Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Although the human immunodeficiency virus type 1 (HIV-1) nef gene still has no precisely defined function, in vivo studies have demonstrated that Nef is an important pathogenic determinant of HIV. In order to identify cellular proteins capable of binding to Nef, the HIV-1LAI nef gene product was expressed in the bacterial vector pGEX-2T as a glutathione S-transferase (GST)-Nef fusion protein. Deletion mutants corresponding to 86 and 35 N-terminal residues of the Nef protein were prepared. The GST-Nef constructs were used to identify cellular kinases capable of interacting with Nef. After incubation with a Jurkat cell lysate, the GST-Nef constructs immobilized on glutathione-agarose beads bound to cellular kinase(s) and were phosphorylated at three sites in vitro: one on threonine at position 15, one on serine between residues 1 and 35, and one on threonine between residues 36 and 86. The Nef-phosphorylating activity was inhibited by protein kinase C (PKC)-selective inhibitors. Cell fractionation showed that this Nef-binding kinase was mainly in the membrane-associated fraction. These results suggest that kinase(s) of the PKC family are specifically bound to and phosphorylate Nef in vitro. The interaction of Nef with cellular kinases and its phosphorylation may be important in mediating the effects of Nef in HIV-1 pathogenesis.
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PMID:In vitro binding and phosphorylation of human immunodeficiency virus type 1 Nef protein by serine/threonine protein kinase. 754 Jan 94

Most neurotransmitter receptors examined to date are either regulated by phosphorylation or contain consensus sequences for phosphorylation by protein kinases. The nicotinic acetylcholine receptor (AChR), which mediates depolarization at the neuromuscular junction, has served as a model for the study of the structure, function, and regulation of ligand-gated ion channels. The AChR is phosphorylated by protein kinase A, protein kinase C, and an unidentified protein tyrosine kinase. Tyrosine phosphorylation of the AChR is correlated with a modulation of the rate of receptor desensitization and is associated with AChR clustering. We showed that agrin, a neuronally derived extracellular matrix protein, induces AChR clustering and tyrosine phosphorylation. In addition, we identified two protein tyrosine kinases, Fyn and Fyk, that appear to be involved in the regulation of synaptic transmission at the neuromuscular junction by phosphorylating the AChR. The two kinases are highly expressed in Torpedo electric organ, a tissue enriched in synaptic components including the AChR. As demonstrated by coimmunoprecipitation, Fyn and Fyk associate with the AChR. Furthermore, the AChR is phosphorylated in Fyn and Fyk immunoprecipitates. We investigated the molecular basis for the association of the AChR with Fyn and Fyk using fusion proteins derived from the kinases. The AChR bound specifically to the SH2 domain fusion proteins of Fyn and Fyk. The association of the AChR with the SH2 domains is dependent on the state of AChR tyrosine phosphorylation and is mediated by the delta subunit of the receptor. These data provide evidence that the protein tyrosine kinases Fyn and Fyk may act to phosphorylate the AChR in vivo.
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PMID:Phosphorylation of the nicotinic acetylcholine receptor by protein tyrosine kinases. 754 72

The significance of site-specific phosphorylation of cardiac troponin I (TnI) by protein kinase C and protein kinase A in the regulation of Ca(2+)-stimulated MgATPase of reconstituted actomyosin S-1 was investigated. The TnI mutants used were T144A, S43A/S45A, and S43A/S45A/T144A (in which the identified protein kinase C phosphorylation sites, Thr-144 and Ser-43/ Ser-45, were, respectively, substituted by Ala) and S23A/S24A and N32 (in which the protein kinase A phosphorylation sites Ser-23/Ser-24 were either substituted by Ala or deleted). The mutations caused subtle changes in the kinetics of phosphorylation by protein kinase C, and all mutants were maximally phosphorylated to various extents (1.3-2.7 mol of phosphate/mol of protein). Protein kinase C could cross-phosphorylate protein kinase A sites but the reverse essentially could not occur. Compared to wild-type TnI and T144A, un-phosphorylated S43A/S45A, S43A/S45A/T144, S23A/ S24A, and N32 caused a decreased Ca2+ sensitivity of Ca(2+)-stimulated MgATPase of reconstituted actomyosin. S-1. Phosphorylation by protein kinase C of wild-type and all mutants except S43A/S45A and S43A/S45A/T144A caused marked reductions in both the maximal activity of Ca(2+)-stimulated MgATPase and apparent affinity of myosin S-1 for reconstitued (regulated) actin. It was further noted that protein kinase C acted in an additive manner with protein kinase A by phosphorylating Ser-23/Ser-24 to bring about a decreased Ca2+ sensitivity of the myofilament. It is suggested that Ser-43/Ser-45 and Ser-23/Ser-24 in cardiac TnI are important for normal Ca2+ sensitivity of the myofilament, and that phosphorylation of Ser-43/Ser-45 and Ser-23/Ser-24 is primarily involved in the protein kinase C regulation of the activity and Ca2+ sensitivity, respectively, of actomyosin S-1 MgATPase.
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PMID:Cardiac troponin I mutants. Phosphorylation by protein kinases C and A and regulation of Ca(2+)-stimulated MgATPase of reconstituted actomyosin S-1. 759 12

