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 multifunctional Ca2+/calmodulin-dependent protein kinase (multifunctional CaM kinase) may be an important mediator for neurotransmitters and hormones that utilize Ca2+ as a "second messenger." We examined the ability of autophosphorylation to convert the multifunctional CaM kinase to a Ca2+/calmodulin-independent (autonomous) form to determine whether autophosphorylation is a mechanism for short- or long-term enhancement of Ca2+ action. As the kinase incorporates phosphate during continuous stimulation by Ca2+/calmodulin, its ability to phosphorylate exogenous substrates becomes increasingly autonomous. Withdrawal of Ca2+ after a critical level of phosphate incorporation is reached leads to a "burst" or rapid increase in Ca2+-independent autophosphorylation. The "burst" of autophosphorylation is distinct from the initial Ca2+-dependent autophosphorylation, however, since it inhibits substrate phosphorylation. Both Ca2+-dependent and Ca2+-independent substrate phosphorylation are inhibited by this autonomous autophosphorylation. Thus, autophosphorylation has a dual role in modulating the activity of multifunctional CaM kinase. It initially enables the kinase to continue phosphorylating substrates after Ca2+ levels decline, but it eventually suppresses this autonomous activity. Tryptic phosphopeptide mapping demonstrates that appearance of phosphothreonine-containing peptides is common to several conditions used to generate an autonomous enzyme. Sequencing reveals the critical "autonomy" site to be threonine286. The inhibitory mode of autophosphorylation involves 3 additional phosphopeptides containing a serine and a threonine residue.
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PMID:Distinct autophosphorylation sites sequentially produce autonomy and inhibition of the multifunctional Ca2+/calmodulin-dependent protein kinase. 254 84

Phospholamban is the major membrane protein of the heart phosphorylated in response to beta-adrenergic stimulation. In cell-free systems, cAMP-dependent protein kinase catalyzes exclusive phosphorylation of serine 16 of phospholamban, whereas Ca2+/calmodulin-dependent protein kinase gives exclusive phosphorylation of threonine 17 (Simmerman, H. K. B., Collins, J. H., Theibert, J. L., Wegener, A. D., and Jones, L. R. (1986) J. Biol. Chem. 261, 13333-13341). In this work we have localized the sites of phospholamban phosphorylation in intact ventricles treated with the beta-adrenergic agonist isoproterenol. Isolation of phosphorylated phospholamban from 32P-perfused guinea pig ventricles, followed by partial acid hydrolysis and phosphoamino acid analysis, revealed phosphorylation of both serine and threonine residues. At steady state after isoproterenol exposure, phospholamban contained approximately equimolar amounts of these two phosphoamino acids. Two major tryptic phosphopeptides containing greater than 90% of the incorporated radioactivity were obtained from phospholamban labeled in intact ventricles. The amino acid sequences of these two tryptic peptides corresponded exactly to residues 14-25 and 15-25 of canine cardiac phospholamban, thus localizing the sites of in situ phosphorylation to serine 16 and threonine 17. Phosphorylation of phospholamban at two sites in heart perfused with isoproterenol was supported by detection of 11 distinct mobility forms of the pentameric protein by use of the Western blotting method, consistent with each phospholamban monomer containing two phosphorylation sites, and with each pentamer containing from 0 to 10 incorporated phosphates. Our results localize the sites of in situ phospholamban phosphorylation to serine 16 and threonine 17 and, furthermore, are consistent with the phosphorylations of these 2 residues being catalyzed by cAMP- and Ca2+/calmodulin-dependent protein kinases, respectively.
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PMID:Phospholamban phosphorylation in intact ventricles. Phosphorylation of serine 16 and threonine 17 in response to beta-adrenergic stimulation. 254 95

