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)

Purified pig brain Ca(2+)-calmodulin (CaM)-dependent protein kinase Ia kinase (Lee, J. C., and Edelman, A. M. (1994) J. Biol. Chem. 269, 2158-2164) enhances, by up to 24-fold, the activity of recombinant CaM kinase IV in a reaction also requiring Ca(2+)-CaM and MgATP. The addition of brain extract, although capable of activating CaM kinase IV by itself, provides no further activation beyond that induced by purified CaM kinase Ia kinase, consistent with the lack of a requirement of additional components for activation. Activation is accompanied by the development of significant (38%) Ca(2+)-CaM-independent CaM kinase IV activity. In parallel fashion to its activation, CaM kinase IV is phosphorylated in a CaM kinase Ia kinase-, Ca(2+)-CaM-, and MgATP-dependent manner. Phosphorylation occurs on multiple serine and threonine residues with a Ser-P:Thr-P ratio of approximately 3:1. The identical requirements for phosphorylation and activation and a linear relationship between extent of phosphorylation of CaM kinase IV and its activation state indicate that CaM kinase IV activation is induced by its phosphorylation. Replacement of Thr-196 of CaM kinase IV with a nonphosphorylatable alanine by site-directed mutagenesis abolishes both the phosphorylation and activation of CaM kinase IV, demonstrating that Thr-196 phosphorylation is essential for activation.
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PMID:Phosphorylation and activation of Ca(2+)-calmodulin-dependent protein kinase IV by Ca(2+)-calmodulin-dependent protein kinase Ia kinase. Phosphorylation of threonine 196 is essential for activation. 761 69

Human Ca(2+)-calmodulin (CaM) dependent protein kinase I (CaMKI) encodes a 370 amino acid protein with a calculated M(r) of 41,337. The 1.5 kb CaMKI mRNA is expressed in many different human tissues and is the product of a single gene located on human chromosome 3. CaMKI 1-306, was unable to bind Ca(2+)-CaM and was completely inactive thereby defining an essential component of the CaM-binding domain to residues C-terminal to 306. CaMKI 1-294 did not bind CaM but was fully active in the absence of Ca(2+)-CaM, indicating that residues 295-306 are sufficient to maintain CaMKI in an auto-inhibited state. CaMKI was phosphorylated on Thr177 and its activity enhanced approximately 25-fold by CaMKI kinase in a Ca(2+)-CaM dependent manner. Replacement of Thr177 with Ala or Asp prevented both phosphorylation and activation by CaMKI kinase and the latter replacement also led to partial activation in the absence of CaMKI kinase. Whereas CaMKI 1-306 was unresponsive to CaMKI kinase, the 1-294 mutant was phosphorylated and activated by CaMKI kinase in both the presence and absence of Ca(2+)-CaM although at a faster rate in its presence. These results indicate that the auto-inhibitory domain in CaMKI gates, in a Ca(2+)-CaM dependent fashion, accessibility of both substrates to the substrate binding cleft and CaMKI kinase to Thr177. Additionally, CaMKI kinase responds directly to Ca(2+)-CaM with increased activity.
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PMID:Human calcium-calmodulin dependent protein kinase I: cDNA cloning, domain structure and activation by phosphorylation at threonine-177 by calcium-calmodulin dependent protein kinase I kinase. 764 87

Glutamate receptor ion channels are colocalized in postsynaptic densities with Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II), which can phosphorylate and strongly enhance non-N-methyl-D-aspartate (NMDA) glutamate receptor current. In this study, CaM-kinase II enhanced kainate currents of expressed glutamate receptor 6 in 293 cells and of wild-type glutamate receptor 1, but not the Ser-627 to Ala mutant, in Xenopus oocytes. A synthetic peptide corresponding to residues 620-638 in GluR1 was phosphorylated in vitro by CaM-kinase II but not by cAMP-dependent protein kinase or protein kinase C. The 32P-labeled peptide map of this synthetic peptide appears to be the same as the two-dimensional peptide map of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) glutamate receptors phosphorylated in cultured hippocampal neurons by CaM-kinase II described elsewhere. This CaM-kinase II regulatory phosphorylation site is conserved in all AMPA/kainate-type glutamate receptors, and its phosphorylation may be important in enhancing postsynaptic responsiveness as occurs during synaptic plasticity.
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PMID:Identification of a Ca2+/calmodulin-dependent protein kinase II regulatory phosphorylation site in non-N-methyl-D-aspartate glutamate receptors. 787 86

