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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)

Regulatory mechanisms of rat brain Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) were probed using a synthetic peptide (CaMK-(281-309] corresponding to residues 281-309 (alpha-subunit) which contained the calmodulin (CaM)-binding and inhibitory domains and also the initial autophosphorylation site (Thr286). Kinetic analyses indicated that inhibition of a completely Ca2+/CaM-independent form of CaM-kinase II by CaMK-(281-309) was noncompetitive with respect to peptide substrate (syntide-2) but was competitive with respect to ATP. Interaction of CaMK-(281-309) with the ATP-binding site was independently confirmed since inactivation of proteolyzed CaM-kinase II by phenylglyoxal (t1/2 = 7 min) was blocked by ATP analog plus Mg2+ or by CaMK-(281-309). In the presence of Ca2+/CaM, CaMK-(281-309) no longer protected against phenylglyoxal inactivation, consistent with our previous observations (Colbran, R.J., Fong, Y.-L., Schworer, C.M., and Soderling, T.R. (1988) J. Biol. Chem. 263, 18145-18151) that binding of Ca2+/CaM to CaMK-(281-309) 1) blocks its inhibitory property, and 2) enhances its phosphorylation at Thr 286. The present study also showed that phosphorylation of CaMK-(281-309) decreased its inhibitory potency at least 10-fold without affecting its Ca2+/CaM-binding ability. Thus, CaM-kinase II is inactive in the absence of Ca2+/CaM because an inhibitory domain within residues 281-309 interacts with the catalytic domain and blocks ATP binding. Autophosphorylation of Thr286 results in a Ca2+/CaM-independent form of the kinase by disrupting the inhibitory interaction with the catalytic domain.
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PMID:Regulatory domain of calcium/calmodulin-dependent protein kinase II. Mechanism of inhibition and regulation by phosphorylation. 253 62

Tryptophan hydroxylase is activated in a crude extract by addition of ATP and Mg2+. This activation is reversible and requires in addition both Ca2+ and calmodulin. Thus, phosphorylation by an endogenous calmodulin-dependent protein kinase has long been suspected. Now that we have prepared a specific polyclonal antibody to rat brain tryptophan hydroxylase, we have been able to prove that this hypothesis is correct. After incubation of purified tryptophan hydroxylase with Ca2+/calmodulin-dependent protein kinase together with [gamma-32P]ATP, Mg2+, Ca2+, and calmodulin, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotting of the enzymes onto nitrocellulose sheets, we could label the band of tryptophan hydroxylase by the antiserum and the peroxidase technique and show by autoradiography that 32P was incorporated into this band. By measuring the radioactivity, we calculated that about 1 mol of phosphate was incorporated per 8 mol of subunits of the enzyme (2 mol of native enzyme). Because the concentration of ATP which we employed (50 microM) gives about half-maximal activation in crude extract compared to saturating ATP conditions (about 1 mM), this result indicates that the incorporation of at least 1 mol of phosphate/mol of tetramer of native tryptophan hydroxylase is required for maximal activation.
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PMID:Formal demonstration of the phosphorylation of rat brain tryptophan hydroxylase by Ca2+/calmodulin-dependent protein kinase. 254 52

Autophosphorylation plays an essential role in proteolytic activation of the type II calmodulin-dependent protein kinase (CaM kinase II). Limited proteolysis of CaM kinase II by trypsin, alpha-chymotrypsin, and Ca2+-stimulated neutral protease (calpain) yielded a catalytically active kinase fragment only when the holoenzyme was autophosphorylated prior to proteolysis. Slightly larger, inactive fragments were obtained from nonphosphorylated CaM kinase II, regardless of whether Ca2+/calmodulin or Mg2+/ATP were present or absent. The active fragment exhibited Ca2+/calmodulin-dependent kinase activity with kinetic parameters identical with those of the activated holoenzyme. The key autophosphorylation site of CaM kinase II was absent from the active fragment which indicates that proteolysis can effectively uncouple the activation state and Ca2+/calmodulin independence of the kinase from the action of phosphoprotein phosphatases. Because autophosphorylation exerts such a tight control over this irreversible process, proteolytic activation of CaM kinase II by intracellular proteases offers an attractive mechanism for prolonging the effects of Ca2+ at the synapse.
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PMID:Autophosphorylation of the type II calmodulin-dependent protein kinase is essential for formation of a proteolytic fragment with catalytic activity. Implications for long-term synaptic potentiation. 255 54

