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)

Phosphorylation of three physiological substrates of protein kinase C (PKC), MARCKS, neuromodulin (Nm), and neurogranin (Ng), was analyzed to determine their relative efficacy as substrates of PKC alpha, beta, and gamma and sensitivities to inhibition by calmodulin (CaM) and S100. Comparison of the Vmax/Km of the phosphorylation of each individual substrate indicated the order of efficacy as PKC substrate was MARCKS > Nm > Ng. Phosphorylation of these proteins in a mixture by PKC beta and gamma was indistinguishable from that when each individual substrate was phosphorylated by these two isozymes. In contrast, the rates of PKC alpha-catalyzed phosphorylation of Nm and Ng in a mixture also containing MARCKS were significantly reduced as compared to that when Nm or Ng was individually phosphorylated by this isozyme. When these substrates were present in a mixture, both CaM and S100 inhibited the PKC-catalyzed phosphorylation of MARCKS to a higher degree than that of Nm or Ng. Protease-activated catalytic fragment of PKC (PKM) was used to determine the effects of Ca2+ and phospholipid on the CaM and S100-mediated inhibition of PKC substrate phosphorylation. CaM and S100 inhibited the PKM-catalyzed phosphorylation of MARCKS only in the presence of Ca2+ and addition of phosphatidylserine (PS)/dioleoylglycerol (DG) did not influence the inhibitory effect. Phosphorylation of Nm or Ng by PKM was inhibited by CaM to a higher degree in the absence than in the presence of Ca2+. S100 was ineffective in inhibiting the phosphorylation of Nm and Ng without Ca2+ and only poorly effective in the presence of Ca2+. The CaM-mediated inhibition of Nm or Ng phosphorylation by PKM was also not affected by PS/DG either with or without Ca2+. The results presented here demonstrate that MARCKS is a preferred substrate of PKC and its phosphorylation by PKC is most sensitive to inhibition by regulatory proteins such as CaM and S100.
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PMID:Differential responses of protein kinase C substrates (MARCKS, neuromodulin, and neurogranin) phosphorylation to calmodulin and S100. 784 Jun 34

We have investigated mechanisms by which intracellular signals act to restructure the spatial organization of the cytoskeleton as the mammalian egg is converted into the zygote. Four distinct approaches (one cytological, two biochemical, and one pharmacological) demonstrate protein kinase C (PKC) and its cytosolic active counterpart, PKM, act in succession at the time of egg activation. PKM serves to remodel the internal cytoskeleton. The cytological approach mapped the distribution of kinase over time using the PKC reporter dye, Rim-1, which demonstrated a temporal shift in kinase distribution from its initial site of activation at the plasma membrane to its subsequent association with a cross-linked network of intermediate filaments referred to as sheets. The first of two biochemical analyses, Western blot analysis, demonstrated that eggs activated with calcium ionophore contained PKC in the detergent-soluble fraction and PKM in the sheet enriched fraction. Prior to egg activation, PKM is not detected in the sheet-enriched fraction; only PKC is detected in the detergent-soluble fraction of these eggs. The second biochemical analysis demonstrated, via [32P]ATP labeling of a known PKC substrate, that the PKM generated from activation of PKC in response to activation of eggs with calcium ionophore is an active kinase and is located in the sheet-enriched fraction. In addition, purified forms of PKM, but not PKC, could be shown to act on the internal cytoskeleton when perfused into a permeabilized egg system. Pharmacological treatments demonstrate that elevation of [Ca2+]i does not act directly to alter the internal cytoskeleton of the egg. Our results suggest that this kinase is employed at the time of fertilization to provide an internal chronometer acting first at the cell periphery as PKC and subsequently in the cell interior as PKM.
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PMID:Protein kinase M, the cytosolic counterpart of protein kinase C, remodels the internal cytoskeleton of the mammalian egg during activation. 787 73

In area CA1 of the hippocampus, the induction of long term potentiation (LTP) requires activation of either N-methyl-D-aspartate receptors (NMDA receptor-dependent LTP) or voltage-gated Ca2+ channels (NMDA receptor-independent LTP). We have investigated biochemical sequelae of NMDA receptor-independent LTP induction. We find that a persistent increase in second messenger-independent protein kinase C activity is associated with the maintenance phase of NMDA receptor-independent LTP. This increase in protein kinase C activity is prevented by blocking LTP with nifedipine, a Ca2+ channel antagonist, or kynurenic acid, a nonselective glutamate receptor antagonist. Additionally, we find an increase in the catalytic fragment of protein kinase C (PKM) in the maintenance phase of NMDA receptor-independent LTP, indicating that proteolytic activation of protein kinase C may account for its autonomous activation. This increase in the catalytic fragment of protein kinase C is also prevented by blocking LTP induction. These results are the first to demonstrate that persistent protein kinase C activation is a possible mechanism for the maintenance of NMDA receptor-independent LTP.
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PMID:Autonomously active protein kinase C in the maintenance phase of N-methyl-D-aspartate receptor-independent long term potentiation. 796 28

