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

Rat 6 fibroblasts that stably overexpress cDNA for the beta 1 isozyme of protein kinase C (PKC3 cells) were used to determine the effect of protein kinase C (PKC) overexpression on hormonal stimulation of phospholipid hydrolysis. In control Rat 6 cells, inositol trisphosphate levels (InsP3) were increased 9-fold in 15 s in response to 10 nM alpha-thrombin, compared with only a 2-fold increase in PKC3 cells. PKC overexpression also inhibited thrombin-stimulated production of 1,2-diacylglycerol, the other product of phosphatidylinositol 4,5-bisphosphate hydrolysis, by 73% at 15 s. In permeabilized cells, PKC overexpression greatly reduced guanosine thiotriphosphate-stimulated InsP3 accumulation, but did not affect InsP3 stimulation by increased free calcium concentration. These data suggest that desensitization of thrombin-stimulated phosphoinositide-phospholipase C is enhanced by PKC-beta 1 overexpression and may involve modulation of G-protein/phospholipase C coupling. In contrast, thrombin was 4.5-fold more effective in stimulation of phosphatidylcholine-phospholipase D activity in PKC3 cells than in control cells, as determined by phosphatidylethanol formation. In permeabilized cells, guanosine thiotriphosphate also stimulated phospholipase D activity more effectively in PKC3 cells than in control cells, suggesting that upregulation of phospholipase D activity by PKC overexpression occurs distal to the thrombin receptor. These results suggest that PKC may act as a switch to up-regulate phosphatidylcholine-phospholipase D and down-regulate phosphoinositide-phospholipase C stimulations.
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PMID:Differential regulation of phosphoinositide and phosphatidylcholine hydrolysis by protein kinase C-beta 1 overexpression. Effects on stimulation by alpha-thrombin, guanosine 5'-O-(thiotriphosphate), and calcium. 131 71

The effects of protein kinase C (PKC) activators on gamma-aminobutyric acidA (GABAA) receptor function were studied by two-electrode voltage-clamp in Xenopus oocytes expressing brain mRNA or subunit cDNAs and in isolated mouse brain cerebellar membrane vesicles (microsacs), using 36Cl- uptake. Both oocytes and microsacs showed transient (desensitizing) and sustained (nondesensitizing) GABAA receptor responses. In oocytes expressing brain mRNA, the PKC activator phorbol myristoyl acetate (PMA), but not the inactive analog phorbol 12-monomyristate, inhibited both transient and sustained GABA-gated chloride currents. The inhibition by PMA was concentration dependent, with an EC50 of approximately 5 nM, and resulted in a decrease in the efficacy, but not the potency, of GABA. Additionally, PMA inhibited GABA-gated chloride currents in oocytes expressing alpha 1 beta 1 gamma 2L subunit cDNAs. The effect of PMA on recombinant receptors was significantly antagonized by PKC inhibitory peptide (PKCI). In the microsac preparation, the PKC activators (-)-7-octylindolactam V and PMA inhibited the sustained phase of 36Cl- flux without altering the transient phase. The action of PMA was blocked by kinase inhibitors and by depletion of Mg-ATP and was mimicked by protein phosphatase inhibitors. These results demonstrate that activation of PKC inhibits GABAA receptor function, and the results from the microsac experiments suggest that PKC-dependent phosphorylation preferentially inactivates a nondesensitized form or state of the receptor.
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PMID:Activation of protein kinase C selectively inhibits the gamma-aminobutyric acidA receptor: role of desensitization. 131 47

