EC:2.7.11.13 - FACTA Search

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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)

We reported previously that recombinant myristoylated, alanine-rich protein kinase C substrate (MARCKS) expressed in Escherichia coli as well as MARCKS purified from rat brain specifically bound to phosphatidylserine (PS) in a calcium-independent manner and that the binding was regulated through phosphorylation of MARCKS (Nakaoka, T., Kojima, N., Hamamoto, T., Kurosawa, N., Lee, Y. C., Kawasaki, H., Suzuki, K., and Tsuji, S. (1993) J. Biochem. (Tokyo) 114, 449-452). In this study, to identify the minimum PS-binding region of MARCKS and the regulatory phosphorylation site, the binding of MARCKS to PS was examined in deletion mutants producing glutathione S-transferase (GST) fusion proteins. The mutant proteins GST-6-180 and GST-127-160 had almost the same ability to bind to immobilized PS as MARCKS purified from rat brain, whereas GST-127-152 did not bind to it. In addition, the binding of GST-6-156 to immobilized PS was 62% of that of GST-6-180, but that of GST-6-152 was only 8% and that of GST-6-135 was not detected. The effect of phosphorylation by protein kinase C was examined in several mutants of GST-6-180 whose serine residues were substituted with alanine. After phosphorylation, the mutants GST-6-180[S156A and S163A], GST-6-180]S156A], and GST-6-180[S163A] did not bind to immobilized PS like native MARCKS and GST-6-180. However, even after phosphorylation, GST-6-180-[S152A] and GST-6-180[S152A and S156A] could bind to immobilized PS. These results strongly suggest that MARCKS binds to PS molecules in the inner leaflet of the plasma membrane through residues 127-156, with residues 153-156 (FKKS) being particularly important in the binding of MARCKS to PS, and that the binding is regulated through the protein kinase C-catalyzed phosphorylation of the serine at residue 152.
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PMID:Characterization of the phosphatidylserine-binding region of rat MARCKS (myristoylated, alanine-rich protein kinase C substrate). Its regulation through phosphorylation of serine 152. 774 64

Long-term potentiation (LTP) is a well known experimental model for studying the activity-dependent enhancement of synaptic plasticity, and because of its long duration and its associative properties, it has been proposed as a system to investigate the molecular mechanisms of memory formation. At present, there are several lines of evidence that indicate that pre- and postsynaptic kinases and their specific substrates are involved in molecular mechanisms underlying LTP. Many studies focus on the involvement of protein kinase C (PKC). One way to investigate the role of PKC in long-term potentiation is to determine the degree of phosphorylation of its substrates after in situ phosphorylation in hippocampal slices. Two possible targets are the presynaptic membrane-associated protein B-50 (a.k.a. GAP 43, neuromodulin and F1), which has been implicated in different forms of synaptical plasticity in the brain such as neurite outgrowth, hippocampal LTP and neurotransmitter release, and the postsynaptic protein neurogranin (a.k.a. RC3, BICKS and p17) which function remains to be determined. This review will focus on the protein kinase C activity in pre- and postsynaptic compartment during the early phase of LTP and the possible involvement of its substrates B-50 and neurogranin.
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PMID:Long-term potentiation and synaptic protein phosphorylation. 775 99

The phosphorylation state of two identified neuralspecific protein kinase C substrates (the presynaptic protein B-50 and the postsynaptic protein neurogranin) was monitored after the induction of long term potentiation in the CA1 field of rat hippocampus slices by quantitative immunoprecipitation following 32Pi labeling in the recording chamber. B-50 phosphorylation was increased from 10 to 60 min, but no longer at 90 min after long term potentiation had been induced, neurogranin phosphorylation only at 60 min. Increased phosphorylation was not found when long term potentiation was blocked with the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonovalerate, when only low frequency stimulation was applied or tetanic stimulation failed to induce long term-potentiation. Our data show that both B-50 and neurogranin phosphorylation are increased following the induction of long term potentiation, thus providing strong evidence for pre- and postsynaptic protein kinase C activation during narrow, partially overlapping, time windows after the induction of long term potentiation.
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PMID:Temporal differences in the phosphorylation state of pre- and postsynaptic protein kinase C substrates B-50/GAP-43 and neurogranin during long-term potentiation. 777 48

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

Neurogranin, neuromodulin, and MARCKS are among the most prominent substrates of protein kinase C (PKC) in the mammalian brain. These phosphoproteins were dephosphorylated by three isoforms of rat brain calcineurin, also known as calmodulin (CaM)-dependent protein phosphatase (CaMPP). The three CaMPP isozymes dephosphorylate neurogranin, the most favorable substrate among the three tested, with subtle differences in their responses to divalent metal ions, Mn2+ and Ni2+. Dephosphorylation of neurogranin by all three CaMPP isozymes, CaMPP-1, -2, and -3, were stimulated to a higher extent by Mn2+ than by Ni2+ in the presence of CaM and Ca2+. The Km values of neurogranin in the presence of Mn2+ were lower than those in the presence of Ni2+ for CaMPP-1 and -2, but that for CaMPP-3 was comparable with either divalent metal ion. The Vmax values were higher in the presence of Mn2+ than those of Ni2+ for all three isozymes. Neurogranin and neuromodulin, both phosphorylated by PKC at a single site, were dephosphorylated completely by CaMPP; however, MARCKS, phosphorylated by PKC at three sites, was partially dephosphorylated by this phosphatase. A higher extent of dephosphorylation of MARCKS could be achieved by the combination of CaMPP and protein phosphatase 2A and a complete dephosphorylation of this protein was observed with protein phosphatase 1. Protein phosphatase 1 and 2A were also effective in a complete dephosphorylation of neurogranin and neuromodulin. Amino acid sequence analysis of the tryptic phosphopeptides derived from MARCKS dephosphorylated by CaMPP and protein phosphatase 2A revealed that the former preferentially dephosphorylated Ser155 and the latter Ser162 of rat brain MARCKS. Both phosphatases dephosphorylated poorly of Ser151. Because of the high concentration of CaMPP in the brain and the colocalization of this phosphatase with major PKC substrates in the various brain regions, it is likely that CaMPP is a phosphatase with potential to reverse the action of PKC.
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PMID:Dephosphorylation of protein kinase C substrates, neurogranin, neuromodulin, and MARCKS, by calcineurin and protein phosphatases 1 and 2A. 786 22

