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)

Mouse monoclonal B-50 antibodies (Mabs) were screened to select a Mab that may interfere with suggested functions of B-50 (GAP-43), such as involvement in neurotransmitter release. Because the Mab NM2 reacted with peptide fragments of rat B-50 containing the unique protein kinase C (PKC) phosphorylation site at serine-41, it was selected and characterized in comparison with another Mab NM6 unreactive with these fragments. NM2, but not NM6, recognized neurogranin (BICKS), another PKC substrate, containing a homologous sequence to rat B-50 (34-52). To narrow down the epitope domain synthetic B-50 peptides were tested in ELISAs. In contrast to NM6, NM2 immunoreacted with B-50 (39-51) peptide, but not with B-50 (43-51) peptide or a C-terminal B-50 peptide. Preabsorption by B-50 (39-51) peptide of NM2 inhibited the binding of NM2 to rat B-50 in contrast to NM6. NM2 selectively inhibited phosphorylation of B-50 during endogenous phosphorylation of synaptosomal plasma membrane proteins. Preabsorption of NM2 by B-50 (39-51) peptide abolished this inhibition. In conclusion, NM2 recognizes the QASFR peptide in B-50 and neurogranin. Therefore, NM2 may be a useful tool in physiological studies of the role of PKC-mediated phosphorylation and calmodulin binding of B-50 and neurogranin.
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PMID:Monoclonal antibody NM2 recognizes the protein kinase C phosphorylation site in B-50 (GAP-43) and in neurogranin (BICKS). 811 10

Repeated, intermittent treatment of rats with amphetamine results in a sensitization of locomotor and stereotyped behaviors that is accompanied by an enhancement in stimulus-induced dopamine release. Increased phosphorylation of the neural specific calmodulin-binding protein, neuromodulin (GAP-43, B-50, F1) has been demonstrated in other forms of synaptic plasticity and plays a role in neurotransmitter release. To determine whether neuromodulin phosphorylation was altered during amphetamine sensitization, the in vivo phosphorylated state of neuromodulin was examined in rat striatum in a post hoc phosphorylation assay. Female, Holtzman rats received saline or 2.5 mg/kg amphetamine twice weekly for 5 weeks. One week after the last dose of amphetamine, rats were challenged with either 1 mg/kg or 2.5 mg/kg amphetamine or saline and the rats were sacrificed 30 min later. Purified synaptic plasma membranes were prepared in the presence of EGTA and okadaic acid to inhibit dephosphorylation, and were subsequently phosphorylated in the presence of purified protein kinase C and [gamma-32P]ATP. The protein kinase C-mediated post hoc phosphorylation of neuromodulin was significantly reduced in groups that received either acute or repeated amphetamine suggesting that neuromodulin in those groups contained more endogenous phosphate. The acute, challenge dose of amphetamine increased neuromodulin phosphorylation in the saline-treated controls but not in the repeated amphetamine-pretreated group. Anti-neuromodulin immunoblots showed no change in neuromodulin levels in any group. There was no significant change in protein kinase C activity in any treatment group. To further investigate the effect of acute amphetamine, the ability of amphetamine to alter neuromodulin phosphorylation in 32Pi-preincubated Percoll-purified rat striatal synaptosomes was examined. Amphetamine (10 microM) significantly increased phosphorylation of a 53 kDa band that migrated with authentic neuromodulin in the synaptosomes by 22% while 500 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) increased neuromodulin phosphorylation by 45%. These data suggest that one injection of amphetamine can increase neuromodulin phosphorylation in rat striatum and that this increase is maintained for at least 1 week following a repeated, sensitizing regimen of amphetamine. Since sensitization can be induced with one dose of amphetamine, it is possible that enhanced neuromodulin phosphorylation could contribute to neurochemical events leading to enhanced release of dopamine and/or behavioral sensitization.
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PMID:Phosphorylation of neuromodulin in rat striatum after acute and repeated, intermittent amphetamine. 811 16