Gamma-aminobutyric acid Type A (GABAA) receptors are the major sites of synaptic inhibition in the central nervous system. These receptors are thought to be pentameric complexes of homologous transmembrane glycoproteins. Molecular cloning has revealed a multiplicity of different GABAA receptor subunits divided into five classes, alpha, beta, gamma, delta, and rho, based on sequence homology. Within the proposed major intracellular domain of these subunits, there are numerous potential consensus sites for protein phosphorylation by a variety of protein kinases. We have used purified fusion proteins of the major intracellular domain of GABAA receptor subunits produced in Escherichia coli to examine the phosphorylation of these subunits by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). The purified fusion protein of the intracellular domain of the beta 1 subunit was an excellent substrate for both PKA and PKC. PKA and PKC phosphorylated the beta 1 subunit fusion protein on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 409 in the intracellular domain of the beta 1 subunit to an alanine residue eliminated the phosphorylation of the beta 1 subunit fusion protein by both protein kinases. The purified fusion proteins of the major intracellular domain of the gamma 2S and gamma 2L subunits of the GABAA receptor were rapidly and stoichiometrically phosphorylated by PKC but not by PKA. The phosphorylation of the gamma 2S subunit occurred on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 327 of the gamma 2S subunit fusion protein to an alanine residue eliminated the phosphorylation of the gamma 2S fusion protein by PKC. The gamma 2L subunit is an alternatively spliced form of the gamma 2S subunit that differs by the insertion of 8 amino acids (LLRMFSFK) within the major intracellular domain of the gamma 2S subunit. The PKC phosphorylation of the gamma 2L subunit occurred on serine residues on two tryptic phosphopeptides. Site-specific mutagenesis of serine 343 within the 8-amino acid insert to an alanine residue eliminated the PKC phosphorylation of the novel site in the gamma 2L subunit. No phosphorylation of a purified fusion protein of the major intracellular loop of the alpha 1 subunit was observed with either PKA or PKC. These results identify the specific amino acid residues within GABAA receptor subunits that are phosphorylated by PKA and PKC and suggest that protein phosphorylation of these sites may be important in regulating GABAA receptor function.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Identification of the cAMP-dependent protein kinase and protein kinase C phosphorylation sites within the major intracellular domains of the beta 1, gamma 2S, and gamma 2L subunits of the gamma-aminobutyric acid type A receptor. 132 Nov 50

Treatment of rat submandibular acinar cell extracts with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused the dose-dependent activation of protein kinase C (PKC), assessed by the phosphorylation of a novel and highly specific substrate. This effect was duplicated by a diacylglycerol, but not by the 4 alpha-phorbol ester 4 alpha-phorbol 12,13-didecanoate. The TPA elevation of PKC was blocked by the PKC inhibitors H-7 and sangivamycin. In intact cells, TPA caused the translocation of PKC from cytosol to membrane, consistent with its known mode of activation. The beta-adrenergic agonist, isoproterenol, stimulated cAMP levels which were significantly reduced by preactivation of PKC. This inhibitory PKC effect was reversed by H-7. When cAMP was stimulated at the post-receptor level, however, by forskolin, NaF or GTP[gamma S], PKC did not inhibit, but rather enhanced the cyclic nucleotide response. Since PKC phosphorylated an endogenous protein of 55 kDa, the size of the beta 1 receptor, these findings indicate that, as in other cell types, PKC can desensitize adenylate cyclase by direct phosphorylation of the beta receptor, but potentiate the cAMP response by a post-receptor mechanism. In mucin secretion studies in the model, TPA alone caused the cAMP-independent release of up to 44% total mucin, which was much less than additive with the isoproterenol response. When the cAMP-mucosecretory response was stimulated at the adenylate cyclase level by forskolin, however, the TPA + forskolin effects were additive. These findings on the modulation of cAMP by PKC indicate cross-talk regulation in the phosphoinositide-cAMP signal transduction pathways in submandibular acinar cells.
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PMID:Regulation of the cAMP signal transduction pathway by protein kinase C in rat submandibular cells. 132 9