The cAMP response element-binding protein (CREB) has been shown to mediate transcriptional activation of genes in response to both cAMP and calcium influx signal transduction pathways. The roles of two multifunctional calcium/calmodulin-dependent protein kinases, CaMKIV and CaMKII, were examined in transient transfection studies that utilized either the full-length or the constitutively active forms of these kinases. The results indicate that CaMKIV is much more potent than CaMKII in activating CREB in three different cell lines. It was also found in these studies that Ser133 of CREB is essential for its activation by CaMKIV. Because both CaMKII and CaMKIV can phosphorylate CREB, we pursued further the mechanism by which CaMKII and CaMKIV differentially regulate CREB activity. Mutagenesis studies and phosphopeptide mapping analysis demonstrated that in vitro, CaMKIV phosphorylates CREB at Ser133 only, whereas CaMKII phosphorylates CREB at Ser133 and a second site, Ser142. Transient transfection studies revealed that phosphorylation of Ser142 by CaMKII blocks the activation of CREB that would otherwise occur when Ser133 is phosphorylated. When Ser142 was mutated to alanine, CREB was activated by CaMKII, as well as by CaMKIV. Furthermore, mutation of Ser142 to alanine enhanced the ability of Ca2+ influx to activate CREB, suggesting a physiological role for the phosphorylation of Ser142 in modulation of CREB activity. These data provide evidence for a new mechanism for regulation of CREB activity involving phosphorylation of a negative regulatory site in the transcriptional activation domain. The studies also provide new insights into possible interactions between the cAMP and Ca2+ signaling pathways in the regulation of transcription. In particular, changes in intracellular Ca2+ have the potential to either inhibit or augment the ability of cAMP to stimulate transcription, depending on the presence of specific forms of Ca2+/calmodulin-dependent protein kinases.
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PMID:Differential activation of CREB by Ca2+/calmodulin-dependent protein kinases type II and type IV involves phosphorylation of a site that negatively regulates activity. 795 15

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

Autophosphorylation of CaM kinase II on Thr286 is known to occur by an intraholoenzyme mechanism, but it is not known whether this reaction is intra- or intersubunit-catalyzed in the native heteromeric enzyme containing 10-12 alpha/beta subunits. In this study inactive CaM kinase II beta subunit, generated by mutation of Lys43 to Ala, and active kinase alpha subunit were expressed separately (homomeric kinases) or co-expressed (heteromeric kinase) using the baculovirus/Sf9 cell expression system and purified on CaM-Sepharose. Ca2+/CaM-dependent autophosphorylation of heteromeric alpha/beta kinase, which activated the enzyme, produced rapid autophosphorylation on Thr286 in both the active alpha and inactive beta subunits; the latter could only occur by intersubunit catalysis. Ca2+/CaM-independent autophosphorylation of nonactivated heteromeric kinase was slow, resulted in partial loss of total kinase activity, occurred only in the alpha subunit, and existed on Thr306 but not Thr286. This result demonstrates intrasubunit catalysis of Thr306 autophosphorylation. These observations that regulatory autophosphorylations of Thr286 and Thr306 were inter- and intrasubunit-catalyzed, respectively, have important consequences for structure/function models of CaM kinase II and for involvement of CaM kinase II autophosphorylation and activation during synaptic plasticity in neural systems.
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PMID:Regulation of Ca2+/calmodulin-dependent protein kinase II by inter- and intrasubunit-catalyzed autophosphorylations. 818 49

We have expressed the rat brain Ca2+/calmodulin (CaM)-dependent protein kinase type IV in insect cells. The recombinant enzyme is produced as a single polypeptide that migrates on SDS-polyacrylamide gel electrophoresis at 61 kDa. Recombinant CaM kinase IV undergoes slow CaM-dependent autophosphorylation. The autophosphorylation of CaM kinase IV occurs on serine residues but is not accompanied by the generation of a CaM-independent activity, as previously reported for the cerebellar enzyme. Comparison of peptide and protein phosphorylation by the recombinant CaM kinase IV and the cerebellar enzyme showed differences in their catalytic activities. The deduced primary sequence of CaM kinase IV contained a domain, 315Phe-Asn-Ala-Arg-Arg-Lys-Leu-Lys323, also found in the regulatory domain of CaM kinase II alpha (residues 293-300). Truncation of CaM kinase IV at Leu313 (at a position analogous to Leu290 in CaM kinase II alpha) generated a fully active, CaM-independent enzyme. This truncated enzyme no longer bound CaM. These data confirm that CaM kinase IV demonstrates intrasteric regulation by an autoinhibitory domain and provides insight into a potentially common mechanism for the regulation of the CaM-dependent multifunctional protein kinases. A number of synthetic peptides were examined for their phosphorylation by both CaM kinase II and IV. These studies showed that several peptides derived from phospholamban were preferential substrates for CaM kinase II whereas a peptide derived from S6 ribosomal protein was selectively phosphorylated by CaM kinase IV. Kinetic analysis of several peptide substrates suggests that while both CaM kinase II and IV recognize the sequence motif represented by R-X-X-T/S, other structural features are also involved in defining the unique substrate specificity of CaM kinase IV.
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PMID:Biochemical characterization of the multifunctional Ca2+/calmodulin-dependent protein kinase type IV expressed in insect cells. 825 36

