phosphorylated and non-phosphorylated plant protein species with standard immunoblot analysis, and optimize sample preparation, efficient electrophoretic separation and transfer. We tested variants of the method including extraction buffers suitable for preservation of phosphorylated protein species in crude extracts from plants and we addressed the importance of the cation (Mn2+ or Zn2+) used in the gel recipe for efficient transfer to PVDF membranes for further immunoblot analysis. We demonstrate the monitoring of Medicago sativa stress-induced mitogen activated protein kinase (SIMK) in stress-treated wild type plants and transgenic SIMKK RNAi line. We further show the hyperosmotically-induced phosphorylation of the previously uncharacterized HvMPK4 of barley. The method is validated using inducible phosphorylation of barley and wheat α-tubulin and of Arabidopsis MPK6. Acrylamide pendant Phos-Tag™offers a flexible tool for studying protein phosphorylation in crops and Arabidopsis circumventing radioactive labeling and the use of phosphorylation specific antibodies. PMID:26029234

  14. Synaptic activation of ribosomal protein S6 phosphorylation occurs locally in activated dendritic domains.

    PubMed

    Pirbhoy, Patricia Salgado; Farris, Shannon; Steward, Oswald

    2016-06-01

    Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6) locally near active synapses. Using antibodies specific for phosphorylation at different sites (ser235/236 versus ser240/244), we show that strong synaptic activation led to dramatic increases in immunostaining throughout postsynaptic neurons with selectively higher staining for p-ser235/236 in the activated dendritic lamina. Following LTP induction, phosphorylation at ser235/236 was detectable by 5 min, peaked at 30 min, and was maintained for hours. Phosphorylation at both sites was completely blocked by local infusion of the NMDA receptor antagonist, APV. Despite robust induction of p-rpS6 following high frequency stimulation, assessment of protein synthesis by autoradiography revealed no detectable increases. Exploration of a novel environment led to increases in the number of p-rpS6-positive neurons throughout the forebrain in a pattern reminiscent of immediate early gene induction and many individual neurons that were p-rpS6-positive coexpressed Arc protein. Our results constrain hypotheses about the possible role of rpS6 phosphorylation in regulating postsynaptic protein synthesis during induction of synaptic plasticity. PMID:27194793

  15. Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

    PubMed Central

    Mijakovic, Ivan; Petranovic, Dina; Macek, Boris; Cepo, Tina; Mann, Matthias; Davies, Julian; Jensen, Peter R.; Vujaklija, Dusica

    2006-01-01

    Single-stranded DNA-binding proteins (SSBs) are required for repair, recombination and replication in all organisms. Eukaryotic SSBs are regulated by phosphorylation on serine and threonine residues. To our knowledge, phosphorylation of SSBs in bacteria has not been reported. A systematic search for phosphotyrosine-containing proteins in Streptomyces griseus by immunoaffinity chromatography identified bacterial SSBs as a novel target of bacterial tyrosine kinases. Since genes encoding protein-tyrosine kinases (PTKs) have not been recognized in streptomycetes, and SSBs from Streptomyces coelicolor (ScSSB) and Bacillus subtilis (BsSSB) share 38.7% identity, we used a B.subtilis protein-tyrosine kinase YwqD to phosphorylate two cognate SSBs (BsSSB and YwpH) in vitro. We demonstrate that in vivo phosphorylation of B.subtilis SSB occurs on tyrosine residue 82, and this reaction is affected antagonistically by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine phosphorylation of SSBs is a conserved process of post-translational modification in taxonomically distant bacteria. PMID:16549871

  16. Interactions between two fission yeast serine/arginine-rich proteins and their modulation by phosphorylation.

    PubMed Central

    Tang, Zhaohua; Käufer, Norbert F; Lin, Ren-Jang

    2002-01-01

    The unexpected low number of genes in the human genome has triggered increasing attention to alternative pre-mRNA splicing, and serine/arginine-rich (SR) proteins have been correlated with the complex alternative splicing that is a characteristic of metazoans. SR proteins interact with RNA and splicing protein factors, and they also undergo reversible phosphorylation, thereby regulating constitutive and alternative splicing in mammals and Drosophila. However, it is not clear whether the features of SR proteins and alternative splicing are present in simple and genetically tractable organisms, such as yeasts. In the present study, we show that the SR-like proteins Srp1 and Srp2, found in the fission yeast Schizosaccharomyces pombe, interact with each other and the interaction is modulated by protein phosphorylation. By using Srp1 as bait in a yeast two-hybrid analysis, we specifically isolated Srp2 from a random screen. This Srp interaction was confirmed by a glutathione-S-transferase pull-down assay. We also found that the Srp1-Srp2 complex was phosphorylated at a reduced efficiency by a fission yeast SR-specific kinase, Dis1-suppression kinase (Dsk1). Conversely, Dsk1-mediated phosphorylation inhibited the formation of the Srp complex. These findings offer the first example in fission yeast for interactions between SR-related proteins and the modulation of the interactions by specific protein phosphorylation, suggesting that a mammalian-like SR protein function may exist in fission yeast. PMID:12186627

  17. Serum Protein Profile Alterations in Hemodialysis Patients

    SciTech Connect

    Murphy, G A; Davies, R W; Choi, M W; Perkins, J; Turteltaub, K W; McCutchen-Maloney, S L; Langlois, R G; Curzi, M P; Trebes, J E; Fitch, J P; Dalmasso, E A; Colston, B W; Ying, Y; Chromy, B A

    2003-11-18

    Background: Serum protein profiling patterns can reflect the pathological state of a patient and therefore may be useful for clinical diagnostics. Here, we present results from a pilot study of proteomic expression patterns in hemodialysis patients designed to evaluate the range of serum proteomic alterations in this population. Methods: Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOFMS) was used to analyze serum obtained from patients on periodic hemodialysis treatment and healthy controls. Serum samples from patients and controls were first fractionated into six eluants on a strong anion exchange column, followed by application to four array chemistries representing cation exchange, anion exchange, metal affinity and hydrophobic surfaces. A total of 144 SELDI-TOF-MS spectra were obtained from each serum sample. Results: The overall profiles of the patient and control samples were consistent and reproducible. However, 30 well-defined protein differences were observed; 15 proteins were elevated and 15 were decreased in patients compared to controls. Serum from one patient exhibited novel protein peaks suggesting possible additional changes due to a secondary disease process. Conclusion: SELDI-TOF-MS demonstrated dramatic serum protein profile differences between patients and controls. Similarity in protein profiles among dialysis patients suggests that patient physiological responses to end-stage renal disease and/or dialysis therapy have a major effect on serum protein profiles.

  18. Remodeled respiration in ndufs4 with low phosphorylation efficiency suppresses Arabidopsis germination and growth and alters control of metabolism at night.

    PubMed

    Meyer, Etienne H; Tomaz, Tiago; Carroll, Adam J; Estavillo, Gonzalo; Delannoy, Etienne; Tanz, Sandra K; Small, Ian D; Pogson, Barry J; Millar, A Harvey

    2009-10-01

    Respiratory oxidative phosphorylation is a cornerstone of cellular metabolism in aerobic multicellular organisms. The efficiency of this process is generally assumed to be maximized, but the presence of dynamically regulated nonphosphorylating bypasses implies that plants can alter phosphorylation efficiency and can benefit from lowered energy generation during respiration under certain conditions. We characterized an Arabidopsis (Arabidopsis thaliana) mutant, ndufs4 (for NADH dehydrogenase [ubiquinone] fragment S subunit 4), lacking complex I of the respiratory chain, which has constitutively lowered phosphorylation efficiency. Through analysis of the changes to mitochondrial function as well as whole cell transcripts and metabolites, we provide insights into how cellular metabolism flexibly adapts to reduced phosphorylation efficiency and why this state may benefit the plant by providing moderate stress tolerance. We show that removal of the single protein subunit NDUFS4 prevents assembly of complex I and removes its function from mitochondria without pleiotropic effects on other respiratory components. However, the lack of complex I promotes broad changes in the nuclear transcriptome governing growth and photosynthetic function. We observed increases in organic acid and amino acid pools in the mutant, especially at night, concomitant with alteration of the adenylate content. While germination is delayed, this can be rescued by application of gibberellic acid, and root growth assays of seedlings show enhanced tolerance to cold, mild salt, and osmotic stress. We discuss these observations in the light of recent data on the knockout of nonphosphorylating respiratory bypass enzymes that show opposite changes in metabolites and stress sensitivity. Our data suggest that the absence of complex I alters the adenylate control of cellular metabolism. PMID:19675153

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

    PubMed

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

    1997-06-13

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

  20. Correlation between persistent forms of zeaxanthin-dependent energy dissipation and thylakoid protein phosphorylation.

    PubMed

    Ebbert, V; Demmig-Adams, B; Adams, W W; Mueh, K E; Staehelin, L A

    2001-01-01

    High light stress induced not only a sustained form of xanthophyll cycle-dependent energy dissipation but also sustained thylakoid protein phosphorylation. The effect of protein phosphatase inhibitors (fluoride and molybdate ions) on recovery from a 1-h exposure to a high PFD was examined in leaf discs of Parthenocissus quinquefolia (Virginia creeper). Inhibition of protein dephosphorylation induced zeaxanthin retention and sustained energy dissipation (NPQ) upon return to low PFD for recovery, but had no significant effects on pigment and Chl fluorescence characteristics under high light exposure. In addition, whole plants of Monstera deliciosa and spinach grown at low to moderate PFDs were transferred to high PFDs, and thylakoid protein phosphorylation pattern (assessed with anti-phosphothreonine antibody) as well as pigment and Chl fluorescence characteristics were examined over several days. A correlation was obtained between dark-sustained D1/D2 phosphorylation and dark-sustained zeaxanthin retention and maintenance of PS II in a state primed for energy dissipation in both species. The degree of these dark-sustained phenomena was more pronounced in M. deliciosa compared with spinach. Moreover, M. deliciosa but not spinach plants showed unusual phosphorylation patterns of Lhcb proteins with pronounced dark-sustained Lhcb phosphorylation even under low PFD growth conditions. Subsequent to the transfer to a high PFD, dark-sustained Lhcb protein phosphorylation was further enhanced. Thus, phosphorylation patterns of D1/D2 and Lhcb proteins differed from each other as well as among plant species. The results presented here suggest an association between dark-sustained D1/D2 phosphorylation and sustained retention of zeaxanthin and energy dissipation (NPQ) in light-stressed, and particularly 'photoinhibited', leaves. Functional implications of these observations are discussed. PMID:16228317

  1. Bioactives from Artemisia dracunculus L. Enhance Insulin Sensitivity via Modulation of Skeletal Muscle Protein Phosphorylation

    PubMed Central

    Kheterpal, Indu; Scherp, Peter; Kelley, Lauren; Wang, Zhong; Johnson, William; Ribnicky, David; Cefalu, William T.

    2014-01-01

    A botanical extract from Artemisia dracunculus L., termed PMI 5011, has been shown previously to improve insulin sensitivity by increasing cellular insulin signaling in in vitro and in vivo studies. These studies suggest that PMI 5011 effects changes in phosphorylation levels of proteins involved in insulin signaling. To explore effects of this promising botanical extract on the human skeletal muscle phosphoproteome, changes in site-specific protein phosphorylation levels in primary skeletal muscle cultures from obese, insulin resistant individuals were evaluated with and without insulin stimulation. Insulin resistance is a condition in which a normal or elevated insulin level results in an abnormal biologic response, e.g., glucose uptake. Using isobaric tagging for relative and absolute quantification (iTRAQ™) followed by phosphopeptide enrichment and liquid chromatography – tandem mass spectrometry, 125 unique phosphopeptides and 159 unique phosphorylation sites from 80 unique proteins were identified and quantified. Insulin stimulation of primary cultured muscle cells from insulin resistant individuals resulted in minimal increase in phosphorylation, demonstrating impaired insulin action in this condition. Treatment with PMI 5011 resulted in significant up regulation of 35 phosphopeptides that were mapped to proteins participating in the regulation of transcription, translation, actin cytoskeleton signaling, caveolae translocation and GLUT4 transport. These data further showed that PMI 5011 increased phosphorylation levels of specific amino acids in proteins in the insulin resistant state that are normally phosphorylated by insulin (thus, increasing cellular insulin signaling) and PMI 5011 also increased the abundance of phosphorylation sites of proteins regulating anti-apoptotic effects. Thus, the phosphoproteomics analysis demonstrated conclusively that PMI 5011 effects changes in phosphorylation levels of proteins and identified novel pathways by which

  2. Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition

    PubMed Central

    Chang, Ching Ching; Few, Ling Ling; Konrad, Manfred; See Too, Wei Cun

    2016-01-01

    Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the ckβ gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme’s activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKβ by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis. PMID:27149373

  3. Protein kinase A alterations in endocrine tumors.

    PubMed

    Yu, B; Ragazzon, B; Rizk-Rabin, M; Bertherat, J

    2012-09-01

    Various molecular and cellular alterations of the cyclic adenosine monophosphate (cAMP) pathway have been observed in endocrine tumors. Since protein kinase A (PKA) is a central key component of the cAMP pathway, studies of the alterations of PKA subunits in endocrine tumors reveal new aspects of the mechanisms of cAMP pathway alterations in human diseases. So far, most alterations have been observed for the regulatory subunits, mainly PRKAR1A and to a lower extent, PRKAR2B. One of the best examples of such alteration today is the multiple neoplasia syndrome Carney complex (CNC). The most common endocrine gland manifestations of CNC are pituitary GH-secreting adenomas, thyroid tumors, testicular tumors, and ACTH-independent Cushing's syndrome due to primary pigmented nodular adrenocortical disease (PPNAD). Heterozygous germline inactivating mutations of the PKA regulatory subunit RIα gene (PRKAR1A) are observed in about two-third of CNC patients, and also in patients with isolated PPNAD. PRKAR1A is considered as a tumor suppressor gene. Interestingly, these mutations can also be observed as somatic alterations in sporadic endocrine tumors. More than 120 different PRKAR1A mutations have been found today. Most of them lead to an unstable mutant mRNA, which will be degraded by nonsense mediated mRNA decay. In vitro and in vivo functional studies are in progress to understand the mechanisms of endocrine tumor development due to PKA regulatory subunits inactivation. PRKAR1A mutations stimulate in most models PKA activity, mimicking in some way cAMP pathway constitutive activation. Cross-talks with other signaling pathways summarized in this review have been described and might participate in endocrine tumorigenesis. PMID:22752956

  4. Effects of anti-Alzheimer drugs on phosphorylation and assembly of microtubules from brain microtubular proteins.

    PubMed

    Shevtsov, P N; Shevtsova, E F; Burbaeva, G Sh; Bachurin, S O

    2014-04-01

    We studied the effects of anti-Alzheimer drugs (tacrine, amiridine, and memantine) on phosphorylation of tubulin and microtubule-associated proteins isolated from rat brain, evaluated the capacity of these proteins to polymerize into microtubules after addition of study pharmacological agents, and analyzed the structure of generated microtubules. It was shown that test substances impair assembly of microtubules to a different extent. Dose-dependent effects of these agents on phosphorylation of tubulin and microtubule-associated proteins were observed. Triazolam (not approved for clinical use as anti-Alzheimer drug) in the same concentrations was used as the reference substance in the same tests. It was observed that this substance even in minimal concentration induced the most pronounced changes in microtubule structure. A direct correlation between the capacity of the test substances to modulate tubulin phosphorylation and to impair microtubule structure was found: the more the substance inhibited tubulin phosphorylation, the more it disordered microtubule structure. PMID:24824692

  5. Phosphopeptide Enrichment with TiO2-Modified Membranes and Investigation of Tau Protein Phosphorylation

    PubMed Central

    Tan, Yu-Jing; Sui, Dexin; Wang, Wei-Han; Kuo, Min-Hao; Reid, Gavin E.; Bruening, Merlin L.

    2013-01-01

    Selective enrichment of phosphopeptides prior to their analysis by mass spectrometry (MS) is vital for identifying protein phosphorylation sites involved in cellular regulation. This study describes modification of porous nylon substrates with TiO2 nanoparticles to create membranes that rapidly enrich phosphopeptides. Membranes with a 22-mm diameter bind 540 nmol of phosphoangiotensin and recover 70% of the phosphopeptides in mixtures with a 15-fold excess of non-phosphorylated proteins. Recovery is 90% for a pure phosphopeptide. Insertion of small membrane disks into HPLC fittings allows rapid enrichment of 5 mL of 1 fmol/μL phosphoprotein digests and concentration into small-volume (10’s of μL) eluates. The combination of membrane enrichment with tandem mass spectrometry reveals seven phosphorylation sites from in vivo phosphorylated tau (p-tau) protein, which is associated with Alzheimer’s disease. PMID:23638980

  6. Connexin47 protein phosphorylation and stability in oligodendrocytes depend on expression of Connexin43 protein in astrocytes.

    PubMed

    May, Dennis; Tress, Oliver; Seifert, Gerald; Willecke, Klaus

    2013-05-01

    Panglial networks are essential for normal physiology in the CNS, and the function of distinct connexins participating in these networks is not well understood. We generated Connexin32 (Cx32)-deficient mice with additional deletion of astrocytic Cx43 to explore the role of both connexins in panglial networks. Cx43/Cx32 double knock-out (dKO) mice revealed strong microglial activation in corpus callosum and cingulum along with severe astrogliosis and scar formation. In addition, most of the fine myelinated fibers projecting from the corpus callosum into the cortex were lost. Myelin loss was caused by a strong decrease of oligodendrocytes in the cingulum of Cx43/Cx32dKO mice. Immunoblot analyses using newly generated specific Cx47 antibodies revealed that oligodendrocytic Cx47 is phosphorylated in vivo depending on astrocytic Cx43 expression. In Cx43-deficient mice, Cx47 protein levels were strongly decreased, whereas Cx47 mRNA levels were not altered. Using Cx43G138R/Cx30KO mice, we show that Cx47 expression depends on the presence of astrocytic Cx43 protein and that its gap junctional channel function is not necessary for Cx47 stabilization. In consequence, Cx43/Cx32dKO mice additionally lack Cx47 expression and therefore cannot form oligodendrocytic gap junctions, which explains the phenotypic similarities to Cx32/Cx47dKO mice. Our findings provide strong evidence that phosphorylation and stability of oligodendrocytic Cx47 proteins is dependent on astrocytic Cx43 expression. These results further unravel the complexity of panglial networks and show that results of previous studies using astrocytic Cx43-deficient mice have to be reconsidered. PMID:23637189

  7. Protein kinase D negatively regulates hepatitis C virus secretion through phosphorylation of oxysterol-binding protein and ceramide transfer protein.

    PubMed

    Amako, Yutaka; Syed, Gulam H; Siddiqui, Aleem

    2011-04-01

    Hepatitis C virus (HCV) RNA replicates its genome on specialized endoplasmic reticulum modified membranes termed membranous web and utilizes lipid droplets for initiating the viral nucleocapsid assembly. HCV maturation and/or the egress pathway requires host sphingolipid synthesis, which occur in the Golgi. Ceramide transfer protein (CERT) and oxysterol-binding protein (OSBP) play a crucial role in sphingolipid biosynthesis. Protein kinase D (PKD), a serine/threonine kinase, is recruited to the trans-Golgi network where it influences vesicular trafficking to the plasma membrane by regulation of several important mediators via phosphorylation. PKD attenuates the function of both CERT and OSBP by phosphorylation at their respective Ser(132) and Ser(240) residues (phosphorylation inhibition). Here, we investigated the functional role of PKD in HCV secretion. Our studies show that HCV gene expression down-regulated PKD activation. PKD depletion by shRNA or inhibition by pharmacological inhibitor Gö6976 enhanced HCV secretion. Overexpression of a constitutively active form of PKD suppressed HCV secretion. The suppression by PKD was subverted by the ectopic expression of nonphosphorylatable serine mutant CERT S132A or OSBP S240A. These observations imply that PKD negatively regulates HCV secretion/release by attenuating OSBP and CERT functions by phosphorylation inhibition. This study identifies the key role of the Golgi components in the HCV maturation process. PMID:21285358

  8. Effect of Pseudomonas aeruginosa on sperm capacitation and protein phosphorylation of boar spermatozoa.

    PubMed

    Sepúlveda, Lilian; Bussalleu, Eva; Yeste, Marc; Bonet, Sergi

    2016-05-01

    Several studies have reported the detrimental effects that bacteriospermia causes on boar sperm quality, but little is known about its effects on IVC. Considering that, the present study sought to evaluate the effects of different concentrations of Pseudomonas aeruginosa on different indicators of capacitation status (sperm viability, membrane lipid disorder, sperm motility kinematics, and protein phosphorylation of boar spermatozoa) after IVC. Flow cytometry and computer assisted sperm analysis (CASA) revealed that the presence of P aeruginosa in boar sperm samples, mostly at concentrations greater than 10(6) CFU/mL, is associated with a significant (P < 0.05) decrease in the percentages of both sperm membrane integrity and sperm with low membrane lipid disorder, and also with a reduction in sperm motility kinetic parameters when compared with results obtained from the control sample, which presented the typical motility pattern of capacitated-like boar spermatozoa. Moreover, Western blot results also showed significant (P < 0.05) changes in the levels of tyrosine, serine, and threonine protein phosphorylation because of bacterial contamination, the decrease in phosphotyrosine levels of p32, a well-known marker of IVC achievement in boar sperm, being the most relevant. Indeed, after 3 hours of IVC, phosphotyrosine levels of p32 in the control sample were 3.13 ± 0.81, whereas in the tubes with 10(6) and 10(8) CFU/mL were 1.05 ± 0.20 and 0.36 ± 0.07, respectively. Therefore, the present study provides novel data regarding the effects of bacterial contamination on boar sperm, suggesting that the presence of P aeruginosa affects the fertilizing ability of boar sperm by altering its ability to accomplish IVC. PMID:26810830

  9. The viral transactivator HBx protein exhibits a high potential for regulation via phosphorylation through an evolutionarily conserved mechanism

    PubMed Central

    2012-01-01

    Background Hepatitis B virus (HBV) encodes an oncogenic factor, HBx, which is a multifunctional protein that can induce dysfunctional regulation of signaling pathways, transcription, and cell cycle progression, among other processes, through interactions with target host factors. The subcellular localization of HBx is both cytoplasmic and nuclear. This dynamic distribution of HBx could be essential to the multiple roles of the protein at different stages during HBV infection. Transactivational functions of HBx may be exerted both in the nucleus, via interaction with host DNA-binding proteins, and in the cytoplasm, via signaling pathways. Although there have been many studies describing different pathways altered by HBx, and its innumerable binding partners, the molecular mechanism that regulates its different roles has been difficult to elucidate. Methods In the current study, we took a bioinformatics approach to investigate whether the viral protein HBx might be regulated via phosphorylation by an evolutionarily conserved mechanism. Results We found that the phylogenetically conserved residues Ser25 and Ser41 (both within the negative regulatory domain), and Thr81 (in the transactivation domain) are predicted to be phosphorylated. By molecular 3D modeling of HBx, we further show these residues are all predicted to be exposed on the surface of the protein, making them easily accesible to these types of modifications. Furthermore, we have also identified Yin Yang sites that might have the potential to be phosphorylated and O-β-GlcNAc interplay at the same residues. Conclusions Thus, we propose that the different roles of HBx displayed in different subcellular locations might be regulated by an evolutionarily conserved mechanism of posttranslational modification, via phosphorylation. PMID:23079056

  10. S6 kinase inactivation impairs growth and translational target phosphorylation in muscle cells maintaining proper regulation of protein turnover.

    PubMed

    Mieulet, Virginie; Roceri, Mila; Espeillac, Catherine; Sotiropoulos, Athanassia; Ohanna, Mickael; Oorschot, Viola; Klumperman, Judith; Sandri, Marco; Pende, Mario

    2007-08-01

    A defect in protein turnover underlies multiple forms of cell atrophy. Since S6 kinase (S6K)-deficient cells are small and display a blunted response to nutrient and growth factor availability, we have hypothesized that mutant cell atrophy may be triggered by a change in global protein synthesis. By using mouse genetics and pharmacological inhibitors targeting the mammalian target of rapamycin (mTOR)/S6K pathway, here we evaluate the control of translational target phosphorylation and protein turnover by the mTOR/S6K pathway in skeletal muscle and liver tissues. The phosphorylation of ribosomal protein S6 (rpS6), eukaryotic initiation factor-4B (eIF4B), and eukaryotic elongation factor-2 (eEF2) is predominantly regulated by mTOR in muscle cells. Conversely, in liver, the MAPK and phosphatidylinositol 3-kinase pathways also play an important role, suggesting a tissue-specific control. S6K deletion in muscle mimics the effect of the mTOR inhibitor rapamycin on rpS6 and eIF4B phosphorylation without affecting eEF2 phosphorylation. To gain insight on the functional consequences of these modifications, methionine incorporation and polysomal distribution were assessed in muscle cells. Rates and rapamycin sensitivity of global translation initiation are not altered in S6K-deficient muscle cells. In addition, two major pathways of protein degradation, autophagy and expression of the muscle-specific atrophy-related E3 ubiquitin ligases, are not affected by S6K deletion. Our results do not support a role for global translational control in the growth defect due to S6K deletion, suggesting specific modes of growth control and translational target regulation downstream of mTOR. PMID:17494629

  11. Thermal sensitivity of mitochondrial respiration efficiency and protein phosphorylation in the clam Mercenaria mercenaria.

    PubMed

    Ulrich, P N; Marsh, A G

    2009-01-01

    The mitochondria of intertidal invertebrates continue to function when organisms are exposed to rapid substantial shifts in temperature. To test if mitochondrial physiology of the clam Mercenaria mercenaria is compromised under elevated temperatures, we measured mitochondrial respiration efficiency at 15 degrees C, 18 degrees C, and 21 degrees C using a novel, high-throughput, microplate respirometry methodology developed for this study. Though phosphorylating (state 3) and resting (state 4) respiration rates were unaffected over this temperature range, respiratory control ratios (RCRs: ratio of state 3 to state 4 respiration rates) decreased significantly above 18 degrees C (p < 0.05). The drop in RCR was not associated with reduction of phosphorylation efficiency, suggesting that, while aerobic scope of mitochondrial respiration is limited at elevated temperatures, mitochondria continue to efficiently produce adenosine triphosphate. We further investigated the response of clam mitochondria to elevated temperatures by monitoring phosphorylation of mitochondrial protein. Three proteins clearly demonstrated significant time- and temperature-specific phosphorylation patterns. The protein-specific patterns of phosphorylation may suggest that a suite of protein kinases and phosphatases regulate mitochondrial physiology in response to temperature. Thus, while aerobic scope of clam mitochondrial respiration is reduced at moderate temperatures, specific protein phosphorylation responses reflect large shifts in function that are initiated within the organelle at higher temperatures. PMID:19194752

  12. Lectin-induced activation of platelets may require only limited phosphorylation of the 47K protein

    SciTech Connect

    Ganguly, C.; Chelladurai, M.; Ganguly, P.

    1986-05-01

    Wheat germ agglutinin (WGA) is an N-acetylglucosamine (Glc-NAc) specific lectin which can activate platelets. Like thrombin, stimulation of platelets by WGA is accompanied by enhanced phosphorylation of two polypeptides of M/sub r/ 47K and 20K. Addition of GlcNAc at different time intervals arrested that aggregation of platelets by WGA and paralleled the modification of phosphorylation of the 47K polypeptide. So, the phosphorylation of the 47K polypeptide may regulate the WGA-receptor mediated stimulation of platelets. However, the ratio of phosphoserine to phosphothreonine in the 47K protein was markedly different in WGA-activated than thrombin-stimulated platelets. Thus, the molecular mechanism of action of thrombin and WGA could be different. To explore this idea, /sup 32/P/sub i/-labeled platelets were stimulated with WGA and the activation arrested with N-acetyl-glucosamine at different times. Two-dimensional gel electrophoresis of total protein at 5s showed only two phosphorylated species of 47K protein. At 60s, maximally four phosphorylated species were noted. In contrast, with thrombin using the same technique, seven to nine phosphorylated components have been reported. These results suggest that the different activators of platelets may act by different mechanisms. In addition, activation of platelets may require only limited levels of phosphorylation of the 47K polypeptide.