The binding of a variety of agonists to their receptors leads to the breakdown of membrane phospholipids and the formation of intracellular second messengers. Hydrolysis of inositol phospholipids by phospholipase C results in the formation of two second messengers, inositol-1,4,5-trisphosphate which mobilizes intracellular calcium and the neutral lipid diacylglycerol (DAG) which binds to and activates protein kinase C (PKC). PKC is actually a family of homologous serine/threonine protein kinases which play a central role in regulation of growth, differentiation and secretion reactions in a variety of cell types. In addition to these feedforward roles of PKC, it is thought to play an important feedback role, regulating early events in signal transduction. To explore these feedback functions we have examined the effect of PKC inhibitors on second messenger formation in thrombin-stimulated human platelets (a rapidly responding system) and the effect of PKC overexpression on second messenger formation and mitogenesis in rat fibroblasts (a system where sustained signaling occurs). Treatment of platelets with inhibitors of PKC potentiates DAG mass formation in response to thrombin while prior activation of PKC with phorbol esters blocks DAG mass formation, consistent with PKC playing a negative feedback role, inhibiting inositol phospholipid breakdown. DAG can also be formed by the sequential hydrolysis of phosphatidylcholine by phospholipase D and phosphatidic acid phosphohydrolase. This is a minor reaction in the rapidly responding platelet system, but may play a role in sustained signaling events. We have found that fibroblasts which overexpress the beta 1 isozyme of PKC display greatly enhanced DAG formation and phospholipase D activation in response to phorbol ester treatment. Upon stimulation of fibroblasts with thrombin, phospholipase D activation is also enhanced by PKC overexpression while formation of inositol phosphates is suppressed. These data suggest that PKC may act as a switch, terminating inositol phospholipid hydrolysis and activating the hydrolysis of phosphatidylcholine. Furthermore, we have observed a strong correlation between activation of phospholipase D and mitogenesis, suggesting an important role for this enzyme in long-term cellular responses to activation.
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PMID:Regulation of phospholipid hydrolysis and second messenger formation by protein kinase C. 132 4

For immune surveillance and function to be effective, T lymphocytes constantly recirculate via lymph and blood between lymphoid organs and body tissues. To enable efficient cell movement and migration, cell adhesion to components of the basement membrane and the extracellular matrix (ECM) must be a rapid and transitory process. Whether phosphorylation and dephosphorylation of cellular proteins are involved in this phenomena was explored by monitoring the adhesion of T cells to immobilized ECM proteins. A short exposure of 51Cr-labeled human CD4+ T cells to phorbol esters in vitro induced a rapid beta 1-integrin-mediated adhesion to both fibronectin and laminin, as determined by inhibition with anti-integrin antibodies. Adhesion was reversible; detachment from the immobilized ECM ligands occurred between 20 and 120 min without further intervention. This T cell adhesion was regulated by the activation of protein kinase C because (a) staurosporine and H-7 inhibitors of protein kinase C suppressed T cell adhesion, and (b) PMA-induced down-regulation of intracellular levels of protein kinase C was associated with the abrogation of the T cell adhesiveness to fibronectin and laminin. Furthermore, inhibition of protein phosphatases activity by okadaic acid delayed the detachment of the T cells from fibronectin or laminin. Thus, we suggest that T cell-ECM interactions such as adhesion and detachment are regulated, respectively, by protein kinase C and protein phosphatases.
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PMID:Involvement of a protein kinase C and protein phosphatases in adhesion of CD4+ T cells to and detachment from extracellular matrix proteins. 132 39

The mammalian GABAA receptor is a multisubunit protein containing a variety of binding sites for psychotropic agents. One of the most widely used of these drugs, ethanol, enhances the function of GABAA receptors in certain circumstances but not others. Previous studies have demonstrated that alternative splicing of the gamma 2L GABA subunit results in an ethanol sensitive and an ethanol-insensitive form, when combined with alpha and beta subunits. We have used in vitro mutagenesis and expression in Xenopus oocytes to show that the consensus site for phosphorylation by protein kinase C contained in the gamma 2L insert is critical for modulation by ethanol but not benzodiazepines, and manipulation of the phosphorylating enzymes in oocytes containing alpha 1 beta 1 gamma 2L can prevent ethanol enhancement. It is likely that phosphorylation or dephosphorylation of a specific site on the GABAA receptor protein can act as a control mechanism for neuronal responses to alcohol exposure.
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PMID:Ethanol potentiation of GABAA receptors requires phosphorylation of the alternatively spliced variant of the gamma 2 subunit. 133 Jul 1