The synthetic peptide neurogranin(28-43), the sequence of which is homologous to the phosphorylation site of the brain specific protein kinase C (PKC) substrates neurogranin and neuromodulin, was tested for its utility as a PKC substrate in crude tissue homogenates. The phosphorylation of neurogranin(28-43) shows time- and protein concentration-dependency. In prolonged incubations, the addition of the protein phosphatase inhibitor sodium pyrophosphate results in increased phosphorylation of neurogranin(28-43). The phosphorylation of neurogranin(28-43) was compared to that of another widely used PKC substrate, S6(229-249). Neurogranin(28-43) is as potent as S6(229-249) and more selective than S6(229-249) as a PKC substrate. Greater than 95% of phosphate incorporation into neurogranin(28-43) can be inhibited by a selective PKC inhibitor, PKC(19-36). Kinetic analysis of neurogranin(28-43) phosphorylation in hippocampal homogenate revealed an apparent Km of 147 nM, virtually identical to previously published Km observed for phosphorylation of the substrate by purified PKC. In addition, we assayed several neuronal and nonneuronal tissues using neurogranin(28-43) as substrate in the presence or absence of detergent. We show that the relative PKC activity assayed with neurogranin(28-43) correlates well to the relative amount of PKC known to be present in various neuronal and nonneuronal tissues. Overall, this report shows that neurogranin(28-43) can be used to selectively assay PKC, even in tissue containing low PKC activity.
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PMID:Use of the synthetic peptide neurogranin(28-43) as a selective protein kinase C substrate in assays of tissue homogenates. 812 77

1. The effects on catecholamine secretion of activation of protein kinase C and clostridial neurotoxins were examined in digitonin-permeabilized bovine adrenal chromaffin cells. 2. The enhancement by phorbol esters increased only the initial rate of secretion; later rates were unaffected. This enhancement was present over a wide range of Ca2+ concentrations and was elicited at 18 as well as at 27 degrees C. 3. Tetanus toxin inhibited both ATP-dependent and ATP-independent secretion, indicating that the tetanus toxin target is important during the final steps in the pathway. 4. Prior activation of protein kinase C by the phorbol ester 12-O-tetradecanoyl phorbol acetate rendered the primed state more sensitive to inhibition by tetanus toxin. The data indicate that a phosphorylated protein kinase C substrate is either identical to or closely associated with the tetanus toxin target protein at the final steps in the pathway. 5. The interaction between the effect of protein kinase activation and that of tetanus toxin suggests that protein kinase C activation does not stimulate a separate pathway of secretion but, rather, modulates the activity of the ongoing pathway. 6. The enhancement of secretion by protein kinase C is caused, at least in part, by a qualitative change in the characteristics of the primed state. This is indicated by the increased sensitivity of primed secretion to inhibition by tetanus toxin and a threefold increase in sensitivity of primed secretion to Ca2+. 7. Because activation of protein kinase C does not increase the later rates of secretion that are limited by ATP-dependent priming reactions, it is unlikely that enhancement of the maximal rate of secretion by TPA is due to an increased amount of the primed state. Instead, protein kinase C activation may increase the efficacy with which Ca2+ stimulates secretion at all Ca2+ concentrations.
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PMID:Protein kinase C and clostridial neurotoxins affect discrete and related steps in the secretory pathway. 819 81