  13. E1A Blocks Hyperphosphorylation of p130 and p107 without Affecting the Phosphorylation Status of the Retinoblastoma Protein

    PubMed Central

    Parreño, Matilde; Garriga, Judit; Limón, Ana; Mayol, Xavier; Beck, George R.; Moran, Elizabeth; Graña, Xavier

    2000-01-01

    The phosphorylation status of the pRB family of growth suppressor proteins is regulated in a cell cycle entry-, progression-, and exit-dependent manner in normal cells. We have shown previously that p130, a member of this family, exhibits patterns of phosphorylated forms associated with various cell growth and differentiation stages. However, human 293 cells, which are transformed cells that express the adenoviral oncoproteins E1A and E1B, exhibit an abnormal pattern of p130 phosphorylated forms. Here we report that, unlike pRB, the phosphorylation status of both p130 and p107 is not modulated during the cell cycle in 293 cells as it is in other cells. Conditional overexpression of individual G1/S cyclins in 293 cells does not alter the phosphorylation status of p130, suggesting that the expression of E1A and/or E1B blocks hyperphosphorylation of p130. In agreement with these observations, transient cotransfection of vectors expressing E1A 12S, but not E1B, in combination with pocket proteins into U-2 OS cells blocks hyperphosphorylation of both p130 and p107. However, the phosphorylation status of pRB is not altered by cotransfection of E1A 12S vectors. Moreover, MC3T3-E1 preosteoblasts stably expressing E1A 12S also exhibit a block in hyperphosphorylation of endogenous p130 and p107. Direct binding of E1A to p130 and p107 is not required for the phosphorylation block since E1A 12S mutants defective in binding to the pRB family also block hyperphosphorylation of p130 and p107. Our data reported here identify a novel function of E1A, which affects p130 and p107 but does not affect pRB. Since E1A does not bind the hyperphosphorylated forms of p130, this function of E1A might prevent the existence of “free” hyperphosphorylated p130, which could act as a CDK inhibitor. PMID:10708433

  14. Insulin rapidly stimulates phosphorylation of a 46-kDa membrane protein on tyrosine residues as well as phosphorylation of several soluble proteins in intact fat cells

    SciTech Connect

    Haering, H.U.; White, M.F.; Machicao, F.; Ermel, B.; Schleicher, E.; Obermaier, B.

    1987-01-01

    It is speculated that the transmission of an insulin signal across the plasma membrane of cells occurs through activation of the tyrosine-specific receptor kinase, autophosphorylation of the receptor, and subsequent phosphorylation of unidentified substrates in the cell. In an attempt to identify possible substrates, the authors labeled intact rat fat cells with (/sup 32/P)orthophosphate and used an antiphosphotyrosine antibody to identify proteins that become phosphorylated on tyrosine residues in an insulin-stimulated way. In the membrane fraction of the fat cells, they found, in addition to the 95-kDa ..beta..-subunit of the receptor, a 46-kDa phosphoprotein that is phosphorylated exclusively on tyrosine residues. This protein is not immunoprecipitated by antibodies against different regions of the insulin receptor and its HPLC tryptic peptide map is different from the tryptic peptide map of the insulin receptor, suggesting that it is not derived from the receptor ..beta..-subunit. Insulin stimulates the tyrosine phosphorylation of the 46-kDa protein within 150 sec in the intact cell 3- to 4-fold in a dose-dependent way at insulin concentrations between 0.5 nM and 100 nM. Insulin (0.5 nM, 100 nM) stimulated within 2 min the /sup 32/P incorporation into a 116-kDa band, a 62 kDa band, and three bands between 45 kDa and 50 kDa 2- to 10-fold. They suggest that the 46-kDa membrane protein and possibly also the soluble proteins are endogenous substrates of the receptor tyrosine kinase in fat cells and that their phosphorylation is an early step in insulin signal transmission.

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

    PubMed

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

    2007-07-01

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

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

    PubMed Central

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

    2007-01-01

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

  17. Homeodomain-interacting protein kinase (Hipk) phosphorylates the small SPOC family protein Spenito.

    PubMed

    Dewald, D N; Steinmetz, E L; Walldorf, U

    2014-12-01

    The Drosophila homeodomain-interacting protein kinase (Hipk) is a versatile regulator involved in a variety of pathways, such as Notch and Wingless signalling, thereby acting in processes including the promotion of eye development or control of cell numbers in the nervous system. In vertebrates, extensive studies have related its homologue HIPK2 to important roles in the control of p53-mediated apoptosis and tumour suppression. Spenito (Nito) belongs to the group of small SPOC family proteins and has a role, amongst others, as a regulator of Wingless signalling downstream of Armadillo. In the present study, we show that both proteins have an enzyme-substrate relationship, adding a new interesting component to the broad range of Hipk interactions, and we map several phosphorylation sites of Nito. Furthermore, we were able to define a preliminary consensus motif for Hipk target sites, which will simplify the identification of new substrates of this kinase. PMID:25040100

  18. Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis

    PubMed Central

    Kohansal-Nodehi, Mahdokht; Chua, John JE; Urlaub, Henning; Jahn, Reinhard; Czernik, Dominika

    2016-01-01

    Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity. DOI: http://dx.doi.org/10.7554/eLife.14530.001 PMID:27115346

  19. A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase II.

    PubMed Central

    Guilfoyle, T J

    1989-01-01

    A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase IIA has been partially purified and characterized. The kinase has a native molecular weight of about 200 kilodaltons. This kinase utilizes Mg2+ and ATP and transfers about 20 phosphates to the heptapeptide repeats Pro-Thr-Ser-Pro-Ser-Tyr-Ser in the carboxyl-terminal domain of the 220-kilodalton subunit of soybean RNA polymerase II. This phosphorylation results in a mobility shift of the 220-kilodalton subunits of a variety of eukaryotic RNA polymerases to polypeptides ranging in size from greater than 220 kilodaltons to 240 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels. The phosphorylation is highly specific to the heptapeptide repeats since a degraded subunit polypeptide of 180 kilodaltons that lacks the heptapeptide repeats is poorly phosphorylated. Synthetic heptapeptide repeat multimers inhibit the phosphorylation of the 220-kilodalton subunit. PMID:2535525

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

    PubMed

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

    2005-01-01

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

  1. Development of bispecific molecules for the in situ detection of protein-protein interactions and protein phosphorylation.

    PubMed

    van Dieck, Jan; Schmid, Volker; Heindl, Dieter; Dziadek, Sebastian; Schraeml, Michael; Gerg, Michael; Massoner, Petra; Engel, Alfred M; Tiefenthaler, Georg; Vural, Serhat; Stritt, Simon; Tetzlaff, Fabian; Soukupova, Monika; Kopetzki, Erhard; Bossenmaier, Birgit; Thomas, Marlene; Klein, Christian; Mertens, Alfred; Heller, Astrid; Tacke, Michael

    2014-03-20

    Investigation of protein-protein interactions (PPIs) and protein phosphorylation in clinical tissue samples can offer valuable information about the activation status and function of proteins involved in disease progression. However, existing antibody-based methods for phosphorylation detection have been found to lack specificity, and methods developed for examining PPIs in vitro cannot be easily adapted for tissues samples. In this study, we eliminated some of these limitations by developing a specific immunohistochemical staining method that uses "dual binders" (DBs), which are bispecific detection agents consisting of two Fab fragment molecules joined by a flexible linker, to detect PPIs and protein phosphorylation. We engineered DBs by selecting Fab fragments with fast off-rate kinetics, which allowed us to demonstrate that stable target binding was achieved only upon simultaneous, cooperative binding to both epitopes. We show that DBs specifically detect the activated HER2/HER3 complex in formalin-fixed, paraffin-embedded cancer cells and exhibit superior detection specificity for phospho-HER3 compared to the corresponding monoclonal antibody. Overall, the performance of DBs makes them attractive tools for future development for clinical applications. PMID:24529991

  2. Distinct and Site-Specific Phosphorylation of the Retinoblastoma Protein at Serine 612 in Differentiated Cells

    PubMed Central

    Hattori, Takayuki; Uchida, Chiharu; Takahashi, Hirotaka; Yamamoto, Naoki; Naito, Mikihiko; Taya, Yoichi

    2014-01-01

    The retinoblastoma susceptibility protein (pRB) is a phosphoprotein that regulates cell cycle progression at the G1/S transition. In quiescent and early G1 cells, pRB predominantly exists in the active hypophosphorylated form. The cyclin/cyclin-dependent protein kinase complexes phosphorylate pRB at the late G1 phase to inactivate pRB. This event leads to the dissociation and activation of E2F family transcriptional factors. At least 12 serine/threonine residues in pRB are phosphorylated in vivo. Although there have been many reports describing bulk phosphorylation of pRB, detail research describing the function of each phosphorylation site remains unknown. Besides its G1/S inhibitory function, pRB is involved in differentiation, prevention of cell death and control of tissue fate. To uncover the function of phosphorylation of pRB in various cellular conditions, we have been investigating phosphorylation of each serine/threonine residue in pRB with site-specific phospho-serine/threonine antibodies. Here we demonstrate that pRB is specifically phosphorylated at Ser612 in differentiated cells in a known kinase-independent manner. We also found that pRB phosphorylated at Ser612 still associates with E2F-1 and tightly binds to nuclear structures including chromatin. Moreover, expression of the Ser612Ala mutant pRB failed to induce differentiation. The findings suggest that phosphorylation of Ser612 provides a distinct function that differs from the function of phosphorylation of other serine/threonine residues in pRB. PMID:24466208

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

    PubMed Central

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

    2015-01-01

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

  4. Analysis of Phosphorylation of the Receptor-Like Protein Kinase HAESA during Arabidopsis Floral Abscission

    PubMed Central

    Taylor, Isaiah; Wang, Ying; Seitz, Kati; Baer, John; Bennewitz, Stefan; Mooney, Brian P.; Walker, John C.

    2016-01-01

    Receptor-like protein kinases (RLKs) are the largest family of plant transmembrane signaling proteins. Here we present functional analysis of HAESA, an RLK that regulates floral organ abscission in Arabidopsis. Through in vitro and in vivo analysis of HAE phosphorylation, we provide evidence that a conserved phosphorylation site on a region of the HAE protein kinase domain known as the activation segment positively regulates HAE activity. Additional analysis has identified another putative activation segment phosphorylation site common to multiple RLKs that potentially modulates HAE activity. Comparative analysis suggests that phosphorylation of this second activation segment residue is an RLK specific adaptation that may regulate protein kinase activity and substrate specificity. A growing number of RLKs have been shown to exhibit biologically relevant dual specificity toward serine/threonine and tyrosine residues, but the mechanisms underlying dual specificity of RLKs are not well understood. We show that a phospho-mimetic mutant of both HAE activation segment residues exhibits enhanced tyrosine auto-phosphorylation in vitro, indicating phosphorylation of this residue may contribute to dual specificity of HAE. These results add to an emerging framework for understanding the mechanisms and evolution of regulation of RLK activity and substrate specificity. PMID:26784444

  5. Vasoactive intestinal peptide stimulates protein phosphorylation in a colonic epithelial cell line

    SciTech Connect

    Cohn, J.A.

    1987-09-01

    The T/sub 84/ colonic epithelial cell line was used to examine protein phosphorylation during neurohumoral stimulation of ion transport. T/sub 84/ cell monolayers grown on collagen-coated filters were mounted in Ussing chambers to measure ion transport stimulated by vasoactive intestinal peptide. Maximal stimulation of active secretion occurred after 8-10 min of stimulation. Protein phosphorylation events accompanying stimulated secretion were detected using two-dimensional gel electrophoresis to resolve phosphoproteins from monolayers previously labeled using /sup 32/P/sub i/. Within 8 min of exposure to vasoactive intestinal peptide, several phosphorylation events were detected, including a two- to fivefold increase in /sup 32/P incorporation into four soluble proteins with apparent molecular weights of 17,000, 18,000, 23,000, and 37,000. The same phosphorylation response occurs in monolayers stimulated by dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP), suggesting that cAMP mediates these intracellular events. This study indicates that changes in protein phosphorylation accompany the secretory action of vasocactive intestinal peptide and suggests that T/sub 84/ cells offer a useful model for studying the possibility that such phosphorylation events regulate enterocyte ion transport.

  6. Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells.

    PubMed Central

    Gard, D L; Lazarides, E

    1982-01-01

    The intermediate filament proteins desmin and vimentin and the muscle tropomyosins were the major protein phosphate acceptors in 8-day-old myotubes incubated for 4 h in medium containing radiolabeled phosphate. The addition of isoproterenol or 8-bromo-cyclic AMP (BrcAMP) resulted in a two- to threefold increase in incorporation of 32PO4 into both desmin and vimentin, whereas no changes in the incorporation of 32PO4 into tropomyosin or other cellular proteins were observed. The BrcAMP- or hormonally induced increase in 32PO4 incorporation into desmin and vimentin was independent of protein synthesis and was not caused by stimulation of protein phosphate turnover. In addition, BrcAMP did not induce significant changes in the specific activity of the cellular ATP pool. These data suggest that the observed increase in 32PO4 incorporation represented an actual increase in phosphorylation of the intermediate filament proteins desmin and vimentin. Two-dimensional tryptic analysis of desmin from 8-day-old myotubes revealed five phosphopeptides of which two showed a 7- to 10-fold increase in 32PO4 incorporation in BrcAMP-treated myotubes. Four of the phosphopeptides identified in desmin labeled in vivo were also observed in desmin phosphorylated in vitro by bovine heart cAMP-dependent protein kinase. Although phosphorylation of desmin and vimentin was apparent in myogenic cells at all stages of differentiation, BrcAMP- and isoproterenol-induced increases in phosphorylation of these proteins were restricted to mature myotubes. These data strongly suggest that in vivo phosphorylation of the intermediate filament proteins desmin and vimentin is catalyzed by the cAMP-dependent protein kinases and that such phosphorylation may be regulated during muscle differentiation. Images PMID:6294504

  7. Global protein phosphorylation dynamics during deoxynivalenol-induced ribotoxic stress response in the macrophage

    SciTech Connect

    Pan, Xiao; Whitten, Douglas A.; Wu, Ming; Chan, Christina; Wilkerson, Curtis G.; Pestka, James J.

    2013-04-15

    Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium that commonly contaminates food, is capable of activating mononuclear phagocytes of the innate immune system via a process termed the ribotoxic stress response (RSR). To encapture global signaling events mediating RSR, we quantified the early temporal (≤ 30 min) phosphoproteome changes that occurred in RAW 264.7 murine macrophage during exposure to a toxicologically relevant concentration of DON (250 ng/mL). Large-scale phosphoproteomic analysis employing stable isotope labeling of amino acids in cell culture (SILAC) in conjunction with titanium dioxide chromatography revealed that DON significantly upregulated or downregulated phosphorylation of 188 proteins at both known and yet-to-be functionally characterized phosphosites. DON-induced RSR is extremely complex and goes far beyond its prior known capacity to inhibit translation and activate MAPKs. Transcriptional regulation was the main target during early DON-induced RSR, covering over 20% of the altered phosphoproteins as indicated by Gene Ontology annotation and including transcription factors/cofactors and epigenetic modulators. Other biological processes impacted included cell cycle, RNA processing, translation, ribosome biogenesis, monocyte differentiation and cytoskeleton organization. Some of these processes could be mediated by signaling networks involving MAPK-, NFκB-, AKT- and AMPK-linked pathways. Fuzzy c-means clustering revealed that DON-regulated phosphosites could be discretely classified with regard to the kinetics of phosphorylation/dephosphorylation. The cellular response networks identified provide a template for further exploration of the mechanisms of trichothecenemycotoxins and other ribotoxins, and ultimately, could contribute to improved mechanism-based human health risk assessment. - Highlights: ► Mycotoxin deoxynivalenol (DON) induces immunotoxicity via ribotoxic stress response. ► SILAC phosphoproteomics using

  8. Thrombin Ca(2+)-dependently stimulates protein tyrosine phosphorylation in BC3H1 muscle cells.

    PubMed Central

    Offermanns, S; Bombien, E; Schultz, G

    1993-01-01

    The proteinase thrombin, known to act via heptahelical G-protein-coupled receptors, is a mitogenic agent for different cell types, including the mouse muscle cell line BC3H1. In this study, the effect of thrombin on tyrosine phosphorylation was examined using anti-phosphotyrosine antibodies. Thrombin was found to induce phosphorylation of 65-70 and 110-120 kDa proteins in BC3H1 cells. The effect of thrombin was concentration-dependent, being half-maximal and maximal at concentrations of 0.03 and 1 unit/ml respectively. The thrombin-induced increase in phosphorylation was rapid (< or = 10 s) and transient, with a peak response after about 1-2 min. The effect of thrombin could be mimicked by the thrombin receptor agonist peptide SFLLRN-NH2. Preincubation of cells with pertussis toxin (PT) had no effect on thrombin-induced tyrosine phosphorylation. Epidermal growth factor, platelet-derived growth factor and insulin stimulated tyrosine phosphorylation of different proteins, among which were 65-70 and 110-120 kDa proteins. The phorbol ester 12-myristate 13-acetate (PMA) as well as the Ca2+ ionophore A23187 both stimulated tyrosine phosphorylation of proteins identical to those phosphorylated by thrombin, suggesting that activation of protein kinase C (PKC) and elevation of the cytosolic Ca2+ concentration alone are sufficient to induce tyrosine phosphorylation. However, calphostin C and other PKC inhibitors, which completely inhibited tyrosine phosphorylation induced by PMA, had no influence on the effect of thrombin, whereas loading of cells with the intracellular Ca2+ chelator bis-(O-aminophenoxy)ethane-NNN'N'-tetra-acetic acid totally blocked thrombin-stimulated tyrosine phosphorylation. Thus tyrosine phosphorylation stimulated by thrombin is an early PT-insensitive cellular response which is either directly mediated by elevation of cytosolic Ca2+ concentration or by a presently unknown mechanism that requires an elevated cytosolic Ca2+ concentration. Images Figure 1

  9. dbPAF: an integrative database of protein phosphorylation in animals and fungi.

    PubMed

    Ullah, Shahid; Lin, Shaofeng; Xu, Yang; Deng, Wankun; Ma, Lili; Zhang, Ying; Liu, Zexian; Xue, Yu

    2016-01-01

    Protein phosphorylation is one of the most important post-translational modifications (PTMs) and regulates a broad spectrum of biological processes. Recent progresses in phosphoproteomic identifications have generated a flood of phosphorylation sites, while the integration of these sites is an urgent need. In this work, we developed a curated database of dbPAF, containing known phosphorylation sites in H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. pombe and S. cerevisiae. From the scientific literature and public databases, we totally collected and integrated 54,148 phosphoproteins with 483,001 phosphorylation sites. Multiple options were provided for accessing the data, while original references and other annotations were also present for each phosphoprotein. Based on the new data set, we computationally detected significantly over-represented sequence motifs around phosphorylation sites, predicted potential kinases that are responsible for the modification of collected phospho-sites, and evolutionarily analyzed phosphorylation conservation states across different species. Besides to be largely consistent with previous reports, our results also proposed new features of phospho-regulation. Taken together, our database can be useful for further analyses of protein phosphorylation in human and other model organisms. The dbPAF database was implemented in PHP + MySQL and freely available at http://dbpaf.biocuckoo.org. PMID:27010073

  10. dbPAF: an integrative database of protein phosphorylation in animals and fungi

    PubMed Central

    Ullah, Shahid; Lin, Shaofeng; Xu, Yang; Deng, Wankun; Ma, Lili; Zhang, Ying; Liu, Zexian; Xue, Yu

    2016-01-01

    Protein phosphorylation is one of the most important post-translational modifications (PTMs) and regulates a broad spectrum of biological processes. Recent progresses in phosphoproteomic identifications have generated a flood of phosphorylation sites, while the integration of these sites is an urgent need. In this work, we developed a curated database of dbPAF, containing known phosphorylation sites in H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. pombe and S. cerevisiae. From the scientific literature and public databases, we totally collected and integrated 54,148 phosphoproteins with 483,001 phosphorylation sites. Multiple options were provided for accessing the data, while original references and other annotations were also present for each phosphoprotein. Based on the new data set, we computationally detected significantly over-represented sequence motifs around phosphorylation sites, predicted potential kinases that are responsible for the modification of collected phospho-sites, and evolutionarily analyzed phosphorylation conservation states across different species. Besides to be largely consistent with previous reports, our results also proposed new features of phospho-regulation. Taken together, our database can be useful for further analyses of protein phosphorylation in human and other model organisms. The dbPAF database was implemented in PHP + MySQL and freely available at http://dbpaf.biocuckoo.org. PMID:27010073

  11. AMP-activated protein kinase modulates tau phosphorylation and tau pathology in vivo

    PubMed Central

    Domise, Manon; Didier, Sébastien; Marinangeli, Claudia; Zhao, Haitian; Chandakkar, Pallavi; Buée, Luc; Viollet, Benoit; Davies, Peter; Marambaud, Philippe; Vingtdeux, Valérie

    2016-01-01

    Neurofibrillary tangles (NFTs) are the pathological hallmark of neurodegenerative diseases commonly known as tauopathies. NFTs result from the intracellular aggregation of abnormally and hyperphosphorylated tau proteins. Tau functions, which include the regulation of microtubules dynamics, are dependent on its phosphorylation status. As a consequence, any changes in tau phosphorylation can have major impacts on synaptic plasticity and memory. Recently, it has been demonstrated that AMP-activated protein kinase (AMPK) was deregulated in the brain of Alzheimer’s disease (AD) patients where it co-localized with phosphorylated tau in pre-tangle and tangle-bearing neurons. Besides, it was found that AMPK was a tau kinase in vitro. Here, we find that endogenous AMPK activation in mouse primary neurons induced an increase of tau phosphorylation at multiple sites, whereas AMPK inhibition led to a rapid decrease of tau phosphorylation. We further show that AMPK mice deficient for one of the catalytic alpha subunits displayed reduced endogenous tau phosphorylation. Finally, we found that AMPK deficiency reduced tau pathology in the PS19 mouse model of tauopathy. These results show that AMPK regulates tau phosphorylation in mouse primary neurons as well as in vivo, and thus suggest that AMPK could be a key player in the development of AD pathology. PMID:27230293

  12. AMP-activated protein kinase modulates tau phosphorylation and tau pathology in vivo.

    PubMed

    Domise, Manon; Didier, Sébastien; Marinangeli, Claudia; Zhao, Haitian; Chandakkar, Pallavi; Buée, Luc; Viollet, Benoit; Davies, Peter; Marambaud, Philippe; Vingtdeux, Valérie

    2016-01-01

    Neurofibrillary tangles (NFTs) are the pathological hallmark of neurodegenerative diseases commonly known as tauopathies. NFTs result from the intracellular aggregation of abnormally and hyperphosphorylated tau proteins. Tau functions, which include the regulation of microtubules dynamics, are dependent on its phosphorylation status. As a consequence, any changes in tau phosphorylation can have major impacts on synaptic plasticity and memory. Recently, it has been demonstrated that AMP-activated protein kinase (AMPK) was deregulated in the brain of Alzheimer's disease (AD) patients where it co-localized with phosphorylated tau in pre-tangle and tangle-bearing neurons. Besides, it was found that AMPK was a tau kinase in vitro. Here, we find that endogenous AMPK activation in mouse primary neurons induced an increase of tau phosphorylation at multiple sites, whereas AMPK inhibition led to a rapid decrease of tau phosphorylation. We further show that AMPK mice deficient for one of the catalytic alpha subunits displayed reduced endogenous tau phosphorylation. Finally, we found that AMPK deficiency reduced tau pathology in the PS19 mouse model of tauopathy. These results show that AMPK regulates tau phosphorylation in mouse primary neurons as well as in vivo, and thus suggest that AMPK could be a key player in the development of AD pathology. PMID:27230293

  13. Phospholipid transfer protein (PLTP) reduces phosphorylation of tau in human neuronal cells (HCN2)

    PubMed Central

    Dong, Weijiang; Albers, John J.; Vuletic, Simona

    2009-01-01

    Tau function is regulated by phosphorylation, and abnormal tau phosphorylation in neurons is one of the key processes associated with development of Alzheimer’s disease and other tauopathies. In this study we provide evidence that phospholipid transfer protein (PLTP), one of the main lipid transfer proteins in the brain, significantly reduces levels of phosphorylated tau, and increases levels of the inactive form of glycogen synthase kinase-3β (GSK3β) in HCN2 cells. Furthermore, inhibition of the phosphatidylinositol-3 kinase (PI3K) reversed the PLTP-induced increase in levels of GSK3β phosphorylated at serine 9 (pGSK3βSer9) and partially reversed the PLTP-induced reduction in tau phosphorylation. We provide evidence that the PLTP-induced changes are not due to activation of Disabled-1 (Dab1), since PLTP reduced levels of total and phosphorylated Dab1 in HCN2 cells. We have also shown that inhibition of tyrosine kinase activity of insulin receptor (IR) and/or insulin-like growth factor 1 (IGF1) receptor (IGFR) reverses PLTP-induced increase in levels of phosphorylated Akt (pAktThr308 and pAktSer473), suggesting that PLTP-mediated activation of the PI3K/Akt pathway is dependent on IR/IGFR receptor tyrosine kinase activity. Our study suggests that PLTP may be an important modulator of signal transduction pathways in human neurons. PMID:19472218

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

    PubMed Central

    Magron, Audrey; Elowe, Sabine; Carreau, Madeleine

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  17. Optical tweezers reveal how proteins alter replication

    NASA Astrophysics Data System (ADS)

    Chaurasiya, Kathy

    Single molecule force spectroscopy is a powerful method that explores the DNA interaction properties of proteins involved in a wide range of fundamental biological processes such as DNA replication, transcription, and repair. We use optical tweezers to capture and stretch a single DNA molecule in the presence of proteins that bind DNA and alter its mechanical properties. We quantitatively characterize the DNA binding mechanisms of proteins in order to provide a detailed understanding of their function. In this work, we focus on proteins involved in replication of Escherichia coli (E. coli ), endogenous eukaryotic retrotransposons Ty3 and LINE-1, and human immunodeficiency virus (HIV). DNA polymerases replicate the entire genome of the cell, and bind both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) during DNA replication. The replicative DNA polymerase in the widely-studied model system E. coli is the DNA polymerase III subunit alpha (DNA pol III alpha). We use optical tweezers to determine that UmuD, a protein that regulates bacterial mutagenesis through its interactions with DNA polymerases, specifically disrupts alpha binding to ssDNA. This suggests that UmuD removes alpha from its ssDNA template to allow DNA repair proteins access to the damaged DNA, and to facilitate exchange of the replicative polymerase for an error-prone translesion synthesis (TLS) polymerase that inserts nucleotides opposite the lesions, so that bacterial DNA replication may proceed. This work demonstrates a biophysical mechanism by which E. coli cells tolerate DNA damage. Retroviruses and retrotransposons reproduce by copying their RNA genome into the nuclear DNA of their eukaryotic hosts. Retroelements encode proteins called nucleic acid chaperones, which rearrange nucleic acid secondary structure and are therefore required for successful replication. The chaperone activity of these proteins requires strong binding affinity for both single- and double-stranded nucleic

  18. Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly*

    PubMed Central

    Shim, Su Yeon; de Castro, Ignacio Perez; Neumayer, Gernot; Wang, Jian; Park, Sang Ki; Sanada, Kamon; Nguyen, Minh Dang

    2015-01-01

    The human ortholog of the targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a cytoskeletal protein that plays a major role in spindle assembly and is required for mitosis. During spindle morphogenesis, TPX2 cooperates with Aurora A kinase and Eg5 kinesin to regulate microtubule organization. TPX2 displays over 40 putative phosphorylation sites identified from various high-throughput proteomic screenings. In this study, we characterize the phosphorylation of threonine 72 (Thr72) in human TPX2, a residue highly conserved across species. We find that Cdk1/2 phosphorylate TPX2 in vitro and in vivo. Using homemade antibodies specific for TPX2 phosphorylated at Thr72, we show that this phosphorylation is cell cycle-dependent and peaks at M phase. Endogenous TPX2 phosphorylated at Thr72 does not associate with the mitotic spindle. Furthermore, ectopic GFP-TPX2 T72A preferentially concentrates on the spindle, whereas GFP-TPX2 WT distributes to both spindle and cytosol. The T72A mutant also increases the proportion of cells with multipolar spindles phenotype. This effect is associated with increased Aurora A activity and abnormally elongated spindles, indicative of higher Eg5 activity. In summary, we propose that phosphorylation of Thr72 regulates TPX2 localization and impacts spindle assembly via Aurora A and Eg5. PMID:25688093