Migration of medial smooth muscle cells (SMC) into the intima is important in intimal thickening of atherosclerotic tissues. To study the functions of three isoforms of platelet-derived growth factor (PDGF) in atherosclerosis, we investigated their effects on SMC migration by Boyden's chamber method. Although PDGF-AB and PDGF-BB enhanced SMC migration dose-dependently, PDGF-AA did not enhance SMC migration, but instead inhibited SMC migration induced by PDGF-AB or PDGF-BB. PDGF-AA also inhibited SMC migration induced by two other migration factors, fibronectin and SMC-derived migration factor. PDGF-AA is considered to be coexpressed with transforming growth factor (TGF)-beta 1 in atherosclerotic tissues. Treatment of SMC with TGF-beta 1 reduced an autocrine migration activity from SMC. Studies using anti-PDGF antibody revealed that an increased secretion of PDGF-AA by TGF-beta 1 caused the reduced migration activity. cAMP increase by forskolin and dibutyryl cAMP suppressed SMC migration, whereas cAMP decrease by pertussis toxin had no effects on PDGF-AA-suppressed migration. In contrast, staurosporine, an inhibitor of protein kinase C, enhanced SMC migration and neutralized the inhibitory effect of PDGF-AA. These findings suggest that PDGF-AA regulates SMC migration in intimal thickening in atheroma formation and that protein kinase C may play an important role in the inhibitory mechanism of PDGF-AA.
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PMID:Regulatory effects of platelet-derived growth factor-AA homodimer on migration of vascular smooth muscle cells. 133 Oct 68

The human promyelocytic cell line NB4 exhibited a weak adhesion capacity for bone marrow-derived stromal cells and their extracellular matrices (5-15% of adherent cells). Adhesion was enhanced by pulse-treatment of cells with phorbolester (PMA 10(-7) M). Adhesion was induced within minutes, was fibronectin-specific, and affected up to 100% of the treated cells. This biological response to PMA resulted from the activation of protein kinase C (PKC), since PKC inhibitors (staurosporine, sphingosine, CGP 41251, and calphostin C) prevented the phenomenon. Phenotypical analysis of integrin receptor expression (particularly FN receptors VLA-4 and VLA-5) at the membrane of untreated or PMA-treated cells revealed that PMA induced no significant modification of the level of expression of these receptors. However, inhibition studies carried out with anti-VLA monoclonal antibodies demonstrated that the FN-specific adhesion triggered by PKC involved the alpha 5 beta 1 FN-specific receptors (VLA-5). We showed that the binding of NB4 cells to fibronectin was RGD-dependent. PMA-induced adhesion was not correlated to phosphorylation of the VLA-5 receptor. These findings may partially explain the malignant behaviour of these cells: The loss of their capacity to adhere to stromal cells may arrest differentiation and explain the large number of leukemic cells in the circulation.
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PMID:Phorbol ester-induced promyelocytic leukemia cell adhesion to marrow stromal cells involves fibronectin specific alpha 5 beta 1 integrin receptors. 138 76

Tumor-cell interaction with the vessel wall during metastasis involves adhesion, induction of endothelial-cell retraction and spreading on the exposed sub-endothelial matrix. The signals for initiation of tumor-cell spreading and the receptors involved are unknown. A protocol was developed to distinguish between initial tumor-cell (B16 amelanotic melanoma; B16a) adhesion to and spreading on fibronectin. The time for maximum spreading was 50 min. Treatment with a lipoxygenase metabolite of arachidonic acid [12(S)-HETE] resulted in maximum spreading in 15 min (max. effect approx. 0.1 microM). Other lipoxygenase metabolites were ineffective. 12(S)-HETE treatment induced a rearrangement of F-actin, vinculin, vimentin intermediate filaments and integrin alpha IIb beta 3, but not integrin alpha 5 beta 1. Antibodies to alpha IIb beta 3 but not alpha 5 beta 1 blocked the 12(S)-HETE effect on B16a spreading. B16a-cell attachment to fibronectin resulted in increased metabolism of arachidonic acid to 12(S)-HETE, which was inhibited by lipoxygenase but not by cyclo-oxygenase inhibitors. Accordingly, lipoxygenase inhibitors but not cyclo-oxygenase inhibitors blocked spontaneous B16a-cell spreading. The protein-kinase-C inhibitors calphostin C, H7 and staurosporine also inhibited spreading, while the protein-kinase-A inhibitor H8 was ineffective. These data suggest that B16a-cell spreading on fibronectin is initiated by a lipoxygenase metabolite [12(S)-HETE] of arachidonic acid and is mediated by protein kinase C.
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PMID:The lipoxygenase metabolite 12(S)-HETE promotes alpha IIb beta 3 integrin-mediated tumor-cell spreading on fibronectin. 139 43


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