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

The subcellular distribution of the endogenous phosphodiesterase activator and its release from membranes by a cyclic AMP-dependent ATP:protein phosphotransferase was studied in fractions and subfractions of rat brain homogenate. These fractions were obtained by differential centrifugation and sucrose density gradient; their identity was ascertained by electron microscopy and specific enzyme markers. In the subcellular particulate fractions, the concentration of activator is highest in the microsomal fraction, followed by the mitochondrial and nuclear fractions. Gradient centrifugation of the main mitochondrial subfraction revealed that activator was concentrated in those fractions containing mainly synaptic membranes. Activator was releasted from membranes by a cyclic AMP-dependent phosphorylation of membrane protein. The release of activator occurred mainly from the mitochondrial subfractions containing synaptic membranes and synaptic vesicles. The data support the view that a release of activator from membranes may be important in normalizing the elevated concentration of cyclic AMP following persistent transsynaptic activation of adenylate cyclase.
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PMID:Release of the phosphodiesterase activator by cyclic AMP-dependent ATP:protein phosphotransferase from subcellular fractions of rat brain. 19 Oct 91

To investigate the role of calmodulin in stimulus-secretion coupling in pancreatic acinar cells, we studied the effects of W-7, a calmodulin inhibitor, and KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II), on amylase secretion from rat pancreatic acini. Calmodulin inhibitor (W-7, 100 microM) and Ca2+/CaM kinase II inhibitor (KN-62, 10 microM) reduced amylase secretion stimulated by cholecystokinin (CCK) or carbachol. W-7 and KN-62 also inhibited amylase secretion stimulated by both calcium ionophore (A23187) and phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA). To clarify the role of calmodulin in the interaction of intracellular mediators, pancreatic acini were permeabilized with streptolysin O. Following permeabilization, amylase secretion was stimulated by submicromolar free Ca2+, and this Ca(2+)-dependent amylase secretion was enhanced by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), TPA or cyclic adenosine 3',5'-monophosphate (cAMP). W-7 and KN-62 had no effects on amylase secretion stimulated by Ca2+ alone, but inhibited the enhancement in Ca(2+)-dependent amylase secretion by GTP gamma S, TPA or cAMP. These data suggest that calmodulin plays an important role in Ca(2+)-dependent amylase secretion from pancreatic acinar cells and in the interaction between Ca2+ and other intracellular messengers.
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PMID:Effects of calmodulin inhibitors on amylase secretion from rat pancreatic acini. 128 79

We report the production of an antibody specific for Ca2+/calmodulin-dependent protein kinase II (CaM-KII) autophosphorylated only at Thr-286 of the alpha subunit. Peptide Y-66 [sequence MHRQETVDC (Met-281 to Cys-289 of alpha subunit of CaM-KII)] was synthesized and phosphorylated by the CaM-KII endogenous to synaptic cytoskeleton (postsynaptic density-enriched fraction); the phosphorylated amino acid residue threonine corresponds to Thr-286 in the kinase alpha subunit. The phosphorylated Y-66 peptide was separated from the unphosphorylated peptide by HPLC and used as an immunogen after being coupled to hemocyanin. The antibodies that reacted with hemocyanin and unphosphorylated Y-66 peptide were adsorbed, and then IgG was purified. ELISA proved that the IgG obtained reacted specifically with phosphorylated Y-66 peptide. Immunoblot analysis showed that the antibody reacted specifically to the autophosphorylated CaM-KII both in purified and synaptic cytoskeleton-associated form. Appearance of CaM-KII subunits immunoreactive to anti-phosphorylated Y-66 antibody paralleled the generation of Ca(2+)-independent kinase activity. Immunocytochemical experiments clearly showed expression of the Thr-286- or Thr-287-autophosphorylated form of CaM-KII in cultured hippocampal cells treated with N-methyl-D-aspartate. Thus, this antibody could be extremely useful for studying the biological functions of CaM-KII.
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PMID:Antibody specific for the Thr-286-autophosphorylated alpha subunit of Ca2+/calmodulin-dependent protein kinase II. 130 2

