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:2.7.11.17 (CaMKII)
4,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ca2+/calmodulin-dependent protein kinase (Ca2+/CaM kinase I), which phosphorylates site I of synapsin I, has been highly purified from bovine brain. The physical properties and substrate specificity of Ca2+/CaM kinase I were distinct from those of all other known Ca2+/CaM kinases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified enzyme preparation consisted of two major polypeptides of Mr 37,000 and 39,000 and a minor polypeptide of Mr 42,000. In the presence of Ca2+ and calmodulin (CaM), all three polypeptides bound CaM, were autophosphorylated on threonine residues, and were labeled by the photoaffinity label 8-azido-ATP. Peptide maps of the three autophosphorylated polypeptides were very similar. The Stokes radius and the sedimentation coefficient of the enzyme were, respectively, 31.8 A and 3.25 s. A molecular weight of 42,400 and a frictional ratio of 1.38 were calculated from the above values, suggesting that Ca2+/CaM kinase I is a monomer. It is possible that the polypeptides of lower molecular weight are derived from the polypeptide of Mr 42,000 by proteolysis; alternatively, the polypeptides may represent isozymes of Ca2+/CaM kinase I. Synapsin I (site I) was the best substrate tested (Km, 2-4 microM) for Ca2+/CaM kinase I. Of many additional proteins tested, only protein III (a phosphoprotein related to synapsin I) and smooth muscle myosin light chain were phosphorylated. Ca2+/CaM kinase I was found in highest concentration in brain, where it showed widespread regional and subcellular distributions. In addition, the enzyme had a widespread and predominantly cytosolic tissue distribution. The widespread neuronal and tissue distribution of Ca2+/CaM kinase I suggests that other substrates might exist for this enzyme in both neuronal and non-neuronal tissues.
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PMID:Purification and characterization of Ca2+/calmodulin-dependent protein kinase I from bovine brain. 310 51

Synapsin I is a neuronal phosphoprotein comprised of two closely related polypeptides with apparent molecular weights of 78,000 and 76,000. It is found in association with the small vesicles clustered at the presynaptic junction. Its precise role is unknown, although it probably participates in vesicle clustering and/or release. Synapsin I is known to associate with vesicle membranes, microtubules, and neurofilaments. We have examined the interaction of purified phosphorylated and unphosphorylated bovine and human synapsin I with tubulin and actin filaments, using cosedimentation, viscometric, electrophoretic, and morphologic assays. As purified from brain homogenates, synapsin I decreases the steady-state viscosity of solutions containing F-actin, enhances the sedimentation of actin, and bundles actin filaments. Phosphorylation by cAMP-dependent kinase has minimal effect on this interaction, while phosphorylation by brain extracts or by purified calcium- and calmodulin-dependent kinase II reduces its actin-bundling and -binding activity. Synapsin's microtubule-binding activity, conversely, is stimulated after phosphorylation by the brain extract. Two complementary peptide fragments of synapsin generated by 2-nitro-5-thiocyanobenzoic cleavage and which map to opposite ends of the molecule participate in the bundling process, either by binding directly to actin or by binding to other synapsin I molecules. 2-Nitro-5-thiocyanobenzoic peptides arising from the central portion of the molecule demonstrate neither activity. In vivo, synapsin I may link small synaptic vesicles to the actin-based cortical cytoskeleton, and coordinate their availability for release in a Ca++-dependent fashion.
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PMID:Synapsin I: an actin-bundling protein under phosphorylation control. 311 96

We report here the results of immunocytochemical and biochemical studies on the localization of synapsin I, a nerve terminal--specific phosphoprotein, at the frog neuromuscular junction. Our results show that in this in situ synapse synapsin I is concentrated in the presynaptic compartment, where it appears to be associated with the synaptic vesicle membrane. Double immunoprecipitated synapsin I from homogenates of frog cutaneous pectoris muscles could be phosphorylated by the catalytic subunit of cyclic adenosine 5'-monophosphate-dependent protein kinase after gel electrophoresis and blotting onto nitrocellulose and could be subsequently identified by an immunoperoxidase technique. Experiments carried out in frog brain preparations indicate that frog synapsin I, like the mammalian protein, can be phosphorylated at different sites by exogenously added catalytic subunit of cyclic adenosine 5'-monophosphate-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase II prepared from mammalian sources. The phosphorylation sites of frog synapsin I, as judged by phosphopeptide mapping, are somewhat different from those of mammalian synapsin I. The study of synapsin I and of the regulation of its state of phosphorylation at the neuromuscular junction may provide important information on its role in synaptic function, since at the present time this is one of the few systems in which a correlation among biochemical, immunocytochemical and electrophysiological results is possible.
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PMID:Localization of synapsin I at the frog neuromuscular junction. 312 73

