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)

Sodium oleate is able to activate soluble protein kinase C (Murakami, K., Chan, S. Y., and Routtenberg, A. (1986) J. Biol. Chem. 261, 15424-15429) but is unable to activate membrane-bound enzyme (El Touny, S., Khan, W., and Hannun, Y. (1990) J. Biol. Chem. 265, 16437-16443). Because physiologic interactions of fatty acids with protein kinase C occur in the presence of membranes, the following studies were conducted to evaluate the effects of surfaces (detergent micelles or platelet membranes) on the activation of protein kinase C by oleate. At concentrations at or above the critical micellar concentration (CMC) of Triton X-100, oleate was present primarily in Triton X-100/oleate-mixed micelles, as determined by gel permeation chromatography and equilibrium dialysis binding studies. At concentrations slightly below the CMC for Triton X-100, the presence of oleate caused the formation of a limited number of mixed micelles. Studies of the dose-dependent activation of purified platelet protein kinase C by sodium oleate in the presence of different concentrations of Triton X-100 indicated that only unbound oleate was able to activate protein kinase C. Platelet protein kinase C was resolved into two major isoenzymes (types II (beta) and III (alpha)) which displayed nearly identical interaction with oleate. Activation of protein kinase C by oleate in a physiologic setting employing platelet substrates and endogenous platelet protein kinase C was investigated. Oleate potently activated protein kinase C in the cytosolic compartment. In platelet homogenates as well as in a reconstituted platelet cytosol and membrane system, the dose dependence of protein kinase C on oleate showed a significant shift to the right. Approximately 30% of oleate was associated with platelet cytosol and 70% was associated with platelet membranes. Partitioning of oleate into the two platelet compartments showed little change with pH, temperature, or duration of incubation. When corrected for free oleate concentration, activation of protein kinase C by oleate showed identical dose dependence in cytosol and homogenate. Arachidonate, a potential physiologic activator of protein kinase C, showed similar behavior as oleate although only 30% of arachidonate partitioned into platelet membranes with the majority of arachidonate (70%) remaining in the cytosolic fraction.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Activation of protein kinase C by oleic acid. Determination and analysis of inhibition by detergent micelles and physiologic membranes: requirement for free oleate. 174 Apr 12

Synaptosomes prepared from brain tissues are known to retain morphological and functional characteristics of the nerve ending. Little information is available, however, as to the biochemical events underlying synaptogenesis and transmitter release. Increasing body of evidence suggests that protein kinase C (PKC) plays crucially important roles through phosphorylation of membrane proteins such as GAP-43 (for 43-kDa growth-associated protein) and 87-kDa MARCKS (for myristoylated, alanine-rich C kinase substrate) in many aspects of the neuronal function. Among them, arrangement of membrane cytoskeletal protein is proposed to be one of the primary sites of PKC action. The present study is an attempt to isolate and characterize PKC associated with synaptosomal membrane cytoskeleton. Rat brain synaptosomal Triton X-100 insoluble elements (cytoskeleton) contains specific [3H]phorbol dibutyrate binding activity and 78-kDa protein which reacts with an antibody against beta II-PKC subspecies. Although 78-kDa protein could not be solublized by the treatment with various ionic and non-ionic detergents and/or high concentrations of salts such as NaCl and LiBr, the fragment of 78-kDa protein was produced and solublized from cytoskeleton by limited proteolysis with calpain II, which cleaves PKC at one or two specific sites of the enzyme to produce catalytic and regulatory fragments. The solubilized 46-kDa fragment was identical with the catalytic fragment of beta II-PKC. The results indicate that this PKC subspecies is tightly associated with the cytoskeletal network of synaptic membranes.
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PMID:Isolation and characterization of protein kinase C from rat brain synaptosome cytoskeleton. 174 42

A small fraction (approximately 5%) of protein kinase C (PKC) in the adult rat brain synaptosomes is tightly associated with Triton X-100-insoluble components (most likely membrane-skeleton elements), and is solubilized only after denaturation with sodium dodecyl sulfate. The kinase domain of this PKC can be released as a soluble form after limited proteolysis with calpain, whereas the regulatory domain which binds phorbol ester remains insoluble. The PKC in this fraction was identified as the beta II-subspecies or its related molecule. Presumably, this enzyme subspecies is responsible for the phosphorylation of a major PKC substrate protein, growth-associated protein-43, which is located in nerve endings as well as in growth cones in association with the membrane-skeleton elements.
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PMID:Protein kinase C in rat brain synaptosomes. Beta II-subspecies as a major isoform associated with membrane-skeleton elements. 183 69