We surveyed rabbit brain cytosol for a new Ca2+/calmodulin (CaM)-dependent kinase. The renaturation blotting assay (RBA) exploits the ability of blotted SDS-denatured proteins to regain enzymic activity after guanidine treatment. Using RBA, we found that the eluate of rabbit brain cytosol from a CaM affinity column contains at least four electrophoretically distinct protein kinase bands which were autophosphorylated in a Ca2+/CaM-dependent manner. The 49 kDa band and the 60 kDa band were alpha and beta subunit of CaM kinase II, and the 42 kDa band was presumed to be CaM kinase I, but the 80 kDa band could not be attributed to any reported Ca2+/CaM-dependent protein kinases. The 80 kDa protein kinase was isolated by three-step chromatography. We examined the phosphorylation of exogenous substrates by 80 kDa protein kinase, and histone IIIs and myosin light chain were phosphorylated in a Ca2+/CaM-dependent manner. W-7, a specific inhibitor for calmodulin, inhibited this kinase activity, but KN-62, a specific inhibitor for CaM kinase II, had no effect on this protein kinase activity. Autoradiography using boiled rabbit brain homogenate as substrate showed three intrinsic substrates (80 kDa, 60 kDa and 42 kDa), which were phosphorylated in a Ca2+/CaM-dependent manner. These findings suggest that a new Ca2+/CaM-dependent protein kinase could be identified by the RBA.
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PMID:Identification of a 80 kDa calmodulin-binding protein as a new Ca2+/calmodulin-dependent kinase by renaturation blotting assay (RBA). 131 May 91

Forebrain ischemia in gerbils, produced by brief bilateral carotid occlusion, induced the dramatic loss of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) as determined by both kinase activity assays and western blot analysis. In cortex and hippocampus, cytosolic CaM-kinase II was completely lost within 2-5 min of ischemia. Particulate CaM-kinase II was more stable and decreased in level approximately 40% after 10 min of ischemia followed by 2 h of reperfusion. CaM-kinase II in cerebellum, which does not become ischemic, was not affected. The rapid loss of CaM-kinase II within 2-5 min was quite specific because cytosolic cyclic AMP kinase and protein kinase C in hippocampus were not affected. These data indicate that cytosolic CaM-kinase II is one of the most rapidly degraded proteins after brief ischemia. Because the multifunctional CaM-kinase II has been implicated in the regulation of numerous neuronal functions, its loss may destine the neuronal cell for death.
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PMID:Ischemia-induced loss of brain calcium/calmodulin-dependent protein kinase II. 131 Jul 19

The Na+/H+ exchanger is a pH-regulatory protein that extrudes one H+ ion in exchange for one Na+ ion when intracellular pH declines. A number of studies have shown phorbol ester stimulation of activity in intact cells, leading to the idea that the exchanger is regulated by protein kinase C-mediated phosphorylation in vivo. cDNA encoding the protein has been cloned, and a recent model suggests a large internal cytoplasmic C-terminal domain that may be a site of regulation of the exchanger [Sardet, Franchi & Pouyssegur (1989) Cell 56, 271-280]. We examined this region of the protein using a rabbit cardiac Na+/H+ exchanger cDNA clone. cDNA of the Na+/H+ exchanger, coding for the C-terminal 178 amino acid residues, was cloned into the expression vector pEX-1 and expressed as a fusion protein with beta-galactosidase. The fusion protein reacted with an antibody produced against a synthetic peptide of the C-terminal 13 amino acid residues of the Na+/H+ exchanger, confirming the identity of the expressed protein. Control and experimental pEX-1-Na+/H+ exchanger protein was purified on a p-aminophenyl beta-D-thiogalactopyranoside-agarose column. Purified Ca2+/calmodulin-dependent protein kinase II readily phosphorylated the Na+/H+ exchanger protein in a Ca(2+)- and calmodulin-dependent manner in vitro, but this region of the protein was not a substrate for purified protein kinase C or for the catalytic subunit of cyclic AMP-dependent protein kinase. Control-expressed beta-galactosidase was phosphorylated to a maximal level of 0.77 +/- 0.17 mol of Pi/mol (mean +/- S.E.M., n = 6) whereas the fusion protein was phosphorylated to a maximal level of 4.09 +/- 0.39 mol of Pi/mol (n = 6), suggesting one site of phosphorylation in beta-galactosidase and three in the C-terminal domain of the Na+/H+ exchanger. Examination of the deduced amino acid sequence of this part of the exchanger reveals three consensus sequences for Ca2+/calmodulin-dependent protein kinase II. These results suggest that the exchanger may be directly regulated in vivo by calmodulin-dependent protein kinase II but not by protein kinase C or cyclic AMP-dependent protein kinase.
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PMID:Phosphorylation of the C-terminal domain of the Na+/H+ exchanger by Ca2+/calmodulin-dependent protein kinase II. 131 52