We have analyzed Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) localization, activity, and endogenous protein substrates during differentiation and synaptogenesis in cultured hippocampal neurons. Primary cultures from hippocampi from 18 d embryonic rats are composed primarily of pyramidal neurons, with minimal contamination by nonneuronal cells. We have used monoclonal (Mab) and affinity-purified polyclonal antibodies that recognize either or both of the subunits of CaM-kinase II in order to localize the enzyme at progressive stages of neuronal differentiation. Diffuse but specific binding, determined by indirect immunofluorescence analyses, was first detected in cell bodies and growth cones of pyramidal neurons after 4 d in culture. Immunoreactivity increased during the next 3 d of culture, at which time fluorescent, labeling was patchy along neuritic processes. By 10 d, intensely fluorescent, discrete spots were observed along processes and on cell bodies. Astrocyte cultures prepared from newborn rat cortex showed no detectable immunofluorescence with anti-CaM-kinase II antibodies. Cytosolic and particulate fractions from cultured pyramidal neurons and astrocytes were analyzed using immunoblot, in vitro phosphorylation, 2-dimensional gel electrophoresis, and phosphopeptide mapping techniques. Although pure astrocyte cultures contained low levels of Ca2+/CaM-stimulated protein kinase activity, they did not display detectable levels of immunoreactive 50 kDa subunit nor 50 and 60 kDa phosphoproteins analogous to the autophosphorylated subunits of CaM-kinase II. Immunoblot analysis detected the 60 kDa kinase subunit in particulate and cytosolic fractions from 2 d neurons. By contrast, the 50 kDa subunit of CaM-kinase II was not detected in cytosolic or particulate fractions of pyramidal neurons before 4 d in culture. In 2 d pyramidal neuron cultures, only low levels of Ca2+/CaM-stimulated protein phosphorylation were observed. Ca2+/CaM-dependent phosphorylation of 10 d pyramidal cell proteins was 3-5-fold greater than that of 2 d cultures, and included major phosphoproteins of 48, 50, 56, 58/60, 80-86, 90, 120, 138, 175, and 190 kDa. Phosphopeptide maps of 58/60 and 50 kDa phosphoproteins gave patterns very similar to those of the autophosphorylated 60 and 50 kDa subunits, respectively, of purified CaM-kinase II. A phosphoprotein doublet of 83 kDa was identified as synapsin I. Developmental changes in Ca2+/CaM-dependent phosphorylation in pyramidal neuron cultures were very similar to those previously described in subcellular fractions from postnatal rat forebrain.
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PMID:Developmental changes in Ca2+/calmodulin-dependent protein kinase II in cultures of hippocampal pyramidal neurons and astrocytes. 334 14

Caldesmon, a major actin- and calmodulin-binding protein of smooth muscle, has been implicated in regulation of the contractile state of smooth muscle. The isolated protein can be phosphorylated by a co-purifying Ca2+/calmodulin-dependent protein kinase, and phosphorylation blocks inhibition of the actomyosin ATPase by caldesmon [Ngai & Walsh (1987) Biochem. J. 244, 417-425]. We have examined the phosphorylation of caldesmon in more detail. Several lines of evidence indicate that caldesmon itself is a kinase and the reaction is an intermolecular autophosphorylation: (1) caldesmon (141 kDa) and a 93 kDa proteolytic fragment of caldesmon can be separated by ion-exchange chromatography: both retain caldesmon kinase activity, which is Ca2+/calmodulin-dependent; (2) chymotryptic digestion of caldesmon generates a Ca2+/calmodulin-independent form of caldesmon kinase; (3) caldesmon purified to electrophoretic homogeneity retains caldesmon kinase activity, and elution of enzymic activity from a fast-performance-liquid-chromatography ion-exchange column correlates with caldesmon of Mr 141,000; (4) caldesmon is photoaffinity-labelled with 8-azido-[alpha-32P]ATP; labelling is inhibited by ATP, GTP and CTP, indicating a lack of nucleotide specificity; (5) caldesmon binds tightly to Affi-Gel Blue resin, which recognizes proteins having a dinucleotide fold. Autophosphorylation of caldesmon occurs predominantly on serine residues (83.3%), with some threonine (16.7%) and no tyrosine phosphorylation. Autophosphorylation is site-specific: 98% of the phosphate incorporated is recovered in a 26 kDa chymotryptic peptide. Complete tryptic/chymotryptic digestion of this phosphopeptide followed by h.p.l.c. indicates three major phosphorylation sites. Caldesmon exhibits a high degree of substrate specificity: apart from autophosphorylation, brain synapsin I is the only good substrate among many potential substrates examined. These observations indicate that caldesmon may regulate its own function (inhibition of the actomyosin ATPase) by Ca2+/calmodulin-dependent autophosphorylation. Furthermore, caldesmon may regulate other cellular processes, e.g. neurotransmitter release, through the Ca2+/calmodulin-dependent phosphorylation of other proteins such as synapsin I.
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PMID:Autophosphorylation of smooth-muscle caldesmon. 341 67