  19. Protein-tyrosine-phosphatase 2C is phosphorylated and inhibited by 44-kDa mitogen-activated protein kinase.

    PubMed Central

    Peraldi, P; Zhao, Z; Filloux, C; Fischer, E H; Van Obberghen, E

    1994-01-01

    Protein-tyrosine-phosphatase 2C (PTP2C, also named SHPTP2, SHPTP3, or PTP1D) is a cytosolic enzyme with two Src homology 2 domains. We have investigated its regulation by phosphorylation in PC12 rat pheochromocytoma cells. In untreated cells, PTP2C was phosphorylated predominantly on serine residues. A 5-min treatment with epidermal growth factor (EGF) induced an increase in phosphorylation on threonine and, to a lesser degree, on serine. After 45 min of exposure to EGF, PTP2C phosphorylation returned to basal levels. Using an in vitro kinase assay, we found that the 44-kDa mitogen-activated protein kinase, p44mapk, phosphorylated PTP2C on serine and threonine residues. This phosphorylation resulted in a pronounced inhibition of PTP2C enzyme activity measured with phosphorylated EGF receptors as substrate. Moreover, in intact PC12 cells, PTP2C was also inhibited following a short EGF treatment, but its activity returned to normal when the exposure to EGF was maintained for 45 min. The profile of this response to EGF can be inversely correlated to that of the stimulatory action of EGF on p44mapk. These data suggest that the EGF-induced regulation of PTP2C activity is mediated by p44mapk. These findings provide evidence for an additional role of the mitogen-activated protein kinase cascade--namely, the regulation of a PTP. Images PMID:8197172

  20. Effect of zinc sulphate on gelling properties of phosphorylated protein isolate from yellow stripe trevally.

    PubMed

    Arfat, Yasir Ali; Benjakul, Soottawat

    2013-12-01

    Impacts of zinc sulphate (ZnSO4) (0-140 μmol/kg) on gel properties of yellow stripe trevally surimi added with sodium tripolyphosphate (STPP) (0.25% and 0.5%, w/w) and protein isolate phosphorylated with STPP at 0.25% and 0.5% (w/w) were studied. Gels from surimi added with 60 μmol ZnSO4/kg in the absence and presence of 0.5% STPP had the increases in breaking force and deformation by 20.9% and 33.3%, and 11.6% and 18.6%, respectively, compared with the control surimi gel (without additives). Gel of protein isolate phosphorylated with 0.5% STPP containing 100 μmol ZnSO4/kg had the increases in breaking force and deformation by 14.87% and 5.6%, respectively, compared with the gel from non-phosphorylated protein isolate at the same ZnSO4 level, suggesting that the phosphorylated protein isolate was more crosslinked by Zn(2+). The addition of ZnSO4 at the suitable level lowered the expressible moisture content, but increased whiteness of surimi or protein isolate gels (P<0.05). Non-covalent bonds, more likely salt bridge and ionic interactions, played a major role in cross-linking of proteins in both surimi and protein isolate added with ZnSO4, regardless of phosphates incorporated. Microstructure study revealed that a gel having highly interconnected and denser network with smaller voids was formed when protein isolate phosphorylated with 0.5% STPP was added with ZnSO4 at a level of 100 μmol/kg. Thus, gel with improved properties could be obtained from protein isolate from yellow stripe trevally phosphorylated with STPP in conjunction with addition of ZnSO4 at an appropriate level. PMID:23871033

  1. Human TNF-α induces differential protein phosphorylation in Schistosoma mansoni adult male worms.

    PubMed

    Oliveira, Katia C; Carvalho, Mariana L P; Bonatto, José Matheus C; Schechtman, Debora; Verjovski-Almeida, Sergio

    2016-02-01

    Schistosoma mansoni and its vertebrate host have a complex and intimate connection in which several molecular stimuli are exchanged and affect both organisms. Human tumor necrosis factor alpha (hTNF-α), a pro-inflammatory cytokine, is known to induce large-scale gene expression changes in the parasite and to affect several parasite biological processes such as metabolism, egg laying, and worm development. Until now, the molecular mechanisms for TNF-α activity in worms are not completely understood. Here, we aimed at exploring the effect of hTNF-α on S. mansoni protein phosphorylation by 2D gel electrophoresis followed by a quantitative analysis of phosphoprotein staining and protein identification by mass spectrometry. We analyzed three biological replicates of adult male worms exposed to hTNF-α and successfully identified 32 protein spots with a statistically significant increase in phosphorylation upon in vitro exposure to hTNF-α. Among the differentially phosphorylated proteins, we found proteins involved in metabolism, such as glycolysis, galactose metabolism, urea cycle, and aldehyde metabolism, as well as proteins related to muscle contraction and to cytoskeleton remodeling. The most differentially phosphorylated protein (30-fold increase in phosphorylation) was 14-3-3, whose function is known to be modulated by phosphorylation, belonging to a signal transduction protein family that regulates a variety of processes in all eukaryotic cells. Further, 75% of the identified proteins are known in mammals to be related to TNF-α signaling, thus suggesting that TNF-α response may be conserved in the parasite. We propose that this work opens new perspectives to be explored in the study of the molecular crosstalk between host and pathogen. PMID:26547565

  2. ADIPOCYTES FROM WOMEN WITH POLYCYSTIC OVARY SYNDROME DEMONSTRATE ALTERED PHOSPHORYLATION AND ACTIVITY OF GLYCOGEN SYNTHASE KINASE 3

    PubMed Central

    Chang, Wendy; Goodarzi, Mark O.; Williams, Heith; Magoffin, Denis A.; Pall, Marita; Azziz, Ricardo

    2009-01-01

    Objective To test the hypothesis that an abnormality in glycogen synthase kinase-3 (GSK3) is a pathogenic factor in PCOS. Design Prospective experimental study (adipocytes). Setting Tertiary care academic medical center and teaching hospital Patients Patients with PCOS and healthy controls. Interventions Blood sampling, physical exam, biopsy of subcutaneous lower abdominal fat. Main Outcome Measure(s) Glucose transport and protein levels and phosphorylation state of GSK3α and GSK3β in adipocytes, assessment of GSK3β activity. Results Basal protein levels of glycogen synthase kinase (GSK3α and GSK3β) did not differ between controls and women with PCOS, nor did basal or insulin-stimulated levels of serine phosphorylated GSK3α. However, in adipocytes of PCOS women insulin stimulation was not associated with increased serine phosphorylation of GSK3β, in contrast to controls. Tyrosine phosphorylation of GSK3β was also higher in PCOS compared to controls. Consistent with the phosphorylation data, GSK3β activity was elevated in PCOS adipocytes. Conclusions These data suggest GSK3β is hyperactivated and resistant to downregulation by insulin in PCOS. Using physiologic approaches, we demonstrated that abnormal GSK3β regulation is a potential mechanism for the insulin resistance seen in some women with PCOS, which may contribute to their development of the syndrome. PMID:18178198

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

    PubMed

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

    1989-05-01

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

  4. Reconstitution of LHC phosphorylation by a protein kinase isolated from spinach thylakoids

    SciTech Connect

    Hind, G.; Coughlan, S.

    1986-01-01

    Protein kinase activity is responsible for phosphorylating the (LHC) light-harvesting chlorophyll a/b protein complex of photosystem II, leading to its migration in the thylakoid membrane, the fractional redistribution of excitation energy between photosystems II and I, and the phenomenon of state transition. Previous work from this laboratory described the purification to homogeneity of a thylakoid protein kinase which catalyzes the phosphorylation of isolated LHC at 1-10% of a rate estimated for this enzyme and substrate when resident together in the thylakoid membrane. In this communication, we report rates of LHC phosphorylation that are close to physiological, in a system comprised of isolated purified protein kinase (LHCK) and native LHC. 9 refs., 1 fig., 2 tabs.

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

    PubMed

    Gerbeth, Carolin; Mikropoulou, Despina; Meisinger, Chris

    2013-10-01

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

  6. Protein kinase CK2 interacts with Chk2 and phosphorylates Mre11 on serine 649

    SciTech Connect

    Kim, Seong-Tae . E-mail: stkim@med.skku.ac.kr

    2005-05-27

    The Mre11-Rad50-Nbs1 protein complex has been known to be involved in a variety of DNA metabolic events that involve DNA double-strand breaks (DSBs). The phosphorylation of Mre11 is increased in response to ionizing radiation, which suggests that phosphorylation of Mre11 may be an important regulatory mechanism of this complex. Mre11-phosphorylating kinase activities were observed in Chk2 immunoprecipitates and HeLa nuclear extracts. Through the tandem affinity tagging system and conventional chromatography, this kinase was purified and identified as protein kinase CK2. CK2 phosphorylates Mre11 in vitro. In vitro kinase assay with a series of truncated Mre11 proteins as substrates for CK2 and site-directed mutagenesis showed that serine 649 of Mre11 is mainly phosphorylated by CK2 in vitro. In vivo labeling and phosphopeptide mapping analysis revealed that this phosphorylation occurs in vivo. These data implicate CK2 as a potential upstream regulator of Mre11 function.

  7. Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling.

    PubMed Central

    Denhardt, D T

    1996-01-01

    The features of three distinct protein phosphorylation cascades in mammalian cells are becoming clear. These signalling pathways link receptor-mediated events at the cell surface or intracellular perturbations such as DNA damage to changes in cytoskeletal structure, vesicle transport and altered transcription factor activity. The best known pathway, the Ras-->Raf-->MEK-->ERK cascade [where ERK is extracellular-signal-regulated kinase and MEK is mitogen-activated protein (MAP) kinase/ERK kinase], is typically stimulated strongly by mitogens and growth factors. The other two pathways, stimulated primarily by assorted cytokines, hormones and various forms of stress, predominantly utilize p21 proteins of the Rho family (Rho, Rac and CDC42), although Ras can also participate. Diagnostic of each pathway is the MAP kinase component, which is phosphorylated by a unique dual-specificity kinase on both tyrosine and threonine in one of three motifs (Thr-Glu-Tyr, Thr-Phe-Tyr or Thr-Gly-Tyr), depending upon the pathway. In addition to activating one or more protein phosphorylation cascades, the initiating stimulus may also mobilize a variety of other signalling molecules (e.g. protein kinase C isoforms, phospholipid kinases, G-protein alpha and beta gamma subunits, phospholipases, intracellular Ca2+). These various signals impact to a greater or lesser extent on multiple downstream effectors. Important concepts are that signal transmission often entails the targeted relocation of specific proteins in the cell, and the reversible formation of protein complexes by means of regulated protein phosphorylation. The signalling circuits may be completed by the phosphorylation of upstream effectors by downstream kinases, resulting in a modulation of the signal. Signalling is terminated and the components returned to the ground state largely by dephosphorylation. There is an indeterminant amount of cross-talk among the pathways, and many of the proteins in the pathways belong to families

  8. Expression and phosphorylation of a MARCKS-like protein in gastric chief cells: further evidence for modulation of pepsinogen secretion by interaction of Ca2+/calmodulin with protein kinase C.

    PubMed

    Raufman, J P; Malhotra, R; Xie, Q; Raffaniello, R D

    1997-03-01

    In gastric chief cells, agents that activate protein kinase C (PKC) stimulate pepsinogen secretion and phosphorylation of an acidic 72-kDa protein. The isoelectric point and molecular mass of this protein are similar to those for a common PKC substrate; the MARCKS (for Myristoylated Alanine-Rich C Kinase Substrate) protein. We examined expression and phosphorylation of the MARCKS-like protein in a nearly homogeneous suspension of chief cells from guinea pig stomach. Western blotting of fractions from chief cell lysates with a specific MARCKS antibody resulted in staining of a myristoylated 72-kDA protein (pp72), associated predominantly with the membrane fraction. Using permeabilized chief cells, we examined the effect of PKC activation (with the phorbol ester PMA), in the presence of basal (100 nM) or elevated cellular calcium (1 microM), on pepsinogen secretion and phosphorylation of the 72-KDa MARCKS-like protein. Secretion was increased 2.3-, 2.6-, and 4.5-fold by incubation with 100 nM PMA, 1 microM calcium, and PMA plus calcium, respectively. A PKC inhibitor (1 microM CGP 41 251) abolished PMA-induced secretion, but did not alter calcium-induced secretion. This indicates that calcium-induced secretion is independent of PKC activation. Chief cell proteins were labeled with 32P-orthophosphate and phosphorylation of pp72 was detected by autoradiography of 2-dimensional polyacrylamide gels. In the presence of basal calcium, PMA (100 nM) caused a > two-fold increase in phosphorylation of pp72. Without PMA, calcium did not alter phosphorylation of pp72. However, 1 microM calcium caused an approx. 50% attenuation of PMA-induced phosphorylation of pp72. Experiments with a MARCKS "phosphorylation/calmodulin binding domain peptide" indicated that calcium/calmodulin inhibits phosphorylation of pp72 by binding to the phosphorylation/calmodulin binding domain and not by inhibiting PKC activity. These observations support the hypothesis that, in gastric chief cells

  9. Phosphorylation of protein synthesis initiation factor 2 (elF-2) in the yeast Saccharomyces cerevisiae

    SciTech Connect

    Romero, D.P.

    1986-01-01

    Initiation Factor 2 (elF-2) in the yeast Saccharomyces cerevisiae is comprised of 3 subunits. The control of protein synthesis in mammalian cells have been shown to involve the phosphorylation of the small (alpha) subunit by a specific protein kinase. Phosphorylation results in an inhibition of protein synthesis. In order to determine whether or not an analogous system is operative in yeast, the phosphorylation state of the alpha subunit of elF-2 in Saccharomyces was determined during various growth and nongrowth conditions. Cells were radiolabelled with /sup 32/P and /sup 35/S, and the whole cell lysates were analyzed by two dimensional gel electrophoresis. These experiments revealed that the smallest subunit (alpha, M/sub r/ = 31,000) is a phosphoprotein in vivo under a variety of growth and nongrowth conditions. This is in direct contrast to the pattern exhibited in mammalian cells. The fact that the small subunit of elF-2 in yeast is phosphorylated under a variety of physiological conditions indicates that such a covalent modification is important for some aspects of elF-2 function. In order to investigate this problem further, a protein kinase that specifically labels the alpha subunit of elF-2 in vitro was isolated. The kinase is not autophosphorylating, utilizes ATP as a phosphate donor, phosphorylates an exogenous protein, casein, modifies serine residues in elF-2, is cyclic nucleotide-independent, and is strongly inhibited by heparin.

  10. Quantitative profiling of spreading-coupled protein tyrosine phosphorylation in migratory cells

    PubMed Central

    Xie, Yajun; Wang, Jinlong; Zhang, Yuanya; Liu, Xiaofei; Wang, Xiaorong; Liu, Kehui; Huang, Xiahe; Wang, Yingchun

    2016-01-01

    Protein tyrosine phosphorylation is an important mechanism that regulates cytoskeleton reorganization and cell spreading of migratory cells. A number of cytoskeletal proteins are known to be tyrosine phosphorylated (pY) in different cellular processes. However, the profile of pY proteins during different stages of cell spreading has not been available. Using immunoafffinity enrichment of pY proteins coupled with label free quantitative proteomics, we quantitatively identified 447 pY proteins in the migratory ECV-304 cells at the early spreading (adhesion) and the active spreading stages. We found that pY levels of the majority of the quantified proteins were significantly increased in the active spreading stage compared with the early spreading stage, suggesting that active cell spreading is concomitant with extra tyrosine phosphorylation. The major categories of proteins impacted by tyrosine phosphorylation are involved in cytoskeleton and focal adhesion regulation, protein translation and degradation. Our findings, for the first time, dissect the cell spreading-specific pY signals from the adhesion induced pY signals, and provide a valuable resource for the future mechanistic research regarding the regulation of cell spreading. PMID:27554326

  11. Controllability of protein-protein interaction phosphorylation-based networks: Participation of the hub 14-3-3 protein family.

    PubMed

    Uhart, Marina; Flores, Gabriel; Bustos, Diego M

    2016-01-01

    Posttranslational regulation of protein function is an ubiquitous mechanism in eukaryotic cells. Here, we analyzed biological properties of nodes and edges of a human protein-protein interaction phosphorylation-based network, especially of those nodes critical for the network controllability. We found that the minimal number of critical nodes needed to control the whole network is 29%, which is considerably lower compared to other real networks. These critical nodes are more regulated by posttranslational modifications and contain more binding domains to these modifications than other kinds of nodes in the network, suggesting an intra-group fast regulation. Also, when we analyzed the edges characteristics that connect critical and non-critical nodes, we found that the former are enriched in domain-to-eukaryotic linear motif interactions, whereas the later are enriched in domain-domain interactions. Our findings suggest a possible structure for protein-protein interaction networks with a densely interconnected and self-regulated central core, composed of critical nodes with a high participation in the controllability of the full network, and less regulated peripheral nodes. Our study offers a deeper understanding of complex network control and bridges the controllability theorems for complex networks and biological protein-protein interaction phosphorylation-based networked systems. PMID:27195976

  12. Controllability of protein-protein interaction phosphorylation-based networks: Participation of the hub 14-3-3 protein family

    PubMed Central

    Uhart, Marina; Flores, Gabriel; Bustos, Diego M.

    2016-01-01

    Posttranslational regulation of protein function is an ubiquitous mechanism in eukaryotic cells. Here, we analyzed biological properties of nodes and edges of a human protein-protein interaction phosphorylation-based network, especially of those nodes critical for the network controllability. We found that the minimal number of critical nodes needed to control the whole network is 29%, which is considerably lower compared to other real networks. These critical nodes are more regulated by posttranslational modifications and contain more binding domains to these modifications than other kinds of nodes in the network, suggesting an intra-group fast regulation. Also, when we analyzed the edges characteristics that connect critical and non-critical nodes, we found that the former are enriched in domain-to-eukaryotic linear motif interactions, whereas the later are enriched in domain-domain interactions. Our findings suggest a possible structure for protein-protein interaction networks with a densely interconnected and self-regulated central core, composed of critical nodes with a high participation in the controllability of the full network, and less regulated peripheral nodes. Our study offers a deeper understanding of complex network control and bridges the controllability theorems for complex networks and biological protein-protein interaction phosphorylation-based networked systems. PMID:27195976

  13. Analysis of the Protein Phosphotome of Entamoeba histolytica Reveals an Intricate Phosphorylation Network

    PubMed Central

    Anwar, Tamanna; Gourinath, Samudrala

    2013-01-01

    Phosphorylation is the most common mechanism for the propagation of intracellular signals. Protein phosphatases and protein kinases play a dynamic antagonistic role in protein phosphorylation. Protein phosphatases make up a significant fraction of eukaryotic proteome. In this article, we report the identification and analysis of protein phosphatases in the intracellular parasite Entamoeba histolytica. Based on an in silico analysis, we classified 250 non-redundant protein phosphatases in E. histolytica. The phosphotome of E. histolytica is 3.1% of its proteome and 1.3 times of the human phosphotome. In this extensive study, we identified 42 new putative phosphatases (39 hypothetical proteins and 3 pseudophosphatases). The presence of pseudophosphatases may have an important role in virulence of E. histolytica. A comprehensive phosphotome analysis of E. histolytica shows spectacular low similarity to human phosphatases, making them potent candidates for drug target. PMID:24236039

  14. The cytotoxicity of the parvovirus minute virus of mice nonstructural protein NS1 is related to changes in the synthesis and phosphorylation of cell proteins.

    PubMed Central

    Anouja, F; Wattiez, R; Mousset, S; Caillet-Fauquet, P

    1997-01-01

    Autonomous parvoviruses exert lytic and cytostatic effects believed to contribute to their antineoplastic activity. Studies with inducible clones have demonstrated a direct involvement of parvovirus nonstructural proteins (NS) in oncolysis. Human and rat fibroblasts have been stably transfected with MVM(p) (minute virus of mice prototype strain) NS genes cloned under the control of a hormone-inducible promoter. Dexamethasone-induced synthesis of the NS proteins in sensitive transformed cells results in cell killing within a few days. From these sensitive cell lines have been isolated some NS-resistant clones that also prove resistant to MVM(p) infection, suggesting that cell factors modulate NS cytotoxicity. We have previously reported that factors involved in cell cycle regulation may contribute to this modulation, since NS toxicity requires cell proliferation and correlates with a cell cycle perturbation leading to an arrest in phase S/G2. In addition to its role in cytotoxicity, NS1 can regulate transcription driven by parvovirus and nonparvovirus promoters. Since phosphorylation is a critical event in controlling the activity of many proteins, notably transcription factors and cell cycle-regulated proteins, we have examined the effect of NS1 on the synthesis and phosphorylation of cell proteins. Our results indicate that NS1 interferes, within 7 h of induction, with phosphorylation of a protein of about 14 kDa (p14). Cell synchronization has enabled us to show that phosphorylation of this protein occurs in early S phase and is prevented when NS1 is induced. This early effect of NS1 on p14 phosphorylation may be directly linked to cytotoxicity and is probably related to the previously reported inhibition of cell DNA synthesis. Late in the induction period (24 h), NS1 also alters the synthesis of a 50-kDa protein and a 35-kDa protein (p50 and p35, respectively). Microsequencing of p35 reveals sequence homology with beta-tubulin. These effects of NS1, observed

  15. Immunoprecipitation of Plasma Membrane Receptor-Like Kinases for Identification of Phosphorylation Sites and Associated Proteins.

    PubMed

    Kadota, Yasuhiro; Macho, Alberto P; Zipfel, Cyril

    2016-01-01

    Membrane proteins are difficult to study for numerous reasons. The surface of membrane proteins is relatively hydrophobic and sometimes very unstable, additionally requiring detergents for their extraction from the membrane. This leads to challenges at all levels, including expression, solubilization, purification, identification of associated proteins, and the identification of post-translational modifications. However, recent advances in immunoprecipitation technology allow to isolate membrane proteins efficiently, facilitating the study of protein-protein interactions, the identification of novel associated proteins, and to identify post-translational modifications, such as phosphorylation. Here, we describe an optimized immunoprecipitation protocol for plant plasma membrane receptor-like kinases. PMID:26577786

  16. Predicting protein phosphorylation from gene expression: top methods from the IMPROVER Species Translation Challenge

    PubMed Central

    Biehl, Michael; Bilal, Erhan; Hormoz, Sahand; Meyer, Pablo; Norel, Raquel; Rhrissorrakrai, Kahn; Bhanot, Gyan; Luo, Feng; Tarca, Adi L.

    2015-01-01

    Motivation: Using gene expression to infer changes in protein phosphorylation levels induced in cells by various stimuli is an outstanding problem. The intra-species protein phosphorylation challenge organized by the IMPROVER consortium provided the framework to identify the best approaches to address this issue. Results: Rat lung epithelial cells were treated with 52 stimuli, and gene expression and phosphorylation levels were measured. Competing teams used gene expression data from 26 stimuli to develop protein phosphorylation prediction models and were ranked based on prediction performance for the remaining 26 stimuli. Three teams were tied in first place in this challenge achieving a balanced accuracy of about 70%, indicating that gene expression is only moderately predictive of protein phosphorylation. In spite of the similar performance, the approaches used by these three teams, described in detail in this article, were different, with the average number of predictor genes per phosphoprotein used by the teams ranging from 3 to 124. However, a significant overlap of gene signatures between teams was observed for the majority of the proteins considered, while Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched in the union of the predictor genes of the three teams for multiple proteins. Availability and implementation: Gene expression and protein phosphorylation data are available from ArrayExpress (E-MTAB-2091). Software implementation of the approach of Teams 49 and 75 are available at http://bioinformaticsprb.med.wayne.edu and http://people.cs.clemson.edu/∼luofeng/sbv.rar, respectively. Contact: gyanbhanot@gmail.com or luofeng@clemson.edu or atarca@med.wayne.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25061067

  17. Soluble amyloid precursor protein alpha inhibits tau phosphorylation through modulation of GSK3β signaling pathway.

    PubMed

    Deng, Juan; Habib, Ahsan; Obregon, Demian F; Barger, Steven W; Giunta, Brian; Wang, Yan-Jiang; Hou, Huayan; Sawmiller, Darrell; Tan, Jun

    2015-11-01

    We recently found that sAPPα decreases amyloid-beta generation by directly associating with β-site amyloid precursor protein (APP)-converting enzyme 1 (BACE1), thereby modulating APP processing. Because inhibition of BACE1 decreases glycogen synthase kinase 3 beta (GSK3β)-mediated Alzheimer's disease (AD)-like tau phosphorylation in AD patient-derived neurons, we determined whether sAPPα also reduces GSK3β-mediated tau phosphorylation. We initially found increased levels of inhibitory phosphorylation of GSK3β (Ser9) in primary neurons from sAPPα over-expressing mice. Further, recombinant human sAPPα evoked the same phenomenon in SH-SY5Y cells. Further, in SH-SY5Y cells over-expressing BACE1, and HeLa cells over-expressing human tau, sAPPα reduced GSK3β activity and tau phosphorylation. Importantly, the reductions in GSK3β activity and tau phosphorylation elicited by sAPPα were prevented by BACE1 but not γ-secretase inhibition. In accord, AD mice over-expressing human sAPPα had less GSK3β activity and tau phosphorylation compared with controls. These results implicate a direct relationship between APP β-processing and GSK3β-mediated tau phosphorylation and further define the central role of sAPPα in APP autoregulation and AD pathogenesis. PMID:26342176

  18. Tyrosine phosphorylation of band 3 protein in Ca2+/A23187-treated human erythrocytes.

    PubMed Central

    Minetti, G; Piccinini, G; Balduini, C; Seppi, C; Brovelli, A

    1996-01-01

    Human erythrocytes were induced to release membrane vesicles by treatment with Ca2+ and ionophore A23187. In addition to the biochemical changes already known to accompany loading of human erythrocytes with Ca2+, the present study reveals that tyrosine phosphorylation of the anion exchanger band 3 protein also occurs. The relationship between tyrosine phosphorylation of band 3 and membrane vesiculation was analysed using quinine (a non-specific inhibitor of the Ca(2+)-activated K+ channel, and the only known inhibitor of Ca(2+)-induced vesiculation) and charybdotoxin, a specific inhibitor of the apamin-insensitive K(+)-channel. Both inhibitors suppressed tyrosine phosphorylation of band 3. In the presence of quinine, membrane vesiculation was also suppressed. In contrast, at the concentration of charybdotoxin required to suppress tyrosine phosphorylation of band 3, membrane vesiculation was only mildly inhibited (16-23% inhibition), suggesting that tyrosine phosphorylation of band 3 is not necessary for membrane vesiculation. Phosphorylation of band 3 was in fact observed when erythrocytes were induced to shrink in a Ca(2+)-independent manner, e.g. by treatment with the K+ ionophore valinomycin or with hypertonic solutions. These observations suggest that band 3 tyrosine phosphorylation occurs when cell volume regulation is required. PMID:8973551

  19. Phosphorylation at intrinsically disordered regions of PAM2 motif-containing proteins modulates their interactions with PABPC1 and influences mRNA fate.