Abnormal phosphorylation of the microtubule associated protein tau component of neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) may result from alterations in protein kinase expression. Calcium/calmodulin dependent protein kinase II (CaM kinase II) has been shown to phosphorylate tau in vitro in such a way to decrease its electrophoretic mobility. A68, apparently a modified form of tau in AD brain, also shows abnormal phosphorylation and slower mobility than tau. To further examine the role of CaM kinase II in AD, in situ hybridization studies were performed on tissues from rat, monkey and human to examine and compare the patterns of CaM kinase II mRNA expression in different brain regions. The most notable differences among the three species were observed in dendrites in layer I of isocortex, in the molecular layer of the dentate gyrus and stratum radiatum and stratum lacunosum-moleculare in hippocampus, where hybridization was detected in rat, but not in monkey or human brain. In addition, comparisons between tau and CaM kinase II mRNA expression were made in tissue from normal aged adults and AD patients, especially in areas prone to NFT formation. CaM kinase II and tau mRNAs were co-expressed in many neuronal populations, both those which are prone to NFT formation as well as those which are rarely affected by AD changes. No major differences in the relative abundance of either CaM kinase II or tau mRNA within particular neuronal populations was noted between normal aged and AD brain. Diminished hybridization was associated with serve neuronal pathology and cell loss.
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PMID:In situ hybridization of calcium/calmodulin dependent protein kinase II and tau mRNAs; species differences and relative preservation in Alzheimer's disease. 131 9

We have investigated regional and temporal alterations in Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and calcineurin (Ca2+/calmodulin-dependent protein phosphatase) after transient forebrain ischemia. Immunoreactivity and enzyme activity of CaM kinase II decreased in regions CA1 and CA3, and in the dentate gyrus, of the hippocampus early (6-12 h) after ischemia, but the decrease in immunoreactivity gradually recovered over time, except in the CA1 region. Furthermore, the increase in Ca2+/calmodulin-independent activity was detected up to 3 days after ischemia in all regions tested, suggesting that the concentration of intracellular Ca2+ increased. In contrast to CaM kinase II, as immunohistochemistry and regional immunoblot analysis revealed, calcineurin was preserved in the CA1 region until 1.5 days and then lost with the increase in morphological degeneration of neurons. Immunoblot analysis confirmed the findings of the immunohistochemistry. These results suggest that there is a difference between CaM kinase II and calcineurin in regional and temporal loss after ischemia and that imbalance of Ca2+/calmodulin-dependent protein phosphorylation-dephosphorylation may occur.
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PMID:Regional and temporal alterations in Ca2+/calmodulin-dependent protein kinase II and calcineurin in the hippocampus of rat brain after transient forebrain ischemia. 131 54

Localization of the gamma and delta types of mRNAs for Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) was determined in the rat brain, making use of in situ hybridization histochemistry. The gamma and delta mRNAs as well as the alpha and beta mRNAs for CaM-kinase II were heterogeneously and distinctly distributed. In the Purkinje cell layer of the cerebellum, alpha, beta, and gamma mRNAs but not delta mRNA were present, whereas beta, gamma, and delta mRNAs were present in the locus coeruleus. These findings provide evidence that CaM-kinase II exists in a variety of forms in different cells composed of a variable number and type of subunits.
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PMID:Evidence for distinct neuronal localization of gamma and delta subunits of Ca2+/calmodulin-dependent protein kinase II in the rat brain. 131 58

Multifunctional calcium-calmodulin-dependent protein kinase (CaM kinase) transduces transient elevations in intracellular calcium into changes in the phosphorylation state and activity of target proteins. By fluorescence emission anisotropy, the affinity of CaM kinase for dansylated calmodulin was measured and found to increase 1000 times after autophosphorylation of the threonine at position 286 of the protein. Autophosphorylation markedly slowed the release of bound calcium-calmodulin; the release time increased from less than a second to several hundred seconds. In essence, calmodulin is trapped by autophosphorylation. The shift in affinity does not occur in a site-directed mutant in which threonine at position 286 has been replaced by a non-phosphorylatable amino acid. These experiments demonstrate the existence of a new state in which calmodulin is bound to CaM kinase even though the concentration of calcium is basal. Calmodulin trapping provides for molecular potentiation of calcium transients and may enable detection of their frequency.
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PMID:Calmodulin trapping by calcium-calmodulin-dependent protein kinase. 131 63


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