The mRNA levels of protein F1 (also known as GAP-43), and protein kinase C (PKC) subtypes were measured 3 days after the induction of long-term enhancement (also known as long-term potentiation) in the hippocampus of chronically prepared conscious rats by quantitative in situ hybridization. Altered mRNA levels correlated significantly with alternations in synaptic efficacy; such correlations have not been reported previously. Rats with greater synaptic enhancement had lower gene expression in the CA3 subfield of F1/GAP-43 and both beta-PKC and gamma-PKC, but not alpha-PKC. For microtubule-associated protein 2 (MAP-2), neurogranin, and the glutamate receptor subtype B-flip, no correlation was observed in any cell field between synaptic enhancement and hybridization to the mRNA. To our surprise, alterations in mRNA levels of F1/GAP-43 and gamma-PKC were highly correlated (r = +0.928, P < 0.001), suggesting coordinate regulation. Since F1/GAP-43 is associated with neurite growth, its lowered expression at 3 days would reduce potential growth, leading to synaptic stabilization. We propose that long-term synaptic change is mediated by gene expression of the very same proteins initially modified posttranslationally.
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PMID:Protein kinase C and F1/GAP-43 gene expression in hippocampus inversely related to synaptic enhancement lasting 3 days. 826 69

RC3 (neurogranin) is a neuron-specific substrate of protein kinase C (PKC) that accumulates predominantly in dendritic spines of forebrain neurons and undergoes long-term potentiation (LTP)-associated increases in PKC-phosphorylation in hippocampal slices. Here the hypothesis that RC3 functions by modulating the IP3/DAG second messenger pathway after its phosphorylation by DAG-activated PKC was tested by heterologous expression in Xenopus oocytes. Acetylcholine-evoked inward chloride (Cl-) currents, dependent on both IP3 release and intracellular calcium (Ca2+), were 2- to 3-fold higher in RC3-injected oocytes than in uninjected control oocytes. RC3-oocytes did not exhibit enhanced currents when preincubated with the protein kinase inhibitor H-7 or when a glycine residue was substituted for serine, the PKC phosphorylation site of RC3. Activation of endogenous oocyte PKC by phorbol esters generated inward Cl- currents in RC3 oocytes but not in control oocytes. RC3-dependent Cl- currents were also elicited by phorbol ester in Ca(2+)-free media. We propose that PKC-phosphorylated RC3 is capable of enhancing the mobilization of intracellular Ca2+ in Xenopus oocytes and, by inference, may play a role in Ca2+ homeostasis in dendrites of forebrain neurons.
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PMID:Functional consequences of expression of the neuron-specific, protein kinase C substrate RC3 (neurogranin) in Xenopus oocytes. 829 95

The mechanism by which norepinephrine (NE) down-regulates alpha 1B-adrenergic receptor (alpha-AR) mRNA was studied in rabbit aortic smooth muscle cells. NE, phorbol esters, and bradykinin each decreased alpha-AR mRNA levels by 70-80%. The protein kinase C inhibitor (+)-1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) abolished the effects of phorbol esters and NE and decreased basal mRNA levels by 52 +/- 3%. Neither ryanodine nor EGTA inhibited down-regulation of alpha-AR mRNA by NE. Actinomycin D caused alpha-AR mRNA level to decrease with a half-life of 3.2 +/- 0.4 h and blocked the effect of H-7 to decrease basal alpha-AR mRNA level. Both NE and phorbol esters increased the rate of alpha-AR mRNA degradation. In NE-desensitized cells, phorbol esters and bradykinin each caused the expected down-regulation of alpha-AR mRNA. The protein phosphatase inhibitor okadaic acid prolonged the normally transient effect of NE for at least 24 h. We conclude that protein kinase C exerts two opposing effects on alpha-AR mRNA levels, 1) a decrease in the stability of the mRNA that requires the sustained phosphorylation of a protein kinase C substrate and 2) a permissive effect on alpha-AR gene transcription.
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PMID:Phorbol esters and norepinephrine destabilize alpha 1B-adrenergic receptor mRNA in vascular smooth muscle cells. 829 18