    PubMed

    Huang, Kai-Lieh; Chadee, Amanda B; Chen, Chyi-Ying A; Zhang, Yueqiang; Shyu, Ann-Bin

    2013-03-01

    Cytoplasmic poly(A)-binding protein (PABP) C1 recruits different interacting partners to regulate mRNA fate. The majority of PABP-interacting proteins contain a PAM2 motif to mediate their interactions with PABPC1. However, little is known about the regulation of these interactions or the corresponding functional consequences. Through in silico analysis, we found that PAM2 motifs are generally embedded within an extended intrinsic disorder region (IDR) and are located next to cluster(s) of potential serine (Ser) or threonine (Thr) phosphorylation sites within the IDR. We hypothesized that phosphorylation at these Ser/Thr sites regulates the interactions between PAM2-containing proteins and PABPC1. In the present study, we have tested this hypothesis using complementary approaches to increase or decrease phosphorylation. The results indicate that changing the extent of phosphorylation of three PAM2-containing proteins (Tob2, Pan3, and Tnrc6c) alters their ability to interact with PABPC1. Results from experiments using phospho-blocking or phosphomimetic mutants in PAM2-containing proteins further support our hypothesis. Moreover, the phosphomimetic mutations appreciably affected the functions of these proteins in mRNA turnover and gene silencing. Taken together, these results provide a new framework for understanding the roles of intrinsically disordered proteins in the dynamic and signal-dependent control of cytoplasmic mRNA functions. PMID:23340509

  20. Regulatory phosphorylation of the p34cdc2 protein kinase in vertebrates.

    PubMed Central

    Norbury, C; Blow, J; Nurse, P

    1991-01-01

    The p34cdc2 protein kinase is a conserved regulator of the eukaryotic cell cycle. Here we show that residues Thr14 and Tyr15 of mouse p34cdc2 become phosphorylated as mouse fibroblasts proceed through the cell cycle. We have mutated these residues and measured protein kinase activity of the p34cdc2 variants in a Xenopus egg extract. Phosphorylation of residues 14 and 15, which lie within the presumptive ATP-binding region of p34cdc2, normally restrains the protein kinase until it is specifically dephosphorylated and activated at the G2/M transition. Regulation by dephosphorylation of Tyr15 is conserved from fission yeast to mammals, while an extra level of regulation of mammalian p34cdc2 involves Thr14 dephosphorylation. In the absence of phosphorylation on these two residues, the kinase still requires cyclin B protein for its activation. Inhibition of DNA synthesis inhibits activation of wild-type p34cdc2 in the Xenopus system, but a mutant which cannot be phosphorylated at residues 14 and 15 escapes this inhibition, suggesting that these phosphorylation events form part of the pathway linking completion of DNA replication to initiation of mitosis. Images PMID:1655417

  1. Calcium ion binding properties and the effect of phosphorylation on the intrinsically disordered Starmaker protein.

    PubMed

    Wojtas, Magdalena; Hołubowicz, Rafał; Poznar, Monika; Maciejewska, Marta; Ożyhar, Andrzej; Dobryszycki, Piotr

    2015-10-27

    Starmaker (Stm) is an intrinsically disordered protein (IDP) involved in otolith biomineralization in Danio rerio. Stm controls calcium carbonate crystal formation in vivo and in vitro. Phosphorylation of Stm affects its biomineralization properties. This study examined the effects of calcium ions and phosphorylation on the structure of Stm. We have shown that CK2 kinase phosphorylates 25 or 26 residues in Stm. Furthermore, we have demonstrated that Stm's affinity for calcium binding is dependent on its phosphorylation state. Phosphorylated Stm (StmP) has an estimated 30 ± 1 calcium binding sites per protein molecule with a dissociation constant (KD) of 61 ± 4 μM, while the unphosphorylated protein has 28 ± 3 sites and a KD of 210 ± 22 μM. Calcium ion binding induces a compaction of the Stm molecule, causing a significant decrease in its hydrodynamic radius and the formation of a secondary structure. The screening effect of Na(+) ions on calcium binding was also observed. Analysis of the hydrodynamic properties of Stm and StmP showed that Stm and StmP molecules adopt the structure of native coil-like proteins. PMID:26445027

  2. An MRM-based Workflow for Quantifying Cardiac Mitochondrial Protein Phosphorylation in Murine and Human Tissue

    PubMed Central

    Lam, Maggie P.Y.; Scruggs, Sarah B.; Kim, Tae-Young; Zong, Chenggong; Lau, Edward; Wang, Ding; Ryan, Christopher M.; Faull, Kym F.; Ping, Peipei

    2012-01-01

    The regulation of mitochondrial function is essential for cardiomyocyte adaptation to cellular stress. While it has long been understood that phosphorylation regulates flux through metabolic pathways, novel phosphorylation sites are continually being discovered in all functionally distinct areas of the mitochondrial proteome. Extracting biologically meaningful information from these phosphorylation sites requires an adaptable, sensitive, specific and robust method for their quantification. Here we report a multiple reaction monitoring-based mass spectrometric workflow for quantifying site-specific phosphorylation of mitochondrial proteins. Specifically, chromatographic and mass spectrometric conditions for 68 transitions derived from 23 murine and human phosphopeptides, and their corresponding unmodified peptides, were optimized. These methods enabled the quantification of endogenous phosphopeptides from the outer mitochondrial membrane protein VDAC, and the inner membrane proteins ANT and ETC complexes I, III and V. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of mitochondrial protein phosphorylation in cardiac physiology and pathophysiology. PMID:22387130

  3. Phosphorylation of the proapoptotic BH3-only protein bid primes mitochondria for apoptosis during mitotic arrest.

    PubMed

    Wang, Pengbo; Lindsay, Jennefer; Owens, Thomas W; Mularczyk, Ewa J; Warwood, Stacey; Foster, Fiona; Streuli, Charles H; Brennan, Keith; Gilmore, Andrew P

    2014-05-01

    Mitosis is a moment of exquisite vulnerability for a metazoan cell. Failure to complete mitosis accurately can lead to aneuploidy and cancer initiation. Therefore, if the exit from mitosis is delayed, normal cells are usually removed by apoptosis. However, how failure to complete mitosis activates apoptosis is still unclear. Here, we demonstrate that a phosphorylated form of the BH3-only protein Bid regulates apoptosis if mitotic exit is delayed. Bid is phosphorylated on serine 66 as cells enter mitosis, and this phosphorylation is lost during the metaphase-to-anaphase transition. Cells expressing a nonphosphorylatable version of Bid or a BH3-domain mutant were resistant to mitotic-arrest-induced apoptosis. Thus, we show that Bid phosphorylation primes cells to undergo mitochondrial apoptosis if mitotic exit is delayed. Avoidance of this mechanism may explain the selective pressure for cancer cells to undergo mitotic slippage. PMID:24767991

  4. Mitochondrial Protein Phosphorylation as a Regulatory Modality: Implications for Mitochondrial Dysfunction in Heart Failure

    PubMed Central

    O’Rourke, Brian; Van Eyk, Jennifer E.; Foster, D. Brian

    2014-01-01

    Phosphorylation of mitochondrial proteins has been recognized for decades, and the regulation of pyruvate- and branched-chain α-ketoacid dehydrogenases by an atypical kinase/phosphatase cascade is well established. More recently, the development of new mass spectrometry-based technologies has led to the discovery of many novel phosphorylation sites on a variety of mitochondrial targets. The evidence suggests that the major classes of kinase and several phosphatases may be present at the mitochondrial outer membrane, intermembrane space, inner membrane, and matrix, but many questions remain to be answered as to the location, timing, and reversibility of these phosphorylation events and whether they are functionally relevant. The authors review phosphorylation as a mitochondrial regulatory strategy and highlight its possible role in the pathophysiology of cardiac hypertrophy and failure. PMID:22103918

  5. The Binding of Plasmodium falciparum Adhesins and Erythrocyte Invasion Proteins to Aldolase Is Enhanced by Phosphorylation.

    PubMed

    Diaz, Suraya A; Martin, Stephen R; Howell, Steven A; Grainger, Munira; Moon, Robert W; Green, Judith L; Holder, Anthony A

    2016-01-01

    Aldolase has been implicated as a protein coupling the actomyosin motor and cell surface adhesins involved in motility and host cell invasion in the human malaria parasite Plasmodium falciparum. It binds to the cytoplasmic domain (CTD) of type 1 membrane proteins of the thrombospondin-related anonymous protein (TRAP) family. Other type 1 membrane proteins located in the apical organelles of merozoites, the form of the parasite that invades red blood cells, including apical membrane antigen 1 (AMA1) and members of the erythrocyte binding ligand (EBL) and reticulocyte binding homologue (RH) protein families have been implicated in host cell binding and invasion. Using a direct binding method we confirm that TRAP and merozoite TRAP (MTRAP) bind aldolase and show that the interaction is mediated by more than just the C-terminal six amino acid residues identified previously. Single amino acid substitutions in the MTRAP CTD abolished binding to aldolase. The CTDs of AMA1 and members of the EBL and RH protein families also bound to aldolase. MTRAP competed with AMA1 and RH4 for binding to aldolase, indicating overlapping binding sites. MTRAP CTD was phosphorylated in vitro by both calcium dependent kinase 1 (CDPK1) and protein kinase A, and this modification increased the affinity of binding to aldolase by ten-fold. Phosphorylation of the CTD of members of the EBL and RH protein families also increased their affinity for aldolase in some cases. To examine whether or not MTRAP expressed in asexual blood stage parasites is phosphorylated, it was tagged with GFP, purified and analysed, however no phosphorylation was detected. We propose that CTD binding to aldolase may be dynamically modulated by phosphorylation, and there may be competition for aldolase binding between different CTDs. The use and efficiency of alternate invasion pathways may be determined by the affinity of adhesins and cell invasion proteins for aldolase, in addition to their host ligand specificity. PMID

  6. Nerve Agent Exposure Elicits Site-Specific Changes in Protein Phosphorylation in Mouse Brain

    PubMed Central

    Zhu, Hongwen; O’Brien, Jennifer J.; O’Callaghan, James P.; Miller, Diane B.; Zhang, Qiang; Rana, Minal; Tsui, Tiffany; Peng, Youyi; Tomesch, John; Hendrick, Joseph P.; Wennogle, Lawrence P; Snyder, Gretchen L.

    2010-01-01

    Organophosphorus (OP) compounds cause toxic symptoms, including convulsions, coma, and death, as the result of irreversible inhibition of acetylcholinesterase (AChE). The development of effective treatments to block these effects and attenuate long-term cognitive and motor disabilities that result from OP intoxication is hampered by a limited understanding of the CNS pathways responsible for these actions. We employed a candidate method (called CNSProfile™) to identify changes in the phosphorylation state of key neuronal phosphoproteins evoked by the OP compound, diisopropyl fluorophosphate (DFP). Focused microwave fixation was used to preserve the phosphorylation state of phosphoproteins in brains of DFP-treated mice; hippocampus and striatum were analyzed by immunoblotting with a panel of phospho-specific antibodies. DFP exposure elicited comparable effects on phosphorylation of brain phosphoproteins in both C57BL/6 and FVB mice. DFP treatment significantly altered phosphorylation at regulatory residues on glutamate receptors, including Serine897 (S897) of the NR1 NMDA receptor. NR1 phosphorylation was bi-directionally regulated after DFP in striatum versus hippocampus. NR1 phosphorylation was reduced in striatum, but elevated in hippocampus, compared with controls. DARPP-32 phosphorylation in striatum was selectively increased at the Cdk5 kinase substrate, Threonine75 (T75). Phencynonate hydrochloride, a muscarinic cholinergic antagonist, prevented seizure-like behaviors and the observed changes in phosphorylation induced by DFP. The data reveal region-specific effects of nerve agent exposure on intracellular signaling pathways that correlate with seizure-like behavior and which are reversed by the muscarinic receptor blockade. This approach identifies specific targets for nerve agents, including substrates for Cdk5 kinase, which may be the basis for new anti-convulsant therapies. PMID:20423708

  7. The virion N protein of infectious bronchitis virus is more phosphorylated than the N protein from infected cell lysates

    SciTech Connect

    Jayaram, Jyothi; Youn, Soonjeon; Collisson, Ellen W. . E-mail: ecollisson@cvm.tamu.edu

    2005-08-15

    Because phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid protein (N) may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. {sup 32}P-orthophosphate labeling and Western blot analyses confirmed that N was the only viral protein that was phosphorylated. Pulse labeling with {sup 32}P-orthophosphate indicated that the IBV N protein was phosphorylated in the virion, as well as at all times during infection in either chicken embryo kidney cells or Vero cells. Pulse-chase analyses followed by immunoprecipitation of IBV N proteins using rabbit anti-IBV N polyclonal antibody demonstrated that the phosphate on the N protein was stable for at least 1 h. Simultaneous labeling with {sup 32}P-orthophosphate and {sup 3}H-leucine identified a 3.5-fold increase in the {sup 32}P:{sup 3}H counts per minute (cpm) ratio of N in the virion as compared to the {sup 32}P:{sup 3}H cpm ratio of N in the cell lysates from chicken embryo kidney cells, whereas in Vero cells the {sup 32}P:{sup 3}H cpm ratio of N from the virion was 10.5-fold greater than the {sup 32}P:{sup 3}H cpm ratio of N from the cell lysates. These studies are consistent with the phosphorylation of the IBV N playing a role in assembly or maturation of the viral particle.

  8. Genetic mapping of a major site of phosphorylation in adenovirus type 2 E1A proteins

    SciTech Connect

    Tsukamotot, A.S.; Ponticelli, A.; Berk, A.J.; Gaynor, R.B.

    1986-07-01

    Adenovirus early region 1A (E1A) encodes two acidic phosphoproteins which are required for transactivation of viral transcription, efficient viral DNA replication in phase G/sub 0/-arrested human cells, and oncogenic transformation of rodent cells. Biochemical analysis of in vivo /sup 32/P-labeled adenovirus type 2 E1A proteins purified with monoclonal antibodies demonstrated that these proteins were phosphorylated at multiple serine residues. Two-dimensional phosphotryptic peptide maps of wild-type and mutant E1A proteins were used to locate a major site of E1A protein phosphorylation at serine-219 of the large E1A protein. Although this serine fell within a consensus sequence for phosphorylation by the cyclic AMP-dependent protein kinases, experiments with mutant CHO cells defective in these enzymes indicated that it was not. Oligonucleotide-directed mutagenesis was used to substitute an alanine for serine-219. This mutation prevented phosphorylation at this site. Nonetheless, the mutant was indistinguishable from the wild type for early gene transactivation, replication on G/sub 0/-arrested WI-38 cells, and transformation of cloned rat embryo fibroblast cells.

  9. Effects of Yiqi Tongyang on HCN4 Protein Phosphorylation in Damaged Rabbit Sinoatrial Node Cells

    PubMed Central

    Liu, Jinfeng; Liu, Ruxiu; Peng, Jie; Wang, Yanli

    2016-01-01

    The hyperpolarization-activated cyclic nucleotide-gated cation channel (If) is closely associated with sinoatrial node pacing function. The present study aimed to investigate the molecular mechanisms involved in pacing function improvements of damaged sinoatrial node cells and the consequent treatment effects on sick sinus syndrome (SSS) after the use of Yiqi Tongyang. HCN4 channel protein expression and phosphorylation were measured by immunoblotting and fluorescent quantitation. After ischemia-reperfusion injury (model group), the HCN4 protein and the optical density (OD) of the phosphorylated HCN4 protein as well as intracellular PKA activity in the sinoatrial node cells decreased significantly. However, the OD values and PKA activity increased to different degrees after treatment with serum containing different doses of Yiqi Tongyang; in contrast, no significant improvement was seen in the control group compared to the model group. These findings demonstrated that the use of the traditional Chinese medicine Yiqi Tongyang could increase HCN4 protein expression and phosphorylation as well as PKA activity within sinoatrial node cells damaged by ischemia-reperfusion. The HCN4 protein is involved in the If-related ion channel. Here, we speculated that these effects could be associated with upregulation of HCN4 protein phosphorylation, which consequently improved cell automaticity, increased heart rate, and had treatment effects on SSS. PMID:27069490

  10. Phosphorylation and calcium antagonistically tune myosin-binding protein C's structure and function.

    PubMed

    Previs, Michael J; Mun, Ji Young; Michalek, Arthur J; Previs, Samantha Beck; Gulick, James; Robbins, Jeffrey; Warshaw, David M; Craig, Roger

    2016-03-22

    During each heartbeat, cardiac contractility results from calcium-activated sliding of actin thin filaments toward the centers of myosin thick filaments to shorten cellular length. Cardiac myosin-binding protein C (cMyBP-C) is a component of the thick filament that appears to tune these mechanochemical interactions by its N-terminal domains transiently interacting with actin and/or the myosin S2 domain, sensitizing thin filaments to calcium and governing maximal sliding velocity. Both functional mechanisms are potentially further tunable by phosphorylation of an intrinsically disordered, extensible region of cMyBP-C's N terminus, the M-domain. Using atomic force spectroscopy, electron microscopy, and mutant protein expression, we demonstrate that phosphorylation reduced the M-domain's extensibility and shifted the conformation of the N-terminal domain from an extended structure to a compact configuration. In combination with motility assay data, these structural effects of M-domain phosphorylation suggest a mechanism for diminishing the functional potency of individual cMyBP-C molecules. Interestingly, we found that calcium levels necessary to maximally activate the thin filament mitigated the structural effects of phosphorylation by increasing M-domain extensibility and shifting the phosphorylated N-terminal fragments back to the extended state, as if unphosphorylated. Functionally, the addition of calcium to the motility assays ablated the impact of phosphorylation on maximal sliding velocities, fully restoring cMyBP-C's inhibitory capacity. We conclude that M-domain phosphorylation may have its greatest effect on tuning cMyBP-C's calcium-sensitization of thin filaments at the low calcium levels between contractions. Importantly, calcium levels at the peak of contraction would allow cMyBP-C to remain a potent contractile modulator, regardless of cMyBP-C's phosphorylation state. PMID:26908872

  11. POSSIBLE LINK BETWEEN METHIONINE OXIDATION AND PROTEIN PHOSPHORYLATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aerobic metabolism leads to the production of reactive oxygen species that may damage proteins. Methionine residues in proteins are particularly susceptible to oxidation to methionine sulfoxide (MetSO) converting its side chain from hydrophobic to hydrophilic. We postulated that this could have a si...

  12. STATE TRANSITION7-Dependent Phosphorylation Is Modulated by Changing Environmental Conditions, and Its Absence Triggers Remodeling of Photosynthetic Protein Complexes1

    PubMed Central

    Bergner, Sonja Verena; Scholz, Martin; Trompelt, Kerstin; Barth, Johannes; Gäbelein, Philipp; Steinbeck, Janina; Xue, Huidan; Clowez, Sophie; Fucile, Geoffrey; Goldschmidt-Clermont, Michel; Fufezan, Christian; Hippler, Michael

    2015-01-01

    In plants and algae, the serine/threonine kinase STN7/STT7, orthologous protein kinases in Chlamydomonas reinhardtii and Arabidopsis (Arabidopsis thaliana), respectively, is an important regulator in acclimation to changing light environments. In this work, we assessed STT7-dependent protein phosphorylation under high light in C. reinhardtii, known to fully induce the expression of LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3) and a nonphotochemical quenching mechanism, in relationship to anoxia where the activity of cyclic electron flow is stimulated. Our quantitative proteomics data revealed numerous unique STT7 protein substrates and STT7-dependent protein phosphorylation variations that were reliant on the environmental condition. These results indicate that STT7-dependent phosphorylation is modulated by the environment and point to an intricate chloroplast phosphorylation network responding in a highly sensitive and dynamic manner to environmental cues and alterations in kinase function. Functionally, the absence of the STT7 kinase triggered changes in protein expression and photoinhibition of photosystem I (PSI) and resulted in the remodeling of photosynthetic complexes. This remodeling initiated a pronounced association of LHCSR3 with PSI-LIGHT HARVESTING COMPLEX I (LHCI)-ferredoxin-NADPH oxidoreductase supercomplexes. Lack of STT7 kinase strongly diminished PSII-LHCII supercomplexes, while PSII core complex phosphorylation and accumulation were significantly enhanced. In conclusion, our study provides strong evidence that the regulation of protein phosphorylation is critical for driving successful acclimation to high light and anoxic growth environments and gives new insights into acclimation strategies to these environmental conditions. PMID:25858915

  13. Interplay between lysine methylation and Cdk phosphorylation in growth control by the retinoblastoma protein

    PubMed Central

    Carr, Simon M; Munro, Shonagh; Kessler, Benedikt; Oppermann, Udo; La Thangue, Nicholas B

    2011-01-01

    As a critical target for cyclin-dependent kinases (Cdks), the retinoblastoma tumour suppressor protein (pRb) controls early cell cycle progression. We report here a new type of regulation that influences Cdk recognition and phosphorylation of substrate proteins, mediated through the targeted methylation of a critical lysine residue in the Cdk substrate recognition site. In pRb, lysine (K) 810 represents the essential and conserved basic residue (SPXK) required for cyclin/Cdk recognition and phosphorylation. Methylation of K810 by the methyltransferase Set7/9 impedes binding of Cdk and thereby prevents subsequent phosphorylation of the associated serine (S) residue, retaining pRb in the hypophosphorylated growth-suppressing state. Methylation of K810 is under DNA damage control, and methylated K810 impacts on phosphorylation at sites throughout the pRb protein. Set7/9 is required for efficient cell cycle arrest, and significantly, a mutant derivative of pRb that cannot be methylated at K810 exhibits compromised cell cycle arrest. Thus, the regulation of phosphorylation by Cdks reflects the combined interplay with methylation events, and more generally the targeted methylation of a lysine residue within a Cdk-consensus site in pRb represents an important point of control in cell cycle progression. PMID:21119616

  14. Quantitative Phosphoproteomics Reveals the Role of Protein Arginine Phosphorylation in the Bacterial Stress Response*

    PubMed Central

    Schmidt, Andreas; Trentini, Débora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

    2014-01-01

    Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response. PMID:24263382

  15. The selective phosphorylation of a guanine nucleotide-binding regulatory protein

    SciTech Connect

    Carlson, K.E.

    1989-01-01

    Receptor-activated signal transduction pathways regulate the responsiveness of cells to external stimuli. These transduction pathways themselves are subject to regulation, most commonly by phosphorylation. Guanine nucleotide-binding regulatory proteins (G Proteins), as requisite signal transducing elements for many plasma membrane receptors, are considered likely targets for regulation by phosphorylation. Protein kinase C (PKC) has been shown to phosphorylate the {alpha} subunit of G{sub i} and other G proteins in solution. However, the occurrence of the phosphorylation of G{sub 1} within intact cells in response to activation of PKC has not been rigorously demonstrated. In this thesis, the extent to which the {alpha} subunits of G{sub i} undergo phosphorylation within human platelets in response to activation of PKC was examined by means of radiolabeling and immunoprecipitation. Incubation of platelets with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, promoted the phosphorylation of several proteins within saponin-permeabilized and intact platelets incubated with ({gamma}{sup 32}P)ATP and ({sup 32}P)H{sub 3}PO{sub 4}, respectively. None of the phosphoproteins, however, were precipitated by either of two antisera containing antibodies differing in specificities for epitopes within G{sub i{alpha}}-despite precipitation of a substantial fraction of the subunit itself. In contrast, other antisera, containing antibodies specific for the recently describe G{sub z{alpha}}, or antibodies for both G{sub z{alpha}} and G{sub i{alpha}}, precipitated a 40-kDa phosphoprotein.

  16. Global Detection of Protein Kinase D-dependent Phosphorylation Events in Nocodazole-treated Human Cells*

    PubMed Central

    Franz-Wachtel, Mirita; Eisler, Stephan A.; Krug, Karsten; Wahl, Silke; Carpy, Alejandro; Nordheim, Alfred; Pfizenmaier, Klaus; Hausser, Angelika; Macek, Boris

    2012-01-01

    Protein kinase D (PKD) is a cytosolic serine/threonine kinase implicated in regulation of several cellular processes such as response to oxidative stress, directed cell migration, invasion, differentiation, and fission of the vesicles at the trans-Golgi network. Its variety of functions must be mediated by numerous substrates; however, only a couple of PKD substrates have been identified so far. Here we perform stable isotope labeling of amino acids in cell culture-based quantitative phosphoproteomic analysis to detect phosphorylation events dependent on PKD1 activity in human cells. We compare relative phosphorylation levels between constitutively active and kinase dead PKD1 strains of HEK293 cells, both treated with nocodazole, a microtubule-depolymerizing reagent that disrupts the Golgi complex and activates PKD1. We identify 124 phosphorylation sites that are significantly down-regulated upon decrease of PKD1 activity and show that the PKD target motif is significantly enriched among down-regulated phosphorylation events, pointing to the presence of direct PKD1 substrates. We further perform PKD1 target motif analysis, showing that a proline residue at position +1 relative to the phosphorylation site serves as an inhibitory cue for PKD1 activity. Among PKD1-dependent phosphorylation events, we detect predominantly proteins with localization at Golgi membranes and function in protein sorting, among them several sorting nexins and members of the insulin-like growth factor 2 receptor pathway. This study presents the first global detection of PKD1-dependent phosphorylation events and provides a wealth of information for functional follow-up of PKD1 activity upon disruption of the Golgi network in human cells. PMID:22496350

  17. In vitro and in vivo protein phosphorylation in Avena sativa L. coleoptiles: effects of Ca2+, calmodulin antagonists, and auxin

    NASA Technical Reports Server (NTRS)

    Veluthambi, K.; Poovaiah, B. W.

    1986-01-01

    In vitro and in vivo protein phosphorylations in oat (Avena sativa L.) coleoptile segments were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by two-dimensional gel electrophoresis. In vitro phosphorylation of several polypeptides was distinctly promoted at 1 to 15 micromolar free Ca2+ concentrations. Ca2(+)-stimulated phosphorylation was markedly reduced by trifluoperazine, chlorpromazine, and naphthalene sulfonamide (W7). Two polypeptides were phosphorylated both under in vitro and in vivo conditions, but the patterns of phosphorylation of several other polypeptides were different under the two conditions indicating that the in vivo phosphorylation pattern of proteins is not truly reflected by in vitro phosphorylation studies. Trifluoperazine, W7, or ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) + calcium ionophore A23187 treatments resulted in reduced levels of in vivo protein phosphorylation of both control and auxin-treated coleoptile segments. Analysis by two-dimensional electrophoresis following in vivo phosphorylation revealed auxin-dependent changes of certain polypeptides. A general inhibition of phosphorylation by calmodulin antagonists suggested that both control and auxin-treated coleoptiles exhibited Ca2+, and calmodulin-dependent protein phosphorylation in vivo.

  18. Effects of experiences on synaptic protein phosphorylation in vitro

    SciTech Connect

    Lyn-Cook, B.D.; Wilson, J.E.

    1983-06-01

    In vitro transfer of /sup 32/P from (gamma-/sup 32/P)-ATP into proteins of particulate fractions from osmotically shocked preparations enriched in rat brain synaptosomes was studied. Phosphate incorporation into protein bands of apparent molecular weights (MW) 44,000, 24,000, 21,000, and 19,000 was affected by the prior experiences of the rats from which the particulate fractions were prepared. Incorporation into all four proteins was increased in particulate fraction from previously naive rats that received active avoidance training. Handling of the subjects prior to training prevented the response of the 24,000 MW protein to training. Phosphate incorporation into 24,000 and 19,000 MW proteins was increased in preparations from previously naive rats that underwent a yoked experience, while incorporation into the 21,000 MW protein was slightly decreased. The yoked experience did not affect in vitro phosphate incorporation into any of these proteins in particulate fractions from previously handled rats.

  19. Cyclin-dependent kinase 5 phosphorylation of familial prion protein mutants exacerbates conversion into amyloid structure.

