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)

Earlier reports from this laboratory have shown that protein kinase C (PKC) is cleaved with Ca2(+)-dependent neutral protease (calpain) I or II to produce a catalytically active fragment, and that calpain I, which is active in the micromolar range of Ca2+, may react preferentially with the active form of PKC that is associated with membranes. Subsequently, PKC is shown to exist as a large family of multiple subspecies with subtle individual characteristics. Three types of PKC designated types I, II, and III are purified from rat brain cytosol, which are shown to correspond to the cDNA clones gamma, beta, and alpha, respectively. The aim of the present study was to characterize the proteolysis of each PKC subspecies with calpain I and II. All types of PKC (82 kDa) were converted to two major fragments: a 47-49-kDa catalytic and a 36-kDa regulatory fragments by the cleavage with either calpain I or II. Analysis of the NH2-terminal sequence of the resulting catalytic fragments indicated that both calpain I and II cleaved at one or two specific sites in the variable region (V3) of each PKC molecule of which structure was clearly different among PKC subspecies. From kinetic studies, the cleavage of PKC subspecies with calpain I, and to a lesser extent, with calpain II (active in the millimolar range of Ca2+), was remarkably enhanced by the simultaneous presence of phospholipid and diacylglycerol or phorbol ester, suggesting that the active forms of PKC subspecies were the preferred targets for proteolysis. Whereas, stimulatory abilities of the lipids were variable among PKC subspecies and inactive form of type I PKC was cleaved with calpain I at a significant rate. Quantitative analysis with a fixed amount of calpain under comparable conditions showed that the susceptibilities of the PKC subspecies were distinctly different one another; the relative rates of cleavage of types I, II, and III PKC with calpain I and II were approximately 100:16:2 and 100:48:23, respectively. These results indicated that within the cell various PKC subspecies might be cleaved at different rates under different physiological conditions.
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PMID:Studies on proteolysis of protein kinase C with calpain I and II. 223 31

Two major protein kinase C (PKC) isozymes, accounting for approximately 95% of the total activity in human neutrophils, were separated by hydroxyapatite chromatography and were identified as beta-PKC (60% of the total) and alpha-PKC (35% of the total). No gamma-PKC was detected. A minor Ca2+/phospholipid requiring kinase that eluted from hydroxyapatite after alpha-PKC did not react significantly with any of the specific antisera employed for identification. Modification of beta-PKC or the minor PKC isozyme by calpain yielded Ca2+/phospholipid-independent forms (PKM) that retained only 50% of the original activities. In contrast, PKM formed from alpha-PKC retained full catalytic activity. For each native isozyme the rate of conversion by calpain was accelerated in the presence of Ca2+ and the lipid effectors, and the PKM form generated in each case was resistant to further digestion by calpain. All three PKC isozymes were also modified by a neutral serine proteinase isolated from human neutrophils, with this proteinase the major effect being loss of kinase activity, via a transient production of a Ca2+/phospholipid-independent form. This neutral serine proteinase appears to be localized at sites of interaction of cytoskeletal proteins with the cell membrane. Following stimulation of intact neutrophils with phorbol 12-myristate 13-acetate complete loss of native cytosolic kinase activity was observed, with recovery of approximately 30% of the original activity as a cytosolic Ca+/phospholipid independent form, presumably PKM. Loss of native PKC activity was greatest for the beta-isozyme. In cells stimulated by fMet-Leu-Phe approximately 60% of the original PKC activity was recovered as native cytosolic PKC and 30% as cytosolic PKM. Inhibitors of calpain reduced the extent of down-regulation of PKC, increased the proportion of PKC that remained associated with the plasma membrane and significantly reduced the proteolytically generated fully active PKM. Taken together, the in vitro and in vivo results suggest that calpain is involved primarily in the conversion of the PKC isozymes to the irreversibly activated PKM forms, and that the neutral serine proteinase may be the enzyme responsible for down-regulation, possibly via PKM as an intermediate.
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PMID:Isozymes of protein kinase C in human neutrophils and their modification by two endogenous proteinases. 229 14

Protein kinase C activity has been measured in extracts of larval brain of Drosophila melanogaster, with the synthetic nonapeptide substrate Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide. Protein kinase C activity in such extracts is abolished in a Ca2+-dependent manner at 18 degrees C, and partly converted to a form independent of effectors. The decay of protein kinase C activity can be prevented by leupeptin or a crude calpastatin preparation isolated from fly heads, indicating the presence of the Ca2+-dependent neutral protease, calpain, in larval brains. The total protein kinase C levels were nearly the same in wild type and three different dunce "memory-mutant" strains. In contrast, the soluble/particulate activity ratios were different: wild-type, 0.91; dunce M11, 0.46; dunce M11/Df(1)dm75e19, 1.23; dunce2, 0.88. These data suggest that the membrane adherence of protein kinase C in larval brain is governed by the actor of genes other than dunce.
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PMID:Protein kinase C in larval brain of wild-type and dunce memory-mutant Drosophila. 250 May 6

