Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.17 (CaMKII)
 4,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) may play a key role in the regulation of insulin secretion. We obtained evidence for the presence of CaM kinase II and its substrate, a 84-kilodalton (kDa) protein, in mouse insulinoma MIN6 cells. CaM kinase II from MIN6 cells has one subunit of 55 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is autophosphorylated in a Ca2+/CaM-dependent manner, and phosphorylates several substrates that serve for rat brain CaM kinase II. In the membrane fraction of MIN6 cells, we identified a 84-kDa protein that was immunoreactive with the antirat brain synapsin I antibody. One-dimensional phosphopeptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed the sites of the phosphorylation by cAMP-dependent protein kinase (cAMP kinase) and that by CaM kinase II to be site 1 (10 kDa) and site 2 (30 kDa), respectively, therefore, the same as for rat brain synapsin I. In this context, we tentatively termed it synapsin I-like protein. In 32P-labeled cells, nonfuel insulin secretagogues, such as ionomycin, KCl, and tolbutamide, and a fuel secretagogue, glucose, stimulated autophosphorylation of CaM kinase II and the phosphorylation of synapsin I-like protein. These secretagogues potentiated the Ca(2+)-independent activity of CaM kinase II and secretion of insulin from MIN6 cells. The 84-kDa protein is apparently a newly identified member of the synapsin family. We suggest that CaM kinase II regulates insulin secretion via phosphorylation of synapsin I-like protein.
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PMID:Ca2+/calmodulin-dependent protein kinase II and synapsin I-like protein in mouse insulinoma MIN6 cells. 764 85

Synapsin I is a synaptic vesicle-associated protein which inhibits neurotransmitter release, an effect which is abolished upon its phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Based on indirect evidence, it was suggested that this effect on neurotransmitter release may be achieved by the reversible anchoring of synaptic vesicles to the actin cytoskeleton of the nerve terminal. Using video-enhanced microscopy, we have now obtained experimental evidence in support of this model: the presence of dephosphorylated synapsin I is necessary for synaptic vesicles to bind actin; synapsin I is able to promote actin polymerization and bundling of actin filaments in the presence of synaptic vesicles; the ability to cross-link synaptic vesicles and actin is specific for synapsin I and is not shared by other basic proteins; the cross-linking between synaptic vesicles and actin is specific for the membrane of synaptic vesicles and does not reflect either a non-specific binding of membranes to the highly surface active synapsin I molecule or trapping of vesicles within the thick bundles of actin filaments; the formation of the ternary complex is virtually abolished when synapsin I is phosphorylated by CaM kinase II. The data indicate that synapsin I markedly affects synaptic vesicle traffic and cytoskeleton assembly in the nerve terminal and provide a molecular basis for the ability of synapsin I to regulate the availability of synaptic vesicles for exocytosis and thereby the efficiency of neurotransmitter release.
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PMID:Dephosphorylated synapsin I anchors synaptic vesicles to actin cytoskeleton: an analysis by videomicroscopy. 787 13

Induction of long-term potentiation in the CA1 region of hippocampal slices is associated with increased activity of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) (Fukunaga, K., Stoppini, L., Miyamoto, E., and Muller, D. (1993) J. Biol. Chem. 268, 7863-7867). Here we report that application of high but not low frequency stimulation to two groups of afferents in the CA1 region of 32P-labeled slices resulted in the phosphorylation of two major substrates of this enzyme, synapsin I and microtubule-associated protein 2, as well as in the autophosphorylation of CaM kinase II. Furthermore, immunoblotting analysis revealed that long term potentiation induction was associated with an increase in the amount of CaM kinase II in the same region. All these changes were prevented when high frequency stimulation was applied in the presence of the N-methyl-D-aspartate receptor antagonist, D-2-amino-5-phosphonopentanoate. These results indicate that activation of CaM kinase II is involved in the induction of synaptic potentiation in both the postsynaptic and presynaptic regions.
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PMID:Increased phosphorylation of Ca2+/calmodulin-dependent protein kinase II and its endogenous substrates in the induction of long-term potentiation. 789 Jul 45