The beta 1 and gamma 2L subunits of the gamma-aminobutyric acid type A receptor (GABAR) contain phosphorylation sites for PKC. To determine the effect of PKC on GABAR function, whole-cell recordings were obtained from mouse fibroblasts expressing recombinant alpha 1 beta 1 gamma 2L receptors, and catalytically active PKC (PKM) was applied via the recording pipette. The first experiment was a population study. Intracellular application of PKM increased GABAR currents, and the enhancement was antagonized by coapplication of the PKC inhibitory peptide. No acceleration or deceleration of GABAR desensitization was observed. The second experiment was a reimpalement study in which paired recordings were made successively from individual cells. Enhancement of GABAR currents by PKM was again obtained. PKM increased GABAR currents at high (> 10 microM) but not at low (< 10 microM) GABA concentrations, resulting in increases in both EC50 and maximal GABAR current. Thus, PKC phosphorylation enhanced recombinant alpha 1 beta 1 gamma 2L GABAR current by increasing maximal current without increasing the affinity of GABA for the GABARs.
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PMID:Protein kinase C enhances recombinant bovine alpha 1 beta 1 gamma 2L GABAA receptor whole-cell currents expressed in L929 fibroblasts. 799 33

Proteolytic cleavage of protein kinase C (PKC) under cell-free conditions generates a co-factor independent, free catalytic subunit (PKM). However, the difficulty in visualizing PKM in intact cells has generated controversy regarding its physiological relevance. In the present study, treatment of SH-SY-5Y cells with 2-O-tetradecanoylphorbol 13-acetate resulted in complete down-regulation of PKC within 24 h without detection of PKM. By contrast, low levels of PKM were transiently detected following ionophore-mediated calcium influx under conditions which induced no detectable PKC loss. PKM was not detected during rapid cell-free degradation of partially purified SH-SY-5Y PKC alpha by purified human brain mM calpain. However, when the kinetics of PKC degradation were slowed by lowering levels of calpain, PKM was transiently detected. PKM was also only transiently observed following calpain-mediated degradation of purified rat brain PKC alpha. Densitometric analyses indicated that, once formed, PKM was degraded approximately 10 times faster than PKC. These data provide an explanation as to why PKM is difficult to observe in situ, and indicate that PKM should not be considered as an 'unregulated' kinase, since its persistence is apparently strictly regulated by proteolysis.
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PMID:Degradation of protein kinase C alpha and its free catalytic subunit, protein kinase M, in intact human neuroblastoma cells and under cell-free conditions. Evidence that PKM is degraded by mM calpain-mediated proteolysis at a faster rate than PKC. 807 May 69

A 7.5-kDa heat- and acid-stable rat brain protein kinase C (PKC) substrate was purified to near homogeneity by a two-step procedure using DEAE-cellulose and hydroxylapatite column chromatography. This 78-amino-acid protein has a sequence identical to that deduced from rat brain RC3 cDNA identified with a cortex-minus-cerebellum subtracted cDNA probe (J. B. Watson et al., J. Neurosci. Res. 26, 397-408, 1990) and exhibits extensive sequence identity to bovine brain neurogranin (J. Baudier et al., J. Biol. Chem. 266, 229-237, 1991). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis this protein, RC3, migrated as a M(r) 15-18K species in the presence of reducing agent and as heterogeneous species of M(r) 13-28K in the absence of reducing agent. Phosphorylation of RC3 by PKC alpha, beta, or gamma was stimulated by Ca2+, phospholipid, and diacylglycerol. A single site, Ser36, which is adjacent to the predicted calmodulin (CaM)-binding domain, was phosphorylated by these enzymes. Phosphorylation of RC3 by PKC or PKM, a protease-degraded PKC, was inhibited by CaM. The effect of CaM apparently targets at RC3, as phosphorylation of protamine sulfate by PKM was not inhibited by CaM. In the absence of Ca2+, RC3 formed a stoichiometric complex with CaM as evidenced by an increase in the M(r) determined by gel filtration chromatography. In the presence of Ca2+, the affinity of RC3 toward CaM is greatly reduced and Ca2+/CaM becomes less inhibitory of the PKM-catalyzed phosphorylation of RC3. Phosphorylation of RC3 by PKM prevented the interaction of this protein with CaM even in the absence of Ca2+. A 20-amino-acid synthetic peptide (AS-20F-W) containing the PKC phosphorylation site and CaM-binding domain of RC3 (Ala29-Ser48) with a substitution of Phe37 with tryptophan was used to monitor the interaction of this peptide with CaM by spectrofluorometry. In the absence of Ca2+, CaM caused negligible change in tryptophan fluorescence of the peptide; however, an enhancement and blue-shift of the emission fluorescence was observed in the presence of Ca2+. It seems that this synthetic peptide, as well as RC3 holoprotein, interacts with CaM through electrostatic interaction in the absence of Ca2+ but through hydrophobic interaction in the presence of Ca2+. In rat brain homogenate, RC3 formed a stable complex with CaM in the presence of Ca2+, as demonstrated by immunoblot analysis following gel filtration chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization of a 7.5-kDa protein kinase C substrate (RC3 protein, neurogranin) from rat brain. 808 Apr 73