Calpain-catalysed hydrolysis of platelet substrates such as cytoskeletal and calmodulin-binding proteins, and of protein kinase C, is assumed to contribute to platelet aggregation. We have measured calpain I activation by immunoblotting, and [Ca2+]i (cytoplasmic Ca2+ concn.) by fura-2 fluorescence, in parallel with measurement of aggregation, in stirred human platelets treated at different [Ca2+]ext (extend Ca2+ concns.) with A23187, leupeptin, phorbol ester and thrombin. Hydrolysis of actin-binding protein, and [3H]5-hydroxytryptamine release, were also measured in some cases. A rise in [Ca2+]i, platelet aggregation and calpain activation often occurred together. With some combinations of agonists and [Ca2+]ext, however, this correlation was clearly not maintained. It was shown: (a) that activation of calpain and its hydrolysis of platelet substrates were not strictly necessary conditions for platelet secretion and aggregation; (b) conversely, that calpain activation could occur without aggregation.
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PMID:Calpain I activation is not correlated with aggregation in human platelets. 252 46

Limited proteolysis of three distinct subspecies of protein kinase C (Ca2+/phospholipid-dependent enzyme, PKC), types I (gamma), II (beta I and beta II), and III (alpha), with Ca2+-dependent neutral proteases I and II (calpains I and II) was studied. All forms of PKC (82 kDa) were converted to two major fragments: a 45-49-kDa catalytic fragment and a 36-kDa regulatory fragment. The cleavage of these PKC subspecies by calpain I (active in the micromolar range Ca2+/ion concentration), and, to a lesser extent, by calpain II (active in the millimolar range), was enhanced by the simultaneous presence of phospholipid and diacylglycerol or phorbol ester, suggesting that the activated form of PKC is the preferred target for proteolysis. Analysis of the NH2-terminal sequence of the resulting catalytic fragments indicated that both calpains I and II cleave at one or two specific sites in the third variable region (V3) of each PKC molecule. Under comparable conditions with calpains I and II, the relative rates of cleavage of types I, II, and III PKC were approximately 100:16:2 and 100:48:23, respectively. The results imply that within the cell various PKC subspecies may be cleaved at different rates under different physiological conditions.
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PMID:Limited proteolysis of protein kinase C subspecies by calcium-dependent neutral protease (calpain). 253 3

The calmodulin and C-kinase antagonists melittin, calmidazolium, N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W7), and trifluoperazine (TFP) also inhibit the activity of the human erythrocyte Ca2+-dependent protease, calpain I. W-5, the nonchlorinated derivative of W-7, was ineffective as an inhibitor of calpain I just as it is for calmodulin and protein kinase C. Dose response studies provided the following IC50 values: melittin, 2.6 microM; calmidazolium, 6.2 microM; trifluoperazine, 130 microM; W-7, 251 microM. These IC50 values indicate that the compounds have affinities 10 to 600 fold less for calpain I than for calmodulin; however, the affinities of the inhibitory compounds are comparable for calpain I and protein kinase C. Kinetic analysis indicates that the compounds are competitive inhibitors of calpain I with respect to substrate.
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PMID:Calmodulin and protein kinase C antagonists also inhibit the Ca2+-dependent protein protease, calpain I. 253 50

To identify the protein kinase that is responsible for catalyzing phosphorylation of actin-binding protein (ABP) in platelets, we have examined the effects of protein kinase C and cAMP-dependent protein kinase on this process. We found that purified platelet protein kinase C from platelets was unable to phosphorylate ABP in vitro. However, a crude platelet kinase preparation phosphorylated ABP in the presence of cAMP, but not in the presence of Ca2+/phosphatidylserine. Fresh platelet plasma membranes incubated with [gamma-32P]ATP phosphorylated ABP in the presence of cAMP and the process was blocked by a cAMP-dependent protein kinase inhibitor; ABP phosphorylation induced by prostaglandin E1 (PGE1) appeared to be reduced by the subsequent addition of thrombin. These results strongly suggest that in situ ABP is phosphorylated by activated cAMP-dependent protein kinase when platelet function is inhibited by PGE1. Furthermore, in the PGE1-treated platelets, ABP was proteolyzed at a slower rate than in control platelets when they were lysed with Triton in the absence of EGTA. Partially purified ABP was proteolyzed by calpain in vitro at a slower rate as well. It was demonstrated that ABP from PGE1-treated platelets recovered its sensitivity to calpain after ABP was incubated with a protein phosphatase that had been purified from platelets. We postulate that ABP is stabilized against proteolysis in response to cAMP-elevating agents and that this blocks cytoskeleton reorganization.
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PMID:In situ phosphorylation of platelet actin-binding protein by cAMP-dependent protein kinase stabilizes it against proteolysis by calpain. 254 93

