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)

The involvement of protein kinase C in the Ca2+-dependent phosphorylation of a 29 000-Mr insulin-granule membrane protein prepared from a rat insulinoma was investigated. Protein kinase C activity towards exogenous lysine-rich histone was detected in a cytosolic fraction prepared from an insulinoma homogenate in the presence of EGTA. This activity bound reversibly to insulin granules in a Ca2+-dependent manner. Phosphatidylserine liposomes removed both protein kinase C activity and the 29 000-Mr protein-phosphorylating activity from the cytosolic fraction in a Ca2+-dependent fashion. Protein kinase C activity and the enzymic activity responsible for the phosphorylation of the 29 000-Mr granule protein behaved identically on sucrose-density-gradient centrifugation, ion-exchange chromatography, (NH4)2SO4 fractionation and gel filtration of the cytosolic fraction. These results are consistent with protein kinase C being the enzyme responsible for the phosphorylation of the 29 000-Mr insulin-granule membrane protein.
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PMID:Involvement of protein kinase C in the phosphorylation of an insulin-granule membrane protein. 633 10

Isolated sheep thyroid follicles release insulin-like growth factors (IGF)-I and -II together with IGF-binding proteins (IGFBPs). We previously showed that TSH suppresses the biosynthesis and release of IGFBPs in vitro which may increase the tissue availability of IGFs, allowing a synergy with TSH which potentiates both thyroid growth and function. Many of the actions of TSH on thyroid cell function are dependent upon activation of adenylate cyclase, although increased synthesis of inositol trisphosphate and activation of protein kinase C (PKC) have also been implicated. We have now examined whether probable changes in intracellular cyclic adenosine monophosphate (cAMP) or PKC are involved in TSH-mediated suppression of IGFBP release. Confluent primary cultures of ovine thyroid cells were maintained in serum-free Ham's modified F-12M medium containing transferrin, somatostatin and glycyl-histidyl-lysine (designated 3H), and further supplemented with sodium iodide (10(-8)-10(-3) mol/l), dibutyryl cAMP (0.25-1 mmol/l), forskolin (5-20 mumol/l) or 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-11)-10(-6) mol/l), with or without exposure to TSH (200 microU/ml). The uptake and organification of Na [125I] by cells was examined after test incubations of up to 48 h, and IGFBPs in conditioned media were analysed by ligand blot using 125I-labelled IGF-II. The PKC activity in the cytosol and plasma membrane fractions of cells was measured by phosphorylation of histone using [gamma-32P]ATP, and PKC immunoreactivity was visualized by Western immunoblot analysis. While dibutyryl cAMP or forskolin largely reproduced the stimulatory effect of TSH on iodine organification, they did not mimic the inhibitory effect of TSH on the secretion of IGFBPs of 43, 34, 28 and 19 kDa. Incubation with physiological or pharmacological concentrations of iodide (10(-6)-10(-3) mol/l) for up to 48 h significantly decreased TSH action on iodide uptake and organification but did not alter the inhibitory action of TSH on IGFBP release. Incubation of cells with 10(-11)-10(-6) mol TPA/l for 24 h inhibited the subsequent ability of TSH both to potentiate iodine organification and to suppress IGFBP release. In 3H medium, PKC activity was predominantly recovered from the membrane fraction but, following incubation for 48 h with TSH, the enzyme was no longer translocated to the membrane and was recovered predominantly from the cytosol.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Role of 3', 5' cyclic adenosine monophosphate and protein kinase C in the regulation of insulin-like growth factor-binding protein secretion by thyroid-stimulating hormone in isolated ovine thyroid cells. 751 34

