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:2.7.11.13 (protein kinase C)
49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The neuron-specific, calmodulin-binding protein B-50 (also known as GAP-43, F1, or neuromodulin) is an endogenous substrate of protein kinase C (PKC). PKC exclusively phosphorylates Ser residues in B-50. As potential phosphorylation sites for PKC, Ser41, Ser110, and Ser122 were indicated, of which Ser41 is contained in the sequence ASF, which matches with the sequence of a synthetic PKC substrate. N-terminally 35S-labeled B-50, produced from cDNA, was subjected to digestion with Staphylococcus aureus V8 protease (SAP). Consecutively, 35S-labeled 28- and 15-kDa fragments were formed, similar to those after digestion of 32P-labeled B-50. In a previous study, we showed that the 32P-labeled 15-kDa SAP fragment contains all 32P radioactivity. The present data indicate that it contains the N-terminus of B-50 as well. The 15-kDa fragment, with a calculated length ranging from amino acid residue 1 to 65, contains only one potential PKC phosphorylation site, at Ser41. Mutagenesis of Ser41 into Thr or Ala resulted in recombinant B-50 products with mobilities on two-dimensional electrophoresis similar to those of the nonmutated recombinant B-50 and the rat brain B-50. Only [Ser41]B-50 was phosphorylated by PKC, whereas [Thr41]- or [Ala41]B-50 did not show any phosphorylation at the positions indicated on the immunoblots. This leads us to the conclusion that Ser41 is the sole phosphorylation site for PKC in vitro.
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PMID:Mutation of serine 41 in the neuron-specific protein B-50 (GAP-43) prohibits phosphorylation by protein kinase C. 214 85

Recently, we have shown that stimulation of [3H]-noradrenaline release from hippocampal slices by 4-aminopyridine (4-AP) is accompanied by an enhancement of the phosphorylation of B-50, a major presynaptic substrate of protein kinase C (PKC). PKC has been implicated in the regulation of transmitter release. In this study, we investigated the effects of 4-AP on B-50 phosphorylation in synaptosomes from rat brain and compared the effects of 4-AP with those of depolarization with K+, in order to gain more insight into the mechanism of action of 4-AP. B-50 phosphorylation was stimulated by incubation with 4-AP for 2 minutes at concentrations ranging from 10 microM to 5 mM. 4-AP (100 microM) stimulated B-50 phosphorylation already within 15 seconds; longer incubations revealed a sustained increase in the presence of 4-AP. B-50 phosphorylation was also stimulated by depolarization with 30 mM K+ for 15 seconds. The effects of both 4-AP or K+ depolarization on B-50 phosphorylation were abolished at low extracellular Ca2+ concentrations. The increase in B-50 phosphorylation induced by 4-AP seemed to be dependent on the state of depolarization, since the effect of 4-AP was largest under nondepolarizing conditions. Comparing the effects of 4-AP and K+ depolarization on B-50 phosphorylation suggests that a different mechanism of action is involved. These results indicate that the stimulation of B-50 phosphorylation by 4-AP in hippocampal slices can be attributed to a direct action of 4-AP on presynaptic terminals. In addition, our results support the hypothesis that B-50 phosphorylation by PKC is involved in Ca2(+)-dependent transmitter release evoked by 4-AP.
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PMID:4-Aminopyridine stimulates B-50 (GAP-43) phosphorylation in rat synaptosomes. 214 57

