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)

Regulation of the cAMP-activated apical membrane Cl- conductance (GaCl) in Necturus gallbladder (NGB) epithelial cells was investigated with intracellular-microelectrode techniques. GaCl was increased by exposure to 8-Br-cAMP, theophylline or forskolin. Neither 8-Br-cGMP nor elevation of intracellular [Ca2+] using ionomycin had effects on GaCl or interfered with activation of GaCl by forskolin. N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide (H8), an inhibitor of cAMP-dependent protein kinase (PKA), slowed but did not prevent the GaCl response to 8-Br-cAMP. Phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), stimulated GaCl but had no effects on intracellular [cAMP]. GaCl was unaffected by 4 alpha-phorbol, a PMA analog which does not activate PKC. Okadaic acid (OA), an inhibitor of protein phosphatases (PP) types 1 and 2A, slowed the activation of GaCl by 8-Br-cAMP, hastened the return of GaCl to basal values following removal of 8-Br-cAMP, and significantly reduced the elevation in intracellular [cAMP] produced by forskolin. OA had no effects on the GaCl changes elicited by theophylline. We conclude that: (a) NGB GaCl can be activated by PKA-mediated phosphorylation of apical membrane Cl- channels or a regulatory protein, (b) GaCl can also be activated via PKC, by a cAMP-independent mechanism, (c) OA-sensitive PP are not required for inactivation of GaCl; OA appears to stimulate phosphodiesterase, which lowers intracellular [cAMP] and affects GaCl activation, and (d) the apical membrane of NGB epithelium lacks a Ca(2+)-activated Cl- conductance.
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PMID:Regulation of cAMP-activated apical membrane chloride conductance in gallbladder epithelium. 816 93

We report that activators and inhibitors of protein kinase C (PKC) and protein phosphatases regulate the activity of a cloned rat brain gamma-aminobutyric acid (GABA) transporter (GAT1) expressed in Xenopus oocytes. Four compounds known to activate PKC increased GABA uptake 2-3.5-fold over basal control levels. Inhibition of PKC by bisindolylmaleimide reduced basal GABA uptake 80% and blocked the phorbol 12-myristate 13-acetate (PMA)-induced stimulation of transport. Okadaic acid, a protein phosphatase inhibitor, stimulated transport 2.5-fold; a 4-fold increase in GABA uptake occurred when oocytes were treated with cyclosporin A, a specific inhibitor of protein phosphatase 2B. Modulation resulted in changes to Vmax but not to Km and was influenced by the functional expression level of the transporter protein; as expression level increased, the ability to up-regulate transporter activity decreased. Down-regulation of transporter activity was independent of expression level. Modulation did not occur through phosphorylation of the three consensus PKC sites predicted by the primary protein sequence since their removal had no effect on the susceptibility of the transporter to modulation by PMA or bisindolylmaleimide. Subcellular fractionation of oocyte membranes demonstrated that under basal level conditions, the majority of GAT1 was targeted to a cytoplasmic compartment corresponding to the trans-Golgi or low density vesicles. Stimulation of PKC with PMA resulted in a translocation of transporters from this compartment to the plasma membrane. At higher expression levels of GAT1 protein, a larger portion of GAT1 was found on the plasma membrane during basal level conditions and treatment with bisindolylmaleimide resulted in removal of these transporters from the plasma membrane. At expression levels demonstrated to be resistant to modulation by PMA, PMA-treatment still resulted in translocation of transporters from the cytoplasm to the plasma membrane. Thus, the inability of PMA to increase uptake at high expression of the GAT1 protein is due to saturation at a step subsequent to translocation. These findings 1) demonstrate the presence of a novel regulated secretory pathway in oocytes and 2) suggest a modulatory mechanism for neurotransmitter transporters that could have significant effects upon synaptic function.
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PMID:Protein kinase C modulates the activity of a cloned gamma-aminobutyric acid transporter expressed in Xenopus oocytes via regulated subcellular redistribution of the transporter. 818 81