This study examined the role of protein tyrosine kinase (PTK) and protein kinase C (PKC) in the signal transduction pathways for lymphocyte activation through IL-2R to generate LAK cells and through TCR-CD3 to generate CD3-AK cells. Two PTK inhibitors [herbimycin A and genistein (PTK-I)] and two PKC inhibitors [calphositin C and staurosporine (PKC-I)] were used in the experiments. It was found that the primary activation pathway through IL-2R was PTK-dependent; that is, generation of both the IL-2-induced proliferative and the cytotoxic responses was completely abrogated by PTK-I and not by PKC-I. Quite different results were obtained with the alpha CD3-induced CD3-AK cell response. First, the alpha CD3-induced proliferation was only partially inhibited by PTK-I or PKC-I alone. Second, generation of CD3-AK cytotoxic response was primarily PKC-dependent; that is, only PKC-I induced significant inhibition. Genistein was found to reduce protein tyrosine phosphorylation in both LAK cells and CD3-AK cells, indicating that CD3-AK cells were also susceptible to PTK-I treatment. Further studies showed that PTK-I and not PKC-I suppressed perforin mRNA expression and N-2-benzyoxycarbonyl-L-lysine thiobeneylester esterase production in LAK cells, and the opposite was true for CD3-AK cells. These results indicate that different pathways were employed in lymphocyte activation through IL-2R and TCR-CD3. The former pathway is primarily PTK-dependent. Activation through TCR-CD3 is a more complex event.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Differential requirement of protein tyrosine kinase and protein kinase C in the generation of IL-2-induced LAK cell and alpha CD3-induced CD3-AK cell responses. 753 36

In a search for new alpha-subunits of trimeric GTP-binding proteins in human platelets, we prepared leucocyte-free platelet concentrates and analyzed total RNA for areas homologous to known alpha-subunits. RT-PCR based on two degenerate primers revealed the expected band of 495 base pairs and an additional band of 540 base pairs reflecting the alternative splice product of Gs alpha. Following subcloning in pGEM-T vector and sequencing, we identified the alpha-subunits Gi alpha-2 and Gs alpha-S of the regulating GTP-binding proteins of adenyl cyclase as well as Gz alpha whose function is unknown, confirming earlier immunological identification. In addition, we identified Gs alpha-L (differing from Gs alpha-S by an insertion of 45 base pairs), G16 alpha, (a member of the pertussis toxin insensitive Gq-family), and two new variants of both Gs alpha-S and Gs alpha-L each containing a C-A-G triplet. With G16 we have identified another candidate for pertussis-toxin insensitive signal transduction in platelets. The C-A-G containing sequences of Gs alpha lead to an insertion of a Ser-residue, which results in the consensus sequence of a phosphorylation site for protein kinase C (Ser-X-Lys), making these variants candidates for protein kinase C-sensitive cyclic AMP formation.
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PMID:Identification of alpha-subunits of trimeric GTP-binding proteins in human platelets by RT-PCR. 754 94

Pleckstrin is a substrate for protein kinase C in activated platelets that contains at its N and C termini two of the pleckstrin homology (PH) domains that have been proposed to mediate protein-protein and protein-lipid interactions. We have recently shown that pleckstrin can inhibit agonist-induced phosphoinositide hydrolysis and that this inhibition requires an intact N-terminal PH domain (residues 6 to 99). In the present studies, we have identified the sites of phosphorylation in pleckstrin and examined their contribution to pleckstrin function. In human platelets activated with thrombin or phorbol esters, and in COS-1 cells expressing pleckstrin, a combination of phosphopeptide analysis and site-directed mutagenesis shows that three residues in the intervening sequence between the two pleckstrin PH domains become phosphorylated: Ser113, Thr114, and Ser117. Replacing all three of these sites with glycine decreased phosphorylation by > 90% and reduced pleckstrin's ability to inhibit phosphoinositide hydrolysis by as much as 80%. Replacing the phosphorylation sites with alanine residues had a similar effect, while substitution with aspartate, glutamate, or lysine residues produced pleckstrin variants that were fully active even in the absence of phosphorylation. These results suggest that phosphorylation enhances pleckstrin's activity by introducing a cluster of charges into a region adjacent to, but not within, the N-terminal PH domain. This may have an allosteric effect on the N-terminal PH domain, regulating its interaction with other molecules necessary for the inhibition of phosphoinositide hydrolysis.
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PMID:Protein kinase C regulates pleckstrin by phosphorylation of sites adjacent to the N-terminal pleckstrin homology domain. 755 87