Conditions that regulate the generation of the Ca2(+)-independent form of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) in cultured rat cerebellar granule cells have been investigated. Under basal conditions, 4-5% of total CaM-kinase II activity, assayed in the presence of Ca2+/CaM, was the Ca2(+)-independent form active in the presence of EGTA. Depolarization with 56 mM K+ produced a transient increase to 9% Ca2+ independence within 15 s followed by a decline to 5-6% at 10 min. The divalent cation ionophore ionomycin elicited 10% Ca2+ independence, which remained elevated. Removal of Ca2+ from the Krebs-Ringer medium reduced basal Ca2+ independence to 1-2% and eliminated the elevation in response to K+ depolarization. Inclusion of 5 microM okadaic acid, a protein phosphatase inhibitor, in the incubation medium potentiated the levels of Ca2(+)-independent activity of CaM-kinase II. Additional studies in granule cell extracts indicated that there were both okadiac acid-sensitive and -insensitive protein phosphatases involved in the reversal of the Ca2+ independence of CaM-kinase II. Phosphopeptide mapping of the CNBr-cleaved 32P-labeled 58-60-kDa subunit of CaM-kinase II revealed that under basal conditions, the kinase contained phosphate in many sites. Conditions that promoted formation of the Ca2(+)-independent form of the kinase increased the 32P incorporation into multiple sites of the kinase. However, there was a good temporal correlation between 32P incorporation into CNBr peptide 1, which contains Thr-287, and generation of the Ca2(+)-independent kinase activity. These results indicate that formation of the Ca2(+)-independent species of CaM-kinase II is dynamically regulated in cerebellar granule cells by Ca2(+)-mobilizing agents and by protein phosphatase activity and is correlated with autophosphorylation of Thr-287.
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PMID:Generation of the Ca2(+)-independent form of Ca2+/calmodulin-dependent protein kinase II in cerebellar granule cells. 255 42

A protein kinase activity was identified in pig brain that co-purified with microtubules through repeated cycles of temperature-dependent assembly and disassembly. The microtubule-associated protein kinase (MTAK) phosphorylated histone H1; this activity was not stimulated by cyclic nucleotides. Ca2+ plus calmodulin, phospholipids or polyamines. MTAK did not phosphorylate synthetic peptides which are substrates for cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase. Ca2+/calmodulin-dependent protein kinase II, protein kinase C or casein kinase II. MTAK activity was inhibited by trifluoperazine [IC50 (median inhibitory concn.) = 600 microM] in a Ca2+-independent fashion. Ca2+ alone was inhibitory [IC50 = 4 mM). MTAK was not inhibited by heparin, a potent inhibitor of casein kinase II, nor a synthetic peptide inhibitor of cyclic AMP-dependent protein kinase. MTAK demonstrated a broad pH maximum (7.5-8.5) and an apparent Km for ATP of 45 microM. Mg2+ was required for enzyme activity and could not be replaced by Mn2+. MTAK phosphorylated serine and threonine residues on histone H1. MTAK is a unique cofactor-independent protein kinase that binds to microtubule structures.
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PMID:Properties of a microtubule-associated cofactor-independent protein kinase from pig brain. 255 23

The high heterogeneity of native rat liver EF-2 prepared from either 105000 x g supernatant or microsome high-salt extract was detected by two-dimensional equilibrium isoelectric focusing-SDS-polyacrylamide gel electrophoresis in the presence of 9.5 M urea. Five spots were always detected, all of Mr 95,000, which were not artefactual for their amount varied when EF-2 was specifically ADP-ribosylated by diphtheria toxin in the presence of NAD+, and/or phosphorylated on a threonine residue by a Ca2+/calmodulin-dependent protein kinase (most likely Ca2+/calmodulin-dependent protein kinase III described by others [(1987) J. Biol. Chem. 262, 17299-17303; (1988) Nature 334, 170-173]). Results of ADP-ribosylation and/or phosphorylation experiments with either unlabeled or labeled reagents ([14C]NAD and [32P]ATP) strongly suggest that our preparation contained native ADP-ribosylated and native phosphorylated forms which could be estimated at about 20% and 40% of the whole EF-2. Phosphorylated and ADP-ribosylated forms of EF-2 could be ADP-ribosylated and phosphorylated, respectively, but a native form both ADP-ribosylated and phosphorylated was not detected. Our results also suggest the existence of a minor native form of EF-2 and of its phosphorylated and ADP-ribosylated derivatives.
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PMID:Heterogeneity of native rat liver elongation factor 2. 279 73