In bovine iris sphincter, myo-inositol 1,4,5-trisphosphate (IP3) 5-phosphatase and myo-inositol 1-phosphate (IP1) monophosphatase are mainly localized in the microsomal and soluble fractions, respectively. Studies on the properties of these enzymes can be summarized as follows. (1) The microsomal IP3 5-phosphatase hydrolyzed IP3 to myo-inositol 1,4-bisphosphate with an apparent Km of 28 microM and Vmax of 32 nmol/min per mg protein. The IP1 monophosphatase in the soluble fraction hydrolyzed IP1 into free inositol with an apparent Km of 89 microM and Vmax of 7 nmol/min per mg protein. (2) IP3 5-phosphatase and IP1 monophosphatase had optimal pH values at 8.0 and 7.0, respectively. (3) Both enzymes required Mg2+ and their highest specific activities were at a cation concentration of 2 mM. (4) Ca2+ (> 0.5 microM) exerted an inhibitory effect on IP3 5-phosphatase activity, and marked inhibition (47%) was observed at a concentration of 10 microM. Higher concentrations of the cation (> 100 microM) were required to inhibit IP1 monophosphatase. (5) IP1 monophosphatase, but not IP3 5-phosphatase, was inhibited by Li+. Li+ had no effect on the contractile response in this smooth muscle. (6) Both enzymes were inhibited by ATP and by the thiol-blocking agent, disulfiram. In addition, thimerosal, a thiol reagent, also inhibited the IP3 5-phosphatase activity. (7) Protein phosphorylation of the microsomal and soluble fractions with PKA or PKC had no effect on the activities of these enzymes. (8) Okadaic acid, a protein phosphatase inhibitor, had no effect on the activity of IP3 5-phosphatase. However, in the intact iris sphincter the toxin significantly reduced the carbachol-induced IP3 production, 1,2-diacylglycerol formation, measured as phosphatidic acid, and caused muscle relaxation.
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PMID:Studies on the properties of myo-inositol-1,4,5-trisphosphate 5-phosphatase and myo-inositol monophosphatase in bovine iris sphincter smooth muscle: effects of okadaic acid and protein phosphorylation. 818 62

Okadaic acid, a newly recognized protein phosphatase inhibitor and a non-TPA type tumor promoter, enhanced 1 alpha 25(OH)2D3(D3)-induced HL-60 cell differentiation into monocyte/macrophage lineage but did not affect dibutyryl cyclic AMP (dbcAMP)-induced differentiation into granulocytic lineage. Okadaic acid alone did not induce any differentiation. The process of D3-induced HL-60 cell differentiation on cultivation in magnesium deficient medium can be divided into two steps namely commitment and phenotypic expression as we have previously reported (J Cell Physiol 1987;131:50; Cell Growth Diff 1991;2:415), and the effect of okadaic acid on each step was studied. The results obtained indicated that okadaic acid inhibited commitment and enhanced phenotypic expression. We have previously shown that PKC has a dual action in the process of differentiation, i.e. as a positive regulatory signal in commitment and as a negative one in phenotypic expression. Thus, although okadaic acid has been reported to enhance the phosphorylation of various proteins that are also phosphorylated by PKC, we found that it mimics the role of PKC inhibitors such as H7 and staurosporine in D3-induced HL-60 cell differentiation.
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PMID:Okadaic acid biologically mimics the role of calcium/phospholipid dependent kinase inhibitors in the process of HL-60 cell differentiation. 821 10

Ca(2+)-dependent and protein kinase C-dependent mechanisms of phospholipase D (PLD) activation were studied in rat hepatocytes by measuring phosphatidylethanol (Peth) formation in the presence of ethanol. Stimulation of Peth formation by 12-O-tetradecanoyl-phorbol 13-acetate (TPA), vasopressin, or A23187 was inhibited by multiple protein kinase C inhibitors or by protein kinase C down-regulation, indicating that this enzyme is involved in the action of all these agents. A controlled elevation of the cytosolic Ca2+ concentration ([Ca2+]cyt) over the range of 0.1-2.0 microM activated Peth formation in the absence of other agonists. Staurosporin potentiated Ca(2+)-induced Peth formation by shifting the [Ca2+]cyt dose-response curve to the left. Other protein kinase C inhibitors (calphostin C, bisindolylmaleimide) inhibited Ca(2+)-mediated Peth formation, but this inhibition was reduced in staurosporin-treated cells. Okadaic acid potentiated PLD activation by TPA, but suppressed PLD activation by elevated [Ca2+]cyt. Desensitization of TPA-induced PLD activity did not affect PLD activation by Ca2+. These data indicate that [Ca2+]cyt and protein kinase C control distinct pathways of PLD activation, but the Ca(2+)-mediated pathway is suppressed by a staurosporin-sensitive protein kinase. Both mechanisms contribute to vasopressin-induced Peth formation in intact hepatocytes. Activation of protein kinase A enhanced vasopressin-induced Peth formation, but not TPA-stimulated or Ca(2+)-stimulated stimulated Peth formation. Protein kinase A acted by enhancing hormonal Ca2+ mobilization, rather than by directly activating PLD, and thereby shifted the balance of Ca(2+)-dependent and protein kinase C-dependent activation mechanisms of PLD in intact cells.
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PMID:The role of cytosolic Ca2+, protein kinase C, and protein kinase A in hormonal stimulation of phospholipase D in rat hepatocytes. 828 38