    PubMed

    Rouget, Raphaël; Sharma, Gyanesh; LeBlanc, Andréa C

    2015-02-27

    Familial prion protein (PrP) mutants undergo conversion from soluble and protease-sensitive to insoluble and partially protease-resistant proteins. Cyclin-dependent kinase 5 (Cdk5) phosphorylation of wild type PrP (pPrP) at serine 43 induces a conversion of PrP into aggregates and fibrils. Here, we investigated whether familial PrP mutants are predisposed to Cdk5 phosphorylation and whether phosphorylation of familial PrP mutants increases conversion. PrP mutants representing three major familial PrP diseases and different PrP structural domains were studied. We developed a novel in vitro kinase reaction coupled with Thioflavin T binding to amyloid structure assay to monitor phosphorylation-dependent amyloid conversion. Although non-phosphorylated full-length wild type or PrP mutants did not convert into amyloid, Cdk5 phosphorylation rapidly converted these into Thioflavin T-positive structures following first order kinetics. Dephosphorylation partially reversed conversion. Phosphorylation-dependent conversion of PrP from α-helical structures into β-sheet structures was confirmed by circular dichroism. Relative to wild type pPrP, most PrP mutants showed increased rate constants of conversion. In contrast, non-phosphorylated truncated PrP Y145X (where X represents a stop codon) and Q160X mutants converted spontaneously into Thioflavin T-positive fibrils after a lag phase of over 20 h, indicating nucleation-dependent polymerization. Phosphorylation reduced the lag phase by over 50% and thus accelerated the formation of the nucleating event. Consistently, phosphorylated Y145X and phosphorylated Q160X exacerbated conversion in a homologous seeding reaction, whereas WT pPrP could not seed WT PrP. These results demonstrate an influence of both the N terminus and the C terminus of PrP on conversion. We conclude that post-translational modifications of the flexible N terminus of PrP can cause or exacerbate PrP mutant conversion. PMID:25572400

  20. Interaction of Ca2+ and protein phosphorylation in the rabbit platelet release reaction.

    PubMed

    Lyons, R M; Shaw, J O

    1980-02-01

    Ca2+ flux and protein phosphorylation have been implicated as playing an important role in the induction of the platelet release reaction. However, the interactions between Ca2+, protein phosphorylation, and the release reaction have been difficult to study because secretion in human platelets is independent of extracellular Ca2+. Thus, we studied rabbit platelets, which, unlike human platelets, require extracellular Ca2+ for serotonin release to occur. Thrombin, basophil platelet-activating factor (PAF), or ionophore A23187 treatment of intact 32PO43--loaded rabbit platelets resulted in a 200-400% increase in phosphorylation of P7P and P9P, respectively. These peptides were similar in all respects to the peptides phosphorylated in thrombin-treated human platelets. When Ca2+ was replaced in the medium by EGTA, (a) thrombin- and PAF-induced rabbit platelet [3H]serotonin release was inhibited by 60-75%, whereas ionophore-induced release was blocked completely; (b) thrombin-, PAF-, or ionophore-induced P9P phosphorylation was inhibited by 60%; and (c) ionophore-induced P7P phosphorylation was decreased by 60%, whereas that caused by thrombin or PAF was decreased by only 20%. At 0.25-0.5 U/ml of thrombin, phosphorylation preceded [3H]serotonin release with the time for half-maximal release being 26.0 +/- 1.3 s SE (n = 3) and the time for half-maximal phosphorylation being 12.3 +/- 1.3 s SE (n = 3) for P7P and 3.7 +/- 0.17 s SE (n = 3) for P9P. P9P phosphorylation was significantly inhibited (P less than 0.015) by removal by Ca2+ from the medium at a time point before any thrombin- or ionophore-induced serotonin release was detectable. Thus, our data suggest that Ca2+ flux precedes the onset of serotonin secretion and that the rabbit platelet is an appropriate model in which to study the effects of Ca2+ on protein phosphorylation during the platelet release reaction. PMID:6985917

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

    PubMed

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

    2016-08-01

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

  2. Role of Ca/sup 2 +/ and calmodulin in phosphorylation of proteins in plants. Progress report

    SciTech Connect

    Cormier, M.J.

    1984-04-04

    The objective of the research is to provide an understanding of in vitro requirements for Ca/sup +2/ dependent protein phosphorylation in plant cells and determine whether this Ca/sup +2/ mediated phosphorylation represents one of the underlying mechanisms by which plants respond to external stimuli. A higher plant model system (soybean cells) has been identified. Both membrane and supernatant fractions were found to contain Ca/sup +2/ dependent protein kinases. Further work will concentrate on the supernatant protein fraction in order to isolate, to homogeneity, the proteins of interest. Casein and lysine-rich histones were found to be good substrates for use in an assay for one of the Ca/sup +2/ dependent kinases. Current work involves isolating and characterizing the Ca/sup +2/ dependent protein kinases, and some of their substrates from the soluble fraction of extracts of soybean cell cultures.

  3. The retinoblastoma protein is phosphorylated on multiple sites by human cdc2.

    PubMed Central

    Lees, J A; Buchkovich, K J; Marshak, D R; Anderson, C W; Harlow, E

    1991-01-01

    The retinoblastoma gene product (pRB) is a nuclear phosphoprotein that is thought to play a key role in the negative regulation of cellular proliferation. pRB is phosphorylated in a cell cycle dependent manner, and studies in both actively dividing and differentiated cells suggest that this modification may be essential for cells to progress through the cell cycle. Using tryptic phosphopeptide mapping we have shown that pRB is phosphorylated on multiple serine and threonine residues in vivo and that many of these phosphorylation events can be mimicked in vitro using purified p34cdc2. Using synthetic peptides corresponding to potential cdc2 phosphorylation sites, we have developed a strategy which has allowed the identification of five sites. S249, T252, T373, S807 and S811 are phosphorylated in vivo, and in each case these sites correspond closely to the consensus sequence for phosphorylation by p34cdc2. This and the observation that pRB forms a specific complex with p34cdc2 in vivo suggests that p34cdc2 or a p34cdc2-related protein is a major pRB kinase. Images PMID:1756735

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

    PubMed

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

    2002-08-01

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

  5. Transforming Growth Factor {beta} Can Stimulate Smad1 Phosphorylation Independently of Bone Morphogenic Protein Receptors.

    PubMed

    Wrighton, Katharine H; Lin, Xia; Yu, Paul B; Feng, Xin-Hua

    2009-04-10

    Transforming growth factor-beta (TGFbeta) superfamily ligands control a diverse set of cellular processes by activating type I and type II serine-threonine receptor kinases. Canonical TGFbeta signaling is mediated via the TbetaRI/ALK5 type I receptor that phosphorylates Smad2 and Smad3 in their SXS motif to facilitate their activation and subsequent role in transcriptional regulation. Canonical bone morphogenic protein (BMP) signaling is mediated via the ALK1/2/3/6 type I receptors that phosphorylate Smad1, Smad5, and Smad8 in their SXS motif. However, studies in endothelial cells have shown that TGFbeta can also lead to the phosphorylation of Smad1, dependent on ALK1 receptor activity. Here we present data showing that TGFbeta can significantly induce Smad1 phosphorylation in several non-endothelial cell lineages. Additionally, by using chemical inhibitors specific for the TGFbeta/activin/nodal (ALK4/5/7) and BMP (ALK1/2/3/6) type I receptors, we show that in some cell types TGFbeta induces Smad1 phosphorylation independently of the BMP type I receptors. Thus, TGFbeta-mediated Smad1 phosphorylation appears to occur via different receptor complexes in a cell type-specific manner. PMID:19224917

  6. A- Kinase Anchoring Protein 150 Controls Protein Kinase C-mediated Phosphorylation and Sensitization of TRPV1

    PubMed Central

    Jeske, Nathaniel A.; Patwardhan, Amol M.; Ruparel, Nikita B.; Akopian, Armen N; Shapiro, Mark S.; Henry, Michael A.

    2009-01-01

    Post-translational modifications on various receptor proteins have significant effects on receptor activation. For the Transient Receptor Potential family V type 1 (TRPV1) receptor, phosphorylation of certain serine/threonine amino acid residues sensitizes the receptor to activation by capsaicin and heat. Although Protein Kinase C (PKC) phosphorylates TRPV1 on certain serine/threonine residues, it is not completely understood how PKC functionally associates with TRPV1. Recent studies have reported that the A-kinase Anchoring Protein 150 (AKAP150) mediates PKA phosphorylation of TRPV1 in several nociceptive models. Here, we demonstrate that AKAP150 also mediates PKC-directed phosphorylation and sensitization of TRPV1. In cultured rat trigeminal ganglia, immunocytochemical analyses demonstrate co-localization of AKAP150 and PKC isoforms α, δ, ε, and γ in TRPV1-positive neurons. Additional biochemical evidence supports immunocytochemical results, indicating that AKAP150 preferentially associates with certain PKC isoforms in rat trigeminal ganglia neurons. Employing siRNA-mediated knock-down of AKAP150 expression, we demonstrate that PKC-mediated phosphorylation of TRPV1 and sensitization to a capsaicin response is dependent upon functional expression of the AKAP150 scaffolding protein. Furthermore, PKC-induced sensitization to a thermal stimulus is abrogated in AKAP150 knock-out animals relative to wild-type. Collectively, results from these studies indicate that the AKAP150 scaffolding protein functionally modulates PKC-mediated phosphorylation and sensitization of the TRPV1 receptor in rat sensory neurons, suggesting the scaffolding protein to be an integral regulator of peripheral inflammatory hyperalgesia. PMID:19767149

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

    SciTech Connect

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

    2005-05-15

    Ezrin-Radixin-Moesin (ERM) binding phosphoprotein 50 (EBP50, a.k.a. NHERF-1) is a scaffold protein essential for the localization and coordinated activity of apical transporters, enzymes and receptors in epithelial cells. EBP50 acts via multiple protein binding interactions, including oligomerization through interactions of its PSD95-Dlg-ZO1 (PDZ) domains. EBP50 can be phosphorylated on multiple sites and phosphorylation of specific sites modulates the extent of oligomerization. The aim of the present study was to test the capacity of protein kinase C (PKC) to phosphorylate EBP50 and to regulate its oligomerization. In vitro experiments showed that the catalytic subunit of PKC directly phosphorylates EBP50. In HEK-293 cells transfected with rat EBP50 cDNA, a treatment with 12 myristate 13-acetate (PMA) induced a translocation of PKC{alpha} and {beta} isoforms to the membrane and increased {sup 32}P incorporation into EBP50. In co-transfection/co-precipitation studies, PMA treatment stimulated EBP50 oligomerization. Mass spectrometry analysis of full-length EBP50 and phosphorylation analyses of specific domains, and of mutated or truncated forms of EBP50, indicated that PKC-induced phosphorylation of EBP50 occurred on the Ser{sup 337}/Ser{sup 338} residue within the carboxyl-tail domain of the protein. Truncation of Ser{sup 337}/Ser{sup 338} also diminished PKC-induced oligomerization of EBP50. These results suggest the PKC signaling pathway can impact EBP50-dependent cellular functions by regulating EBP50 oligomerization.

  8. Quantitative methods for the analysis of protein phosphorylation in drug development.

    PubMed

    Olive, D Michael

    2004-10-01

    Most signal transduction and cell signaling pathways are mediated by protein kinases. Protein kinases have emerged as important cellular regulatory proteins in many aspects of neoplasia. Protein kinase inhibitors offer the opportunity to target diseases such as cancer with chemotherapeutic agents specific for the causative molecular defect. In order to identify possible targets and assess kinase inhibitors, quantitative methods for analyzing protein phosphorylation have been developed. This review examines some of the current formats used for quantifying kinase function for drug development. PMID:15966829

  9. Pr-specific phytochrome phosphorylation in vitro by a protein kinase present in anti-phytochrome maize immunoprecipitates

    NASA Technical Reports Server (NTRS)

    Biermann, B. J.; Pao, L. I.; Feldman, L. J.

    1994-01-01

    Protein kinase activity has repeatedly been found to co-purify with the plant photoreceptor phytochrome, suggesting that light signals received by phytochrome may be transduced or modulated through protein phosphorylation. In this study immunoprecipitation techniques were used to characterize protein kinase activity associated with phytochrome from maize (Zea mays L.). A protein kinase that specifically phosphorylated phytochrome was present in washed anti-phytochrome immunoprecipitates of etiolated coleoptile proteins. No other substrate tested was phosphorylated by this kinase. Adding salts or detergents to disrupt low-affinity protein interactions reduced background phosphorylation in immunoprecipitates without affecting phytochrome phosphorylation, indicating that the protein kinase catalytic activity is either intrinsic to the phytochrome molecule or associated with it by high-affinity interactions. Red irradiation (of coleoptiles or extracts) sufficient to approach photoconversion saturation reduced phosphorylation of immunoprecipitated phytochrome. Subsequent far-red irradiation reversed the red-light effect. Phytochrome phosphorylation was stimulated about 10-fold by a co-immunoprecipitated factor. The stimulatory factor was highest in immunoprecipitates when Mg2+ was present in immunoprecipitation reactions but remained in the supernatant in the absence of Mg2+. These observations provide strong support for the hypothesis that phytochrome-associated protein kinase modulates light responses in vivo. Since only phytochrome was found to be phosphorylated, the co-immunoprecipitated protein kinase may function to regulate receptor activity.

  10. Caldesmon and heat shock protein 20 phosphorylation in nitroglycerin- and magnesium-induced relaxation of swine carotid artery.

    PubMed

    Rembold, C M; O'Connor, M

    2000-03-17

    Nitrovasodilators, high extracellular Mg(2+), and some other relaxing agents can cause smooth muscle relaxation without reductions in myosin regulatory light chain (MRLC) phosphorylation. Relaxations without MRLC dephosphorylation suggest that other regulatory systems, beyond MRLC phosphorylation, are present in smooth muscle. We tested whether changes in caldesmon phosphorylation, heat shock protein 20 (HSP20) phosphorylation, or intracellular pH (pH(i)) could be responsible for relaxation without MRLC dephosphorylation. In unstimulated tissues, caldesmon was phosphorylated 1.02+/-0.10 mol P(i)/mol caldesmon (mean+/-1 S.E.M.), HSP20 was phosphorylated 0.005+/-0.003 mol P(i)/mol HSP20, and estimated pH(i) was 7.21+/-0.07. Histamine stimulation induced a contraction, an intracellular acidosis, but did not significantly change caldesmon or HSP20 phosphorylation. Addition of nitroglycerin induced a relaxation, significantly increased HSP20 phosphorylation to 0.18+/-0.02 mol P(i)/mol HSP20, did not significantly change caldesmon phosphorylation, and pH(i) returned to near unstimulated values. Increase in extracellular Mg(2+) to 10 mM induced a relaxation, but did not significantly change HSP20 or caldesmon phosphorylation. These data suggest that changes in caldesmon phosphorylation, HSP20 phosphorylation, or pH(i) cannot be the sole explanation for relaxation without MRLC dephosphorylation. However, it is possible that HSP20 phosphorylation may be involved in nitroglycerin-induced relaxation without MRLC dephosphorylation. PMID:10699367

  11. Kaposi΄s sarcoma-associated herpesvirus ORF36 protein induces chromosome condensation and phosphorylation of histone H3.

    PubMed

    Kim, Sunmi; Cha, Seho; Jang, Jun Hyeong; Kim, Yejin; Seo, Taegun

    2013-01-01

    Kaposi΄s sarcoma-associated herpesvirus (KSHV) has been known as an agent causing Kaposi΄s sarcoma, primary effusion lymphoma, and multicentric Castleman΄s disease. In the lytic phase of the virus cycle, various viral genes are expressed, which causes host cell dysregulation. Among the lytic genes, viral protein kinase (vPK) encoded by ORF36 is a member of serine/threonine protein kinase (CHPK) family, which is involved in viral gene expression, viral DNA replication and encapsidation, and nuclear egress of virions. Recent studies have shown that the BGLF4 protein of Epstein-Barr virus (EBV), a member of the CHPK family, alters the host cell chromatin structure through phosphorylation of its key regulators. The role of KSHV ORF36 in cellular mitotic events, however, is not yet understood. In the current study, we showed that KSHV ORF36 induced chromosome condensation and phosphorylation of histone H3 on Ser 10, which are known as cellular mitosis markers. These processes have occurred in a kinase activity-dependent manner. PMID:23530827

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

    PubMed Central

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

    2015-01-01

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

  13. Glucose Regulated Protein 78 Phosphorylation in Sperm Undergoes Dynamic Changes during Maturation

    PubMed Central

    Lobo, Vivian; Rao, Parimala; Gajbhiye, Rahul; Kulkarni, Vijay; Parte, Priyanka

    2015-01-01

    GRP78, a resident endoplasmic reticulum (ER) chaperone involved in protein transport, folding and assembly, has been reported in sperm. It is shown to be localized in the neck region of human sperm. We have previously reported GRP78 to be less phosphorylated in asthenozoosperm.The present study aimed to determine whether sperm GRP78 undergoes phosphorylation changes during epididymal maturation and whether there are any differences in GRP78 phosphoforms in asthenozoosperm vis-à-vis normozoosperm. Testicular- and cauda epididymal- sperm from adult male Holtzman rats, and semen ejaculates collected from normal and asthenozoospermic individuals were investigated. DIGE carried out to determine phosphorylation of GRP78 in asthenozoosperm and normal sperm reveals a shift in the location of GRP78 of asthenozoosperm towards the alkaline pH, indicative of reduced GRP78 phosphorylation. Immunoprecipitation studies using antibodies specific to GRP78, serine-, threonine-, and tyrosine phosphorylation and Pan phospho antibody demonstrates GRP78 to be phosphorylated at all three residues in rat spermatozoa. Phosphatase assays using Calf intestinal alkaline phosphatase and Lambda protein phosphatase followed by nanofluidic proteomic immunoassay (NIA) show that in rat, GP4.96, GP4.94 and GP4.85 are the three phosphoforms in mature (caudal) sperm as against two phosphoforms GP4.96and GP4.94in immature (testicular) sperm. In mature human sperm GP5.04, GP4.96, and GP4.94were the 3 phosphoforms observed. GP4.94[P = 0.014]andGP5.04 [P = 0.02] are significantly reduced in asthenozoosperm. Ours is the first report indicating GRP78 in sperm to be phosphorylated at serine, threonine and tyrosine residues contrary to published literature reporting GRP78 not to be tyrosine phosphorylated. We report the presence of GRP78 phosphoforms in rat- and human- sperm and our data suggest that GRP78 phosphorylation in sperm undergoes spatial reorganization during epididymal maturation. Significant

  14. Effect of protein kinase P on phosphorylations catalyzed by the epidermal growth factor.

    PubMed Central

    Abdel-Ghany, M; Kole, H K; Racker, E

    1987-01-01

    Protein kinase P (PK-P) activated by histones or certain other basic compounds has been purified previously from yeast [Yanagita, Y., Abdel-Ghany, M., Raden, D., Nelson, N. & Racker, E. (1987) Proc. Natl. Acad. Sci. USA 84, 925-929]. It is shown here that PK-P is present in solubilized membranes of A-431 carcinoma cells where it changes the epidermal growth factor (EGF) receptor kinase activity. Polylysine, a weak PK-P activator, inhibited the autophosphorylation of the EGF receptor both in the absence and presence of EGF. Increased PK-P activity induced by histone 1, a potent activator, gave rise to increased autophosphorylation of the EGF receptor as well as phosphorylation at tyrosine residues of numerous other endogenous membrane components. The stimulation by histone was particularly striking in the presence of EGF. A similar stimulation was achieved with polylysine and EGF on addition of yeast PK-P. However, addition of yeast PK-P in the presence of histone 1 markedly inhibited the EGF-stimulated phosphorylation of endogenous membrane proteins. We conclude from these results that the effect of PK-P on the EGF receptor takes place in three phases: at low levels PK-P inhibits the autophosphorylation, at intermediate levels it stimulates the autophosphorylation as well as the EGF-dependent phosphorylation of numerous other membrane proteins, and at high levels it inhibits the phosphorylation of these proteins. Images PMID:3501120

  15. Protein kinase A-dependent phosphorylation modulates DNA-binding activity of hepatocyte nuclear factor 4.

    PubMed

    Viollet, B; Kahn, A; Raymondjean, M

    1997-08-01

    Hepatocyte nuclear factor 4 (HNF4), a liver-enriched transcription factor of the nuclear receptor superfamily, is critical for development and liver-specific gene expression. Here, we demonstrate that its DNA-binding activity is modulated posttranslationally by phosphorylation in vivo, ex vivo, and in vitro. In vivo, HNF4 DNA-binding activity is reduced by fasting and by inducers of intracellular cyclic AMP (cAMP) accumulation. A consensus protein kinase A (PKA) phosphorylation site located within the A box of its DNA-binding domain has been identified, and its role in phosphorylation-dependent inhibition of HNF4 DNA-binding activity has been investigated. Mutants of HNF4 in which two potentially phosphorylatable serines have been replaced by either neutral or charged amino acids were able to bind DNA in vitro with affinity similar to that of the wild-type protein. However, phosphorylation by PKA strongly repressed the binding affinity of the wild-type factor but not that of HNF4 mutants. Accordingly, in transfection assays, expression vectors for the mutated HNF4 proteins activated transcription more efficiently than that for the wild-type protein-when cotransfected with the PKA catalytic subunit expression vector. Therefore, HNF4 is a direct target of PKA which might be involved in the transcriptional inhibition of liver genes by cAMP inducers. PMID:9234678

  16. Protein kinase A-dependent phosphorylation modulates DNA-binding activity of hepatocyte nuclear factor 4.

    PubMed Central

    Viollet, B; Kahn, A; Raymondjean, M

    1997-01-01

    Hepatocyte nuclear factor 4 (HNF4), a liver-enriched transcription factor of the nuclear receptor superfamily, is critical for development and liver-specific gene expression. Here, we demonstrate that its DNA-binding activity is modulated posttranslationally by phosphorylation in vivo, ex vivo, and in vitro. In vivo, HNF4 DNA-binding activity is reduced by fasting and by inducers of intracellular cyclic AMP (cAMP) accumulation. A consensus protein kinase A (PKA) phosphorylation site located within the A box of its DNA-binding domain has been identified, and its role in phosphorylation-dependent inhibition of HNF4 DNA-binding activity has been investigated. Mutants of HNF4 in which two potentially phosphorylatable serines have been replaced by either neutral or charged amino acids were able to bind DNA in vitro with affinity similar to that of the wild-type protein. However, phosphorylation by PKA strongly repressed the binding affinity of the wild-type factor but not that of HNF4 mutants. Accordingly, in transfection assays, expression vectors for the mutated HNF4 proteins activated transcription more efficiently than that for the wild-type protein-when cotransfected with the PKA catalytic subunit expression vector. Therefore, HNF4 is a direct target of PKA which might be involved in the transcriptional inhibition of liver genes by cAMP inducers. PMID:9234678

  17. Peptide phosphorylation by calcium-dependent protein kinase from maize seedlings.

    PubMed

    Loog, M; Toomik, R; Sak, K; Muszynska, G; Järv, J; Ek, P

    2000-01-01

    Ca2+-dependent protein kinase (CDPK-1) was purified from maize seedlings, and its substrate specificity studied using a set of synthetic peptides derived from the phosphorylatable sequence RVLSRLHS15VRER of maize sucrose synthase 2. The decapeptide LARLHSVRER was found to be efficiently phosphorylated as a minimal substrate. The same set of peptides were found to be phosphorylated by mammalian protein kinase Cbeta (PKC), but showed low reactivity with protein kinase A (PKA). Proceeding from the sequence LARLHSVRER, a series of cellulose-membrane-attached peptides of systematically modified structure was synthesised. These peptides had hydrophobic (Ala, Leu) and ionic (Arg, Glu) amino acids substituted in each position. The phosphorylation of these substrates by CDPK-1 was measured and the substrate specificity of the maize protein kinase characterised by the consensus sequence motif A/L-5X-4R-3X-2X-1SX+1R+2Z+3R+4, where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine compared with the other three varied amino acids. This motif had a characteristic sequence element RZR at positions +2 to +4 and closely resembled the primary structure of the sucrose synthase phosphorylation site. The sequence surrounding the phosphorylatable serine in this consensus motif was similar to the analogous sequence K/RXXS/TXK/R proposed for mammalian PKC, but different from the consensus motif RRXS/TX for PKA. PMID:10632703

  18. Circles within circles: crosstalk between protein Ser/Thr/Tyr-phosphorylation and Met oxidation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Reversible posttranslational protein modifications such as phosphorylation of Ser/Thr/Tyr and Met oxidation are critical for both metabolic regulation and cellular signalling. Although these modifications are typically studied individually, herein we describe the potential for cross-talk...

  19. Inhibition of protein phosphorylation by synthetic peptides from the Fc region of human IgG during the mixed lymphocyte response

    SciTech Connect

    McClurg, M.R.; Hahn, G.S.; Plummer, J.M.

    1986-03-01

    Certain synthetic peptides derived from the Fc region of human IgG suppressed protein, RNA, and DNA synthesis during mixed lymphocyte reactions. Responder mononuclear cells were incubated with medium or agents that alter phosphorylation of cellular proteins before immunomodulatory Fc peptides and stimulator cells were added. Incubating cells with trifluoperazine which inhibits calcium binding to calmodulin and inhibits protein kinase C (PKC) increased inhibition of the MLR induced by Fc peptides. Conversely, incubating cells with dubutyryl cyclic AMP (DBcAMP), calmodulin, 1,2-diolein, or phorbol myristate acetate (PMA) abolished inhibition of the MLR induced by Fc peptides. Inhibition of the MLR by Fc ..gamma.. peptides was not affected when DBcAMP or PMA was added after peptide addition. The PKC activity of cell homogenates was decreased by 69% when Fc..gamma.. peptides were present during the MLR. The in vitro phosphorylation of histone Hl by partially purified PKC from lymphocytes was inhibited 74% in the presence of Fc..gamma.. peptides. These results indicate that suppression of the MLR induced by Fc..gamma.. peptides is dependent on inhibition of protein phosphorylation by kinases including protein kinase C. The inhibition of phosphorylation may be related to the ability of Fc..gamma.. peptides to reverse animal models of autoimmune disease.

  20. Protein phosphorylation detection using dual-mode field-effect devices and nanoplasmonic sensors

    NASA Astrophysics Data System (ADS)

    Bhalla, Nikhil; di Lorenzo, Mirella; Pula, Giordano; Estrela, Pedro

    2015-03-01

    Phosphorylation by kinases is an important post-translational modification of proteins. It is a critical control for the regulation of vital cellular activities, and its dysregulation is implicated in several diseases. A common drug discovery approach involves, therefore, time-consuming screenings of large libraries of candidate compounds to identify novel inhibitors of protein kinases. In this work, we propose a novel method that combines localized surface plasmon resonance (LSPR) and electrolyte insulator semiconductor (EIS)-based proton detection for the rapid identification of novel protein kinase inhibitors. In particular, the selective detection of thiophosphorylated proteins by LSPR is achieved by changing their resonance properties via a pre-binding with gold nanoparticles. In parallel, the EIS field-effect structure allows the real-time electrochemical monitoring of the protein phosphorylation by detecting the release of protons associated with the kinases activity. This innovative combination of both field-effect and nanoplasmonic sensing makes the detection of protein phosphorylation more reliable and effective. As a result, the screening of protein kinase inhibitors becomes more rapid, sensitive, robust and cost-effective.

  1. In situ phosphorylation of proteins in MCTs microdissected from rat kidney: Effect of AVP

    SciTech Connect

    Homma, S.; Gapstur, S.M.; Yusufi, N.K.; Dousa, T.P. )

    1988-04-01

    Adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP)-dependent protein phosphorylation is considered a key step in the cellular action of vasopressin (AVP) to regulate water permeability in collecting tubules. However, the proteins serving as a substrate(s) for phosphorylation in undisrupted cells have not yet been identified. In the present study, the authors developed a method for investigation of in situ phosphorylation of microdissected segments of medullary collecting tubules (MCT) from rat kidney. Incubation of microdissected MCT segments with low concentrations of saponin, semipermeabilization, increased permeability of the membrane for ATP but did not allow leakage of macromolecules such as lactate dehydrogenase. This treatment also did not cause major disruption of cell structure, or impairment of AVP-sensitive adenylate cyclase. Incubation of semipermeabilized MCT with {gamma}-({sup 32}P)ATP resulted in corporation of {sup 32}P{sub i} into two major protein bands detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent autoradiography. Similar incubation of tubules disrupted by hyposmotic solutions and a stronger detergent Triton X-100 resulted in {sup 32}P{sub i} incorporation into multiple protein bands. These findings demonstrate a novel method for identification of endogenous protein substrate(s) for cAMP-dependent protein kinase and other protein kinases and phosphatases that are probably involved in post-cAMP steps in the cellular action of AVP in the intact cells of collecting tubules.

  2. Phosphorylation Reaction in cAPK Protein Kinase - Free Energy Quantum Mechanic/Molecular Mechanics Simulations.

    SciTech Connect

    Valiev, Marat; Yang, Jie; Adams, Joseph; Taylor, Susan S.; Weare, John H.

    2007-11-29

    Protein kinases catalyze the transfer of the γ-phosphoryl group from ATP, a key regulatory process governing signalling pathways in eukaryotic cells. The structure of the active site in these enzymes is highly conserved implying common catalytic mechanism. In this work we investigate the reaction process in cAPK protein kinase (PKA) using a combined quantum mechanics and molecular mechanics approach. The novel computational features of our work include reaction pathway determination with nudged elastic band methodology and calculation of free energy profiles of the reaction process taking into account finite temperature fluctuations of the protein environment. We find that the transfer of the γ-phosphoryl group in the protein environment is an exothermic reaction with the reaction barrier of 15 kcal/mol.