Synaptic plasma membranes from rat brain cortex possess intrinsic ability to dephosphorylate the endogenous protein B-50. At low concentrations of [gamma-32P]ATP, B-50 phosphorylation in synaptic membranes is maximal at 30 seconds, followed by dephosphorylation for an additional 60 minutes. The dephosphorylation of 32P-labeled B-50 is not sensitive to the protease inhibitor leupeptin and not correlated with a loss of the B-50 content of synaptic membranes as measured with immunoblot analysis. Dephosphorylation of membrane-associated B-50 is stimulated to a small extent by Mg2+ but not by Ca2+. Heat-stable protein phosphatase inhibitors prevent dephosphorylation of 32P-labeled B-50. Dephosphorylation of B-50 in synaptic membranes is stimulated by ATP, ADP, or adenosine 5'-O-thiotriphosphate, but not by adenine, adenosine, other adenine or guanine nucleotides, nonhydrolyzable analogs of ATP or GTP, nor by adenosine 5'-O-(2-thiodiphosphate). B-50, phosphorylated by exogenous protein kinase C and purified to homogeneity, has been used as a substrate to follow the purification of B-50 phosphatase activity. B-50 phosphatase activity can be solubilized from synaptic membranes with 0.5% Triton X-100 and 75 mM KCl. Chromatography of the extract on DEAE-cellulose yields enhanced B-50 phosphatase activity.
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PMID:Dephosphorylation of B-50 in synaptic plasma membranes. 215 32

The role of protein kinase C (PKC) in the formation of memory for a one-trial passive avoidance task in 1-day-old chicks has been studied, following earlier observations that training on this task results in transient and lateralised changes in the phosphorylation state of presynaptic B-50 protein, a PKC substrate. In accord with hypotheses that the activity of PKC is regulated by translocation from cytosol to membrane, a significant increase was found in the fraction of the alpha/beta forms of the enzyme, assayed immunologically, present in a synaptic-membrane-bound, Triton-extractable form in the left intermediate medial hyperstriatum ventrale (IMHV) of chicks 30 min after training on the passive avoidance task. Two inhibitors of PKC, melittin (10 microliters, 120 microM) and H7 (10 microliters, 10 mM), if injected intracerebrally 10 min prior to or 10 min after training, were without effect on the general behaviour of the chicks or their training. However, these injections of the inhibitors produced amnesia in birds tested 3 h later. This effect was lateralised; only left hemisphere injections of the inhibitors produced amnesia. A possible state-dependency interpretation of these results was ruled out. The results are discussed in the context of hypotheses as to the regulatory role of PKC in neural plasticity and memory formation.
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PMID:Memory formation in the chick depends on membrane-bound protein kinase C. 229 19

To determine changes in the degree of phosphorylation of the protein kinase C substrate B-50 in vivo, a quantitative immunoprecipitation assay for B-50 (GAP43, F1, pp46) was developed. B-50 was phosphorylated in intact hippocampal slices with 32Pi or in synaptosomal plasma membranes with [gamma-32P]ATP. Phosphorylated B-50 was immunoprecipitated from slice homogenates or synaptosomal plasma membranes using polyclonal anti-B-50 antiserum. Proteins in the immunoprecipitate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the incorporation of 32P into B-50 was quantified by densitometric scanning of the autoradiogram. Only a single 48-kilodalton phosphoband was detectable in the immunoprecipitate, but this band was absent when preimmune serum was used. The B-50 immunoprecipitation assay was quantitative under the following condition chosen, as (1) recovery of purified 32P-labelled B-50 added to slice homogenates or synaptosomal plasma membranes was greater than 95%; and (2) modulation of B-50 phosphorylation in synaptosomal plasma membranes with adrenocorticotrophic hormone, polymyxin B, or purified protein kinase C in the presence of phorbol diester resulted in EC50 values identical to those obtained without immunoprecipitation. With this immunoprecipitation assay we found that treatment of hippocampal slices with 4 beta-phorbol 12,13-dibutyrate stimulated B-50 phosphorylation, whereas 4 alpha-phorbol 12,13-didecanoate was inactive. Thus, we conclude that the B-50 immunoprecipitation assay is suitable to monitor changes in B-50 phosphorylation in intact neuronal tissue.
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PMID:Determination of changes in the phosphorylation state of the neuron-specific protein kinase C substrate B-50 (GAP43) by quantitative immunoprecipitation. 252 Nov 82