Regulatory domains of the multifunctional Ca2+/calmodulin-dependent protein kinase II were investigated utilizing synthetic peptides. These peptides were derived from the sequence between positions 281 and 319 as translated from the cDNA sequence of the rat brain 50-kDa subunit (Lin, C. R., Kapiloff, M. S., Durgerian, S., Tatemoto, K., Russo, A. F., Hanson, P., Schulman, H., and Rosenfeld, M. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5962-5966), which contain the putative calmodulin-binding region as well as potential autophosphorylation sites. Peptide 290 to 309 was found to be a potent calmodulin antagonist with an IC50 of 52 nM for inhibition of Ca2+/calmodulin-dependent protein kinase II. Neither truncation from the amino terminus (peptide 296-309) nor extension in the carboxyl-terminal direction (peptide 294-319) markedly affected calmodulin binding, whereas shortening the peptide from the carboxyl terminus (peptide 290-302) or from both ends (peptide 295-304) resulted in the elimination of this activity. Peptide 281-290 did not bind calmodulin, but was a good substrate for the enzyme, being phosphorylated at Thr-286. Several of the peptides inhibited the kinase in a partially competitive, substrate-directed manner, but were not themselves phosphorylated. These studies identify domains within Ca2+/calmodulin-dependent protein kinase II which may be involved in 1) inhibition of the kinase in the absence of calmodulin, 2) binding of calmodulin, and 3) the resulting activation. Additionally, it is suggested that phosphorylation of residues flanking these domains may be responsible for the known regulatory effects of autophosphorylation on the properties of the kinase.
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PMID:Calcium/calmodulin-dependent protein kinase II. Characterization of distinct calmodulin binding and inhibitory domains. 283 67

Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II converts the enzyme to a Ca2+-independent form. The time course for this conversion correlates with the autophosphorylation of a threonine residue located within a thermolytic phosphopeptide common to the alpha and beta/beta' subunits. In the present study, this site was identified in the alpha subunit. After autophosphorylation under conditions that produced near-maximal Ca2+-independent activity, the alpha and beta/beta' subunits were separated by NaDodSO4/PAGE, and the alpha subunit was cleaved with cyanogen bromide. The major phosphopeptide (CB-1), containing phosphothreonine as the only radiolabeled amino acid, was purified by reverse-phase high performance liquid chromatography and subjected to automated gas-phase Edman degradation. The sequence obtained, Xaa-Arg-Gln-Glu-Thr-Val-Asp-Xaa-Leu-Lys-Lys-Phe-Asn-Ala-Arg-Arg-Lys-Leu, represented the NH2-terminal 18 residues (residues 282-299) of a 26-amino acid cyanogen bromide peptide predicted from the deduced primary structure of the alpha subunit and contained a consensus sequence for Ca2+/calmodulin-dependent kinase II phosphorylation that included Thr-286. The sequences obtained for two phosphopeptides derived from secondary chymotryptic digestion of CB-1 confirmed that Thr-286 was the phosphorylated residue.
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PMID:Ca2+/calmodulin-dependent protein kinase II: identification of threonine-286 as the autophosphorylation site in the alpha subunit associated with the generation of Ca2+-independent activity. 284 67

The autophosphorylation of purified Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) on a threonine-containing phosphopeptide common to both the alpha and beta subunits was previously shown to convert this enzyme into a catalytically active Ca2+-independent species. We now have examined the phosphorylation and activation of Ca2+/CaM kinase II in synaptosomes, a Ca2+-dependent neurosecretory system consisting of isolated nerve terminals. Synaptosomes were prelabeled with 32Pi and the alpha subunit of Ca2+/CaM kinase II was immunoprecipitated. Under basal incubation conditions the alpha subunit was phosphorylated. Depolarization of synaptosomes produced a rapid (2-5 s) Ca2+-dependent increase of about 50% in the state of phosphorylation of the alpha subunit. This was followed by a slower increase in the 32P content of the alpha subunit over the next 5 min of depolarization. The enhanced phosphorylation was characterized by an initial rise (2 s) and subsequent decrease (30 s) in the phosphothreonine content of the alpha subunit. In contrast, the phosphoserine content of the alpha subunit slowly increased during the course of depolarization. Thermolytic two-dimensional phosphopeptide maps of the alpha subunit demonstrated that depolarization stimulated the labeling of a phosphopeptide associated with autoactivation. In parallel experiments, unlabeled synaptosomes were depolarized, and lysates of these synaptosomes were assayed for Ca2+/CaM kinase II activity. Depolarization produced a rapid (less than or equal to 2 s) increase in Ca2+-independent Ca2+/CaM kinase II activity. This activity returned to basal levels by 60 s. Thus, depolarization of intact synaptosomes is associated with the transient phosphorylation of Ca2+/CaM kinase II on threonine residues, presumably involving an autophosphorylation mechanism and concomitantly the transient generation of the Ca2+-independent form of Ca2+/CaM kinase II.
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PMID:Autophosphorylation and activation of Ca2+/calmodulin-dependent protein kinase II in intact nerve terminals. 284 57