In previous studies, we described a soluble Ca2+/calmodulin-dependent protein kinase which is the major Ca2+/calmodulin-dependent microtubule-associated protein 2 (MAP-2) kinase in rat brain [Schulman, H. (1984) J. Cell Biol. 99, 11-19; Kuret, J. A., & Schulman, H. (1984) Biochemistry 23, 5495-5504]. We now demonstrate that this protein kinase has broad substrate specificity. Consistent with a multifunctional role in cellular physiology, we show that in vitro the enzyme can phosphorylate numerous substrates of both neuronal and nonneuronal origin including vimentin, ribosomal protein S6, synapsin I, glycogen synthase, and myosin light chains. We have used MAP-2 to purify the enzyme from rat lung and show that the brain and lung kinases have nearly indistinguishable physical and biochemical properties. A Ca2+/calmodulin-dependent protein kinase was also detected in rat heart, rat spleen, and in the ring ganglia of the marine mollusk Aplysia californica. Partially purified MAP-2 kinase from each of these three sources displayed endogenous phosphorylation of a 54 000-dalton protein. Phosphopeptide analysis reveals a striking homology between this phosphoprotein and the 53 000-dalton autophosphorylated subunit of the major rat brain Ca2+/calmodulin-dependent protein kinase. The enzymes phosphorylated MAP-2, synapsin I, and vimentin at peptides that are identical with those phosphorylated by the rat brain kinase. This enzyme may be a multifunctional Ca2+/calmodulin-dependent protein kinase with a widespread distribution in nature which mediates some of the effects of Ca2+ on microtubules, intermediate filaments, and other cellular constituents in brain and other tissues.
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PMID:Ca2+/calmodulin-dependent microtubule-associated protein 2 kinase: broad substrate specificity and multifunctional potential in diverse tissues. 407 98

Calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase found in several tissues, is highly concentrated in mammalian brain. In an attempt to identify endogenous brain substrates for calcineurin, kinetic analyses of the dephosphorylation of several well-characterized phosphoproteins purified from brain were performed. The proteins studied were: G-substrate, a substrate for cyclic GMP-dependent protein kinase; DARPP-32, a substrate for cyclic AMP-dependent protein kinase; Protein K.-F., a substrate for a cyclic nucleotide- and Ca2+-independent protein kinase; and synapsin I, a substrate for cyclic AMP-dependent (site I) and a Ca2+/calmodulin-dependent protein kinase (site II). Calcineurin dephosphorylated each of these proteins in a Ca2+/calmodulin-dependent manner. Similar Km values were obtained for each substrate: G-substrate, 3.8 microM; DARPP-32, 1.6 microM; Protein K.-F., approximately 3 microM (S0.5); synapsin I (site I), 7.0 microM; synapsin I (site II), 4.4 microM. However, significant differences were obtained for the maximal rates of dephosphorylation. The kcat values were: G-substrate, 0.41 s-1; DARPP-32, 0.20 s-1; Protein K.-F., 0.7 s-1; synapsin I (site I), 0.053 s-1; synapsin I (site II), 0.040 s-1. Comparisons of the catalytic efficiency (kcat/Km) for each substrate indicated that DARPP-32, G-substrate, and Protein K.-F. are all potential substrates for calcineurin in vivo.
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PMID:Mammalian brain phosphoproteins as substrates for calcineurin. 633 98

