Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We studied the molecular mechanism of noradrenaline release from the presynaptic terminal and the involvement of the protein kinase C substrate B-50 (GAP-43) in this process. To gain access to the interior of the presynaptic terminal, we searched for conditions to permeate rat brain synaptosomes by the bacterial toxin streptolysin O. A crude synaptosomal/mitochondrial preparation was preloaded with [3H]noradrenaline. After permeation with 0.8 IU/ml streptolysin O, noradrenaline efflux could be induced in a concentration-dependent manner by elevating the free Ca2+ concentration from 10(-8) to 10(-5) M. Efflux of the cytosolic marker protein lactate dehydrogenase was not affected by this increase in Ca2+. Ca2(+)-induced efflux of noradrenaline was largely dependent on the presence of exogenous ATP. Changing the Na+/K+ ratio in the buffer did not affect Ca2(+)-induced noradrenaline release. Release of noradrenaline could also be evoked by phorbol esters, indicating the involvement of protein kinase C. Ca2(+)- and phorbol ester-induced release were not additive at higher phorbol ester concentrations (greater than 10(-7) M). We compared the sensitivities of Ca2(+)- and phorbol ester-induced release of noradrenaline to the protein kinase inhibitors H-7 and polymyxin B and to antibodies raised against synaptic protein kinase C substrate B-50. Ca2(+)-induced release was inhibited by B-50 antibodies and polymyxin B, but not by H-7; phorbol ester-induced release was inhibited by polymyxin B and by H-7, but only marginally by antibodies to B-50.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Noradrenaline release from streptolysin O-permeated rat cortical synaptosomes: effects of calcium, phorbol esters, protein kinase inhibitors, and antibodies to the neuron-specific protein kinase C substrate B-50 (GAP-43). 182 43

Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin-binding protein believed to play a role in regulation of neurite outgrowth and neuroplasticity. Neuromodulin is phosphorylated by protein kinase C, and this phosphorylation prevents calmodulin from binding to neuromodulin (Alexander, K. A., Cimler, B. M., Meier, K. E. & Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). The only other protein kinase known to phosphorylate neuromodulin is casein kinase II (Pisano, M. R., Hegazy, M. G., Reimann, E. M. & Dokas, L. A. (1988) Biochem. Biophys. Res. Commun. 155, 1207-1212). Phosphoamino acid analyses revealed that casein kinase II modified serine and threonine residues in both native bovine and recombinant mouse neuromodulin. Two serines located in the C-terminal end of neuromodulin, Ser-192 and Ser-193, were identified as the major casein kinase II phosphorylation sites. Thr-88, Thr-89, or Thr-95 were identified as minor casein kinase II phosphorylation sites. Phosphorylation by casein kinase II did not affect the ability of neuromodulin to bind to calmodulin-Sepharose. However, calmodulin did inhibit the phosphorylation of neuromodulin by casein kinase II with a Ki of 1-2 microM. Calmodulin inhibition of casein kinase II phosphorylation was due to calmodulin binding to neuromodulin rather than to the protein kinase. These data suggest that the minimal secondary and tertiary structure exhibited by neuromodulin may be sufficient to juxtapose its calmodulin-binding domain, located at the N-terminal end, with the neuromodulin casein kinase II phosphorylation sites at the C-terminal end of the protein. We propose that calmodulin regulates casein kinase II phosphorylation of neuromodulin by binding to neuromodulin and sterically hindering the interaction of casein kinase II with its phosphorylation sites on neuromodulin.
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PMID:Phosphorylation of neuromodulin (GAP-43) by casein kinase II. Identification of phosphorylation sites and regulation by calmodulin. 182 73