1. The mechanisms underlying bradykinin (BK)-mediated contractions in strips of guinea-pig gallbladder (GPG) were examined by use of selective bradykinin (BK) receptor agonists and antagonists. 2. Addition of BK and related kinins (0.1 pM-10 microM) after 2 h of equilibration of the preparation caused graded contractions characterized by two distinct phases: high affinity (0.1 pM-1 nM) and low affinity (3 nM-10 microM). The rank order of potency for the first phase (mean EC50, pM) was: BK (1.36) = Hyp3-BK (1.44) = Lys-BK (1.54) > Tyr8-BK (2.72) > Met-Lys-BK (4.30). The rank order of potency for the second phase (mean EC50, nM, at concentration producing 50% of the contraction caused by 80 mM KCl) was: Hyp3-BK (8.95) > Met-Lys-BK (12.78) > Tyr8-BK (33.75) > Lys-BK caused by 80 mM KCl) was: Hyp3-BK (8.95) > Met-Lys-BK (12.78) > Tyr8-BK (33.75) > Lys-BK (60.92) > BK (77.35). The contractile responses (g of tension) to 3 microM of BK (the highest concentration tested) were: Hyp3-BK, 1.76 +/- 0.09; BK, 1.65 +/- 0.12; Lys-BK, 1.45 +/- 0.13; Tyr8-BK, 1.36 +/- 0.15 and Met-Lys-BK, 1.36 +/- 0.15. The selective B1 agonist, des-Arg9-BK, caused only a weak contraction with maximal response (0.21 +/- 0.05 g), which corresponded to approximately 10% of that induced by BK. 3. BK-induced contraction in GPG was inhibited by indomethacin (3 microM) or ibuprofen (30 microM), and was partially reduced by phenidone (30 microM), but was not affected by atropine (1 JM), nicardipine (1 gM),Ca2+-free medium plus EGTA, dazoxiben (30 nM), L-655,240 (10 nM, a selective receptor antagonist ofthromboxane A2), MK-571 (0.1 microM, a selective leukotriene D4 receptor antagonist), tetrodotoxin(0.3microM), CP 96,345 (0.3 microM, a NK1 receptor antagonist), mepyramine (1 microM), glibenclamide (1 microM), H-7(3 microM), staurosporine (100 nM), or phorbol 12-myristate 13-acetate (1 microM). However, BK-induced contractions in GPG maintained in Ca2+-free medium were markedly attenuated by ryanodine (10microM).4. Prostaglandin E2, prostaglandin F2alpha or U46619 (0.1 nM to 100microM), caused concentration-dependent contractions in GPG with mean EC50s of 3.1 microM; 1.7 microM and 0.47 nM and maximal responses of1.36 +/-0.15; 1.32 +/- 0.20 and 0.96 +/- 0.09 g, respectively.5. The selective B2 receptor antagonists, Hoe 140, NPC 17731 and NPC 17761 (0.01 -1 microM), caused concentration-dependent displacements to the right of the contractile concentration-response curve for BK. The selective B1 receptor antagonist, des-Arg9-[Leu8]-BK (1 microM), did not affect BK-induced GPG contraction.6. These data suggest that both high and low affinity BK responses in GPG are mediated by activation of B2 receptors, and that BK-mediated contraction in GPG depends on the release of intracellular Ca2+sources sensitive to ryanodine. In addition, BK-induced contraction in GPG is mediated by release of proinflammatory eicosanoid(s) derived from the cyclo-oxygenase pathway from arachidonic acid metabolism unrelated to thromboxane A2, and seems not to be coupled to activation of a protein kinase C-dependent mechanism.
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PMID:Mechanisms of bradykinin-induced contraction of the guinea-pig gallbladder in vitro. 759 22