Previous studies have purified from brain a Ca2+/calmodulin-dependent protein kinase II (designated CaM-kinase II) that phosphorylates synapsin I, a synaptic vesicle-associated phosphoprotein. CaM-kinase II is composed of a major Mr 50K polypeptide and a minor Mr 60K polypeptide; both bind calmodulin and are phosphorylated in a Ca2+/calmodulin-dependent manner. Recent studies have demonstrated that the 50K component of CaM-kinase II and the major postsynaptic density protein (mPSDp) in brain synaptic junctions (SJs) are virtually identical and that the CaM-kinase II and SJ 60K polypeptides are highly related. In the present study the photoaffinity analog [alpha-32P]8-azido-ATP was used to demonstrate that the 60K and 50K polypeptides of SJ-associated CaM-kinase II each bind ATP in the presence of Ca2+ plus calmodulin. This result is consistent with the observation that these proteins are phosphorylated in a Ca2+/calmodulin-dependent manner. Experiments using 32P-labeled peptides obtained by limited proteolysis of 60K and 50K polypeptides from SJs demonstrated that within each kinase polypeptide the same peptide regions contain both autophosphorylation and 125I-calmodulin binding sites. These results suggested that the autophosphorylation of CaM-kinase II could regulate its capacity to bind calmodulin and, thus, its capacity to phosphorylate substrate proteins. By using 125I-calmodulin overlay techniques and sodium dodecyl sulfate-polyacrylamide gel electrophoresis we found that phosphorylated 50K and 60K CaM-kinase II polypeptides bound more calmodulin (50-70%) than did unphosphorylated kinase polypeptides. Levels of in vitro CaM-kinase II activity in SJs were measured by phosphorylation of exogenous synapsin I. SJs containing highly phosphorylated CaM-kinase II displayed greater activity in phosphorylating synapsin I (300% at 15 nM calmodulin) relative to control SJs that contained unphosphorylated CaM-kinase II. The CaM-kinase II activity in phosphorylated SJs was indistinguishable from control SJs at saturating calmodulin concentrations (300-1,000 nM). These findings show that the degree of autophosphorylation of CaM-kinase II in brain SJs modulates its in vitro activity at low and possibly physiological calmodulin concentrations; such a process may represent a mechanism of regulating this kinase's activity at CNS synapses in situ.
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PMID:Autophosphorylation of calmodulin-kinase II in synaptic junctions modulates endogenous kinase activity. 654 10

To detect potential substrate proteins for Ca2+/calmodulin-dependent protein kinase II outside the central nervous system, antibodies were made to a synthetic peptide corresponding to a sequence within synapsin I which is phosphorylated by this enzyme. In neural tissues, this antibody (212) identified an 86/80 kDa doublet corresponding to synapsin I. In rat liver, intestinal enterocytes and the clone 9 cell line this antibody identified two proteins of 170 and 85 kDa. These proteins were present in the particulate fraction of liver postnuclear supernatant, and were released into the soluble fraction when extracted with 100 mM NaCl. In liver, enterocytes, and clone 9 cells, these antigens were localized by immunocytochemical techniques to small intracellular vesicles. The endocytic compartment of clone 9 cells was labeled by continuous uptake of horseradish peroxidase; antibody 212-labeled vesicles exhibited overlap with the compartment. To confirm the identity of this compartment as endosomal, rat liver endosomes were labeled in vivo by intravenous injection of horseradish peroxidase. Horseradish peroxidase-containing endosomes of approximately 80 nm were recognized by antibody 212. Occasionally, larger endosomes (approximately 300-500 nm) were also labeled. In clone 9 cells, partial overlap was observed between the 212 antigen and a transferrin receptor-positive, brefeldin A-sensitive compartment. In clone 9 cells double-labeled with anti-tubulin and antibody 212, then imaged using confocal microscopy, these vesicles appeared to be associated with microtubules. This antigen has properties similar to that of CLIP-170, a membrane-associated endosomal phosphoprotein. These findings demonstrate that a 170/85 kDa antigen containing an epitope for the Ca2+/calmodulin-dependent protein kinase II phosphorylation sequence is associated with an endocytic compartment.
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PMID:Antibodies to an epitope on synapsin I detect a protein associated with the endocytic compartment in non-neuronal cells. 753 73

The mitogenic activity of several growth factors is mediated by calcium-dependent signal transduction. Calmodulin (CaM) binding proteins such as CaM-dependent protein kinases are important components of this pathway and may be altered in diseases characterized by abnormal cell growth. CaM kinase II is believed to regulate the phosphorylation of microtubular-associated proteins and control the initiation of DNA synthesis. Furthermore, drugs that inhibit CaM-mediated signal transduction also inhibit cellular proliferation and are cytotoxic to numerous malignant cell lines, including those established from malignant gliomas. Yet, little is known about CaM-dependent protein kinases in these tumors. Therefore, we have investigated the activity and distribution of CaM-dependent protein kinase II in normal and malignant glial tissues, a kinase believed to play a critical role in cell cycle regulation. C6 and 9L cells contained kinase activities that were activated by Ca2+/CaM and inhibited by trifluoperazine. Tissue extracts from these cell lines and from rat brain white matter phosphorylated exogenous synapsin I in a pattern consistent with the presence of CaM kinase II activity as determined by phosphopeptide mapping. CaM kinase II activity was confirmed using a specific peptide substrate and inhibitor. An unexpected finding was that glioma lines, but not rat brain white matter, also contained a CaM-dependent protein kinase detected by the phosphorylation of a M(r) 100,000 protein, subsequently identified as elongation factor 2, the only known substrate for CaM kinase III.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Calmodulin-dependent protein kinases in rat glioblastoma. 764 41


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