Human erythrocytes contain cytosolic protein kinase C (PKC) which, when activated by phorbol 12-myristate 13-acetate (PMA), induces the phosphorylation of the membrane skeletal proteins band 4.1, band 4.9 and adducin. We found that brief treatments of erythrocytes with PMA resulted in a decrease in cytosolic PKC content and in the transient appearance in the cytosol of a Ca(2+)- and phospholipid-independent 55 kDa fragment of PKC, called PKM. Prolonged treatment with PMA resulted in the complete and irreversible loss of erythrocyte PKC. To investigate the possible role of calpain in this process, the calpain inhibitors leupeptin and E-64 were sealed inside erythrocytes by reversible haemolysis. Both inhibitors prolonged the lifetime of PKC in PMA-treated cells, and leupeptin was shown to block the PMA-stimulated appearance of PKM in the cytosol. Significantly, leupeptin also completely blocked PMA-stimulated phosphorylation of membrane and cytosolic substrates. This effect was mimicked by other calpain inhibitors (MDL-28170 and calpain inhibitor I), but did not occur when other protease inhibitors such as phenylmethanesulphonyl fluoride, pepstatin A or chymostatin were used. In addition, the phosphorylation of exogenous histone sealed inside erythrocytes was also blocked by leupeptin. Immunoblotting showed that leupeptin did not prevent the PMA-induced translocation of PKC to the erythrocyte membrane. Thus inhibition of PKC phosphorylation of membrane skeletal proteins by calpain inhibitors was not due to inhibition of PKC translocation to the membrane. Our results suggest that PMA treatment of erythrocytes results in the translocation of PKC to the plasma membrane, followed by calpain-mediated cleavage of PKC to PKM. This cleavage, or some other leupeptin-inhibitable process, is a necessary step for the phosphorylation of membrane skeletal substrates, suggesting that the short-lived PKM may be responsible for membrane skeletal phosphorylation. Our results suggest a potential mechanism whereby erythrocyte PKC may be subject to continual down-regulation during the lifespan of the erythrocyte due to repeated activation events, possibly related to transient Ca2+ influx. Such down-regulation may play an important role in erythrocyte survival or pathophysiology.
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PMID:Phorbol 12-myristate 13-acetate-stimulated phosphorylation of erythrocyte membrane skeletal proteins is blocked by calpain inhibitors: possible role of protein kinase M. 828 66

In the model of transient brain ischemia of 6-min duration in gerbils we have estimated: 1. The concentration of brain gangliosides: A significant decrease to about 70% of control was observed selectively in the hippocampus at 3 and 7 d after ischemia. 2. The activity of Na+,K(+)-ATPase: The enzyme activity was not affected in either hippocampus nor in cerebral cortex. 3. The malonaldehyde (MDA) concentration: The levels of MDA had increased at 30 min after ischemia up to 123 and 129% of control in hippocampus and cerebral cortex, respectively. 4. Immunoreactivity of protein kinase C detected by Western blotting: In hippocampus the early translocation toward membranes was followed by a decrease in total enzyme content at 6, 24, 72, and 96 h of postischemic recovery. Also, a sharp increase of 50 kDa isoform (PKM) was noticed immediately and at the early recovery times. The behavior of these biochemical markers of ischemic brain injury in the hippocampus after the short (6 min) insult was contrasted with their reaction in the cerebral cortex as well as after prolongation of the ischemia to 15 min. These results taken together indicate that an early increase in PKC translocation followed by a decrease is the most symptomatic for selective, delayed, postischemic hippocampal injury, resulting from short duration (6 min) ischemia of the gerbil brain.
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PMID:Protein kinase C as an early and sensitive marker of ischemia-induced progressive neuronal damage in gerbil hippocampus. 829 17