Dihydropyridine-sensitive Ca2+ channels exist in many different types of cells and are believed to be regulated by various protein phosphorylation and dephosphorylation reactions. The present study concerns the phosphorylation of a putative component of dihydropyridine-sensitive Ca2+ channels by the calcium and phospholipid-dependent protein kinase, protein kinase C. A skeletal muscle peptide of 165 kDa, which is known to contain receptors for dihydropyridines, phenylalkylamines, and other Ca2+ channel effectors, was found to be an efficient substrate for protein kinase C when the peptide was phosphorylated in its membrane-bound state. Protein kinase C incorporated 1.5-2.0 mol of phosphate/mol of peptide within 2 min into the 165-kDa peptide in incubations carried out at 37 degrees C. In contrast to the membrane-bound peptide, the purified 165-kDa peptide in detergent solution was phosphorylated to a markedly less extent than its membrane-bound counterpart; less than 0.1 mol of phosphate/mol of peptide was incorporated. Preincubation of the membranes with several types of drugs known to be Ca2+ channel activators or inhibitors had no specific effects on the rate and/or extent of phosphorylation of the 165-kDa peptide by protein kinase C. The phosphorylation of the membrane-bound 165-kDa peptide by protein kinase C was compared to that catalyzed by cAMP-dependent protein kinase and was found to be not additive. Prior phosphorylation of the 165-kDa peptide by cAMP-dependent protein kinase prevented subsequent phosphorylation of the peptide by protein kinase C. Phosphoamino acid analysis indicated that protein kinase C phosphorylated the 165-kDa peptide at both serine and threonine residues. Phosphopeptide mapping experiments showed that protein kinase C phosphorylated one unique site in the 165-kDa peptide, and, in addition, other sites that were phosphorylated by either cAMP-dependent protein kinase or a multifunctional Ca2+/calmodulin-dependent protein kinase. The results suggest that the 165-kDa dihydropyridine/phenylalkylamine receptor could serve as a physiological substrate of protein kinase C in intact cells. It is therefore possible that the regulation of dihydropyridine-sensitive Ca2+ channels by activators of protein kinase C may occur at the level of this peptide.
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PMID:Phosphorylation of the 165-kDa dihydropyridine/phenylalkylamine receptor from skeletal muscle by protein kinase C. 284 62

Microtubule-associated protein 2 (MAP2) is an excellent substrate for both cyclic-AMP (cAMP)-dependent and Ca2+/calmodulin-dependent kinases. A recently purified cytosolic Ca2+/calmodulin-dependent kinase (now designated CaM kinase II) phosphorylates MAP2 as a major substrate. We now report that microtubule-associated cAMP-dependent and calmodulin-dependent protein kinases phosphorylate MAP2 on separate sites. Tryptic phosphopeptide digestion and two-dimensional phosphopeptide mapping revealed 11 major peptides phosphorylated by microtubule-associated cAMP-dependent kinase and five major peptide species phosphorylated by calmodulin-dependent kinase. All 11 of the cAMP-dependently phosphorylated peptides were phosphorylated on serine residues, whereas four of five major peptides phosphorylated by the calmodulin-dependent kinase were phosphorylated on threonine. Only one peptide spot phosphorylated by both kinases was indistinguishable by both migration and phosphoamino acid site. The results indicate that cAMP-dependent and calmodulin-dependent kinases may regulate microtubule and cytoskeletal dynamics by phosphorylation of MAP2 at distinct sites.
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PMID:Phosphorylation of microtubule-associated protein 2 at distinct sites by calmodulin-dependent and cyclic-AMP-dependent kinases. 299 17


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