  3. A Broadly Conserved G-Protein-Coupled Receptor Kinase Phosphorylation Mechanism Controls Drosophila Smoothened Activity

    PubMed Central

    Maier, Dominic; Cheng, Shuofei; Faubert, Denis; Hipfner, David R.

    2014-01-01

    Hedgehog (Hh) signaling is essential for normal growth, patterning, and homeostasis of many tissues in diverse organisms, and is misregulated in a variety of diseases including cancer. Cytoplasmic Hedgehog signaling is activated by multisite phosphorylation of the seven-pass transmembrane protein Smoothened (Smo) in its cytoplasmic C-terminus. Aside from a short membrane-proximal stretch, the sequence of the C-terminus is highly divergent in different phyla, and the evidence suggests that the precise mechanism of Smo activation and transduction of the signal to downstream effectors also differs. To clarify the conserved role of G-protein-coupled receptor kinases (GRKs) in Smo regulation, we mapped four clusters of phosphorylation sites in the membrane-proximal C-terminus of Drosophila Smo that are phosphorylated by Gprk2, one of the two fly GRKs. Phosphorylation at these sites enhances Smo dimerization and increases but is not essential for Smo activity. Three of these clusters overlap with regulatory phosphorylation sites in mouse Smo and are highly conserved throughout the bilaterian lineages, suggesting that they serve a common function. Consistent with this, we find that a C-terminally truncated form of Drosophila Smo consisting of just the highly conserved core, including Gprk2 regulatory sites, can recruit the downstream effector Costal-2 and activate target gene expression, in a Gprk2-dependent manner. These results indicate that GRK phosphorylation in the membrane proximal C-terminus is an evolutionarily ancient mechanism of Smo regulation, and point to a higher degree of similarity in the regulation and signaling mechanisms of bilaterian Smo proteins than has previously been recognized. PMID:25009998

  4. New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein

    PubMed Central

    Sehgal, Kapil; Sylvester, Marc; Skubal, Magdalena; Josten, Michele; Steinhäuser, Christian; De Koninck, Paul; Theis, Martin

    2016-01-01

    Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3’UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future. PMID:26915047

  5. Effect of growth hormone on protein phosphorylation in isolated rat hepatocytes

    SciTech Connect

    Yamada, K.; Lipson, K.E.; Marino, M.W.; Donner, D.B.

    1987-02-10

    Hepatocytes from male rats were incubated with (/sup 32/P)P/sub i/ for 40 min at 37/sup 0/C, thereby equilibrating the cellular ATP pool with /sup 32/P. Subsequent exposure to bovine growth hormone for 10 additional min did not change the specific activity of cellular (..gamma..-/sup 32/P)ATP. Two-dimensional gel electrophoresis or chromatofocusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to fractionate phosphoproteins solubilized from control or hormone-stimulated cells. Stimulation of hepatocytes with 5 nM growth hormone for 10 min at 37/sup 0/C affected the phosphorylation of a number of proteins including an M/sub r/ 46,000 species of pI 4.7 whose phosphorylation was augmented (2.65 +/- 0.50)-fold. A significant fraction of the maximal effect of growth hormone on phosphorylation of the M/sub r/ 46,000 species was elicited by 1-5% receptor occupancy. Bovine growth hormone, which binds to somatogenic receptors with great specificity, or recombinant human growth hormone, which is not contaminated with other hormones, affected phosphorylation of hepatic proteins similarly. The M/sub r/ 46,000 phosphoprotein was isolated in a fraction enriched in cytosol after centrifugation of cellular homogenates. Phosphorylation of the M/sub r/ 46,000 phosphoprotein was also increased (1.75 +/- 0.35)-fold and (2.15 +/- 0.50)-fold by insulin and glucagon, respectively. These observations are consistent with the possibility that selective changes in the phosphorylation state of cellular proteins may mediate growth hormone actions in cells.

  6. New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein.

    PubMed

    Kaczmarczyk, Lech; Labrie-Dion, Étienne; Sehgal, Kapil; Sylvester, Marc; Skubal, Magdalena; Josten, Michele; Steinhäuser, Christian; De Koninck, Paul; Theis, Martin

    2016-01-01

    Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3'UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future. PMID:26915047

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

    PubMed

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

    2016-05-01

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

  8. Protein substrates for cGMP-dependent protein phosphorylation in cilia of wild type and atalanta mutants of Paramecium.

    PubMed

    Ann, K S; Nelson, D L

    1995-01-01

    In the ciliated protozoan Paramecium, swimming direction is regulated by voltage-gated Ca2+ channels in the ciliary membrane. In response to depolarizing stimuli, intraciliary Ca2+ rises, triggering reversal of the ciliary power stroke and backward swimming. One class of Ca(2+)-unresponsive behavioral mutants of Paramecium, atalanta mutants, cannot swim backward even though they have functional Ca2+ channels in their ciliary membrane. Several atalanta mutants were characterized with regard to several Ca(2+)-dependent activities, but no significant difference between wild type and the mutants was detected. However, one allelic group, atalanta A (initially characterized by Hinrichsen and Kung [1984: Genet. Res. Camb. 43:11-20]), showed a helical swimming path of opposite handedness from that of wild-type cells when detergent-permeabilized cells ("models") were reactivated with MgATP. When cGMP-dependent protein kinase purified from wild-type cells was added to atalanta A models, the handedness of the swimming path was reversed. Cyclic GMP stimulated in vitro phosphorylation of several proteins in isolated cilia, and the pattern of phosphoproteins was very similar for wild type and atalanta mutants, with one exception: a protein of 59 kDa was phosphorylated much less in the mutant ata A. When ciliary proteins were separated by gel electrophoresis and then phosphorylated "on blot" by purified cGMP-dependent protein kinase, phosphoprotein patterns were similar in wild type and ata mutants except that a 48 kDa protein (p48) from ata A3 was more heavily phosphorylated. This difference in p48 phosphorylation was also observed with cGMP-dependent protein kinase purified from ata A3 mutant cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7796456

  9. Membrane association of sucrose synthase: changes during the graviresponse and possible control by protein phosphorylation

    NASA Technical Reports Server (NTRS)

    Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

    1997-01-01

    Sucrose synthase (SuSy) plays an important role in sucrose degradation and occurs both as a soluble and as a membrane-associated enzyme in higher plants. We show that membrane association can vary in vivo in response to gravistimulation, apparently involving SuSy dephosphorylation, and is a reversible process in vitro. Phosphorylation of SuSy has little effect on its activity but decreases its surface hydrophobicity as reported with the fluorescent probe bis-ANS. We postulate that phosphorylation of SuSy (and perhaps other membrane proteins) is involved in the release of the membrane-bound enzyme in part as a result of decreased surface hydrophobicity.

  10. Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1

    NASA Technical Reports Server (NTRS)

    Haynes, J. I. 2nd; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein.

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

    SciTech Connect

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

    1990-07-01

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

  12. Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1.

    PubMed Central

    Haynes, J I; Consigli, R A

    1992-01-01

    The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein. Images PMID:1318417

  13. Identification of four novel phosphorylation sites in estrogen receptor α: impact on receptor-dependent gene expression and phosphorylation by protein kinase CK2

    PubMed Central

    2009-01-01

    Background Estrogen receptor α (ERα) phosphorylation is important for estrogen-dependent transcription of ER-dependent genes, ligand-independent receptor activation and endocrine therapy response in breast cancer. However ERα phosphorylation at the previously identified sites does not fully account for these receptor functions. To determine if additional ERα phosphorylation sites exist, COS-1 cells expressing human ERα were labeled with [32P]H3PO4 in vivo and ERα tryptic phosphopeptides were isolated to identify phosphorylation sites. Results Previously uncharacterized phosphorylation sites at serines 46/47, 282, 294, and 559 were identified by manual Edman degradation and phosphoamino acid analysis and confirmed by mutagenesis and phospho-specific antibodies. Antibodies detected phosphorylation of endogenous ERα in MCF-7, MCF-7-LCC2, and Ishikawa cancer cell lines by immunoblot. Mutation of Ser-282 and Ser-559 to alanine (S282A, S559A) resulted in ligand independent activation of ERα as determined by both ERE-driven reporter gene assays and endogenous pS2 gene expression in transiently transfected HeLa cells. Mutation of Ser-46/47 or Ser-294 to alanine markedly reduced estradiol dependent reporter activation. Additionally protein kinase CK2 was identified as a kinase that phosphorylated ERα at S282 and S559 using motif analysis, in vitro kinase assays, and incubation of cells with CK2 kinase inhibitor. Conclusion These novel ERα phosphorylation sites represent new means for modulation of ERα activity. S559 represents the first phosphorylation site identified in the extreme C-terminus (F domain) of a steroid receptor. PMID:20043841

  14. Activation of the oncogenic potential of the avian cellular src protein by specific structural alteration of the carboxy terminus.

    PubMed Central

    Reynolds, A B; Vila, J; Lansing, T J; Potts, W M; Weber, M J; Parsons, J T

    1987-01-01

    The role of tyrosine phosphorylation in the regulation of tyrosine protein kinase activity was investigated using site-directed mutagenesis to alter the structure and environment of the three tyrosine residues present in the C terminus of avian pp60c-src. Mutations that change Tyr 527 to Phe or Ser activate in vivo tyrosine protein kinase activity and induce cellular transformation of chicken cells in culture. In contrast, alterations of tyrosine residues present at positions 511 or 519 in c-src do not induce transformation or in vivo tyrosine protein kinase activity. Amber mutations, which alter the structure of the pp60c-src C terminus by inducing premature termination of the c-src protein at either residue 518 or 523 also induce morphological transformation and increase in vivo tyrosine phosphorylation, whereas removal of the last four residues of c-src by chain termination at residue 530 does not alter the kinase activity or the biological activity of the resultant c-src protein. We conclude from these studies that C-terminal alterations which either remove or replace Tyr 527 serve to activate the c-src protein resulting in cellular transformation and increased in vivo tyrosine protein kinase activity. Images Fig. 2. Fig. 3. Fig. 4. PMID:2822389

  15. Comparison of phosphorylation kinetics in DNA repair proteins after exposure to high and low LET radiations

    NASA Astrophysics Data System (ADS)

    Okayasu, R.; Okabe, A.; Takakura, K.

    We irradiated plateau phase normal human fibroblasts with 2 Gy X-rays 70 keV um carbon 290MeV n and 200 keV um iron ions 500 MeV n and observed the kinetics of phosphorylation in various proteins associated with DNA double strand break DSB repair GammaH2AX foci a marker for DSBs were detected immediately after irradiation and the peak of phosphorylation was seen 30 to 60 min post-irradiation for three kinds of radiations Disappearance of gamma-H2AX foci was much faster for X-irradiated samples than that for heavy ion irradiated samples the phosphorylation kinetics for carbon and iron ions are similar for gamma-H2AX foci In contrast phosphorylation of an NHEJ protein DNA-PKcs threonine 2609 was significantly delayed in carbon and iron irradiated cells when compared to X-irradiated cells Disappearance of DNA-PKcs sites was much faster in X-irradiated samples than carbon and iron samples which showed a similar pattern as in the case of gamma-H2AX Furthermore in the case of ATM protein phosphorylation serine 1981 iron irradiation alone caused a significant initial delay but the kinetics of disappearance is similar for iron and carbon samples with much higher remaining number of foci in iron samples than those for X-rays and carbon ions These results suggest that 1 high LET irradiation induces complex and or severe DNA DSB damage which affects the function of DSB repair proteins 2 Both ATM and DNA-PKcs may recognize the complexity of DSBs but ATM may be more sensitive to detecting the complexity of DSB damage 3 gamma-H2AX may

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

    PubMed Central

    Ithuralde, Raúl Esteban; Turjanski, Adrián Gustavo

    2016-01-01

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

  17. Phosphorylation of Mycobacterium tuberculosis protein tyrosine kinase A PtkA by Ser/Thr protein kinases.

    PubMed

    Zhou, Peifu; Wong, Dennis; Li, Wu; Xie, Jianping; Av-Gay, Yossef

    2015-11-13

    Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has inflicted about one third of mankind and claims millions of deaths worldwide annually. Signalling plays an important role in Mtb pathogenesis and persistence, and thus represents attractive resource for drug target candidates. Here, we show that protein tyrosine kinase A (PtkA) can be phosphorylated by Mtb endogenous eukaryotic-like Ser/Thr protein kinases (eSTPKs). Kinase assays showed that PknA, PknD, PknF, and PknK can phosphorylate PtkA in dose- and time-dependent manner. Enzyme kinetics suggests that PknA has the highest affinity and enzymatic efficiency towards PtkA. Furthermore, protein-protein interaction assay in surrogate host showed that PtkA interacts with multi-eSTPKs in vivo, including PknA. Lastly, we show that PtkA phosphorylation by eSTPKs occurs on threonine residues and may effect tyrosine phosphorylation levels and thus PtkA activity in vitro. These results demonstrate that PtkA can serve as a substrate to many eSTPKs and suggests that's its activity can be regulated. PMID:26417687

  18. PD Trafficking of Potato Leaf Roll Virus Movement Protein in Arabidopsis Depends on Site-specific Protein Phosphorylation

    PubMed Central

    Sonnewald, Uwe

    2011-01-01

    Many plant viruses encode for specialized movement proteins (MP) to facilitate passage of viral material to and through plasmodesmata (PD). To analyze intracellular trafficking of potato leaf roll virus (PLRV) movement protein (MP17) we performed GFP fusion experiments with distinct deletion variants of MP17. These studies revealed that the C-terminus of MP17 is essential but not sufficient for PD targeting. Interestingly, fusion of GFP to three C-terminal MP17 deletion variants resulted in the accumulation of GFP in chloroplasts. This indicates that MP17 harbors hidden plastid targeting sequences. Previous studies showed that posttranslational protein phosphorylation influences PD targeting of MP and virus spread. Analysis of MP17-derived phospho-peptides by mass spectrometry revealed four phosphorylated serine residues (S71, S79, S137, and S140). Site-directed mutagenesis of S71/S79 and S137/S140 showed that the C-terminal serine residues S137/S140 are dispensable for PD targeting. However, exchange of S71/S79 to A71/A79 abolished PD targeting of the mutated MP17 protein. To mimic phosphorylation of S71/S79 both amino acids were substituted by aspartic acid. The resulting D71/D79 variant of MP17 was efficiently targeted to PD. Further deletion analysis showed that PD targeting of MP17 is dependent on the C-terminus, phosphorylation of S71 and/or S79 and a N-terminal domain. PMID:22645527

  19. The unfolded protein response mediates reversible tau phosphorylation induced by metabolic stress

    PubMed Central

    van der Harg, J M; Nölle, A; Zwart, R; Boerema, A S; van Haastert, E S; Strijkstra, A M; Hoozemans, J JM; Scheper, W

    2014-01-01

    The unfolded protein response (UPR) is activated in neurodegenerative tauopathies such as Alzheimer's disease (AD) in close connection with early stages of tau pathology. Metabolic disturbances are strongly associated with increased risk for AD and are a potent inducer of the UPR. Here, we demonstrate that metabolic stress induces the phosphorylation of endogenous tau via activation of the UPR. Strikingly, upon restoration of the metabolic homeostasis, not only the levels of the UPR markers pPERK, pIRE1α and BiP, but also tau phosphorylation are reversed both in cell models as well as in torpor, a physiological hypometabolic model in vivo. Intervention in the UPR using the global UPR inhibitor TUDCA or a specific small-molecule inhibitor of the PERK signaling pathway, inhibits the metabolic stress-induced phosphorylation of tau. These data support a role for UPR-mediated tau phosphorylation as part of an adaptive response to metabolic stress. Failure to restore the metabolic homeostasis will lead to prolonged UPR activation and tau phosphorylation, and may thus contribute to AD pathogenesis. We demonstrate that the UPR is functionally involved in the early stages of tau pathology. Our data indicate that targeting of the UPR may be employed for early intervention in tau-related neurodegenerative diseases. PMID:25165879

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

    PubMed

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

    2009-06-01

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

  1. Aluminum interaction with human brain tau protein phosphorylation by various kinases

    SciTech Connect

    El-Sebae, A.H.; Zeid, M.M.A.; Saleh, M.A. . Environmental Chemistry and Toxicology Lab.); Abdel-Ghany, M.E.; Shalloway, D. ); Blancato, J. . Environmental Monitoring Systems Lab.)

    1993-01-01

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

  2. Aluminum interaction with human brain tau protein phosphorylation by various kinases

    SciTech Connect

    El-Sebae; Abou Zeid, M.M.; Saleh, M.A. . Environmental Chemistry and Toxicology Lab.); Abdel-Ghany, M.E.; Shalloway, D. . Section of Biochemistry, Mol, and Cell Biology); Blancato, J. . Environmental Monit. Systems Lab.)

    1993-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    2015-01-01

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

  5. Phosphorylation of human respiratory syncytial virus P protein at serine 54 regulates viral uncoating

    SciTech Connect

    Asenjo, Ana; Gonzalez-Armas, Juan C.; Villanueva, Nieves

    2008-10-10

    The human respiratory syncytial virus (HRSV) structural P protein, phosphorylated at serine (S) and threonine (T) residues, is a co-factor of viral RNA polymerase. The phosphorylation of S54 is controlled by the coordinated action of two cellular enzymes: a lithium-sensitive kinase, probably glycogen synthetase kinase (GSK-3) {beta} and protein phosphatase 2A (PP2A). Inhibition of lithium-sensitive kinase, soon after infection, blocks the viral growth cycle by inhibiting synthesis and/or accumulation of viral RNAs, proteins and extracellular particles. P protein phosphorylation at S54 is required to liberate viral ribonucleoproteins (RNPs) from M protein, during the uncoating process. Kinase inhibition, late in infection, produces a decrease in genomic RNA and infectious viral particles. LiCl, intranasally applied to mice infected with HRSV A2 strain, reduces the number of mice with virus in their lungs and the virus titre. Administration of LiCl to humans via aerosol should prevent HRSV infection, without secondary effects.

  6. The Role of Protein Phosphorylation in the Gustatory Cortex and Amygdala During Taste Learning

    PubMed Central

    Adaikkan, Chinnakkaruppan

    2012-01-01

    Protein phosphorylation and dephosphorylation form a major post-translation mechanism that enables a given cell to respond to ever-changing internal and external environments. Neurons, similarly to any other cells, use protein phosphorylation/dephosphorylation to maintain an internal homeostasis, but they also use it for updating the state of synaptic and intrinsic properties, following activation by neurotransmitters and growth factors. In the present review we focus on the roles of several families of kinases, phosphatases, and other synaptic-plasticity-related proteins, which activate membrane receptors and various intracellular signals to promote transcription, translation and protein degradation, and to regulate the appropriate cellular proteomes required for taste memory acquisition, consolidation and maintenance. Attention is especially focused on the protein phosphorylation state in two forebrain areas that are necessary for taste-memory learning and retrieval: the insular cortex and the amygdala. The various temporal phases of taste learning require the activation of appropriate waves of biochemical signals. These include: extracellular signal regulated kinase I and II (ERKI/II) signal transduction pathways; Ca2+-dependent pathways; tyrosine kinase/phosphatase-dependent pathways; brain-derived neurotrophicfactor (BDNF)-dependent pathways; cAMP-responsive element bindingprotein (CREB); and translation-regulation factors, such as initiation and elongation factors, and the mammalian target of rapamycin (mTOR). Interestingly, coding of hedonic and aversive taste information in the forebrain requires activation of different signal transduction pathways. PMID:22792024

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

    PubMed

    Aryal, Bibek; Laurent, Christophe; Geisler, Markus

    2016-04-15

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

  8. A rapid extraction procedure of human hair proteins and identification of phosphorylated species.

    PubMed

    Nakamura, Akira; Arimoto, Makoto; Takeuchi, Keiji; Fujii, Toshihiro

    2002-05-01

    We developed a rapid and convenient extraction procedure of human hair proteins to examine their biochemical properties in detail. This procedure is based upon the fact that the combination of thiourea and urea in the presence of a reductant can effectively remove proteins from the cortex part of human hair. The extracted fraction mainly consisted of hard alpha-keratins with molecular masses of 40-60 kDa, matrix proteins with 12-18kDa, and minor components with 110-115kDa and 125-135kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein phosphorylation in human hair was investigated by immunoblotting with antibodies against phosphoserine, phosphothreonine and phosphotyrosine. We found serine phosphorylation in alpha-keratins and matrix proteins and threonine phosphorylation in alpha-keratins. The extraction was also found to be effective when wool, chicken feathers, rat hair and human nails were used as starting materials. PMID:12033494

  9. Expanding the proteome of an RNA virus by phosphorylation of an intrinsically disordered viral protein.

    PubMed

    Cordek, Daniel G; Croom-Perez, Tayler J; Hwang, Jungwook; Hargittai, Michele R S; Subba-Reddy, Chennareddy V; Han, Qingxia; Lodeiro, Maria Fernanda; Ning, Gang; McCrory, Thomas S; Arnold, Jamie J; Koc, Hasan; Lindenbach, Brett D; Showalter, Scott A; Cameron, Craig E

    2014-08-29

    The human proteome contains myriad intrinsically disordered proteins. Within intrinsically disordered proteins, polyproline-II motifs are often located near sites of phosphorylation. We have used an unconventional experimental paradigm to discover that phosphorylation by protein kinase A (PKA) occurs in the intrinsically disordered domain of hepatitis C virus non-structural protein 5A (NS5A) on Thr-2332 near one of its polyproline-II motifs. Phosphorylation shifts the conformational ensemble of the NS5A intrinsically disordered domain to a state that permits detection of the polyproline motif by using (15)N-, (13)C-based multidimensional NMR spectroscopy. PKA-dependent proline resonances were lost in the presence of the Src homology 3 domain of c-Src, consistent with formation of a complex. Changing Thr-2332 to alanine in hepatitis C virus genotype 1b reduced the steady-state level of RNA by 10-fold; this change was lethal for genotype 2a. The lethal phenotype could be rescued by changing Thr-2332 to glutamic acid, a phosphomimetic substitution. Immunofluorescence and transmission electron microscopy showed that the inability to produce Thr(P)-2332-NS5A caused loss of integrity of the virus-induced membranous web/replication organelle. An even more extreme phenotype was observed in the presence of small molecule inhibitors of PKA. We conclude that the PKA-phosphorylated form of NS5A exhibits unique structure and function relative to the unphosphorylated protein. We suggest that post-translational modification of viral proteins containing intrinsic disorder may be a general mechanism to expand the viral proteome without a corresponding expansion of the genome. PMID:25031324

  10. Phosphorylation of the beta-subunit of CD11/CD18 integrins by protein kinase C correlates with leukocyte adhesion.

    PubMed

    Valmu, L; Autero, M; Siljander, P; Patarroyo, M; Gahmberg, C G

    1991-11-01

    Adhesion of activated leukocytes to cells is of critical functional importance. The adhesion is known to be mediated mainly by the CD11/CD18 integrins, also known as leukocytic cell adhesion molecules, or Leu-CAM. We have now studied the phosphorylation of Leu-CAM by protein kinase C and the correlation of phosphorylation with the generation of the adhesive phenotype among human peripheral blood mononuclear leukocytes during cell activation. We here show that a good correlation exists between the phosphorylation of the beta subunit of Leu-CAM (CD18), and the extent of cell-to-cell adhesion. The phosphorylated CD18 subunit was associated with both CD11a and CD11b. Purified protein kinase C was able to phosphorylate the beta subunit of isolated Leu-CAM in vitro. The phosphorylation occurred mainly on serine residues. PMID:1682156

  11. GTP Cyclohydrolase I Phosphorylation and Interaction with GTP Cyclohydrolase Feedback Regulatory Protein Provide Novel Regulation of Endothelial Tetrahydrobiopterin and Nitric Oxide

    PubMed Central

    Li, Li; Rezvan, Amir; Salerno, John C.; Husain, Ahsan; Kwon, Kihwan; Jo, Hanjoong; Harrison, David G.; Chen, Wei

    2009-01-01

    Rationale GTP cyclohydrolase I (GTPCH-1) is the rate-limiting enzyme involved in de novo biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases and aromatic amino acid hydroxylases. GTPCH-1 undergoes negative feedback regulation by its end-product BH4 via interaction with the GTP cyclohydrolase feedback regulatory protein (GFRP). Such a negative feedback mechanism should maintain cellular BH4 levels within a very narrow range; however, we recently identified a phosphorylation site (S81) on human GTPCH-1 that markedly increases BH4 production in response to laminar shear. Objective To define how S81 phosphorylation alters GTPCH-1 enzyme activity and how this is modulated by GFRP. Methods and Results Using prokaryotically expressed proteins, we found that the GTPCH-1 phospho-mimetic mutant (S81D) has increased enzyme activity, reduced binding to GFRP and resistance to inhibition by GFRP compared to wild-type GTPCH-1. Using siRNA or overexpressing plasmids, GFRP was shown to modulate phosphorylation of GTPCH-1, BH4 levels and nitric oxide (NO) production in human endothelial cells. Laminar, but not oscillatory shear stress caused dissociation of GTPCH-1 and GFRP, promoting GTPCH-1 phosphorylation. We also found that both GTPCH-1 phosphorylation and GFRP down-regulation prevents eNOS uncoupling in response to oscillatory shear. Finally oscillatory shear was associated with impaired GTPCH-1 phosphorylation and reduced BH4 levels in vivo. Conclusion These studies provide a new mechanism for regulation of endothelial GTPCH-1 by its phosphorylation and interplay with GFRP. This mechanism allows for escape from GFRP negative feedback and permits large amounts of BH4 to be produced in response to laminar shear stress. PMID:19926872

  12. Phosphorylation Regulates the Endocytic Function of the Yeast Dynamin-Related Protein Vps1.

    PubMed

    Smaczynska-de Rooij, Iwona I; Marklew, Christopher J; Allwood, Ellen G; Palmer, Sarah E; Booth, Wesley I; Mishra, Ritu; Goldberg, Martin W; Ayscough, Kathryn R

    2016-03-01

    The family of dynamin proteins is known to function in many eukaryotic membrane fusion and fission events. The yeast dynamin-related protein Vps1 functions at several stages of membrane trafficking, including Golgi apparatus to endosome and vacuole, peroxisomal fission, and endocytic scission. We have previously shown that in its endocytic role, Vps1 functions with the amphiphysin heterodimer Rvs161/Rvs167 to facilitate scission and release of vesicles. Phosphoproteome studies of Saccharomyces cerevisiae have identified a phosphorylation site in Vps1 at serine 599. In this study, we confirmed this phosphorylation event, and we reveal that, like Rvs167, Vps1 can be phosphorylated by the yeast cyclin-associated kinase Pho85 in vivo and in vitro. The importance of this posttranslational modification was revealed when mutagenesis of S599 to a phosphomimetic or nonphosphorylatable form caused defects in endocytosis but not in other functions associated with Vps1. Mutation to nonphosphorylatable valine inhibited the Rvs167 interaction, while both S599V and S599D caused defects in vesicle scission, as shown by both live-cell imaging and electron microscopy of endocytic invaginations. Our data support a model in which phosphorylation and dephosphorylation of Vps1 promote distinct interactions and highlight the importance of such regulatory events in facilitating sequential progression of the endocytic process. PMID:26711254

  13. Male-specific phosphorylated SR proteins in adult flies of the Mediterranean Fruitfly Ceratitis capitata

    PubMed Central

    2014-01-01

    Alternative splicing is a widely used mechanism of gene regulation in sex determination pathways of Insects. In species from orders as distant as Diptera, Hymenoptera and Coleoptera, female differentiation relies on the activities of conserved splicing regulators, TRA and TRA-2, promoting female-specific expression of the global effector doublesex (dsx). Less understood is to what extent post-translational modifications of splicing regulators plays a role in this pathway. In Drosophila melanogaster phosphorylation of TRA, TRA-2 and the general RBP1 factor by the LAMMER kinase doa (darkener of apricot) is required for proper female sex determination. To explore whether this is a general feature of the pathway we examined sex-specific differences in phosphorylation levels of SR splicing factors in the dipteran species D. melanogaster, Ceratitis capitata (Medfly) and Musca domestica (Housefly). We found a distinct and reproducible pattern of male-specific phosphorylation on protein extracts enriched for SR proteins in C. capitata suggesting that differential phosphorylation may also contribute to the regulation of sex-specific splicing in the Medfly. PMID:25472723

  14. Dysfunctional conformational dynamics of protein kinase A induced by a lethal mutant of phospholamban hinder phosphorylation

    PubMed Central

    Kim, Jonggul; Masterson, Larry R.; Cembran, Alessandro; Verardi, Raffaello; Shi, Lei; Gao, Jiali; Taylor, Susan S.; Veglia, Gianluigi

    2015-01-01

    The dynamic interplay between kinases and substrates is crucial for the formation of catalytically committed complexes that enable phosphoryl transfer. However, a clear understanding on how substrates modulate kinase structural dynamics to control catalytic efficiency is still missing. Here, we used solution NMR spectroscopy to study the conformational dynamics of two complexes of the catalytic subunit of the cAMP-dependent protein kinase A with WT and R14 deletion phospholamban, a lethal human mutant linked to familial dilated cardiomyopathy. Phospholamban is a central regulator of heart muscle contractility, and its phosphorylation by protein kinase A constitutes a primary response to β-adrenergic stimulation. We found that the single deletion of arginine in phospholamban’s recognition sequence for the kinase reduces its binding affinity and dramatically reduces phosphorylation kinetics. Structurally, the mutant prevents the enzyme from adopting conformations and motions committed for catalysis, with concomitant reduction in catalytic efficiency. Overall, these results underscore the importance of a well-tuned structural and dynamic interplay between the kinase and its substrates to achieve physiological phosphorylation levels for proper Ca2+ signaling and normal cardiac function. PMID:25775607

  15. ATM-dependent Phosphorylation of the Fanconi Anemia Protein PALB2 Promotes the DNA Damage Response.

    PubMed

    Guo, Yingying; Feng, Wanjuan; Sy, Shirley M H; Huen, Michael S Y

    2015-11-13

    The Fanconi anemia protein PALB2, also known as FANCN, protects genome integrity by regulating DNA repair and cell cycle checkpoints. Exactly how PALB2 functions may be temporally coupled with detection and signaling of DNA damage is not known. Intriguingly, we found that PALB2 is transformed into a hyperphosphorylated state in response to ionizing radiation (IR). IR treatment specifically triggered PALB2 phosphorylation at Ser-157 and Ser-376 in manners that required the master DNA damage response kinase Ataxia telangiectasia mutated, revealing potential mechanistic links between PALB2 and the Ataxia telangiectasia mutated-dependent DNA damage responses. Consistently, dysregulated PALB2 phosphorylation resulted in sustained activation of DDRs. Full-blown PALB2 phosphorylation also required the breast and ovarian susceptible gene product BRCA1, highlighting important roles of the BRCA1-PALB2 interaction in orchestrating cellular responses to genotoxic stress. In summary, our phosphorylation analysis of tumor suppressor protein PALB2 uncovers new layers of regulatory mechanisms in the maintenance of genome stability and tumor suppression. PMID:26420486

  16. Phosphorylation Regulates the Endocytic Function of the Yeast Dynamin-Related Protein Vps1

    PubMed Central

    Smaczynska-de Rooij, Iwona I.; Marklew, Christopher J.; Allwood, Ellen G.; Palmer, Sarah E.; Booth, Wesley I.; Mishra, Ritu; Goldberg, Martin W.