The mechanism of protein kinase C (PKC) activation by phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol (PI) was investigated by using Triton X-100 mixed micellar methods. The activation of PKC by PIP2, for which maximal activity was 60% of that elicited by sn-1,2-diacyglycerol (DAG), was similar to activation by DAG in several respects: (1) activation by PIP2 and DAG required phosphatidylserine (PS) as a phospholipid cofactor, (2) PIP2 and DAG reduced the concentration of Ca2+ and PS required for activation, (3) the concentration dependences of activation by PIP2 and DAG depended on the concentration of PS, and (4) PIP2 and DAG complemented one another to achieve maximal activation. On the other hand, PIP2 activation of PKC differed from activation by DAG in several respects. With increasing concentrations of PIP2, (1) the optimal concentration of PS required was constant at 12 mol%, (2) the maximal activity at 12 mol% PS increased, and (3) the cooperativity for PS decreased. PIP2 did not inhibit [3H]phorbol 12,13-dibutyrate (PDBu) binding of PKC at saturating levels of PS; however, at subsaturating levels of PS, PIP2 enhanced [3H]PDBu binding by acting as a phospholipid cofactor. PIP did not function as an activator but served as a phospholipid cofactor in the presence of PS. While PIP2, PIP, and PI did not support DAG-dependent PKC activation as phospholipid cofactors, their presence reduced the amount of PS required for maximal activation to as low as 2 mol% from 8 mol%.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mechanism of protein kinase C activation by phosphatidylinositol 4,5-bisphosphate. 184 57

The aim of this study was to demonstrate the presence of calmodulin-stimulated protein kinase II, protein kinase C, and cyclic AMP-stimulated protein kinase in isolated myenteric ganglia and to characterize the major ganglia phosphoproteins using biochemical and immunochemical techniques. Ganglia from the small intestine of guinea-pigs were isolated, disrupted by sonication in Triton X-100, and phosphorylated. The phosphoprotein patterns obtained were compared with those of synaptosomes from guinea-pig and rat cerebral cortex. Myenteric ganglia were as rich in protein kinase C and cyclic AMP-stimulated protein kinase as brain tissue, but the level of calmodulin-stimulated protein kinase II was relatively lower. The alpha subunit of calmodulin-stimulated protein kinase II was detected by immunoblotting and the beta subunit by autophosphorylation. The ratio of beta to alpha subunit was considerably higher in ganglia than in brain and ganglia beta subunit had a lower apparent molecular weight than the brain enzyme. A number of neuronal phosphoproteins were found in ganglia including the 87,000 mol. wt phosphoprotein, synapsins 1a and 1b, and proteins IIIa and IIIb. A phosphoprotein of 48,000 mol. wt had many of the characteristics of the B-50 protein but was not the same. In addition, a number of other phosphoproteins not previously identified in neurons were found in ganglia including those with apparent molecular weights of 60,000 and 58,000 that were the major calmodulin kinase substrates. The guinea-pig enteric nervous system has been extensively studied but, unlike other parts of the mammalian nervous system, little is known about the intracellular mechanisms underlying its functions. A technique for isolating myenteric ganglia is now available and we have used this preparation to characterize the major protein kinase and phosphoproteins present in this tissue. The results obtained will allow the phosphorylation of the various proteins to be investigated after physiological or pharmacological manipulation of myenteric ganglia in situ and in vivo.
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PMID:Protein phosphorylation in guinea-pig myenteric ganglia and brain: presence of calmodulin kinase II. protein kinase C and cyclic AMP kinase and characterization of major phosphoproteins. 185 Dec 58

The effect of cholinergic stimulation of cellular protein phosphorylation was studied using an intact cell preparation isolated from the avian salt gland. Isolated cells were allowed to incorporate 32Pi into the cellular ATP pool and then challenged with compounds known to induce ion secretion in this tissue. Addition of carbachol resulted in a time- and concentration-dependent (EC50 = 500 nM) increase in 32Pi content of a 170-kDa protein (pp170). The stimulated phosphorylation could be blocked by the inclusion of atropine (100 microM). Subcellular fractionation studies localized pp170 to the plasma membrane fraction of the tissue. The integral nature of this protein was demonstrated by detergent-solubilization experiments with Triton X-100. The possibility that carbachol stimulates phosphorylation of pp170 via activation of protein kinase C (PKC) was investigated. Incubating salt gland cells with 4 beta-phorbol 12-myristate 13-acetate (PMA; 1 microM) or carbachol (100 microM) resulted in a translocation of soluble PKC from the cytosol to a plasma membrane fraction. Addition of either PMA (1 microM) or ionomycin (1 microM) alone did not enhance phosphorylation of pp170. A 4.5-fold increase in the phosphorylation state of pp170 was only observed when PMA and ionomycin were added concurrently. Preincubation of salt gland cells with PKC inhibitors H-7 (50 microM) or staurosporine (10 microM) inhibited the carbachol-stimulated phosphorylation of pp170. These findings suggest that carbachol mediates its secretomimetic effects via activation of PKC and that pp170 may represent a novel integral membrane PKC substrate protein.
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PMID:Carbachol-stimulated phosphorylation of a 170-kDa endogenous protein in avian salt gland cells. 188 75