The phenomenon of long-term potentiation (LTP), a long lasting increase in the strength of synaptic transmission which is due to brief, repetitive activation of excitatory afferent fibres, is one of the most striking examples of synaptic plasticity in the mammalian brain. In the CA1 region of the hippocampus, the induction of LTP requires activation of NMDA (N-methyl-D-aspartate) receptors by synaptically released glutamate with concomitant postsynaptic membrane depolarization. This relieves the voltage-dependent magnesium block of the NMDA-receptor ion channel, allowing calcium to flow into the dendritic spine. Although calcium has been shown to be a necessary trigger for LTP (refs 11, 12), little is known about the immediate biochemical processes that are activated by calcium and are responsible for LTP. The most attractive candidates have been calcium/calmodulin-dependent protein kinase II (CaM-KII) (refs 13-16), protein kinase C (refs 17-19), and the calcium-dependent protease, calpain. Extracellular application of protein kinase inhibitors to the hippocampal slice preparation blocks the induction of LTP (refs 21-23) but it is unclear whether this is due to a pre- and/or postsynaptic action. We have found that intracellular injection into CA1 pyramidal cells of the protein kinase inhibitor H-7, or of the calmodulin antagonist calmidazolium, blocks LTP. Furthermore, LTP is blocked by the injection of synthetic peptides that are potent calmodulin antagonists and inhibit CaM-KII auto- and substrate phosphorylation. These findings demonstrate that in the postsynaptic cell both activation of calmodulin and kinase activity are required for the generation of LTP, and focus further attention on the potential role of CaM-KII in LTP.
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PMID:An essential role for postsynaptic calmodulin and protein kinase activity in long-term potentiation. 254 23

In addition to other proteinases human neutrophils contain two non granular neutral endopeptidases: a serine proteinase and a cysteine Ca2+ dependent proteinase named calpain. Serine proteinase localized in association with the cytoskeleton-membrane proteins, apparently exerts a dual role: it is partially released into the medium during neutrophil stimulation by phorbol myristate acetate (PMA), presumably acting as one of the cytotoxic factors; and in its intracellular localization is presumably involved in the process of down regulation of native protein kinase C (PKC). Calpain, predominantly present in resting conditions in an inactive form, becomes activated in the course of neutrophil stimulation and appears to be involved both in the down regulation of native PKC as well as in the formation of a proteinase-activated kinase form, presumably derived from PKC and defined as PKC-M. Calpain once activated appears to be also involved in cytoskeleton rearrangement, through proteolytic degradation specifically oriented by substrate phosphorylation. Activation, down regulation of PKC, formation of the proteinase-activated kinase, as well as proteolytic processing of cytoskeleton have been demonstrated to be correlated to those biochemical responses which characterize neutrophil activation.
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PMID:The role of intracellular proteinases in human neutrophil activation. 256 40

Purified bovine myocardial sarcolemma vesicles were shown to contain calcium-dependent proteinase inhibitor protein by direct assay and by immunoblot analysis following gel electrophoresis (Western blotting). Calcium-dependent proteinase (calpain, EC 3.4.22.17) was not detected in the sarcolemma vesicles. The inhibitor protein was not solubilized when the vesicles were ruptured by repetitive freezing and thawing. However, a large amount of latent inhibitor activity was exposed after freezing and thawing the sarcolemma, and the inhibitor was much more susceptible to removal by 1.0 M NaCl or proteolysis following this treatment. Since the vesicles were predominantly right-side-out, the latter observations suggested that the inhibitor was associated with the cytoplasmic face of the sarcolemma. The endogenous inhibitor was capable of protecting sarcolemmal protein kinase C from proteolytic conversion to soluble protein kinase M by type I or type II calcium-dependent proteinase. Thus, the inhibitor is probably important in controlling calcium-dependent proteolysis of sarcolemmal proteins.
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PMID:A sarcolemma-associated inhibitor is capable of modulating calcium-dependent proteinase activity. 282 May 3


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