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

Phosphorylation of the neuron-specific substrate of protein kinase C (PKC), B-50 (GAP-43), was studied parallel with noradrenaline release in rat brain synaptosomes. Both could be evoked by treating the synaptosomes with high K+ or veratridine. Phorbol 12,13-dibutyrate enhanced depolarization-induced B-50 phosphorylation and noradrenaline release. To investigate the involvement of PKC-mediated B-50 phosphorylation in noradrenaline release, we applied a variety of kinase inhibitors. Prior to measuring the effects of these inhibitors in intact synaptosomes, we determined their effectivity and specificity in a membrane phosphorylation assay. H-7 most specifically inhibited PKC-dependent phosphorylation, whereas calmidazolium inhibited calmodulin-dependent phosphorylation. Polymyxin B affected both protein kinase systems. Only polymyxin B effectively inhibited noradrenaline release in the intact synaptosomes. We conclude that PKC as well as calmodulin-dependent processes are important for the release event. Data are discussed in view of the presumed function of B-50 as a calmodulin-binding protein.
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PMID:Evidence for a relationship between B-50 (GAP-43) and [3H]noradrenaline release in rat brain synaptosomes. 196 52

We studied the molecular events underlying K(+)-induced phosphorylation of the neuron-specific protein kinase C substrate B-50. Rat cortical synaptosomes were prelabelled with 32P-labelled orthophosphate. B-50 phosphorylation was measured by an immunoprecipitation assay. In this system, various phorbol esters, as well as a synthetic diacylglycerol derivative, enhance B-50 phosphorylation. K+ depolarization induces a transient enhancement of B-50 phosphorylation, which is totally dependent on extracellular Ca2+. Also, the application of the Ca2+ ionophore A23187 induces B-50 phosphorylation, but the magnitude and kinetics of A23187-induced B-50 phosphorylation differ from those induced by depolarization. The protein kinase inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and staurosporine antagonize K(+)- as well as PDB-induced B-50 phosphorylation, whereas trifluoperazine and calmidazolium are ineffective under both conditions. We suggest that elevation of the intracellular Ca2+ level after depolarization is a trigger for activation of protein kinase C, which subsequently phosphorylates its substrate B-50. This sequence of events could be of importance for the mechanism of depolarization-induced transmitter release.
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PMID:Depolarization-induced phosphorylation of the protein kinase C substrate B-50 (GAP-43) in rat cortical synaptosomes. 213 8

The neuronal protein B-50 may be involved in diverse functions including neural development, axonal regeneration, neural plasticity, and synaptic transmission. The rat B-50 sequence contains 226 amino acids which include 14 Ser and 14 Thr residues, all putative sites for phosphorylation by calcium/phospholipid-dependent protein kinase C (PKC). Phosphorylation of the protein appears to be a major factor in its biochemical and possibly its physiological activity. Therefore, we investigated rat B-50 phosphorylation and identified a single phosphorylated site at Ser41. Phosphoamino acid analysis eliminated the 14 Thr residues because only [32P]Ser was detected in an acid hydrolysate of [32P]B-50. Staphylococcus aureus protease peptide mapping produced a variety of radiolabelled [32P]B-50 products, none of which had the same molecular weights or HPLC retention times as several previously characterized fragments. Indirect confirmation of the results was provided by differential phosphorylation of major and minor forms of B-60 that have their N-termini at, or C-terminal to, the Ser41 residue and are the major products of specific B-50 proteolysis. Only those forms of B-60 that contained the Ser41 residue incorporated phosphate label. The results are discussed with reference to the substrate requirements for B-50 phosphorylation by PKC and the proposed structure of the B-50 calmodulin binding domain.
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PMID:Evidence for a single protein kinase C-mediated phosphorylation site in rat brain protein B-50. 280

Various regulators of protein kinase activities were tested for their effects on the in vitro transfer of phosphate from [gamma-32P]ATP to four proteins of rat brain synaptic particulate preparations. One protein, of apparent molecular weight 44,000, accepted 32P in the presence of 8 mM EDTA and no added Mg2+. It was the major phosphoprotein of brain mitochondria. Its phosphorylation was inhibited by pyruvate and stimulated by K+, and it comigrated in electrophoretic gels with authentic alpha-subunit of pyruvate: lipoamide oxidoreductase (decarboxylating) (EC 1.2.4.1) from bovine heart. The major kinase acting on three proteins of apparent molecular weights 24,000, 21,000, and 19,000 was stimulated by Ca2+, by preincubation with phospholipase C, and by 12-tetradecanoyl 4-beta-phorbol 13-acetate. Phosphorylation of these lower-molecular-weight proteins was inhibited by ACTH1-24, by cyclic 3',5'-adenosine monophosphate, and by 50 microM trifluoperazine. The stimulatory effect of Ca2+ was antagonized by calmodulin. The kinase in question appears to be B-50 protein kinase or protein kinase C.
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PMID:Regulation of phosphate incorporation into four brain phosphoproteins that are affected by experience. 298 Dec 89