cDNAs containing the entire coding regions of the alpha and beta subunits of calmodulin-dependent protein kinase II (CaM kinase II) were isolated from a rat cerebrum cDNA library, ligated into an expression vector under the control of SV40 early promoter and introduced into Chinese hamster ovary (CHO) cells. To investigate the role of the alpha and beta subunits and their functional domains in CaM kinase II activity, the properties of the kinases expressed in the transfected cells were studied. CaM kinase II activity was detected in the transfected cells when the alpha and beta cDNAs were introduced into CHO cells simultaneously. RNA transfer blot and protein immunoblot analyses demonstrated the expression of the mRNAs and proteins of both alpha and beta subunits in the cloned cells. When alpha or beta cDNA was introduced into CHO cells separately, a significant level of the enzyme activity was also expressed, indicating that the alpha and beta subunits exhibited enzyme activity individually. The apparent Km values for ATP and MAP 2 were almost the same for the alpha subunit, beta subunit, alpha beta complex, and brain CaM kinase II. However, there was a slight difference in the affinity for calmodulin between the expressed proteins. The alpha and beta subunits expressed in the same cells polymerized to form alpha beta complex of a size similar to that of brain CaM kinase II. The alpha subunit also polymerized to form an oligomer, which showed almost the same S value as that of alpha beta complex and brain CaM kinase II. In contrast, the beta subunit did not polymerize. The alpha subunit, beta subunit, alpha beta complex, and brain CaM kinase II were autophosphorylated with [gamma-32P]ATP in the presence of Ca2+ and calmodulin, which resulted in the appearance of Ca2+-independent activity. The Ca2+-independent activity was 60-75% of the total activity as measured in the presence of Ca2+ plus calmodulin. To examine the functional relationship of peptide domains of the subunits of CaM kinase II, deleted cDNAs were introduced into CHO cells and the properties of the expressed proteins were studied. In cells transfected with alpha or beta cDNA from which the association domain was deleted, a significant level of kinase activity was expressed. However, the expressed proteins showed hardly any autophosphorylation and the appearance of Ca2+-independent enzyme activity was very low, indicating that the association domain was essential for the autophosphorylation and for the appearance of the Ca2+-independent activity.
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PMID:Expression and characterization of calmodulin-dependent protein kinase II from cloned cDNAs in Chinese hamster ovary cells. 255 31

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

Protein phosphatase C was purified 140-fold from bovine brain with 8% yield using histone H1 phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase (cyclic AMP-kinase). Brain protein phosphatase C was considered to consist of 10 and 90%, respectively, of the catalytic subunits of protein phosphatases 1 and 2A on the basis of the effects of ATP and inhibitor-2. Protein phosphatase C dephosphorylated microtubule-associated protein 2 (MAP2), tau factor, and tubulin phosphorylated by a multifunctional Ca2+/calmodulin-dependent protein kinase (calmodulin-kinase) and the catalytic subunit of cyclic AMP-kinase. The properties of dephosphorylation of MAP2, tau factor, and tubulin were compared. The Km values were in the ranges of 1.6-2.7 microM for MAP2 and tau factor. The Km value for tubulin decreased from 25 to 10-12.5 microM in the presence of 1.0 mM Mn2+. No difference in kinetic properties of dephosphorylation was observed between the substrates phosphorylated by the two kinases. Protein phosphatase C did not dephosphorylate the native tubulin, but universally dephosphorylated tubulin phosphorylated by the two kinases. The holoenzyme of protein phosphatase 2A from porcine brain could also dephosphorylate MAP2, tau factor, and tubulin phosphorylated by the two kinases. The phosphorylation of MAP2 and tau factor by calmodulin-kinase separately induced the inhibition of microtubule assembly, and the dephosphorylation by protein phosphatase C removed its inhibitory effect. These data suggest that brain protein phosphatases 1 and 2A are involved in the switch-off mechanism of both Ca2+/calmodulin-dependent and cyclic AMP-dependent regulation of microtubule formation.
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PMID:Dephosphorylation of microtubule proteins by brain protein phosphatases 1 and 2A, and its effect on microtubule assembly. 283 18