Regulation of neurotransmitter release is thought to involve modulation of the release probability by protein phosphorylation. In order to identify novel targets for such regulatory processes, we have studied the phosphorylation of rabphilin-3A in vitro. Rabphilin-3A is a synaptic vesicle protein that interacts with rab3A in a GTP-dependent manner and binds Ca2+ in a phospholipid-dependent manner. Here we show that rabphilin-3A is an efficient substrate for Ca2+/calmodulin-dependent protein kinase II, which phosphorylates rat rabphilin-3A at residue 234 and 274, and for cAMP-dependent protein kinase, which phosphorylates rat rabphilin-3A at residue 234. This identifies the middle region of rabphilin-3A situated between the N-terminal rab3A-binding sequences and the C-terminal C2-domains involved in Ca2+/phospholipid binding as a regulatory domain. Thus, rabphilin-3A is a second phosphoprotein on synaptic vesicles that, similar to synapsin I, may integrate phosphorylation signals from multiple protein kinase signaling pathways in the cell.
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PMID:Phosphorylation of rabphilin-3A by Ca2+/calmodulin- and cAMP-dependent protein kinases in vitro. 789 Nov 74

Annexin VI bound to > 14 species of proteins in the whole homogenate of rat forebrain in a Ca2+/phosphatidylserine- or phosphatidic acid-dependent manner. When the subcellular fractions of rat forebrain were examined with a blot from a sodium dodecyl sulfate-polyacrylamide gel, each annexin VI-binding protein showed a different distribution, suggesting that annexin VI is a multifunctional protein. Of these proteins, the doublets of M(r) 80,000 were enriched in the purified synaptic vesicles and were identified as synapsin I. Annexin VI bound to the head domain of synapsin I. When the binding of annexin VI to synapsin I was characterized in the native state, the affinity of the binding for Ca2+ (KCa) was 12.6 microM, and the affinity for annexin VI (KD) was approximately 270 nM. Phosphorylation of synapsin I by cyclic AMP-dependent protein kinase and by Ca2+/calmodulin-dependent protein kinase II inhibited the annexin VI binding. The mode of the inhibition was different between the two kinases. These results indicate that annexin VI may modulate the function of synapsin I in a Ca(2+)- and phospholipid-dependent manner.
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PMID:Annexin VI binds to a synaptic vesicle protein, synapsin I. 793 47

The substrate recognition determinants of Ca2+/calmodulin-dependent protein kinase Ia were investigated by using peptide analogues based on the amino acid sequence around Ser-9 of synapsin I. The Km and Vmax for the synthetic peptide Leu-Arg-Arg-Arg-Leu-Ser-Asp-Ala-Asn-Phe are 3.9 microM and 18.5 mumol/(min.mg), respectively. Deletion of Leu at the -5 position lowers the Vmax/Km by 470-fold. The requirement for a hydrophobic residue at -5 was confirmed by the 90- to 2400-fold reduction in Vmax/Km produced by Arg, Ala, or Asp substitutions, but only 2.6-fold decrease after Phe substitution at this position. A hydrophobic residue is similarly required at the +4 position. Deletion of Phe at this position produces a 67-fold reduction, and substitution of Ala for Phe a 43-fold reduction in Vmax/Km. In contrast, substitution with Leu increases Vmax/Km by 1.8-fold. Arg at -3 is also required for recognition as shown by an approximately 240-fold decrease in Vmax/Km after Ala substitution at this position. Positions -2, -4, and +1 appear to play secondary roles in substrate recognition. Arg at -2 and -4 are positive determinants, since Ala substitution at these positions decreases Vmax/Km by 4.7- and 11-fold, respectively. Asp at +1 is a negative influence, since Ala and Leu substitutions at this position increase Vmax/Km by 2.3- and 6.3-fold, respectively. Substitution of Ala for Leu at -1 or Thr for Ser at the 0 position has little effect on phosphorylation kinetics. Thus, Ca2+/calmodulin-dependent protein kinase Ia has the minimal substrate recognition motif of Hyd-Xaa-Arg-Xaa-Xaa-(Ser*/Thr*)-Xaa-Xaa-Xaa-Hyd, where Hyd represents a hydrophobic amino acid residue.
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PMID:A requirement of hydrophobic and basic amino acid residues for substrate recognition by Ca2+/calmodulin-dependent protein kinase Ia. 802 98