RS7-3G11 is a murine monoclonal antibody (MAb) raised against human non-small-cell lung carcinoma, and is under clinical evaluation. The epithelial/carcinoma antigen EGP-1, defined by RS7-3G11, was isolated and purified to homogeneity from a cervical carcinoma cell line, ME180. EGP-1 is a glycoprotein with an average molecular mass of 47.8 kDa. Metabolic labeling of the antigen with 32P-orthophosphate and subsequent immunoprecipitation with RS7-3G11 showed that it is a phosphoprotein. Phosphoamino acid analysis of the in vivo phosphorylated EGP-1 revealed that the phosphorylation is on serine. In vitro analysis with purified antigen demonstrated that protein kinase C, and not protein kinase A, is involved in phosphorylating the antigen in vitro. In vitro analysis indicated a stoichiometry of phosphorylation of 0.54 mole of phosphate per mole of EGP-1. Phosphoamino acid analysis and phosphopeptide mapping of the antigen phosphorylated in vitro by protein kinase C showed that phosphorylation occurred on a serine residue, specifically on serine 303, located in the cytoplasmic domain of EGP-1. Treatment of ME180 cells with phorbol ester increased the phosphorylation of EGP-1. The biological function of EGP-1 remains to be elucidated. In this report we elucidate an involvement of protein kinase C in phosphorylating EGP-1, which may signify a role for this antigen in signal transduction across the cell membrane.
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PMID:The epithelial/carcinoma antigen EGP-1, recognized by monoclonal antibody RS7-3G11, is phosphorylated on serine 303. 763 74

In a variety of intact cells, phorbol esters are known to activate phospholipase D. In a cell-free system consisting of plasma membrane and cytosol from human neutrophils, phorbol esters activated phospholipase D in an adenosine nucleotide triphosphate-dependent manner. ATP gamma S (adenosine 5'-O-(thiotriphosphate)) was 2-3-fold more effective than ATP, while ADP and AppNHp (adenyl-5'-yl imidodiphosphate) were ineffective, and activation was blocked by the kinase inhibitor staurosporine. In cytosol deplete of protein kinase C by chromatography on threnoine-Sepharose, phorbol ester-dependent activation was lost, but was restored upon addition of purified rat brain protein kinase C. The target for phosphorylation was shown to be the plasma membrane plasma membrane was phosphorylated using ATP gamma S/phorbol 12,13-dibutyrate and protein kinase C and was reisolated to remove activators. Upon adding nucleotide-depleted cytosol, activator-independent phospholipase D activity was seen. Using this prephosphorylation protocol, PKC-dependent activation of plasma membranes was found to require micromolar calcium, implicating a conventional protein kinase C. Using recombinant isoforms of protein kinase C, only the conventional isoforms showed significant activation, with the following rank order of potency: beta 1 > alpha > gamma; the beta 2, delta, epsilon, eta, and sigma isoforms showed little or no activity. Thus, conventional isoform(s) of protein kinase C activate neutrophil phospholipase D by phosphorylating a target protein located in the plasma membrane.
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PMID:Regulation of phospholipase D by protein kinase C in human neutrophils. Conventional isoforms of protein kinase C phosphorylate a phospholipase D-related component in the plasma membrane. 764 30