The Ca2+ and calmodulin sensitivity of endogenous protein kinase activity in synaptosomal membrane fragments from rat brain was studied in medium containing Ca2+ plus EGTA using a modified computer programme to calculate free Ca2+ concentrations that took into account the effect of all competing cations and chelators. The Ca2+-dependent phosphorylation of 10 major polypeptide acceptors with Mr values ranging from 50 to 360 kilodaltons required calmodulin in reactions that were all equally sensitive to Ca2+; half-maximal phosphorylation required a free Ca2+ concentration of 45 nM and maximal phosphorylation approximately 110 nM. The significance of these values in relation to published data on the intracellular concentration of free Ca2+ in the nervous system is discussed. One acceptor of 45 kilodaltons was phosphorylated in a Ca2+-dependent reaction that did not require calmodulin. This polypeptide appeared to correspond to the B-50 protein, an established substrate of the lipid-dependent protein kinase C. Further study of this phosphorylating system showed that the reaction was only independent of calmodulin at saturating concentrations of Ca2+; at subsaturating concentrations (in the range 50-130 nM), a small but significant stimulation of the enzyme by calmodulin was demonstrated. The possible significance of this finding is discussed.
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PMID:Ca2+ sensitivity of Ca2+-dependent protein kinase activities toward intrinsic proteins in synaptosomal membrane fragments from rat cerebral tissue. 307 18

The phosphoprotein B-50 (GAP-43) was purified from adult rat brain cortex and phosphorylated by casein kinase II. Phosphorylation of B-50 by casein kinase II approached 1.2 mol phosphate/mol B-50. The apparent Km of casein kinase II for B-50 was 4 microM with an apparent Vmax of 13 nmol.min-1.mg-1. A tryptic phosphopeptide map on reversed phase HPLC and phosphoamino acid analysis of [32P]B-50 showed that casein kinase II phosphorylated in serine residue(s) which were located in a single tryptic peptide. Phosphorylation of B-50 by casein kinase II was inhibited more than 90% by 5 micrograms heparin/ml or 2.4 mM peptide substrate specific for casein kinase II (RRREEETEEE). The initial phosphorylation rate was increased about 2-fold by 1 mM spermine.
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PMID:Phosphorylation of protein B-50 (GAP-43) from adult rat brain cortex by casein kinase II. 317 3

Antibodies to the phosphoprotein B-50 of rat brain were used to trace cross-reacting brain proteins of vertebrates. With the SDS-gel-immunoperoxidase method, a cross-reacting protein (CP) of apparent Mr 53,000 was demonstrated in the homogenate and the synaptic plasma membrane fraction of bovine brain. Sequence 1-24 of adrenocorticotropin (ACTH1-24) (10(-5) M and 10(-4) M) inhibited endogenous phosphorylation of CP in synaptic plasma membranes. The protein was partially characterized and purified to homogeneity from bovine brain by procedures previously described for rat B-50. CP was enriched in ammonium sulfate precipitated protein (ASP) fractions and phosphorylated by an endogenous protein kinase. Two-dimensional gel analysis of bovine and rat ASP showed that the cross-reacting protein had an isoelectric point less acidic than B-50. Limited proteolysis by Staphylococcus aureus protease yielded a "peptide map" analogous to B-50. Two major fragments of Mr 30,000 and 17,000 were produced. In addition, CP exhibited other similarities to rat B-50: phosphorylation by rat brain protein kinase C, microheterogeneity observed after isoelectric focusing, and possibly degradation by endogenous proteolysis. Cross-reaction of proteins in brain homogenates of other mammalian species and of chicken was demonstrated: the Mr of the proteins ranged from 47,000 to 53,000. We conclude that (1) the cross-reacting bovine protein is a "B-50 protein," and (2) the Mr of the "B-50 protein" varies from species to species.
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PMID:Cross-reaction of anti-rat B-50: characterization and isolation of a "B-50 phosphoprotein" from bovine brain. 623 84


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