To elucidate the functional role of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in neuronal cells, we studied the phenotypic effects of overexpression of the CaM kinase II wild-type alpha subunit and a mutant enzyme alpha isoform (Ala-286), in which formation of the Ca(2+)-independent form by autophosphorylation is markedly suppressed by replacement of Thr-286 with Ala, using Neuro2a (Nb2a) and NG108-15 neuroblastoma cell lines. The cDNAs inserted into the EcoRI site of pEF321 expression vector were introduced into Nb2a and NG108-15 cells with pEF321-neo (neo). Stable clones were obtained by G418 selection. The specific activities of CaM kinase II in alpha and Ala-286 transfectants were two to four times higher than those in non-transfectants and in cells transfected with neo alone. Indirect immunofluorescence using a monoclonal antibody specific to the CaM kinase II alpha isoform revealed that CaM kinase II was mainly localized in the perikaryal and dendritic cytoplasm of the alpha and Ala-286 transfectants. Immediately after plating, Nb2a and NG108-15 cells transfected with neo, alpha and Ala-286 cDNAs appeared round. Several hours after plating, alpha transfectants showed cell flattening and initiation of neurite outgrowth, and thereafter extended numerous long and branching neurites. Numerous filopodia protruded from flat growth cones, some of which were accompanied by extensive veil formation. Non- and neo transfectants remained round. In Ala-286 transfectants, however, the phenotypic changes were remarkably less than in alpha transfectants.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Overexpression of Ca2+/calmodulin-dependent protein kinase II in Neuro2a and NG108-15 neuroblastoma cell lines promotes neurite outgrowth and growth cone motility. 838 Nov 67

Incubation of either purified rat forebrain Ca2+/calmodulin-dependent protein kinase II (CaMKII) or the purified recombinant, baculovirus-expressed mouse alpha subunit of CaMKII (Brickey, D. A., Colbran, R. J., Fong, Y.-L., and Soderling, T. R. (1990) Biochem. Biophys. Res. Commun. 173, 578-584) with EGTA, magnesium acetate, and [gamma-32P]ATP resulted in the incorporation of up to 1.0 mol of 32PO4/mol of subunit within 60 min at 30 degrees C; both serine and threonine residues became autophosphorylated. The Vmax for this basal autophosphorylation was 0.051 +/- 0.005 mol of 32PO4/mol of subunit/min, and the Km(ATP) was 145 +/- 8 microM. Vmax and Km(ATP) values for Ca2+/calmodulin-dependent autophosphorylation were determined to be 1.3 +/- 0.4 mol/mol/min and 19 +/- 2 microM, respectively. Basal autophosphorylation resulted in inactivation of Ca2+/calmodulin-dependent kinase activity toward exogenous peptide substrate; there was no measurable increase in Ca(2+)-independent CaMKII activity. Inactivation was not observed following preincubation with non-hydrolyzable ATP analogs in place of ATP. CaMKII that had been inactivated by basal autophosphorylation could be fully re-activated by incubation with the purified catalytic subunit of protein phosphatase 2A. Following basal autophosphorylation, the calmodulin-binding ability of CaMKII was also reduced, presumably accounting for the observed inactivation. Both the inactivation and the decrease in calmodulin-binding that resulted from basal autophosphorylation were abrogated by mutation of threonine 306 to alanine, but not by mutation of threonine 305 to alanine. Furthermore, mutation of threonine 306, but not threonine 305, to alanine reduced the extent of basal autophosphorylation at threonine residues. Thus, basal autophosphorylation at threonine 306 blocks calmodulin binding, resulting in inactivation of CaMKII.
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PMID:Inactivation of Ca2+/calmodulin-dependent protein kinase II by basal autophosphorylation. 838

We have previously purified and cloned rat brain Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK), and the 68-kDa recombinant CaM-KK activates in vitro both CaM-kinase IV (CaM-K IV) and CaM-K I (Tokumitsu, H., Enslen, H., and Soderling, T. R. (1995) J. Biol. Chem. 270, 19320-19324). In the present study we have determined that activation of CaM-K IV through phosphorylation of Thr196 by CaM-KK is triggered by elevated intracellular Ca2+ in intact cells and requires binding of Ca2+/CaM to both enzymes. An expressed fragment of CaM-K IV (CaM-K IV178-246), which contains the activating phosphorylation site (Thr196) but not the autoinhibitory domain or the CaM-binding domain, still required Ca2+/CaM for phosphorylation by wild-type CaM-KK. A truncated mutant of CaM-KK (CaM-KK1-434) phosphorylated CaM-K IV178-246 in a Ca2+/CaM-independent manner, but this constitutively active CaM-KK1 434 required Ca2+/CaM for phosphorylation and activation of wild-type CaM-K IV. These results demonstrate that binding of Ca2+/CaM to both CaM-K IV and CaM-KK is required for the CaM-kinase cascade. Both CaM-KK and CaM-K IV appear to have similar Ca2+/CaM requirements with EC50 values of approximately 100 nM. Studies using co-expression of CaM-K IV with CaM-KK in COS-7 cells demonstrated that CaM-KK rapidly activated both total and Ca2+/CaM-independent activities of wild-type CaM-K IV, but not the Thr196 --> Ala mutant, upon ionomycin stimulation.
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PMID:Requirements for calcium and calmodulin in the calmodulin kinase activation cascade. 862 23


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