Paraquat (methyl viologen, PQ) is a widely used herbicide that produces oxygen-derived free radicals and severely injures human lungs. In this study we examined the effects of PQ on the protein kinase C (PKC), ornithine decarboxylase (ODC) and c-jun oncogene expression in WI-38 human lung cells. Exposure of cells to 25-200 microM PQ resulted in an increase of [3H]phorbol dibutyrate (PDBu) binding and PKC redistribution in a dose-dependent manner. Interestingly, a superoxide dismutase mimic, 4-hydroxyl-2,2,6,6-tetramethyl-piperidine-1-oxyl (Tempol, 2.5 mM) and catalase (400 micrograms/ml) could significantly reduce the PQ-stimulated increase of phorbol ester binding and particular PKC phosphorylating activity, but dimethylsulfoxide (DMSO, 1.5%), an effective .OH trapping agent, failed to prevent this stimulation. In addition, an endogenous substrate of PKC, 80 kDa protein, was found to be highly phosphorylated in intact WI-38 cells treated with 50 microM PQ. The increase of phosphorylated proteins could be completely or partly abolished by Tempol or catalase, but only the phosphorylation of 80 kDa protein was diminished by protein kinase C inhibitor, 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7). A maximal peak of ODC activity was observed at 6 h of treatment with 50 microM PQ. PQ induced activity was reduced at the following rates, Tempol 85%, DMSO 80% and catalase 45%, but H-7 failed to do so. Furthermore, we found that the level of c-jun mRNA was transiently increased by PQ and the peak appeared at 1 h of treatment. When correlated with the PKC result, Tempol, catalase and H-7 all effectively blocked PQ-elicited c-jun transcript expression, but DMSO only exhibited a weakly inhibitory effect. We therefore propose that superoxide anion (O2- and H2O2 generated by PQ could activate PKC and lead to induction of c-jun gene expression; on the other hand, O2- and .OH might trigger other kinase pathways to elevate ODC activity. Finally, the sequential expression of c-jun oncogene and ODC may cooperate to relieve the oxidative damages elicited by PQ.
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PMID:Pronounced activation of protein kinase C, ornithine decarboxylase and c-jun proto-oncogene by paraquat-generated active oxygen species in WI-38 human lung cells. 766 13

The regulation of the inhibitor of nuclear factor kappa B (I kappa B) by interleukin 1 (IL1) was investigated in HeLa cells. Two forms of I kappa B were resolved by ion-exchange chromatography. The major form (75%) was identified as MAD3 by specific antisera. IL1 generated rapidly (6 min) an electrophoretically retarded form of MAD3 that was stable in acid and was converted into the unmodified form by phosphatase 2A. It thus corresponded to a phosphorylation of the protein on serine or threonine. IL1 also caused the disappearance of MAD3 from the cells, which was complete 15 min after stimulation and coincided with a 46% reduction of cellular I kappa B activity. Newly-synthesized MAD3 accumulated to pre-stimulation levels between 60 and 90 min after stimulation and this coincided with the down-regulation of the phosphorylating activity. The serine proteinase inhibitors 3,4-dichloroisocoumarin (DCI) and tosylphenylalanyl chloromethylketone (TPCK) prevented phosphorylation and disappearance of MAD3. At the same concentrations (10-100 microM), they also increased basal phosphorylation of the small heat shock protein (hsp27) and prevented the IL1- and phorbol 12-myristate 13-acetate-induced increases of its phosphorylation. The inhibitors were thus interfering with protein kinases when blocking degradation of MAD3. Recombinant MAD3 phosphorylated in vitro by protein kinase C was not electrophoretically retarded, suggesting that MAD3 was phosphorylated by another kinase in IL1-stimulated cells. Our results suggest that the IL1-induced phosphorylation of MAD3 on serine or threonine leads to its degradation. DCI and TPCK blocked phosphorylation mechanisms and it could not be concluded that serine proteinases were involved in the breakdown of MAD3.
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PMID:Interleukin 1-induced phosphorylation of MAD3, the major inhibitor of nuclear factor kappa B of HeLa cells. Interference in signalling by the proteinase inhibitors 3,4-dichloroisocoumarin and tosylphenylalanyl chloromethylketone. 771 87

Phosphorylation of the P proteins of nonsegmented negative-strand RNA viruses is critical for their function as transactivators of the viral RNA polymerases. Using unphosphorylated P protein of human parainfluenza virus type 3 (HPIV3) expressed in Escherichia coli, we have shown that the cellular protein kinase that phosphorylates P in vitro is biochemically and immunologically indistinguishable from cellular protein kinase C isoform zeta (PKC-zeta). Further, PKC-zeta is specifically packaged within the progeny HPIV3 virions and remains tightly associated with the ribonucleoprotein complex. The P protein seems also to be phosphorylated intracellularly by PKC-zeta, as shown by the similar protease digestion pattern of the in vitro and in vivo phosphorylated P proteins. The growth of HPIV3 in CV-1 cells is completely abrogated when a PKC-zeta-specific inhibitor pseudosubstrate peptide was delivered into cells. These data indicate that PKC-zeta plays an important role in HPIV3 gene expression by phosphorylating P protein, thus providing an opportunity to develop antiviral agents against an important human pathogen.
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PMID:Cellular protein kinase C isoform zeta regulates human parainfluenza virus type 3 replication. 776 74