A calcium/calmodulin-dependent protein kinase was partially purified from mouse brain cytosol and compared to a type II calcium/calmodulin-dependent protein kinase (CaM kinase II) previously purified from rat brain. The purification (approximately 200-fold) was followed by the ability of the kinase to phosphorylate the high molecular weight microtubule-associated protein, MAP-2. Approximately 40% of the mouse brain kinase was soluble, and it contained two subunits of 50 kD and 58-60 kD. Both subunits bound [125I]calmodulin in a calcium-dependent manner and demonstrated calmodulin-dependent autophosphorylation. The subunits from whole brain were present in a molar ratio of 3/1. The apparent Km values of the kinase for ATP and calmodulin were 17 microM and 55 nM respectively. The time course, substrate specificity, and subunit phosphopeptide maps were comparable to CaM kinase II from rat brain. Regional distribution studies indicate that the enzyme activity was enriched in hippocampus, cerebral cortex and corpus striatum, whereas activity in cerebellum and pons/medulla was approximately 10-fold lower. All of these characteristics were shared with the rat brain enzyme, indicating that the kinase in mouse brain was a type II calcium/calmodulin-dependent kinase. The mouse may be useful for examining the neuronal localization of CaM kinase II in different brain regions, since this model offers a variety of genetic mutants with well-defined lesions in specific neuronal populations.
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PMID:Identification and regional distribution of a type II calcium/calmodulin-dependent kinase in mouse brain. 283 92

The purified catalytic subunit (C) of cAMP-dependent protein kinase produced a 2-fold activation of the low Km phosphodiesterase in crude microsomes (P-2 pellet) of rat adipocytes. This activation was C subunit concentration-dependent, ATP-dependent, blocked by a specific peptide inhibitor, and lost if the C subunit was first heat denatured. The concentration of ATP necessary for half-maximal activation of the low Km phosphodiesterase was 4.50 +/- 1.1 microM, which was nearly the same as the known Km of C subunit for ATP (3.1 microM) using other substrates. The concentration of C subunit producing half-maximal activation of phosphodiesterase was 0.22 +/- 0.04 microM, slightly less than the measured concentration of total C subunit in adipocytes (0.45 microM). The activation of the low Km phosphodiesterase by C subunit was specific, since on an equimolar basis, myosin light chain kinase, cGMP-dependent protein kinase, or Ca2+/calmodulin-dependent protein kinase II did not activate the enzyme. The percent stimulation of phosphodiesterase by C subunit was about the same as that produced by incubation of adipocytes with a cAMP analog, and the enzyme first activated in vivo with the analog was not activated to the same extent (on a percentage basis) by in vitro treatment with C subunit. Treatment of the crude microsomes with trypsin resulted in transfer of phosphodiesterase catalytic activity from the particulate to the supernatant fraction, but the enzyme in the supernatant was minimally activated by C subunit, suggesting either loss or dislocation of the regulatory component. The C subunit-mediated activation of phosphodiesterase was preserved after either transfer of phosphodiesterase activity to the supernatant fraction by nonionic detergents or partial purification of the transferred enzyme. The present findings are consistent with the suggestion that protein kinase regulates the concentration of cAMP through phosphodiesterase activation and provide direct evidence that the mechanism of activation involves phosphorylation.
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PMID:Activation of the particulate low Km phosphodiesterase of adipocytes by addition of cAMP-dependent protein kinase. 283 86

A monoclonal antibody, A1, was produced against sodium dodecyl sulfate-polyacrylamide gel electrophoresis purified canine phospholamban and isolated from mouse ascites by chromatography on a hydroxylapatite column. Western immunoblotting experiments showed that the antibody was specific for phospholamban and cross-reacted with the protein from a bovine source. Incubation of bovine cardiac sarcoplasmic reticulum (SR) vesicles with the antibody resulted in a marked increase in the ATP-dependent Ca2+ pump activity which was slightly higher than that brought about by cyclic AMP-dependent protein kinase. This observation provides direct proof for the involvement of phospholamban as a SR Ca2+ pump regulatory protein. In addition to stimulating the Ca2+ pump activity, antibody A1 was capable of blocking the phosphorylation of phospholamban by cyclic AMP-dependent protein kinase and by an endogenous SR Ca2+/calmodulin-dependent protein kinase. It was also capable of blocking the dephosphorylation of phosphorylated phospholamban by an endogenous SR protein phosphatase. From these observations, it may be suggested that the antigenic site of A1 antibody is proximal to the phosphorylation sites of phospholamban.
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PMID:Stimulation of bovine cardiac sarcoplasmic reticulum Ca2+ pump and blocking of phospholamban phosphorylation and dephosphorylation by a phospholamban monoclonal antibody. 293 76


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