    2015-01-01

    The family of dynamin proteins is known to function in many eukaryotic membrane fusion and fission events. The yeast dynamin-related protein Vps1 functions at several stages of membrane trafficking, including Golgi apparatus to endosome and vacuole, peroxisomal fission, and endocytic scission. We have previously shown that in its endocytic role, Vps1 functions with the amphiphysin heterodimer Rvs161/Rvs167 to facilitate scission and release of vesicles. Phosphoproteome studies of Saccharomyces cerevisiae have identified a phosphorylation site in Vps1 at serine 599. In this study, we confirmed this phosphorylation event, and we reveal that, like Rvs167, Vps1 can be phosphorylated by the yeast cyclin-associated kinase Pho85 in vivo and in vitro. The importance of this posttranslational modification was revealed when mutagenesis of S599 to a phosphomimetic or nonphosphorylatable form caused defects in endocytosis but not in other functions associated with Vps1. Mutation to nonphosphorylatable valine inhibited the Rvs167 interaction, while both S599V and S599D caused defects in vesicle scission, as shown by both live-cell imaging and electron microscopy of endocytic invaginations. Our data support a model in which phosphorylation and dephosphorylation of Vps1 promote distinct interactions and highlight the importance of such regulatory events in facilitating sequential progression of the endocytic process. PMID:26711254

  17. Membrane protein assembly: two cytoplasmic phosphorylated serine sites of Vpu from HIV-1 affect oligomerization

    PubMed Central

    Chen, Chin-Pei; Lin, Meng-Han; Chan, Ya-Ting; Chen, Li-Chyong; Ma, Che; Fischer, Wolfgang B.

    2016-01-01

    Viral protein U (Vpu) encoded by human immunodeficiency virus type 1 (HIV-1) is a short integral membrane protein which is known to self-assemble within the lipid membrane and associate with host factors during the HIV-1 infectivity cycle. In this study, full-length Vpu (M group) from clone NL4-3 was over-expressed in human cells and purified in an oligomeric state. Various single and double mutations were constructed on its phosphorylation sites to mimic different degrees of phosphorylation. Size exclusion chromatography of wild-type Vpu and mutants indicated that the smallest assembly unit of Vpu was a dimer and over time Vpu formed higher oligomers. The rate of oligomerization increased when (i) the degree of phosphorylation at serines 52 and 56 was decreased and (ii) when the ionic strength was increased indicating that the cytoplasmic domain of Vpu affects oligomerization. Coarse-grained molecular dynamic simulations with models of wild-type and mutant Vpu in a hydrated lipid bilayer supported the experimental data in demonstrating that, in addition to a previously known role in downregulation of host factors, the phosphorylation sites of Vpu also modulate oligomerization. PMID:27353136

  18. Influence of phosphorylation by protein kinase A on CFTR at the cell surface and endoplasmic reticulum.

    PubMed

    Seibert, F S; Chang, X B; Aleksandrov, A A; Clarke, D M; Hanrahan, J W; Riordan, J R

    1999-12-01

    CFTR possesses a large cluster of strict dibasic consensus sites for phosphorylation by protein kinase A (PKA) in the R-domain and an obligatory dependence on phosphorylation is a hallmark of CFTR Cl(-) channel function. Removal of as many as 11 of these sites reduces the conformational change in the R-domain and the degree of channel activation in response to PKA. However, until recently a completely PKA-unresponsive CFTR variant has not been reported, leaving open the possibility that the residual response may be mediated by associating ancillary phosphoproteins. We traced the residual PKA-catalyzed (32)P-labelling of the variant with 11 sites mutagenized (11SA) to distinct CNBr phosphopeptides within the R-domain. Mutagenesis of 4 additional monobasic sites in these segments produced a 15SA variant in which Cl(-) channel response to PKA was abolished. Therefore, it can be concluded that ancillary phosphoproteins do not contribute to CFTR activation by PKA. Notably, however, the 15SA protein did exhibit a low level of constitutive channel activity not dependent on PKA, which might have reflected a down-regulating effect of phosphorylation of one or two of the 15 sites as suggested by others. However, this did not prove to be the case.Since immature CFTR has been claimed to be active in the endoplasmic reticulum (ER), we also examined whether it can be phosphorylated in cells and what influence if any this might have on its susceptibility to degradation. Teleologically, activation by phosphorylation of CFTR Cl(-) channels in the ER might be undesirable to the cell. Using various phosphorylation site mutants and kinase and phosphatase inhibitors in pulse-chase experiments, we have found that although nascent CFTR can be phosphorylated at the ER, this is without effect on its ability to mature and avoid proteolysis. Furthermore, we found that microsomes from cells expressing CFTR processing mutants such as DeltaF508 do not generate Cl(-) active channels when fused

  19. Band 3 Erythrocyte Membrane Protein Acts as Redox Stress Sensor Leading to Its Phosphorylation by p72 Syk

    PubMed Central

    Pantaleo, Antonella; Ferru, Emanuela; Pau, Maria Carmina; Khadjavi, Amina; Mandili, Giorgia; Mattè, Alessandro; Spano, Alessandra; De Franceschi, Lucia; Pippia, Proto; Turrini, Francesco

    2016-01-01

    In erythrocytes, the regulation of the redox sensitive Tyr phosphorylation of band 3 and its functions are still partially defined. A role of band 3 oxidation in regulating its own phosphorylation has been previously suggested. The current study provides evidences to support this hypothesis: (i) in intact erythrocytes, at 2 mM concentration of GSH, band 3 oxidation, and phosphorylation, Syk translocation to the membrane and Syk phosphorylation responded to the same micromolar concentrations of oxidants showing identical temporal variations; (ii) the Cys residues located in the band 3 cytoplasmic domain are 20-fold more reactive than GSH; (iii) disulfide linked band 3 cytoplasmic domain docks Syk kinase; (iv) protein Tyr phosphatases are poorly inhibited at oxidant concentrations leading to massive band 3 oxidation and phosphorylation. We also observed that hemichromes binding to band 3 determined its irreversible oxidation and phosphorylation, progressive hemolysis, and serine hyperphosphorylation of different cytoskeleton proteins. Syk inhibitor suppressed the phosphorylation of band 3 also preventing serine phosphorylation changes and hemolysis. Our data suggest that band 3 acts as redox sensor regulating its own phosphorylation and that hemichromes leading to the protracted phosphorylation of band 3 may trigger a cascade of events finally leading to hemolysis. PMID:27034738

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

    PubMed

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

    2016-05-13

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

  1. Selective Phosphorylation Inhibitor of Delta Protein Kinase C-Pyruvate Dehydrogenase Kinase Protein-Protein Interactions: Application for Myocardial Injury in Vivo.

    PubMed

    Qvit, Nir; Disatnik, Marie-Hélène; Sho, Eiketsu; Mochly-Rosen, Daria

    2016-06-22

    Protein kinases regulate numerous cellular processes, including cell growth, metabolism, and cell death. Because the primary sequence and the three-dimensional structure of many kinases are highly similar, the development of selective inhibitors for only one kinase is challenging. Furthermore, many protein kinases are pleiotropic, mediating diverse and sometimes even opposing functions by phosphorylating multiple protein substrates. Here, we set out to develop an inhibitor of a selective protein kinase phosphorylation of only one of its substrates. Focusing on the pleiotropic delta protein kinase C (δPKC), we used a rational approach to identify a distal docking site on δPKC for its substrate, pyruvate dehydrogenase kinase (PDK). We reasoned that an inhibitor of PDK's docking should selectively inhibit the phosphorylation of only PDK without affecting phosphorylation of the other δPKC substrates. Our approach identified a selective inhibitor of PDK docking to δPKC with an in vitro Kd of ∼50 nM and reducing cardiac injury IC50 of ∼5 nM. This inhibitor, which did not affect the phosphorylation of other δPKC substrates even at 1 μM, demonstrated that PDK phosphorylation alone is critical for δPKC-mediated injury by heart attack. The approach we describe is likely applicable for the identification of other substrate-specific kinase inhibitors. PMID:27218445

  2. Tyrosine phosphorylation and protein degradation control the transcriptional activity of WRKY involved in benzylisoquinoline alkaloid biosynthesis.

    PubMed

    Yamada, Yasuyuki; Sato, Fumihiko

    2016-01-01

    Benzylisoquinoline alkaloids (BIQ) are among the most structurally diverse and pharmaceutically valuable secondary metabolites. A plant-specific WRKY-type transcription factor, CjWRKY1, was isolated from Coptis japonica and identified as a transcriptional activator of BIQ biosynthesis. However, the expression of CjWRKY1 gene alone was not sufficient for the activation of genes encoding biosynthetic enzymes. Here, we report the importance of post-translational regulation of CjWRKY1 in BIQ biosynthesis. First, we detected the differential accumulation of CjWRKY1 protein in two cell lines with similar CjWRKY1 gene expression but different levels of accumulated alkaloids. Further investigation of the WRKY protein identified the phosphorylation of the WRKYGQK core domain at Y115. The CjWRKY(Y115E) phosphorylation-mimic mutant showed loss of nuclear localization, DNA-binding activity, and transactivation activity compared to wild-type CjWRKY1. Rapid degradation of the CjWRKY1 protein was also confirmed following treatment with inhibitors of the 26S proteasome and protease inhibitors. The existence of two independent degradation pathways as well as protein phosphorylation suggests the fine-tuning of CjWRKY1 activities is involved in the regulation of biosynthesis of BIQs. PMID:27552928

  3. Tyrosine phosphorylation and protein degradation control the transcriptional activity of WRKY involved in benzylisoquinoline alkaloid biosynthesis

    PubMed Central

    Yamada, Yasuyuki; Sato, Fumihiko

    2016-01-01

    Benzylisoquinoline alkaloids (BIQ) are among the most structurally diverse and pharmaceutically valuable secondary metabolites. A plant-specific WRKY-type transcription factor, CjWRKY1, was isolated from Coptis japonica and identified as a transcriptional activator of BIQ biosynthesis. However, the expression of CjWRKY1 gene alone was not sufficient for the activation of genes encoding biosynthetic enzymes. Here, we report the importance of post-translational regulation of CjWRKY1 in BIQ biosynthesis. First, we detected the differential accumulation of CjWRKY1 protein in two cell lines with similar CjWRKY1 gene expression but different levels of accumulated alkaloids. Further investigation of the WRKY protein identified the phosphorylation of the WRKYGQK core domain at Y115. The CjWRKYY115E phosphorylation-mimic mutant showed loss of nuclear localization, DNA-binding activity, and transactivation activity compared to wild-type CjWRKY1. Rapid degradation of the CjWRKY1 protein was also confirmed following treatment with inhibitors of the 26S proteasome and protease inhibitors. The existence of two independent degradation pathways as well as protein phosphorylation suggests the fine-tuning of CjWRKY1 activities is involved in the regulation of biosynthesis of BIQs. PMID:27552928

  4. A Pan-specific Antibody for Direct Detection of Protein Histidine Phosphorylation

    PubMed Central

    Kee, Jung-Min; Oslund, Rob C.; Perlman, David H.; Muir, Tom W.

    2013-01-01

    Despite its importance in central metabolism and bacterial cell signaling, protein histidine phosphorylation has remained elusive with respect to its extent and functional roles in biological systems due to the lack of adequate research tools. We report the development of the first pan-pHis antibody using a stable phosphohistidine (pHis) mimetic as the hapten. This antibody was successfully used in ELISA, Western blot, dot blot, immunoprecipitation, and in detection and identification of histidine-phosphorylated proteins from native cell lysates when coupled with mass spectrometric analysis. We also observed that protein pHis levels in E. coli lysates depend on carbon source and nitrogen availability in the growth media. In particular, we found that pHis levels on PpsA are sensitive to nitrogen availability in vivo and that α-ketoglutarate (α-KG) inhibits phosphotransfer from phosphorylated phosphoenolpyruvate synthase (PpsA) to pyruvate. We expect this antibody to open opportunities for investigating other pHis-proteins and their functions. PMID:23708076

  5. Translation elongation factor 1A mutants with altered actin bundling activity show reduced aminoacyl-tRNA binding and alter initiation via eIF2α phosphorylation.

    PubMed

    Perez, Winder B; Kinzy, Terri Goss

    2014-07-25

    Apart from its canonical function in translation elongation, eukaryotic translation elongation factor 1A (eEF1A) has been shown to interact with the actin cytoskeleton. Amino acid substitutions in eEF1A that reduce its ability to bind and bundle actin in vitro cause improper actin organization in vivo and reduce total translation. Initial in vivo analysis indicated the reduced translation was through initiation. The mutant strains exhibit increased levels of phosphorylated initiation factor 2α (eIF2α) dependent on the presence of the general control non-derepressible 2 (Gcn2p) protein kinase. Gcn2p causes downregulation of total protein synthesis at initiation in response to increases in deacylated tRNA levels in the cell. Increased levels of eIF2α phosphorylation are not due to a general reduction in translation elongation as eEF2 and eEF3 mutants do not exhibit this effect. Deletion of GCN2 from the eEF1A actin bundling mutant strains revealed a second defect in translation. The eEF1A actin-bundling proteins exhibit changes in their elongation activity at the level of aminoacyl-tRNA binding in vitro. These findings implicate eEF1A in a feedback mechanism for regulating translation at initiation. PMID:24936063

  6. Evidence for phosphorylation of the major seed storage protein of the common bean and its phosphorylation-dependent degradation during germination.

    PubMed

    López-Pedrouso, María; Alonso, Jana; Zapata, Carlos

    2014-03-01

    Phaseolin is the major seed storage protein of common bean, Phaseolus vulgaris L., accounting for up to 50 % of the total seed proteome. The regulatory mechanisms responsible for the synthesis, accumulation and degradation of phaseolin in the common bean seed are not yet sufficiently known. Here, we report on a systematic study in dormant and 4-day germinating bean seeds from cultivars Sanilac (S) and Tendergreen (T) to explore the presence and dynamics of phosphorylated phaseolin isoforms. High-resolution two-dimensional electrophoresis in combination with the phosphoprotein-specific Pro-Q Diamond phosphoprotein fluorescent stain and chemical dephosphorylation by hydrogen fluoride-pyridine enabled us to identify differentially phosphorylated phaseolin polypeptides in dormant and germinating seeds from cultivars S and T. Phosphorylated forms of the two subunits of type α and β that compose the phaseolin were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and MALDI-TOF/TOF tandem MS. In addition, we found that the levels of phosphorylation of the phaseolin changed remarkably in the seed transition from dormancy to early germination stage. Temporal changes in the extent of phosphorylation in response to physiological and metabolic variations suggest that phosphorylated phaseolin isoforms have functional significance. In particular, this prospective study supports the hypothesis that mobilization of the phaseolin in germinating seeds occurs through the degradation of highly phosphorylated isoforms. Taken together, our results indicate that post-translational phaseolin modifications through phosphorylations need to be taken into consideration for a better understanding of the molecular mechanisms underlying its regulation. PMID:24142381

  7. Nucleophosmin protein expression level, but not threonine 198 phosphorylation, is essential in growth and proliferation

    PubMed Central

    Brady, SN; Maggi, LB; Winkeler, CL; Toso, EA; Gwinn, AS; Pelletier, CL; Weber, JD

    2010-01-01

    Nucleophosmin (NPM), an oligomeric phosphoprotein and nucleolar target of the ARF tumor suppressor, contributes to several critical cellular processes. Previous studies have shown that the human NPM’s phosphorylation by cyclin E–cyclin-dependent kinase 2 (cdk2) on threonine (Thr) 199 regulates its translocation from the centrosome during cell cycle progression. Given our previous finding that ARF directly binds NPM, impeding its transit to the cytoplasm and arresting cells before S-phase entry, we hypothesized that ARF might also inhibit NPM phosphorylation. However, ARF induction did not impair phosphorylation of the cdk2 target residue in murine NPM, Thr198. Furthermore, phosphorylation of Thr198 occurred throughout the cell cycle and was concomitant with increases in overall NPM expression. To investigate the cell’s presumed requirement for NPM-Thr198 phosphorylation in promoting the processes of growth and proliferation, we examined the effects of a non-phosphorylatable NPM mutant, T198A, in a clean cell system in which endogenous NPM had been removed by RNA interference. Here, we show that the T198A mutant is fully capable of executing NPM’s described roles in nucleocytoplasmic shuttling, ribosome export and cell cycle progression. Moreover, the proliferative defects observed with stable NPM knockdown were restored by mutant NPM-T198A expression. Thus, we demonstrate that the reduction in NPM protein expression blocks cellular growth and proliferation, whereas phosphorylation of NPM-Thr198 is not essential for NPM’s capacity to drive cell cycle progression and proliferation. PMID:19561638

  8. Temporal dissociation of frequency-dependent acceleration of relaxation and protein phosphorylation by CaMKII.

    PubMed

    Huke, Sabine; Bers, Donald M

    2007-03-01

    Frequency-dependent acceleration of relaxation (FDAR) is an important intrinsic mechanism that allows for diastolic filling of the ventricle at higher heart rates, yet its molecular mechanism is still not understood. Previous studies showed that FDAR is dependent on functional sarcoplasmic reticulum (SR) and can be abolished by phosphatase or by Ca/CaM kinase (CaMKII) inhibition. Additionally, CaMKII activity/autophosphorylation has been shown to be frequency-dependent. Thus, we tested the hypothesis that CaMKII phosphorylation of SR Ca(2+)-handling proteins (Phospholamban (PLB), Ca(2+) release channel (RyR)) mediates FDAR. Here we show that FDAR occurs abruptly in fluo-4 loaded isolated rat ventricular myocytes when frequency is raised from 0.1 to 2 Hz. The effect is essentially complete within four beats (2 s) with the tau of [Ca(2+)](i) decline decreasing by 42+/-3%. While there is a detectable increase in PLB Thr-17 and RyR Ser-2814 phosphorylation, the increase is quantitatively small (PLB<5%, RyR approximately 8%) and the time-course is clearly delayed with regard to FDAR. The low substrate phosphorylation indicates that pacing of myocytes only mildly activates CaMKII and consistent with this CaMKIIdelta autophosphorylation did not increase with pacing alone. However, in the presence of phosphatase 1 inhibition pacing triggered a net-increase in autophosphorylated CaMKII and also greatly enhanced PLB and RyR phosphorylation. We conclude that FDAR does not rely on phosphorylation of PLB or RyR. Even though CaMKII does become activated when myocytes are paced, phosphatases immediately antagonize CaMKII action, limit substrate phosphorylation and also prevent sustained CaMKII autophosphorylation (thereby suppressing global CaMKII effects). PMID:17239900

  9. Electrogenerated Chemiluminescence Bioassay of Two Protein Kinases Incorporating Peptide Phosphorylation and Versatile Probe.

    PubMed

    Liu, Xia; Dong, Manman; Qi, Honglan; Gao, Qiang; Zhang, Chengxiao

    2016-09-01

    A sensitive electrogenerated chemiluminescence (ECL) bioassay was developed for the detection of two protein kinases incorporating the peptide phosphorylation and a versatile ECL probe. Cyclic adenosine monophosphate-dependent protein kinase (PKA) and casein kinase II (CK2) were used as proof-of-concept targets while a PKA-specific peptide (CLRRASLG) and a CK2-specific peptide (CRRRADDSDDDDD) were used as the recognition substrates. Taking advantage of the ability of protein A binding with the Fc region of a variety of antibodies with high affinity, a ruthenium derivative-labeled protein A was utilized as a versatile ECL probe for bioassay of multiple protein kinases. A specific peptide substrate toward target protein kinase was first self-assembled on the surface of gold electrode and then serine in the specific peptide on the electrode was phosphorylated by target protein kinase in the presence of adenosine-5'-triphosphate. After recognition of the phosphorylated peptide by monoclonal antiphosphoserine antibody, the versatile ECL probe was specifically bound to the antiphosphoserine antibody on the electrode surface. The ECL bioassay was developed successfully in the individual detection of PKA and CK2 with detection limit of 0.005 U/mL and 0.004 U/mL, respectively. In addition, the ECL bioassay was applied to quantitative analysis of the kinase inhibitors and monitoring drug-triggered kinase activation in cell lysates. Moreover, an ECL imaging bioassay using electron-multiplying charged coupled device as detector on the gold electrode array was developed for the simultaneous detection of PKA and CK2 activity from 0.01 U/mL to 0.4 U/mL, respectively, at one time. This work demonstrates that the ingenious design and use of a versatile ECL probe are promising to simultaneous detection of multiple protein kinases and screening of kinase inhibitor. PMID:27518533

  10. Phosphorylation of the Antiviral Protein Interferon-inducible Transmembrane Protein 3 (IFITM3) Dually Regulates Its Endocytosis and Ubiquitination*

    PubMed Central

    Chesarino, Nicholas M.; McMichael, Temet M.; Hach, Jocelyn C.; Yount, Jacob S.

    2014-01-01

    Interferon-inducible transmembrane protein 3 (IFITM3) is essential for innate defense against influenza virus in mice and humans. IFITM3 localizes to endolysosomes where it prevents virus fusion, although mechanisms controlling its trafficking to this cellular compartment are not fully understood. We determined that both mouse and human IFITM3 are phosphorylated by the protein-tyrosine kinase FYN on tyrosine 20 (Tyr20) and that mouse IFITM3 is also phosphorylated on the non-conserved Tyr27. Phosphorylation led to a cellular redistribution of IFITM3, including plasma membrane accumulation. Mutation of Tyr20 caused a similar redistribution of IFITM3 and resulted in decreased antiviral activity against influenza virus, whereas Tyr27 mutation of mouse IFITM3 showed minimal effects on localization or activity. Using FYN knockout cells, we also found that IFITM3 phosphorylation is not a requirement for its antiviral activity. Together, these results indicate that Tyr20 is part of an endocytosis signal that can be blocked by phosphorylation or by mutation of this residue. Further mutagenesis narrowed this endocytosis-controlling region to four residues conforming to a YXXΦ (where X is any amino acid and Φ is Val, Leu, or Ile) endocytic motif that, when transferred to CD4, resulted in its internalization from the cell surface. Additionally, we found that phosphorylation of IFITM3 by FYN and mutagenesis of Tyr20 both resulted in decreased IFITM3 ubiquitination. Overall, these results suggest that modification of Tyr20 may serve in a cellular checkpoint controlling IFITM3 trafficking and degradation and demonstrate the complexity of posttranslational regulation of IFITM3. PMID:24627473

  11. Bisphenol A accelerates capacitation-associated protein tyrosine phosphorylation of rat sperm by activating protein kinase A.

    PubMed

    Wan, Xiaofeng; Ru, Yanfei; Chu, Chen; Ni, Zimei; Zhou, Yuchuan; Wang, Shoulin; Zhou, Zuomin; Zhang, Yonglian

    2016-06-01

    Bisphenol A (BPA) is a synthetic estrogen-mimic chemical. It has been shown to affect many reproductive endpoints. However, the effect of BPA on the mature sperm and the mechanism of its action are not clear yet. Here, our in vitro studies indicated that BPA could accelerate sperm capacitation-associated protein tyrosine phosphorylation in time- and dose-dependent manners. In vivo, the adult male rats exposed to a high dose of BPA could result in a significant increase in sperm activity. Further investigation demonstrated that BPA could accelerate capacitation-associated protein tyrosine phosphorylation even if sperm were incubated in medium devoid of BSA, HCO3 (-), and Ca(2+) However, this action of BPA stimulation could be blocked by H89, a highly selective blocker of protein kinase A (PKA), but not by KH7, a specific inhibitor of adenylyl cyclase. These data suggest that BPA may activate PKA to affect sperm functions and male fertility. PMID:27174873

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

    PubMed Central

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

    2015-01-01

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

  13. Embryonic poly(A)-binding protein (ePAB) phosphorylation is required for Xenopus oocyte maturation.

    PubMed

    Friend, Kyle; Brook, Matthew; Bezirci, F Betül; Sheets, Michael D; Gray, Nicola K; Seli, Emre

    2012-07-01

    Oocyte maturation and early embryonic development require the cytoplasmic polyadenylation and concomitant translational activation of stored maternal mRNAs. ePAB [embryonic poly(A)-binding protein, also known as ePABP and PABPc1-like] is a multifunctional post-transcriptional regulator that binds to poly(A) tails. In the present study we find that ePAB is a dynamically modified phosphoprotein in Xenopus laevis oocytes and show by mutation that phosphorylation at a four residue cluster is required for oocyte maturation. We further demonstrate that these phosphorylations are critical for cytoplasmic polyadenylation, but not for ePAB's inherent ability to promote translation. Our results provide the first insight into the role of post-translational modifications in regulating PABP protein activity in vivo. PMID:22497250

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

    SciTech Connect

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

    1989-04-01

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

  15. The Clinical Significance of Phosphorylated Heat Shock Protein 27 (HSPB1) in Pancreatic Cancer

    PubMed Central

    Okuno, Mitsuru; Adachi, Seiji; Kozawa, Osamu; Shimizu, Masahito; Yasuda, Ichiro

    2016-01-01

    Pancreatic cancer is one of most aggressive forms of cancer. After clinical detection it exhibits fast metastatic growth. Heat shock protein 27 (HSP27; HSPB1) has been characterized as a molecular chaperone which modifies the structures and functions of other proteins in cells when they are exposed to various stresses, such as chemotherapy. While the administration of gemcitabine, an anti-tumor drug, has been the standard treatment for patients with advanced pancreatic cancer, accumulating evidence shows that HSP27 plays a key role in the chemosensitivity to gemcitabine. In addition, phosphorylated HSP27 induced by gemcitabine has been associated with the inhibition of pancreatic cancer cell growth. In this review, we summarize the role of phosphorylated HSP27, as well as HSP27, in the regulation of chemosensitivity in pancreatic cancer. PMID:26805817

  16. The Clinical Significance of Phosphorylated Heat Shock Protein 27 (HSPB1) in Pancreatic Cancer.

    PubMed

    Okuno, Mitsuru; Adachi, Seiji; Kozawa, Osamu; Shimizu, Masahito; Yasuda, Ichiro

    2016-01-01

    Pancreatic cancer is one of most aggressive forms of cancer. After clinical detection it exhibits fast metastatic growth. Heat shock protein 27 (HSP27; HSPB1) has been characterized as a molecular chaperone which modifies the structures and functions of other proteins in cells when they are exposed to various stresses, such as chemotherapy. While the administration of gemcitabine, an anti-tumor drug, has been the standard treatment for patients with advanced pancreatic cancer, accumulating evidence shows that HSP27 plays a key role in the chemosensitivity to gemcitabine. In addition, phosphorylated HSP27 induced by gemcitabine has been associated with the inhibition of pancreatic cancer cell growth. In this review, we summarize the role of phosphorylated HSP27, as well as HSP27, in the regulation of chemosensitivity in pancreatic cancer. PMID:26805817

  17. G Protein-coupled Receptor Kinase 2–mediated Phosphorylation of Ezrin Is Required for G Protein-coupled Receptor–dependent Reorganization of the Actin Cytoskeleton

    PubMed Central

    Cant, Sarah H.; Pitcher, Julie A.