Treatment of human myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (PKC), is associated with induction of monocytic differentiation. Since PKC can act immediately upstream to the cytoplasmic Raf-1 serine/threonine protein kinase, we studied activation of Raf-1 during induction of the differentiated monocytic phenotype. The results demonstrate that Raf-1 is activated during TPA-induced monocytic differentiation of HL-60 cells. In contrast, there was little effect of TPA on this kinase in an HL-60 variant, designated HL-525, which is resistant to TPA-induced differentiation. Treatment of both HL-60 and HL-525 cells with okadaic acid, an inhibitor of serine/threonine protein phosphatases 1 and 2A, was associated with Raf-1 activation and induction of the monocytic phenotype. Since Raf-1 can activate the mitogen-activated protein (MAP) kinases, we also studied the relationship between MAP kinase activation and monocytic differentiation. Treatment of HL-60, but not HL-525, cells with TPA was associated with increased MAP kinase activity as determined by phosphorylation of myelin basic protein and the c-Jun Y peptide. Okadaic acid-induced differentiation of both HL-60 and HL-525 cells was similarly accompanied by increases in MAP kinase activity. These findings indicated that activation of Raf-1/MAP kinase signaling is associated with induction of a differentiated monocytic phenotype and that okadaic acid bypasses a defect in this cascade in TPA-treated HL-525 cells. While recent studies have shown that HL-525 cells are deficient in PKC beta, the present results demonstrate that PKC beta expression is up-regulated in the HL-525 variant by treatment with retinoic acid. The results also demonstrate that retinoic acid-treated HL-525 cells respond to TPA with activation of Raf-1 and MAP kinase, as well as induction of monocytic differentiation. Taken together, the results indicate that activation of Raf-1/MAP kinase signaling is associated with monocytic differentiation and that stimulation of serine/threonine protein phosphorylation by TPA or okadaic acid is sufficient for reversal of the leukemic HL-60 phenotype.
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PMID:Activation of Raf-1 and mitogen-activated protein kinases during monocytic differentiation of human myeloid leukemia cells. 828 41

Group I Burkitt's lymphoma cell lines and the B104 lymphoma cell line which expresses a phenotype of immature B cells undergo apoptosis after cross-linking of their surface immunoglobulin (Ig) receptors or after exposure to a calcium ionophore, while protein kinase C (PKC)-activating phorbol esters prevent such apoptosis. We show here that blockade of the phosphoprotein phosphatase calcineurin or phosphatase 2B by cyclosporin A (CsA) also protects these B cell lines against Ca(2+)-dependent apoptosis but not against apoptosis triggered by the PKC inhibitor chelerythrine or by serum deprivation. Okadaic acid, an inhibitor of phosphatases 1, 2A and 2C was ineffective. Among a series of human cytokines tested, only interferon-alpha and tumor necrosis factor-alpha were shown to protect against Ca(2+)-dependent apoptosis when used alone or in combination with CsA. In contrast to phorbol esters which block the progression into the S/G2 phases of the cell cycle, CsA partially restored the proliferation of cells exposed to the calcium ionophore. Altogether these data provide indirect evidence for the control of B cell apoptosis by the serine/threonine phosphorylation status of yet undefined key cellular substrates.
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PMID:The phosphoprotein phosphatase calcineurin controls calcium-dependent apoptosis in B cell lines. 829 81