Although it is suggested that in the renal proximal tubules, dopamine D1 receptor activation causes inhibition of Na+/K+ATPase via a phospholipase C and protein kinase C coupled pathway, the direct stimulation of protein kinase C by dopamine has not been reported. The present study was designed to examine the effects of dopamine and selective dopamine D1 receptor and dopamine D2 receptor agonists on protein kinase C activity. The renal proximal tubule suspensions were obtained from male Sprague-Dawley rats. The tubules were incubated separately with dopamine and fenoldopam in the presence or absence of dopamine D1 receptor antagonist, SCH 23390 ([(R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3- benzazepine]). The protein kinase C activity was measured by using a kinase target peptide, conjugated to a fluorescent molecule in water. The amino acid sequence of this peptide is, Proline-Leucine-Serine-Arginine-Threonine-Leucine-Serine-Valine-Alanine- Alanine-Lysine(PKSRTLSVAAK). We found that dopamine and fenoldopam [6-chloro-2,3,4,5-tetrahydro-1-(4-hydroxyphenyl)-1H-3-benzazepine-7,8-di ol] produced concentration-dependent increases in protein kinase C activity, which was blocked by SCH 23390. However, the dopamine D2 receptor agonist, bromocriptine [(5' alpha)-2-bromo-12'-hydroxy-2'-(1-methyl-ethyl)-5'-(2-methylpropyl)erg o- taman-3',6',18-trione] failed to stimulate protein kinase C activity at all the concentrations tested. These results provide direct evidence that dopamine stimulates protein kinase C activity via activation of dopamine D1 receptors.
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PMID:Dopamine causes stimulation of protein kinase C in rat renal proximal tubules by activating dopamine D1 receptors. 762 15

To investigate the function of protein kinase C (PKC)-delta, we mutated its ATP binding site by converting the invariant lysine in the catalytic domain (amino acid 376) to an arginine. Expression vectors containing wild type and mutant PKC-delta cDNAs were generated either with or without an influenza virus hemagglutinin epitope tag. After expression in 32D cells by transfection, the PKC-delta ATP binding mutant (PKC-delta K376R) was not able to phosphorylate itself or the PKC-delta pseudosubstrate region-derived substrate, indicating that PKC-delta K376R was an inactive enzyme. PKC activity was inhibited by 67% in 32D cells coexpressing both PKC-delta wild type (PKC-delta WT) and PKC-delta K376R when compared to 32D cells expressing only PKC-delta WT. Mixture of PKC-delta WT and PKC-delta K376R kinase sources in vitro also reduced the enzymatic activity of PKC-delta WT. These results suggest that PKC-delta K376R competes with PKC-delta WT and inhibits PKC-delta WT phosphorylation of its in vitro substrate. While PKC-delta WT overexpressed in 32D cells demonstrated 12-O-tetradecanoylphorbol-13-acetate (TPA)-dependent translocation from the cytosolic to the membrane fraction, PKC-delta K376R was exclusively localized in the membrane fraction even prior to TPA stimulation. Unlike PKC-delta WT which was phosphorylated on tyrosine residue(s) only after TPA treatment, PKC-delta K376R was constitutively phosphorylated on tyrosine residue(s). Although exposure of PKC-delta WT transfectants to TPA induced 32D monocytic differentiation, the 32D/PKC-delta K376R transfectants were resistant to TPA-induced differentiation. Thus, expression of active PKC-delta is required to mediate 32D monocytic differentiation in response to TPA stimulation.
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PMID:Characterization of a protein kinase C-delta (PKC-delta) ATP binding mutant. An inactive enzyme that competitively inhibits wild type PKC-delta enzymatic activity. 771 39