The neuron-specific phosphoprotein B-50/GAP43 has been implicated in axonal outgrowth, since high levels of B-50/GAP43 are found in growth cones and during development of the nervous system. In adult brain, the B-50 levels are decreased. B-50 is primarily found in axons and presynaptic terminals. It is phosphorylated by protein kinase C, and this process has been implicated in the modulation of membrane signal transduction. During the outgrowth of the pyramidal tract, high levels of B-50 have been reported, whereas a low amount of B-50 persists into the adult stage. By immunoelectron microscopy, using immunogold labeling on cryosections and pre-embedding peroxidase labeling, we examined the distribution of B-50 in the pyramidal tract at the third cervical segment in developing 2-d-old and adult 90-d-old rats. B-50 immunoreactivity was found in axons and growth cones of the outgrowing tract. In the adult pyramidal tract, both unmyelinated and myelinated axons contained B-50 immunoreactivity. The immunogold label was predominantly located at the plasma membrane. Since the peroxidase reaction product was observed exclusively intracellularly, we conclude that the B-50 immunoreactivity is predominantly located at the cytoplasmic side of the plasma membrane of axons and growth cones. The high immunoreactivity in growth cones and axons of the outgrowing pyramidal tract further supports the hypothesis that B-50 plays a role in neurite outgrowth. The presence of B-50 in the adult pyramidal tract cannot merely be attributed to transport to the synapse. Therefore, it is suggested that B-50 plays, in addition, a local, growth-associated role in the adult tract.
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PMID:B-50/GAP43 is localized at the cytoplasmic side of the plasma membrane in developing and adult rat pyramidal tract. 253 Dec 16

A characteristic feature of neurite formation is high expression of the phosphoprotein B-50/GAP43. Previous studies with growth cone membranes have indicated that this neuron-specific protein kinase C substrate may be involved in transmembrane signal transduction at the growth cone. We monitored the degree of phosphorylation of B-50 by quantitative B-50 immunoprecipitation from intact nerve growth cones, isolated from 5-day-old rat brain and prelabeled with 32P-orthophosphate. B-50 phosphorylation in nerve growth cones is stimulated by 4 beta-phorbol 12,13-dibutyrate (PDB) and 1,2-dioctanoylglycerol (DOG) in a concentration-dependent manner, but not by 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD). These results confirm that B-50 is a substrate of PKC in intact growth cones. Depolarization induced by 30 mM K+ produces a transient increase in B-50 phosphorylation, which is maximal after 15 sec and declines to basal level within 5 min. This rise in B-50 phosphorylation can be partially blocked by atropine (10(-3)-10(-4) M), suggesting the involvement of muscarinic receptors. Indeed, the cholinergic receptor agonist carbachol enhances B-50 phosphorylation in a concentration-dependent manner (50% at 10(-3) M). Since the effect of carbachol (10(-3) M) can be blocked by atropine (10(-7) M), we conclude that this increase in B-50 phosphorylation is mediated through activation of the muscarinic receptors on the growth cones. The carbachol-induced stimulation is further increased by concurrent K+-depolarization. The effects of carbachol and depolarization are additive. To our knowledge, this is the first report showing receptor-mediated effects on the PKC substrate B-50 in growth cones. Our data support the hypothesis that phosphorylation of B-50 by PKC is involved in signal transduction in nerve growth cones.
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PMID:Muscarinic receptor activation stimulates B-50/GAP43 phosphorylation in isolated nerve growth cones. 253 Dec 15