In vivo phosphorylation of P gamma, an inhibitory subunit of cGMP-phosphodiesterase of frog (Rana catesbeiana) photoreceptor rod outer segments, was investigated using a quick-freezing technique and a newly developed method for the preparation of rod outer segments. Light-dependent phosphorylation of P gamma was observed. Okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A, enhanced the apparent incorporation of 32P into P gamma, suggesting that P gamma is in equilibrium between phosphorylation and dephosphorylation. Neither phorbol ester, a potent activator of protein kinase C, nor changes in the extracellular Ca2+ concentration affected the in vivo phosphorylation of P gamma.
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PMID:Light-dependent in vivo phosphorylation of an inhibitory subunit of cGMP-phosphodiesterase in frog rod photoreceptor outer segments. 830 81

We have recently demonstrated that binding by monoclonal antibody (mAb) 8A2 to regenerating retinal ganglion cell axons in goldfish explants specifically induces a sustained, actin-based retraction response that is similar in most respects to a spontaneous retraction (S.G. Finnegan, V. Lemmon, and E. Koenig, Cell Motil. Cytoskeleton, 1992). Experiments were conducted to evaluate potential signal transduction pathways that may play a role in mediating retraction, using the mAb 8A2 retraction model system. Potential roles of cAMP, elevated intracellular calcium, or calmodulin-dependent processes were probed and the results did not appear to implicate them in either the induction or the maintenance of the axon retraction response. In contrast, treatment with phorbol 12-myristate 13-acetate, but not with inactive phorbol esters, induced a retraction response, although the response was more variable and less robust than that produced by mAb 8A2. However, both forms of induction were blocked by staurosporine, a nonspecific kinase inhibitor. Okadaic acid, a potent serine/threonine phosphatase inhibitor produced a very robust retraction response, and subthreshold doses significantly potentiated the retraction response induced by mAb 8A2. Genistein inhibited the mAb 8A2-induced retraction response at concentrations selective for tyrosine kinase activity in a dose-dependent manner. These findings are consistent with the hypothesis that an augmented phosphorylation state of one or more axonal proteins, perhaps catalyzed in part by protein kinase C, produces a sustained physiological retraction. In addition, tyrosine kinase may be involved in transducing surface-mediated interactions that trigger retraction, including the binding reaction signal of mAb 8A2.
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PMID:Monoclonal antibody 8A2-induced retraction appears to be mediated by protein phosphorylation in goldfish retinal ganglion cell axons. 838 19

Protein kinase C modulates the receptor for tumor necrosis factor (TNF) in wide variety of different cell types. However, there is no information about the role of phosphatases in the regulation of the TNF receptor. In this report, we investigated the effect of okadaic acid, an inhibitor of serine/threonine phosphatases, on TNF receptors in U-937 cells, a human histiocytic lymphoma cell line. In our study okadaic acid induced a dose- and time-dependent down-modulation of TNF receptors. On exposure of cells to 0.5 microM okadaic acid for 60 min at 37 degrees C, a complete down-regulation of the receptors was observed, but no modulation occurred at 4 degrees C. Scatchard analysis of the binding data on U-937 cells revealed that okadaic acid caused a decrease in the high affinity cell surface receptor number without a significant change in the affinity constant. The down-regulation of the TNF receptor by okadaic acid was not specific to U-937 cells, as it was also observed with several other cell types. Okadaic acid had no significant effect on the internalization of the receptor, but it did induce a shedding of the TNF receptor from its cell surface. On exposure of cells to okadaic acid, a dose- and time-dependent increase in the 40-kDa polypeptide was detected in the medium with anti-p80 antibodies by Western blot analysis. The secreted product was also found to bind TNF. The mechanism by which okadaic acid down-modulates the TNF receptor appears to be quite different from that of phorbol ester. First, okadaic acid synergistically potentiated the effect of phorbol ester. Second, the phorbol ester-mediated down-modulation could be blocked by H-7 and staurosporine, well known protein kinase C inhibitors, but these inhibitors had no effect on okadaic acid-mediated response. Third, phorbol ester-mediated down-modulation of the TNF receptor was reversible after removal of the agent, but the response mediated through okadaic acid was irreversible. Fourth, the TNF receptor was down-regulated by okadaic acid but not by phorbol ester in cells depleted of protein kinase C. Thus, overall, our results demonstrate that serine/threonine phosphatases can down-modulate and induce shedding of TNF receptors by a mechanism distinct from that of protein kinase C.
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PMID:Okadaic acid induces down-modulation and shedding of tumor necrosis factor receptors. Comparison with another tumor promoter, phorbol ester. 838 75


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