Sublethal concentrations of reactive oxygen intermediates including H2O2 can alter human T cell function and inhibit proliferative responses but relatively little is known about the effects of low levels of oxidant stress on signaling pathways. In the present study, we investigated whether the exposure of Jurkat T cells to micromolar concentrations of H2O2 might influence the activity of certain serine/threonine kinases and protein phosphatases important for T cell signaling as well as initiation of nuclear events. Jurkat cells treated with 100-200 microM H2O2 exhibited rapid increases in cytosolic protein kinase C (PKC) activity without detectable translocation of PKC to the membrane/particulate compartment. The stimulation of PKC activity by H2O2 was associated with an increase in the activation of kinases phosphorylating myelin basic protein (MBP), a substrate for mitogen-activated protein (MAP) kinase and RRLSSLRA (S6 peptide; a substrate for the approximately 90-kDa ribosomal S6 kinases). Optimal activation of MAP kinase in cells treated with H2O2 was preceded by increases in protein tyrosine phosphorylations and occurred at sublethal concentrations of H2O2 which did not markedly deplete intracellular ATP. Pretreatment of cells with the PKC inhibitors sangivamycin and H7 suppressed but did not block the stimulation of MAP kinase activity in response to H2O2 or phytohemagglutinin. The activities of both protein tyrosine phosphatase (PTP) and protein phosphatase 2A (PP2A) were reduced after H2O2 treatment of intact cells. Furthermore, kinetic studies showed that H2O2 was capable of suppressing the activities of PTP and PP2A before inducing optimal increases in MAP kinase activity. These results demonstrate that the exposure of T cells to sublethal levels of oxidant stress acutely stimulates the MAP kinase cascade and suggest that this activation may involve PKC-dependent and -independent pathways as well as inhibition of certain protein phosphatases.
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PMID:Sublethal levels of oxidant stress stimulate multiple serine/threonine kinases and suppress protein phosphatases in Jurkat T cells. 777 89

The effects of the calcium ionophore, A23187, on human neutrophil activation were studied in relation to the signaling mechanism of cyclic guanosine monophosphate (cGMP)-dependent protein kinase (G-kinase). Immunocytochemistry demonstrated that G-kinase translocated from a diffuse localization in the cytoplasm to the cytoskeleton after stimulation with A23187. Over a period of 5 minutes, G-kinase was transiently colocalized with the intermediate filament protein, vimentin. At 3 minutes' stimulation with A23187, colocalization of G-kinase and vimentin was predominantly confined to filaments that extended into the uropod. The time of colocalization of G-kinase and vimentin was reduced in the A23187-stimulated cell from 3 minutes to 1 minute by 8-Br-cGMP. Coincident with colocalization was an increase in cGMP levels and transient phosphorylation of vimentin in adhered A23187-stimulated cells. Phosphorylation of vimentin was maximal after 3 minutes with A23187, and was essentially over at 5 minutes. The time of phosphorylation of vimentin was also reduced from 3 minutes to 1 minute when cells were preincubated with 8-Br-cGMP and then stimulated with A23187, which suggests that cyclic adenosine monophosphate (cAMP)-dependent protein kinase does not phosphorylate vimentin in A23187-treated neutrophils. Phosphorylation of vimentin was not observed in nonactivated cells treated only with 8-Br-cGMP. The presence of the protein kinase C inhibitors, staurosporine or H-7, did not inhibit vimentin phosphorylation in A23187-treated cells, which provides supportive data that protein kinase C is not the phosphorylating enzyme. These results suggest that vimentin and G-kinase are colocalized in a Ca(2+)-dependent manner in neutrophils, and that vimentin is transiently phosphorylated by G-kinase in response to the colocalization of the two proteins. The transient redistribution of compartmentalized G-kinase represents one type of neutrophil activation mechanism.
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PMID:Cyclic guanosine monophosphate-dependent protein kinase is targeted to intermediate filaments and phosphorylates vimentin in A23187-stimulated human neutrophils. 780 96


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