Limited proteolysis of protein kinase C (PKC) by calcium-activated proteolysis cleaves the regulatory and catalytic subunits of PKC, generating a free, constitutively activated kinase ("PKM") that, unlike the intact parent enzyme, is not calcium-dependent, and is not restricted to the plasma membrane. These latter properties leave open the possibility that PKM may have access to, and may therefore phosphorylate, substrates normally unavailable to intact PKC. We examined the potential involvement of such aberrant phosphorylation in certain aspects of the neurodegeneration accompanying Alzheimer's disease by microinjecting PKC and PKM, along with a rhodamine-conjugated dextran tracer, into undifferentiated NB2a/d1 mouse neuroblastoma cells. After 4 hr, cultures were fixed and processed for immunofluorescence with monoclonal antibodies (PHF-1, ALZ-50, Tau-1, AT8) directed against tau in various phosphorylation states followed by fluorescein-conjugated secondary antibodies. Microinjected cells were localized via co-injected rhodamine-conjugated dextran tracer under rhodamine illumination, after which antibody immunoreactivity was examined under fluorescein illumination. Microdensitometric analyses indicated that microinjection of PKC did not increase basal immunofluorescent intensities of the antibodies; by contrast, microinjection of PKM induced three- and twofold increases in PHF-1 and ALZ-50 levels, respectively. By contrast, no significant alteration was observed in AT8 and Tau-1 immunofluorescence following either PKC or PKM microinjection. Whereas undifferentiated NB2a/d1 cells typically elaborate short, filopodia-like neurites, phase-contrast microscopy revealed the absence of filopodia or neurites on PKM-injected cells, while a similar percentage of PKC-injected cells. Cell-free analyses confirmed the ability of PKC, in the presence of necessary co-factors, and PKM to increase PHF-1 and ALZ-50 immunoreactivity; no change was observed in AT8 or Tau-1 immunoreactivity. These findings underscore the possibility that an abnormal amplification in limited PKC proteolysis to generate PKM could, under certain pathological conditions, contribute to neuronal degeneration.
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PMID:Hyperphosphorylation of Tau and filopodial retraction following microinjection of protein kinase C catalytic subunits. 860 Feb 97

Calcium influx into SH-SY5Y human neuroblastoma cells after ionophore treatment or transient permeabilization in calcium-containing medium increased ALZ-50 immunoreactivity markedly. This increase was prevented by inhibitors active against calpain or against protein kinase C (PKC), suggesting that both of these enzymes were required to mediate the effect of calcium influx on ALZ-50 immunoreactivity. Treatment with PKC activator TPA increased ALZ-50 immunoreactivity in the absence of calcium influx or after intracellular delivery of the specific calpain inhibitor calpastatin, indicating that the influence of PKC was downstream from that of calpain. Calcium influx also resulted in mu-calpain autolysis (one index of calpain activation) and the transient appearance of PKM (i.e., free PKC catalytic subunits, generated by calpain-mediated cleavage of the regulatory and catalytic PKC domains). Inhibition of calpain within intact cells resulted in a dramatic increase in steady-state levels of total tau (migrating at 46-52 kDa) but resulted in a relatively minor increase in 68-kDa ALZ-50-immunoreactive tau isoforms. Although calcium influx into intact cells resulted in accumulation of ALZ-50 immunoreactivity, total tau levels were, by contrast, rapidly depleted. Incubation of isolated fractions with calpain in the presence of calcium indicated that ALZ-50-immunoreactive tau isoforms were more resistant to calpain-mediated proteolysis than were non-ALZ-50 reactive tau isoforms. These data therefore indicate that calpain may regulate tau levels directly via proteolysis and indirectly through PKC activation. A consequence of the latter action is altered tau phosphorylation, perhaps involving one or more kinase cascades, and the preferential accumulation of ALZ-50-immunoreactive tau isoforms due to their relative resistance to degradation. These findings provide a basis for the possibility that disregulation of calcium homeostasis may contribute to the pathological levels of conversion of tau to A68 by hyperactivation of the calpain/PKC system.
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PMID:Calcium influx into human neuroblastoma cells induces ALZ-50 immunoreactivity: involvement of calpain-mediated hydrolysis of protein kinase C. 862 10


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