The unique structures of process-bearing cells in the central nervous system (CNS) present an ideal model with which to study the differential distribution of mRNA. We conducted a side-by-side examination of the intracellular distribution of nine neural mRNAs by in situ hybridization histochemistry in mammalian brain and observed four general types of mRNA distributions. (1) Some mRNA species were confined to cell somas and included those encoding the glial proteins, myelin proteolipid protein and 2'3'-cyclic nucleotide-3'-phosphodiesterase and the neuronal enzymes, neuron-specific enolase and glutamate decarboxylase-67. (2) Some mRNAs were found abundantly within the cell soma and were also located throughout cellular processes. These included myelin basic protein (MBP) mRNA, which was localized to the cell soma and myelin sheaths of oligodendrocytes, and glial fibrillary acidic protein (GFAP) mRNA, which was localized to the cell soma and processes of reactive and some non-reactive astrocytes in the adult brain and radial glia in embryonic brain. (3) Some mRNAs were found primarily in perinuclear cytoplasm but in some cells were also observed in cell processes. These included mRNAs encoding the protein kinase C/calmodulin-binding substrates, RC3 (neurogranin) and GAP-43, which were identified in the somas as well as within the proximal dendritic branches of specific forebrain neurons. (4) Some mRNAs were localized primarily within cell processes. These included MAP2 mRNA, which was identified by deep staining within dendritic fields but by only light staining within neuronal cell bodies. The data also indicated that the stage of cellular development and the regional location of a cell within the CNS had a profound influence on translocation events. MAP2 mRNA was found in the dendritic processes of most neurons but was confined to the soma of neurons in specific brainstem nuclei. MBP mRNA was confined to the perinuclear cytoplasm of immature oligodendrocytes and was then transported into the myelin sheath at a developmental stage corresponding to myelination. The distribution patterns of these mRNAs are likely to reflect the mechanism by which the protein products of these molecules are targeted within neurons and glia. In addition, mRNA movement may be influenced by cellular and regional factors not encoded solely within the structure of the translocated mRNA.
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PMID:Cellular influences on RNA sorting in neurons and glia: an in situ hybridization histochemical study. 787 39

Dictyostelium discoideum spores can be activated to initiate germination either endogenously via a diffusible autoactivator, or exogenously via heat. Following activation, three successive stages of germination occur, the lag stage, spore swelling and amoebal emergence. A previous study [Lydan M. A. and Cotter D. A. (1994) FEBS Lett. 115, 137-142] has shown that spore swelling is dependent on the activity of calmodulin. In this study, the calmodulin antagonists trifluoperazine and calmidazolium inhibited autoactivation, but had no effect upon heat activation. These agents also inhibited amoebal emergence following either form of activation. The effects caused by the anti-calmodulin agents were specific to an inhibition of calmodulin function since agents which modulate the activity of protein kinase C had no effect upon spore germination. A calcium-dependent calmodulin-binding protein of about 64,000 M(r) may be associated with the process of autoactivation since it was only seen in those spores which respond to the autoactivator. Overall, this study provides evidence to show that calmodulin plays a regulatory role during autoactivation and amoebal emergence during spore germination in D. discoideum and provides evidence for the calmodulin-dependent mechanisms which mediate each of these phases of germination.
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PMID:Calmodulin function and calmodulin-binding proteins during autoactivation and spore germination in Dictyostelium discoideum. 788 2

Two neuronal protein kinase C substrates, RC3/neurogranin and GAP-43/neuromodulin, preferentially bind to calmodulin (CaM) when Ca2+ is absent. We examine RC3.CaM and GAP-43.CaM interactions by circular dichroism spectroscopy using purified, recombinant RC3 and GAP-43, sequence variants of RC3 displaying qualitative and quantitative differences in CaM binding affinities, and overlapping peptides that cumulatively span the entire amino acid sequence of RC3. We conclude that CaM stabilizes a basic, amphiphilic alpha-helix within RC3 and GAP-43 under physiological salt concentrations only when Ca2+ is absent. This provides structural confirmation for two binding modes and suggests that CaM regulates the biological activities of RC3 and GAP-43 through an allosteric, Ca(2+)-sensitive mechanism that can be uncoupled by protein kinase C-mediated phosphorylation. More generally, our observations imply an alternative allosteric regulatory role for the Ca(2+)-free form of CaM.
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PMID:Calmodulin stabilizes an amphiphilic alpha-helix within RC3/neurogranin and GAP-43/neuromodulin only when Ca2+ is absent. 789 19

Previous studies have shown that RC3 (neurogranin) is a postsynaptic, protein kinase C (PKC)/calmodulin-binding substrate that accumulates throughout the perikaryal and dendritic cytoplasm and is often closely associated with the postsynaptic density (PSD) in dendritic spines of neostriatal neurons. Here Western immunoblotting studies of rat brain subcellular fractions confirm that RC3 is predominantly a cytosolic protein but is found in lower amounts in membrane-enriched microsomes and synaptosomes. Solubilization of synaptosomes suggests that RC3 may only be loosely associated with the PSD.
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PMID:Localization of RC3 (neurogranin) in rat brain subcellular fractions. 789 18

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

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