Several proteins that are important components of the calcium/phospholipid second messenger system (e.g. phospholipase C, protein kinase C, myristoylated alanine-rich C kinase substrate (MARCKS) and pp60src) contain clusters of basic residues that can interact with acidic lipids on the cytoplasmic surface of plasma membranes. We have studied the membrane binding of MARCKS and pp60src, peptides that mimic the basic regions of these proteins, and simple model peptides. Specifically, we determined how the binding of these model peptides depends on (1) the number of basic residues in the peptide (2) the fraction of acidic lipids in the membrane (3) the ionic strength of the solution (4) the chemical nature of the basic residues (Arg versus Lys) and the acidic phospholipids [phosphatidylglycerol (PG) versus phosphatidylserine (PS)] (5) the pressure and (6) the temperature. The results are consistent with a simple theoretical model: each basic residue in a peptide binds independently to an acidic lipid with an intrinsic microscopic association constant of 1-10 M-1 (binding energy congruent to 1 kcal/mol). The binding is described with a mass action formalism and the non-specific electrostatic accumulation of the peptides in the aqueous diffuse double layer is described with the Gouy-Chapman theory. This Gouy-Chapman/mass action model accounts surprisingly well for the sigmoidal dependence of binding on the percentage of acidic lipids in the membrane (apparent co-operativity or Hill coefficient > 1); the model assumes that the multivalent basic peptides bind > 1 acidic lipids and thus induce or stabilize domain formation.
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PMID:Does the binding of clusters of basic residues to acidic lipids induce domain formation in membranes? 776 86

We have recently reported that the Arg1152-->Gln insulin receptor mutation (QK single mutant) alters a conserved motif (RK motif) immediately next to the key tyrosine phosphorylation sites (Tyr1146, Tyr1150, Tyr1151) of the receptor and constitutively activates its kinase and metabolic signaling. To investigate further the function of the RK motif, we have expressed two additional mutant insulin receptors: a single mutant, in which the second basic residue in the RK motif (Lys1153) was substituted (RA mutant); and a double mutant, in which both the Arg and the Lys residues were replaced with noncharged amino acids (QA mutant). As compared with the transfected wild-type receptors (WT), both the single and the double mutant receptors were normally synthetized and transported to the plasma membrane and bound insulin normally. Whereas the double mutant receptor exhibited preserved insulin-dependent autophosphorylation, kinase activity, and 2-deoxyglucose uptake, all of these functions were grossly impaired in the two single mutant receptors. Two-dimensional analysis of tryptic phosphopeptides from receptor beta-subunits revealed that decreased autophosphorylation of the single mutant receptors mainly involved regulatory Tyr1150,1151 and carboxyl-terminal Tyr1316,1322. At variance with the insulin-stimulated, insulin-independent tyrosine kinase activity toward poly(Glu-Tyr) 4:1 was increased 3-fold in both the double and the single mutants. All mutant receptors induced a 2-fold increase in basal 2-deoxyglucose uptake in NIH-3T3 cells. Treatment of WT transfected cells with 12-O-tetradecanoyl-phorbol-13-acetate or 8-bromo-cAMP increased insulin receptor phosphorylation by 3-fold. No phosphorylation was observed in cells expressing the two single or the double mutant receptor. Consistently, purified preparations of PKC and PKA phosphorylated the WT but not the mutant receptors in vitro. A 17-amino acid synthetic peptide encoding the receptor sequence surrounding the RK motif inhibited phosphorylation of WT insulin receptors by both protein kinases A and C. A mutant peptide in which the RK sequence was replaced by QK (to mimic the mutation in the QK receptor) exhibited no inhibitory effect. Thus, the RK insulin receptor motif is required for insulin receptor phosphorylation by protein kinases C and A and may modulate insulin-independent receptor activity. The RK motif may also have an important structural role in allowing normal insulin regulation of the kinase.
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PMID:Decreased phosphorylation of mutant insulin receptor by protein kinase C and protein kinase A. 779 89


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