A previously developed kinetic theory for lipid-dependent membrane enzymes (Sandermann, H. (1982) Eur. J. Biochem. 127, 123-128) is used to examine the activation of protein kinase C by phosphatidylserine. Hill-coefficients ranging up to 11 have been reported for activation in mixed micelles with Triton X-100. On the basis of this uniquely high degree of cooperativity, protein kinase C has been postulated to represent a new class of lipid-dependent membrane enzymes (Newton, A. and Koshland, D.E., Jr. (1989) J. Biol. Chem. 264, 14909-14915). In contrast, activation in the absence of Triton X-100 has led to Hill-coefficients of only less than or equal to 2.6. In order to resolve the apparent discrepancy, activation is now considered to involve binding of PS monomers to interacting sites on the enzyme, a non-activating PS trapping process also occurring in the presence of Triton X-100. Estimates for trapping are made for several sets of published data for micellar activation. The kinetic model developed here successfully fits each data set using a Hill-coefficient of only 3.0. An influence of Ca2+/ions or of a two-step mechanism of lipid-protein interaction are considered as possible molecular explanations. It is concluded (i) that lipid activation of protein kinase C may proceed without unique cooperativity and (ii) that ligand trapping could provide another means for 'threshold-type' kinetic regulation of membrane enzyme and receptor systems.
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PMID:Lipid-dependent membrane enzymes. Kinetic modelling of the activation of protein kinase C by phosphatidylserine. 193 63

The characteristics of the activation of a histone H4 kinase activity in Triton X-100 lysates of rabbit peritoneal neutrophils pretreated with fMet-Leu-Phe were studied: The activation of the kinase was a) inhibited by the antagonist of formylpeptide, t-Boc-(Phe-Leu)2(-)-Phe, b) completely inhibited by pertussis toxin pretreatment, c) not affected by the pretreatment of neutrophils with an activator of protein kinase C, phorbol-12-myristate-13-acetate, or an inhibitor of protein kinase C, 1-(5-isoquinoline-sulfonyl)-2-methyl-piperazine, and d) not inhibited in the cells preloaded with the intracellular calcium chelators, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra acetic acid acetoxymethyl-ester (BAPTA/AM). These results suggest that the stimulus-induced activation of H4 kinase requires functional receptor and GTP-binding protein but neither calcium mobilization nor protein kinase C activation.
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PMID:Stimulation of a histone H4 protein kinase in Triton X-100 lysates of rabbit peritoneal neutrophils pretreated with chemotactic factors: lack of requirements of calcium mobilization and protein kinase C activation. 196 52

Solid-phase binding assays with protein species purified from cultured rat glioma C6 cells and Ehrlich ascites revealed that plectin bound specifically to lamin B but not to lamins A and C. Lamin B interaction was significantly decreased upon in vitro phosphorylation of either lamin B or plectin with protein kinase A or C. In contrast, phosphorylation of plectin with kinase A increased its binding to vimentin, suggesting a different regulation of plectin interactions by this kinase. 32P-radiolabeling of rat glioma C6 cells revealed plectin as a major in vivo target of protein kinase A and protein kinase C. Plectin, present in lysates of dibutyryladenosine 3',5'-cyclic monophosphate-treated cells, showed a 2.5 times higher binding affinity to vimentin than plectin from phorbol ester-treated cells. Furthermore, the relative amounts of plectin in 1% Triton X-100/high salt-insoluble cell fractions decreased to one-fourth of control values upon treating cells with phorbol esters, whereas vimentin was unaffected. This finding suggested a protein kinase C-dependent weakening of plectin interaction with intermediate filaments in vivo. Taken together, these results point to a role of plectin in interlinking cytoskeletal and nuclear elements and suggest that specific protein kinases are involved in regulating these interactions.
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PMID:Protein kinase A- and protein kinase C-regulated interaction of plectin with lamin B and vimentin. 202 31

TRH and phorbol dibutyrate (PDBu) stimulate PRL secretion and synthesis from GH4C1 rat pituitary cells through activation of protein kinase C (PKC). TRH responses are mediated by increases in cellular levels of two PKC activators, Ca2+ and diacylglycerol (DAG), whereas PDBu acts as a DAG analog. We conducted experiments to compare the effects of Ca2+ and PDBu/DAG on alpha-PKC redistribution and to determine to what components of the particulate fraction activated alpha-PKC associates. Subcellular fractionation experiments demonstrated that TRH and PDBu both caused chelator-stable association of alpha-PKC with the particulate fraction. In contrast, Ca2+-mediated association with the particulate fraction was not chelator stable. Immunocytofluorescence experiments also demonstrated that TRH, PDBu, and increased cytosolic Ca2+ (due to ionomycin or K+ depolarization) caused redistribution. The effect of TRH was rapid and transient, similar to TRH stimulation of phospholipase C. The translocated alpha-PKC in the particulate fraction from TRH- or PDBu-treated cultures was not solubilized with Triton X-100. In comparable studies using an immunofluorescence assay, alpha-PKC immunofluorescence remained in detergent-insoluble preparations from TRH- and PDBu-stimulated, but not resting cells. The association of activated alpha-PKC with chelator- and detergent-insoluble material suggested that activated alpha-PKC may be associated with membrane and cytoskeletal components.
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PMID:Activation of alpha-protein kinase C leads to association with detergent-insoluble components of GH4C1 cells. 210 89


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