Neuromodulin (p57, GAP-43, F1, B-50) is a major neural-specific, calmodulin binding protein found in brain, spinal cord, and retina that is associated with membranes. Phosphorylation of neuromodulin by protein kinase C causes a significant reduction in its affinity for calmodulin (Alexander, K. A., Cimler, B. M., Meirer, K. E., and Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). It has been proposed that neuromodulin may function to bind and concentrate calmodulin at specific sites within neurons and that activation of protein kinase C causes the release of free calmodulin at high concentrations near its target proteins. It was the goal of this study to determine whether bovine brain contains a phosphoprotein phosphatase that will utilize phosphoneuromodulin as a substrate. Phosphatase activity for phosphoneuromodulin was partially purified from a bovine brain extract using DEAE-Sephacel and Sephacryl S-200 gel filtration chromatography. The neuromodulin phosphatase activity was resolved into two peaks by Affi-Gel Blue chromatography. One of these phosphatases, which represented approximately 60% of the total neuromodulin phosphatase activity, was tentatively identified as calcineurin by its requirement for Ca2+ and calmodulin (CaM) and inhibition of its activity by chlorpromazine. Therefore, bovine brain calcineurin was purified to homogeneity and examined for its phosphatase activity against bovine phosphoneuromodulin. Calcineurin rapidly dephosphorylated phosphoneuromodulin in the presence of micromolar Ca2+ and 3 microM CaM. The apparent Km and Vmax for the dephosphorylation of neuromodulin, measured in the presence of micromolar Ca2+ and 2 microM CaM, were 2.5 microM and 70 nmol Pi/mg/min, respectively, compared to a Km and Vmax of 4 microM and 55 nmol Pi/mg/min, respectively, for myosin light chain under the same conditions. Dephosphorylation of neuromodulin by calcineurin was stimulated 50-fold by calmodulin in the presence of micromolar free Ca2+. Half-maximal stimulation was observed at a calmodulin concentration of 0.5 microM. We propose that phosphoneuromodulin may be a physiologically important substrate for calcineurin and that calcineurin and protein kinase C may regulate the levels of free calmodulin available in neurons.
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PMID:Dephosphorylation of neuromodulin by calcineurin. 254 35

We have studied the subcellular distribution of phosphoproteins in intact hippocampal slices and examined factors that regulate their phosphorylation and dephosphorylation in situ. The presence of Ca2+ in slice equilibration and prelabeling buffers and high-K+-induced depolarization markedly increased 32Pi incorporation into endogenous proteins. Ca2+-stimulatory effects were significantly reduced by Ca2+-channel blockers and the calmodulin antagonist W-13. Certain proteins were dephosphorylated in situ, and their dephosphorylation was dependent on both Ca2+ and depolarization. A number of proteins phosphorylated in situ was similar to those previously characterized in synaptic fractions phosphorylated in vitro. Many phosphoproteins were identified on the basis of molecular weight, isoelectric point, immunoreactivity, and phosphopeptide mapping; these included the 87 kDa substrate of protein kinase C, synapsin I, the 50 and 60 kDa subunits of Ca2+/calmodulin-dependent protein kinase II (CKII), tubulin, B-50, the alpha-subunit of pyruvate dehydrogenase and myelin basic proteins. CKII phosphorylation in situ appeared similar but not identical to its in vitro counterpart. Phosphopeptide mapping analysis of in situ labeled substrate proteins indicated that cAMP-, Ca2+/calmodulin-, and Ca2+/phospholipid-dependent protein kinases were all active in slice preparations under basal conditions. Increased 32Pi labeling of hippocampal proteins following tissue depolarization appeared to be associated with increased activity of endogenous protein kinases since depolarization did not result in 32Pi-labeling of any new phosphoproteins.
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PMID:In situ protein phosphorylation in hippocampal tissue slices. 255 35

The convulsant drug 4-aminopyridine (4-AP) stimulates the phosphorylation of the neuron-specific presynaptic protein B-50 in hippocampal slices. This effect could be attenuated by the protein kinase C (PKC) inhibitor staurosporine. Moreover, the endogenous phosphorylation of B-50 was found to be restricted to the 15 kDa Staphylococcus aureus protease fragment of B-50, known to contain the PKC acceptor site. The effect of 4-AP on B-50 phosphorylation was sensitive to the Na+ channel blocker tetrodotoxin. These results indicate that 4-AP stimulates PKC activity in hippocampal slices by a mechanism dependent on Na+ channel activity.
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PMID:Activation of protein kinase C by 4-aminopyridine dependent on Na+ channel activity in rat hippocampal slices. 255 67


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