    2005-01-01

    G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes activated G protein-coupled receptors (GPCRs). Here, we identify ezrin as a novel non-GPCR substrate of GRK2. GRK2 phosphorylates glutathione S-transferase (GST)-ezrin, but not an ezrin fusion protein lacking threonine 567 (T567), in vitro. These results suggest that T567, the regulatory phosphorylation site responsible for maintaining ezrin in its active conformation, represents the principle site of GRK2-mediated phosphorylation. Two lines of evidence indicate that GRK2-mediated ezrin-radixinmoesin (ERM) phosphorylation serves to link GPCR activation to cytoskeletal reorganization. First, in Hep2 cells muscarinic M1 receptor (M1MR) activation causes membrane ruffling. This ruffling response is ERM dependent and is accompanied by ERM phosphorylation. Inhibition of GRK2, but not rho kinase or protein kinase C, prevents ERM phosphorylation and membrane ruffling. Second, agonist-induced internalization of the β2-adrenergic receptor (β2AR) and M1MR is accompanied by ERM phosphorylation and localization of phosphorylated ERM to receptor-containing endocytic vesicles. The colocalization of internalized β2AR and phosphorylated ERM is not dependent on Na+/H+ exchanger regulatory factor binding to the β2AR. Inhibition of ezrin function impedes β2AR internalization, further linking GPCR activation, GRK activity, and ezrin function. Overall, our results suggest that GRK2 serves not only to attenuate but also to transduce GPCR-mediated signals. PMID:15843435

  18. Desiccation enhances phosphorylation of PSII and affects the distribution of protein complexes in the thylakoid membrane.

    PubMed

    Gao, Shan; Gu, Wenhui; Xiong, Qian; Ge, Feng; Xie, Xiujun; Li, Jian; Chen, Weizhou; Pan, Guanghua; Wang, Guangce

    2015-03-01

    Desiccation has significant effects on photosynthetic processes in intertidal macro-algae. We studied an intertidal macro-alga, Ulva sp., which can tolerate desiccation, to investigate changes in photosynthetic performance and the components and structure of thylakoid membrane proteins in response to desiccation. Our results demonstrate that photosystem II (PSII) is more sensitive to desiccation than photosystem I (PSI) in Ulva sp. Comparative proteomics of the thylakoid membrane proteins at different levels of desiccation suggested that there were few changes in the content of proteins involved in photosynthesis during desiccation. Interestingly, we found that both the PSII subunit, PsbS (Photosystem II S subunit) (a four-helix protein in the LHC superfamily), and light-harvesting complex stress-related (LHCSR) proteins, which are required for non-photochemical quenching in land plants and algae, respectively, were present under both normal and desiccation conditions and both increased slightly during desiccation. In addition, the results of immunoblot analysis suggested that the phosphorylation of PSII and LHCII increases during desiccation. To investigate further, we separated out a supercomplex formed during desiccation by blue native-polyacrylamide gel electrophoresis and identified the components by mass spectrometry analysis. Our results show that phosphorylation of the complex increases slightly with decreased water content. All the results suggest that during the course of desiccation, few changes occur in the content of thylakoid membrane proteins, but a rearrangement of the protein complex occurs in the intertidal macro-alga Ulva sp. PMID:25132456

  19. Phenotypically Dormant and Immature Leukaemia Cells Display Increased Ribosomal Protein S6 Phosphorylation

    PubMed Central

    Pallis, Monica; Harvey, Tamsin; Russell, Nigel

    2016-01-01

    Mechanistic/mammalian target of rapamycin (mTOR) activity drives a number of key metabolic processes including growth and protein synthesis. Inhibition of the mTOR pathway promotes cellular dormancy. Since cells from patients with acute myeloid leukaemia (AML) can be phenotypically dormant (quiescent), we examined biomarkers of their mTOR pathway activity concurrently with Ki-67 and CD71 (indicators of cycling cells) by quantitative flow cytometry. Using antibodies to phosphorylated epitopes of mTOR (S2448) and its downstream targets ribosomal protein S6 (rpS6, S235/236) and 4E-BP1 (T36/45), we documented that these phosphorylations were negligible in lymphocytes, but evident in dormant as well as proliferating subsets of both mobilised normal stem cell harvest CD34+ cells and AML blasts. Although mTOR phosphorylation in AML blasts was lower than that of the normal CD34+ cells, p-4E-BP1 was 2.6-fold higher and p-rpS6 was 22-fold higher. Moreover, in contrast to 4E-BP1, rpS6 phosphorylation was higher in dormant than proliferating AML blasts, and was also higher in the immature CD34+CD38- blast subset. Data from the Cancer Genome Atlas show that rpS6 expression is associated with that of respiratory chain enzymes in AML. We conclude that phenotypic quiescence markers do not necessarily predict metabolic dormancy and that elevated rpS6 ser235/236 phosphorylation is characteristic of AML. PMID:26985829

  20. Phosphorylation by the c-Abl protein tyrosine kinase inhibits parkin's ubiquitination and protective function

    PubMed Central

    Ko, Han Seok; Lee, Yunjong; Shin, Joo-Ho; Karuppagounder, Senthilkumar S.; Gadad, Bharathi Shrikanth; Koleske, Anthony J.; Pletnikova, Olga; Troncoso, Juan C.; Dawson, Valina L.; Dawson, Ted M.

    2010-01-01

    Mutations in PARK2/Parkin, which encodes a ubiquitin E3 ligase, cause autosomal recessive Parkinson disease (PD). Here we show that the nonreceptor tyrosine kinase c-Abl phosphorylates tyrosine 143 of parkin, inhibiting parkin's ubiquitin E3 ligase activity and protective function. c-Abl is activated by dopaminergic stress and by dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium (MPP+) in vitro and in vivo by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), leading to parkin inactivation, accumulation of the parkin substrates aminoacyl-tRNA synthetase-interacting multifunctional protein type 2 (AIMP2) (p38/JTV-1) and fuse-binding protein 1 (FBP1), and cell death. STI-571, a c-Abl-family kinase inhibitor, prevents the phosphorylation of parkin, maintaining parkin in a catalytically active and protective state. STI-571’s protective effects require parkin, as shRNA knockdown of parkin prevents STI-571 protection. Conditional knockout of c-Abl in the nervous system also prevents the phosphorylation of parkin, the accumulation of its substrates, and subsequent neurotoxicity in response to MPTP intoxication. In human postmortem PD brain, c-Abl is active, parkin is tyrosine-phosphorylated, and AIMP2 and FBP1 accumulate in the substantia nigra and striatum. Thus, tyrosine phosphorylation of parkin by c-Abl is a major posttranslational modification that inhibits parkin function, possibly contributing to pathogenesis of sporadic PD. Moreover, inhibition of c-Abl may be a neuroprotective approach in the treatment of PD. PMID:20823226

  1. Phenotypically Dormant and Immature Leukaemia Cells Display Increased Ribosomal Protein S6 Phosphorylation.

    PubMed

    Pallis, Monica; Harvey, Tamsin; Russell, Nigel

    2016-01-01

    Mechanistic/mammalian target of rapamycin (mTOR) activity drives a number of key metabolic processes including growth and protein synthesis. Inhibition of the mTOR pathway promotes cellular dormancy. Since cells from patients with acute myeloid leukaemia (AML) can be phenotypically dormant (quiescent), we examined biomarkers of their mTOR pathway activity concurrently with Ki-67 and CD71 (indicators of cycling cells) by quantitative flow cytometry. Using antibodies to phosphorylated epitopes of mTOR (S2448) and its downstream targets ribosomal protein S6 (rpS6, S235/236) and 4E-BP1 (T36/45), we documented that these phosphorylations were negligible in lymphocytes, but evident in dormant as well as proliferating subsets of both mobilised normal stem cell harvest CD34+ cells and AML blasts. Although mTOR phosphorylation in AML blasts was lower than that of the normal CD34+ cells, p-4E-BP1 was 2.6-fold higher and p-rpS6 was 22-fold higher. Moreover, in contrast to 4E-BP1, rpS6 phosphorylation was higher in dormant than proliferating AML blasts, and was also higher in the immature CD34+CD38- blast subset. Data from the Cancer Genome Atlas show that rpS6 expression is associated with that of respiratory chain enzymes in AML. We conclude that phenotypic quiescence markers do not necessarily predict metabolic dormancy and that elevated rpS6 ser235/236 phosphorylation is characteristic of AML. PMID:26985829

  2. In vivo and in vitro phosphorylation and subcellular localization of trypanosomatid cytoskeletal giant proteins.

    PubMed

    Baqui, M M; Milder, R; Mortara, R A; Pudles, J

    2000-09-01

    Promastigote forms of Phytomonas serpens, Leptomonas samueli, and Leishmania tarentolae express cytoskeletal giant proteins with apparent molecular masses of 3,500 kDa (Ps 3500), 2,500 kDa (Ls 2500), and 1,200 kDa (Lt 1200), respectively. Polyclonal antibodies to Lt 1200 and to Ps 3500 specifically recognize similar polypeptides of the same genera of parasite. In addition to reacting with giant polypeptides of the Leptomonas species, anti-Ls 2500 also cross reacts with Ps 3500, and with a 500-kDa polypeptide of Leishmania. Confocal immunofluorescence and immunogold electron microscopy showed major differences in topological distribution of these three proteins, though they partially share a common localization at the anterior end of the cell body skeleton. Furthermore, Ps 3500, Ls 2500, and Lt 1200 are in vivo phosphorylated at serine and threonine residues, whereas, in vitro phosphorylation of cytoskeletal fractions reveal that only Ps 3500 and Ls 2500 are phosphorylated. Heat treatment (100 degrees C) of high salt cytoskeletal extracts demonstrates that Ps 3500 and Ls 2500 remain stable in solution, whereas Lt 1200 is denatured. Kinase assays with immunocomplexes of heat-treated giant proteins show that only Ps 3500 and Ls 2500 are phosphorylated. These results demonstrate the existence of a novel class of megadalton phosphoproteins in promastigote forms of trypanosomatids that appear to be genera specific with distinct cytoskeletal functions. In addition, there is also evidence that Ps 3500 and Ls 2500, in contrast to Lt 1200, seem to be autophosphorylating serine and threonine protein kinases, suggesting that they might play regulatory roles in the cytoskeletal organization. PMID:11002308

  3. Phosphorylation and localization of protein-zero related (PZR) in cultured endothelial cells.

    PubMed

    Kusano, Ken-ichi; Thomas, Tamlyn N; Fujiwara, Keigi

    2008-01-01

    Protein-zero related (PZR) is an immunoglobulin V (IgV)-type immunoreceptor with two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). PZR interacts with Src homology 2 domain-containing tyrosine phosphatase (SHP-2) via its tyrosine-phosphorylated ITIMs, for which c-Src is a putative kinase. Towards elucidating PZR function in endothelial cells (ECs), the authors cloned PZR from bovine aortic endothelial cells (BAECs) and characterized it. Mature bovine PZR had 94.8% and 92.7% sequence identity with canine and human proteins, respectively, and the two ITIM sequences were conserved among higher vertebrates. PZR was expressed in many cell types and was localized to cell contacts and intracellular granules in BAECs and mesothelioma (REN) cells. Coimmunoprecipitation revealed that PZR, Grb-2-associated binder-1 (Gab1), and platelet endothelial cell adhesion molecule-1 (PECAM-1) were three major SHP-2-binding proteins in BAECs. H(2)O(2) enhanced PZR tyrosine phosphorylation and PZR/SHP-2 interaction in ECs in a dose-and time-dependent manner. To see if tyrosine kinases other than Src are also capable of phosphorylating PZR, the authors cotransfected HEK293 cells with PZR and one of several tyrosine kinases and found that c-Src, c-Fyn, c-Lyn, Csk, and c-Abl, but not c-Fes, phosphorylated PZR and increased PZR/SHP-2 interaction. These results suggest that PZR is a cell adhesion protein that may be involved in SHP-2-dependent signaling at interendothelial cell contacts. PMID:18568953

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

    PubMed

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

    2014-09-19

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

  5. Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D

    SciTech Connect

    Waldron, Richard T. . E-mail: rwaldron@mednet.ucla.edu; Whitelegge, Julian P.; Faull, Kym F.; Rozengurt, Enrique

    2007-05-04

    Protein kinase D (PKD) phosphorylates the c-jun amino-terminal in vitro at site(s) distinct from JNK [C. Hurd, R.T. Waldron, E. Rozengurt, Protein kinase D complexes with c-jun N-terminal kinase via activation loop phosphorylation and phosphorylates the c-jun N-terminus, Oncogene 21 (2002) 2154-2160], but the sites have not been identified. Here, metabolic {sup 32}P-labeling of c-jun protein in COS-7 cells indicated that PKD phosphorylates c-jun in vivo at a site(s) between aa 43-93, a region containing important functional elements. On this basis, the PKD-mediated phosphorylation site(s) was further characterized in vitro using GST-c-jun fusion proteins. PKD did not incorporate phosphate into Ser63 and Ser73, the JNK sites in GST-c-jun(1-89). Rather, PKD and JNK could sequentially phosphorylate distinct site(s) simultaneously. By mass spectrometry of tryptic phosphopeptides, Ser58 interposed between the JNK-binding portion of the delta domain and the adjacent TAD1 was identified as a prominent site phosphorylated in vitro by PKD. These data were further supported by kinase reactions using truncations or point-mutations of GST-c-jun. Together, these data suggest that PKD-mediated phosphorylation modulates c-jun at the level of its N-terminal functional domains.

  6. Ser/Thr/Tyr Protein Phosphorylation in the Archaeon Halobacterium salinarum—A Representative of the Third Domain of Life

    PubMed Central

    Gnad, Florian; Reichelt, Peter; Mann, Matthias; Oesterhelt, Dieter

    2009-01-01

    In the quest for the origin and evolution of protein phosphorylation, the major regulatory post-translational modification in eukaryotes, the members of archaea, the “third domain of life”, play a protagonistic role. A plethora of studies have demonstrated that archaeal proteins are subject to post-translational modification by covalent phosphorylation, but little is known concerning the identities of the proteins affected, the impact on their functionality, the physiological roles of archaeal protein phosphorylation/dephosphorylation, and the protein kinases/phosphatases involved. These limited studies led to the initial hypothesis that archaea, similarly to other prokaryotes, use mainly histidine/aspartate phosphorylation, in their two-component systems representing a paradigm of prokaryotic signal transduction, while eukaryotes mostly use Ser/Thr/Tyr phosphorylation for creating highly sophisticated regulatory networks. In antithesis to the above hypothesis, several studies showed that Ser/Thr/Tyr phosphorylation is also common in the bacterial cell, and here we present the first genome-wide phosphoproteomic analysis of the model organism of archaea, Halobacterium salinarum, proving the existence/conservation of Ser/Thr/Tyr phosphorylation in the “third domain” of life, allowing a better understanding of the origin and evolution of the so-called “Nature's premier” mechanism for regulating the functional properties of proteins. PMID:19274099

  7. Intrinsic disorder within an AKAP-protein kinase A complex guides local substrate phosphorylation

    PubMed Central

    Smith, F Donelson; Reichow, Steve L; Esseltine, Jessica L; Shi, Dan; Langeberg, Lorene K; Scott, John D; Gonen, Tamir

    2013-01-01

    Anchoring proteins sequester kinases with their substrates to locally disseminate intracellular signals and avert indiscriminate transmission of these responses throughout the cell. Mechanistic understanding of this process is hampered by limited structural information on these macromolecular complexes. A-kinase anchoring proteins (AKAPs) spatially constrain phosphorylation by cAMP-dependent protein kinases (PKA). Electron microscopy and three-dimensional reconstructions of type-II PKA-AKAP18γ complexes reveal hetero-pentameric assemblies that adopt a range of flexible tripartite configurations. Intrinsically disordered regions within each PKA regulatory subunit impart the molecular plasticity that affords an ∼16 nanometer radius of motion to the associated catalytic subunits. Manipulating flexibility within the PKA holoenzyme augmented basal and cAMP responsive phosphorylation of AKAP-associated substrates. Cell-based analyses suggest that the catalytic subunit remains within type-II PKA-AKAP18γ complexes upon cAMP elevation. We propose that the dynamic movement of kinase sub-structures, in concert with the static AKAP-regulatory subunit interface, generates a solid-state signaling microenvironment for substrate phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.01319.001 PMID:24192038

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

    PubMed Central

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

    2015-01-01

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

  9. Changes in reflectin protein phosphorylation are associated with dynamic iridescence in squid

    PubMed Central

    Izumi, Michi; Sweeney, Alison M.; DeMartini, Daniel; Weaver, James C.; Powers, Meghan L.; Tao, Andrea; Silvas, Tania V.; Kramer, Ryan M.; Crookes-Goodson, Wendy J.; Mäthger, Lydia M.; Naik, Rajesh R.; Hanlon, Roger T.; Morse, Daniel E.

    2010-01-01

    Many cephalopods exhibit remarkable dermal iridescence, a component of their complex, dynamic camouflage and communication. In the species Euprymna scolopes, the light-organ iridescence is static and is due to reflectin protein-based platelets assembled into lamellar thin-film reflectors called iridosomes, contained within iridescent cells called iridocytes. Squid in the family Loliginidae appear to be unique in which the dermis possesses a dynamic iridescent component with reflective, coloured structures that are assembled and disassembled under the control of the muscarinic cholinergic system and the associated neurotransmitter acetylcholine (ACh). Here we present the sequences and characterization of three new members of the reflectin family associated with the dynamically changeable iridescence in Loligo and not found in static Euprymna iridophores. In addition, we show that application of genistein, a protein tyrosine kinase inhibitor, suppresses ACh- and calcium-induced iridescence in Loligo. We further demonstrate that two of these novel reflectins are extensively phosphorylated in concert with the activation of iridescence by exogenous ACh. This phosphorylation and the correlated iridescence can be blocked with genistein. Our results suggest that tyrosine phosphorylation of reflectin proteins is involved in the regulation of dynamic iridescence in Loligo. PMID:19776150

  10. In vivo phosphorylation of the Na,K-ATPase alpha subunit in sciatic nerves of control and diabetic rats: effects of protein kinase modulators.

    PubMed Central

    Borghini, I; Geering, K; Gjinovci, A; Wollheim, C B; Pralong, W F

    1994-01-01

    The phosphorylation state of the Na,K-ATPase alpha subunit has been examined in 32P-labeled sciatic nerves of control and streptozotocin-treated diabetic rats. Intact nerves were challenged with protein kinase (PK) modulators and alpha-subunit 32P labeling was analyzed after immunoprecipitation. In control nerves, the PKC activator phorbol 12-myristate 13-acetate (PMA) had little effect on alpha-subunit 32P labeling. In contrast, staurosporine, a PKC inhibitor, and extracellular calcium omission decreased it. In Ca(2+)-free conditions, PMA restored the labeling to basal levels. The cAMP-raising agent forskolin reduced the 32P labeling of the alpha subunit. The results suggest that nerve Na,K-ATPase is tonically phosphorylated by PKC in a Ca(2+)-dependent manner and that PKA modulates the phosphorylation process. In nerves of diabetic rats, PMA increased 32P labeling of the alpha subunit. In contrast to staurosporine or extracellular calcium omission, the decreased state of phosphorylation seen with forskolin was no longer significant in diabetic nerves. No change in the level of alpha-subunit isoforms (alpha 1 or alpha 2) was detected by Western blot analysis in such nerves. In conclusion, the altered effect of PK activators on Na,K-ATPase phosphorylation state is consistent with the view that a defect in PKC activation exists in diabetic nerves. Images PMID:8016140

  11. Methyl-CpG-binding protein 2 is phosphorylated by homeodomain-interacting protein kinase 2 and contributes to apoptosis.

    PubMed

    Bracaglia, Giorgia; Conca, Barbara; Bergo, Anna; Rusconi, Laura; Zhou, Zhaolan; Greenberg, Michael E; Landsberger, Nicoletta; Soddu, Silvia; Kilstrup-Nielsen, Charlotte

    2009-12-01

    Mutations in the methyl-CpG-binding protein 2 (MeCP2) are associated with Rett syndrome and other neurological disorders. MeCP2 represses transcription mainly by recruiting various co-repressor complexes. Recently, MeCP2 phosphorylation at Ser 80, Ser 229 and Ser 421 was shown to occur in the brain and modulate MeCP2 silencing activities. However, the kinases directly responsible for this are largely unknown. Here, we identify the homeodomain-interacting protein kinase 2 (HIPK2) as a kinase that binds MeCP2 and phosphorylates it at Ser 80 in vitro and in vivo. HIPK2 modulates cell proliferation and apoptosis, and the neurological defects of Hipk2-null mice indicate its role in proper brain functions. We show that MeCP2 cooperates with HIPK2 in induction of apoptosis and that Ser 80 phosphorylation is required together with the DNA binding of MeCP2. These data are, to our knowledge, the first that describe a kinase associating with MeCP2, causing its specific phosphorylation in vivo and, furthermore, they reinforce the role of MeCP2 in regulating cell growth. PMID:19820693

  12. Methyl-CpG-binding protein 2 is phosphorylated by homeodomain-interacting protein kinase 2 and contributes to apoptosis

    PubMed Central

    Bracaglia, Giorgia; Conca, Barbara; Bergo, Anna; Rusconi, Laura; Zhou, Zhaolan; Greenberg, Michael E; Landsberger, Nicoletta; Soddu, Silvia; Kilstrup-Nielsen, Charlotte

    2009-01-01

    Mutations in the methyl-CpG-binding protein 2 (MeCP2) are associated with Rett syndrome and other neurological disorders. MeCP2 represses transcription mainly by recruiting various co-repressor complexes. Recently, MeCP2 phosphorylation at Ser 80, Ser 229 and Ser 421 was shown to occur in the brain and modulate MeCP2 silencing activities. However, the kinases directly responsible for this are largely unknown. Here, we identify the homeodomain-interacting protein kinase 2 (HIPK2) as a kinase that binds MeCP2 and phosphorylates it at Ser 80 in vitro and in vivo. HIPK2 modulates cell proliferation and apoptosis, and the neurological defects of Hipk2-null mice indicate its role in proper brain functions. We show that MeCP2 cooperates with HIPK2 in induction of apoptosis and that Ser 80 phosphorylation is required together with the DNA binding of MeCP2. These data are, to our knowledge, the first that describe a kinase associating with MeCP2, causing its specific phosphorylation in vivo and, furthermore, they reinforce the role of MeCP2 in regulating cell growth. PMID:19820693

  13. Architecture of the cystic fibrosis transmembrane conductance regulator protein and structural changes associated with phosphorylation and nucleotide binding.

    PubMed

    Zhang, Liang; Aleksandrov, Luba A; Zhao, Zhefeng; Birtley, James R; Riordan, John R; Ford, Robert C

    2009-09-01

    We describe biochemical and structural studies of the isolated cystic fibrosis transmembrane conductance regulator (CFTR) protein. Using electron cryomicroscopy, low resolution three-dimensional structures have been obtained for the non-phosphorylated protein in the absence of nucleotide and for the phosphorylated protein with ATP. In the latter state, the cytosolic nucleotide-binding domains move closer together, forming a more compact packing arrangement. Associated with this is a reorganization within the cylindrical transmembrane domains, consistent with a shift from an inward-facing to outward-facing configuration. A region of density in the non-phosphorylated protein that extends from the bottom of the cytosolic regions up to the transmembrane domains is hypothesised to represent the unique regulatory region of CFTR. These data offer insights into the architecture of this ATP-binding cassette protein, and shed light on the global motions associated with nucleotide binding and priming of the chloride channel via phosphorylation of the regulatory region. PMID:19524678

  14. Protein kinase CK2 triggers cytosolic zinc signaling pathways by phosphorylation of zinc channel ZIP7

    PubMed Central

    Taylor, Kathryn M.; Hiscox, Stephen; Nicholson, Robert I.; Hogstrand, Christer; Kille, Peter

    2012-01-01

    The transition element zinc, which has recently been identified as an intracellular second messenger, has been implicated in various signaling pathways, including those leading to cell proliferation. Zinc channels of the ZIP protein family (Solute Carrier Family 39A, SLC39A) transiently increase the cytosolic free zinc (Zn2+) concentration in response to extracellular signals. Here, we show that phosphorylation of evolutionarily conserved residues in zinc transporter ZIP7 is associated with the gated release of Zn2+ from intracellular stores, leading to activation of tyrosine kinases and the phosphorylation of AKT and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Through pharmacological manipulation, proximity assay, and mutagenesis, we identified CK2 as the kinase responsible for ZIP7 activation. Together, the present results show that eukaryotic transition element channels can be activated post-translationally by phosphorylation eliciting a cell signaling cascade. Our study links the regulated release of zinc from intracellular stores to phosphorylation of kinases involved in proliferative responses and cell migration, suggesting a functional role for ZIP7 and zinc signals for these events which are characteristic of cancerous cells. Furthermore, the interaction of ZIP7 with CK2, a kinase that is antiapoptotoc and promotes cell division, highlights the potential for ZIP7 as a target for anti-cancer drug development. PMID:22317921

  15. Phosphoryl transfer reaction snapshots in crystals: Insights into the mechanism of protein kinase a catalytic subunit

    DOE PAGESBeta

    Das, Amit; Gerlits, Oksana O.; Heller, William T.; Kovalevskyi, Andrii Y.; Langan, Paul; Tian, Jianhui

    2015-06-19

    To study the catalytic mechanism of phosphorylation catalyzed by cAMP-dependent protein kinase (PKA) a structure of the enzyme-substrate complex representing the Michaelis complex is of specific interest as it can shed light on the structure of the transition state. However, all previous crystal structures of the Michaelis complex mimics of the PKA catalytic subunit (PKAc) were obtained with either peptide inhibitors or ATP analogs. Here we utilized Ca2+ ions and sulfur in place of the nucleophilic oxygen in a 20-residue pseudo-substrate peptide (CP20) and ATP to produce a close mimic of the Michaelis complex. In the ternary reactant complex, themore » thiol group of Cys-21 of the peptide is facing Asp-166 and the sulfur atom is positioned for an in-line phosphoryl transfer. Replacement of Ca2+ cations with Mg2+ ions resulted in a complex with trapped products of ATP hydrolysis: phosphate ion and ADP. As a result, the present structural results in combination with the previously reported structures of the transition state mimic and phosphorylated product complexes complete the snapshots of the phosphoryl transfer reaction by PKAc, providing us with the most thorough picture of the catalytic mechanism to date.« less

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

    SciTech Connect

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

    2013-02-01

    Highlights: ► AMPK phosphorylates CtBP1 on serine 158. ► AMPK-mediated phosphorylation of CtBP1 causes the ubiquitination and