Ca2+/calmodulin-dependent protein kinase I (CaM kinase I) was previously purified from bovine brain (Nairn, A. C., and Greengard, P. (1987) J. Biol. Chem. 262, 7273-7281) based on its ability to phosphorylate the synaptic vesicle protein, synapsin I at site 1. The cDNA for this protein kinase has now been cloned from both a rat and a bovine brain cDNA library and the complete amino acid sequence of rat CaM kinase I determined. The rat cDNA encoded a protein of 331 amino acids with a calculated M(r) of 37,545, and the encoded kinase was expressed in bacteria as a glutathione S-transferase fusion protein. The resulting fusion protein was purified by Sepharose-CaM affinity chromatography and shown to be totally dependent on Ca2+ and CaM for activity. Furthermore, the purified kinase phosphorylates synapsin I at the same site (site 1) as the endogenous brain enzyme. CaM kinase I is homologous to other known protein kinases and contains all nine invariant amino acids conserved in the catalytic domain of this class of enzymes. CaM kinase I was most identical to CaM kinase II both in the catalytic domain and in a short region at the COOH-terminal that is predicted to be the calmodulin-binding domain. CaM kinase I appeared to be encoded by a single gene. RNase protection assays detected the mRNA encoding CaM kinase I in all tissues examined. High concentrations of the kinase mRNA were found in all regions of the brain with frontal cortex showing the greatest level. CaM kinase I was autophosphorylated in a Ca2+/CaM-dependent manner at a threonyl residue (Thr-177) which is located at a position equivalent to that of the threonyl residue (Thr-197) autophosphorylated in cAMP-dependent protein kinase.
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PMID:Calcium/calmodulin-dependent protein kinase I. cDNA cloning and identification of autophosphorylation site. 825 80

Both Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and protein kinase C (PKC) have been implicated as possible candidates for contributing to the induction of long-term potentiation (LTP) in the hippocampus. The induction of LTP in the CA1 region of the hippocampus, an event which requires postsynaptic Ca2+ influx through NMDA-type glutamate receptors, is blocked by calmodulin antagonists and inhibitors of CaM kinase II and PKC. In the present study, we describe the activation characteristics of CaM kinase II and PKC through the stimulation of glutamate receptors and regulation of the phosphorylation of substrates for CaM kinase II in the hippocampus. In cultured rat hippocampal neurons, glutamate elevated the Ca(2+)-independent activity of CaM kinase II through autophosphorylation, and this response was blocked by specific antagonists of the NMDA receptor. In addition, glutamate stimulated the translocation of PKC from the cytosol to the membrane fraction through the metabotropic glutamate receptor. In the experiments with 32P-labeled cells, the phosphorylation of microtubule-associated protein 2 (MAP2) and synapsin I was stimulated by the exposure to glutamate. Finally, we demonstrated that high, but not low, frequency stimulation applied to two groups of CA1 afferents in the slices resulted in the induction of LTP with concomitant long-lasting increases in the Ca(2+)-independent and total CaM kinase II activities as well as the autophosphorylation. It could be blocked by preincubation of the slices with NMDA-receptor antagonist. These results suggest that glutamate can activate CaM kinase II through NMDA receptors in the induction of LTP and in turn stimulates the phosphorylation of target proteins such as MAP2 and synapsin I.
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PMID:[The role of Ca2+/calmodulin-dependent protein kinase II in the cellular signal transduction]. 828 67

The cytoplasmic domain of synaptotagmin (a synaptic vesicle-specific protein) has a high degree of homology with the Ca(2+)-phospholipid binding domain of protein kinase C. The Ca(2+)-phospholipid binding activity of synaptotagmin has been implicated in the docking and fusion of synaptic vesicles with the presynaptic membrane during Ca(2+)-induced exocytosis. The protein sequence contains potential phosphorylation sites for various protein kinases which could modulate its binding activity. At present there is no clear evidence that the protein is endogenously phosphorylated in intact vesicles. Here it is reported that phospho-synaptotagmin was immunoprecipitated from endogenously phosphorylated synaptic vesicles. The conditions used indicate that synaptotagmin, as synapsin I, is phosphorylated by Ca2+/calmodulin-dependent protein kinase II.
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PMID:Synaptotagmin is endogenously phosphorylated by Ca2+/calmodulin protein kinase II in synaptic vesicles. 838 69

Long-term potentiation (LTP) is a form of synaptic plasticity that may underlie learning and memory. The experiments reported here demonstrate that cocaine blocks the induction of LTP at the excitatory synapses in the CA1 region of the hippocampus, but does not appear to do so by blocking NMDA receptors or channels. Once LTP had been established, however, cocaine had no effect on the potentiated response. Cocaine was also able to block LTP initiated by superfusing slices with 25 mM TEA. The ability to block LTP was shared by the local anesthetics lidocaine and procaine, but not by tetrodotoxin, suggesting that the blockade of sodium channels alone did not disrupt LTP. Biochemical experiments demonstrated that cocaine can inhibit phosphorylation of purified Synapsin I by Ca2+/calmodulin-dependent protein kinase II. This effect, presumably mediated by effects on calmodulin, is a previously unreported action of cocaine, and suggests that cocaine at high dose levels might disrupt types of learning that are mediated by an LTP-like mechanism.
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PMID:Cocaine inhibits hippocampal long-term potentiation. 849 60


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