Differentiated neuroblastoma cells exhibit both the delayed rectifier potassium current (IK) and the M-current (IM). The present study was designed to determine the roles of protein kinase C (PKC) and of the calmodulin-binding protein 80K/MARCKS, a prominent substrate for PKC and possible regulator of these currents. Neuroblastoma x glioma (NG108-15) hybrid cells transfected with m1 muscarinic receptors were grown with 1% fetal bovine serum (FBS) without the prostaglandin E1 (PGE1) and isobutylmethylxanthine (IBMX) usually added in preparation for electrophysiological studies. Under these conditions, the usual pleomorphism was largely abolished, leaving two populations of small cells with stellate and spherically symmetrical geometries. Whole-cell patch clamping indicated that the two cell types had identical electrophysiological properties, displaying: IK, a small current through a "T-like" Ca2+ channel, and no M-current. Stimulation with carbachol shifted the distribution of cells to a more stellate morphology within 24 hr and later (after 48 hr) reduced the PKC substrate 80K/MARCKS by 22 +/- 7%. In contrast to the stimulation of IK observed with cardiac cells, PKC activation produced only a small inhibition of IK, which was independent of carbachol pretreatment. Thus, PKC and 80K/MARCKS can be dissociated from the regulation of IK in neuroblastoma cells.
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PMID:Whole-cell recording of neuroblastoma x glioma cells during downregulation of a major substrate, 80K/MARCKS, of protein kinase C. 832 Jul 19

Previous reports using various protein kinase inhibitors have suggested that protein kinase activity is necessary for both the induction and maintenance of hippocampal long-term potentiation (LTP), a cellular phenomenon likely to contribute to mammalian memory formation. We designed and characterized a selective peptide substrate for protein kinase C (PKC), corresponding to amino acids 28 to 43 of the neuronal protein neurogranin, and used the substrate to obtain direct biochemical evidence for activation of PKC in both the induction and maintenance phases of LTP. As the effect cannot be accounted for by either of two well-known mechanisms for persistent PKC activation, membrane insertion, or proteolysis, the persistent activation of PKC in the maintenance phase of LTP appears to occur via another mechanism. The maintenance phase of LTP is associated with decreased immunoreactivity of PKC, an effect that can be reversed with phosphatase treatment. Thus, PKC appears to be both phosphorylated and persistently activated in the maintenance phase of LTP.
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PMID:Mechanism of protein kinase C activation during the induction and maintenance of long-term potentiation probed using a selective peptide substrate. 810 34

Nerve growth factor (NGF) treatment of PC12 cells led to the rapid phosphorylation of a calmodulin-binding protein of 100 kDa (CaM-BP100) identified on blot overlays with 125I-labeled CaM. The effect was detected as a retardation in the mobility of the protein by an apparent 10 kDa on SDS gels. The mobility shift was complete within 5 min and was maintained for 24 h in the continued presence of NGF. The protein was present in both the soluble and crude particulate fractions, and the gel mobility shift occurred in both fractions. Epidermal growth factor elicited a similar response, but the mobility shift was reversed within 12 h. The gel retardation was due to phosphorylation of CaM-BP100, as it could be reversed if cytoplasmic extracts were held under dephosphorylating conditions at 37 degrees C for 10 min prior to electrophoresis; dephosphorylation was inhibited by okadaic acid but not vanadate, suggesting the participation of a Ser/Thr phosphatase. Treatment with either acid or alkaline phosphatase also reversed the mobility shift. CaM-BP100 phosphorylation was stimulated by 12-O-tetradecanoylphorbol-13-acetate in intact cells, but the effect of NGF did not involve a protein kinase C-dependent process, because it occurred in PC12 cells depleted of protein kinase C. The phosphorylation event appeared to be due to an NGF-stimulated protein kinase, as mixing extracts from NGF-treated cells with extracts from control cells in the presence of ATP and Mg2+ reconstituted the mobility shift in vitro. CaM-BP100 appears to be a minor cellular phosphoprotein, as 32P labeling of the protein could not be detected in crude cell extracts. These results suggest that receptor tyrosine kinases communicate with at least one component of the Ca2+/calmodulin-signaling pathway early in signal transduction.
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PMID:Rapid and sustained phosphorylation of a calmodulin-binding protein (CaM-BP100) in NGF-treated PC12 cells. 839 55


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