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)

Rat liver L-type pyruvate kinase was phosphorylated in vitro by a Ca2+/calmodulin-dependent protein kinase purified from rabbit liver. The calmodulin (CaM)-dependent kinase catalyzed incorporation of up to 1.7 mol of 32P/mol of pyruvate kinase subunit; maximum phosphorylation was associated with a 3.0-fold increase in the K0.5 for P-enolpyruvate. This compares to incorporation of 0.7 to 1.0 mol of 32P/mol catalyzed by the cAMP-dependent protein kinase with a 2-fold increase in K0.5 for P-enolpyruvate. When [32P]pyruvate kinase, phosphorylated by the CaM-dependent protein kinase, was subsequently incubated with 5 mM ADP and cAMP-dependent protein kinase (kinase reversal conditions), 50-60% of the 32PO4 was removed from pyruvate kinase, but the K0.5 for P-enolpyruvate decreased only 20-30%. Identification of 32P-amino acids after partial acid hydrolysis showed that the CaM-dependent protein kinase phosphorylated both threonyl and seryl residues (ratio of 1:2, respectively) whereas the cAMP-dependent protein kinase phosphorylated only seryl groups. The two phosphorylation sites were present in the same 3-4-kDa CNBr fragment located near the amino terminus of the enzyme subunit. These results indicate that the CaM-dependent protein kinase catalyzed phosphorylation of L-type pyruvate kinase at two discrete sites. One site is apparently the same serine which is phosphorylated by the cAMP-dependent protein kinase. The second site is a unique threonine residue whose phosphorylation also inactivates pyruvate kinase by elevating the K0.5 for P-enolpyruvate. These results may account for the Ca2+-dependent phosphorylation of pyruvate kinase observed in isolated hepatocytes.
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PMID:Phosphorylation of L-type pyruvate kinase by a Ca2+/calmodulin-dependent protein kinase. 299 54

Purified phospholamban isolated from canine cardiac sarcoplasmic reticulum vesicles was subjected to proteolysis and peptide mapping to localize the different sites of phosphorylation on the protein and to gain further information on its subunit structure. Five different proteases (trypsin, papain, chymotrypsin, elastase, and Pronase) degraded the oligomeric 27-kDa phosphoprotein into a major 21-22-kDa protease-resistant fragment. No 32P was retained by this protease-resistant fragment, regardless of whether phospholamban had been phosphorylated by cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase, or protein kinase C. Phosphoamino acid analysis and thin-layer electrophoresis of liberated phosphopeptides revealed that 1 threonine and 2 serine residues were phosphorylated in phospholamban and that 1 of these serine residues and the threonine residue were in close proximity. Only serine was phosphorylated by cAMP-dependent protein kinase, whereas Ca2+-calmodulin-dependent protein kinase phosphorylated exclusively threonine. The results demonstrate that phospholamban has a large protease-resistant domain and a smaller protease-sensitive domain, the latter of which contains all of the sites of phosphorylation. The 21-22-kDa protease-resistant domain, although devoid of incorporated 32P, was completely dissociated into identical lower molecular weight subunits by boiling in sodium dodecyl sulfate, suggesting that this region of the molecule promotes the relatively strong interactions that hold the subunits together. The data presented lend further support for a model of phospholamban structure in which several identical low molecular weight subunits are noncovalently bound to one another, each containing one site of phosphorylation for cAMP-dependent protein kinase and another site of phosphorylation for Ca2+/calmodulin-dependent protein kinase.
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PMID:Proteolytic cleavage of phospholamban purified from canine cardiac sarcoplasmic reticulum vesicles. Generation of a low resolution model of phospholamban structure. 300 93

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

The amino acid sequence of the Alzheimer disease amyloid precursor (ADAP) has been deduced from the corresponding cDNA, and hydropathy analysis of the sequence suggests a receptor-like structure with a single transmembrane domain. The putative cytoplasmic domain of ADAP contains potential sites for serine and threonine phosphorylation. In the present study, synthetic peptides derived from this domain were used as model substrates for various purified protein kinases. Protein kinase C rapidly catalyzed the phosphorylation of a peptide corresponding to amino acid residues 645-661 of ADAP [ADAP peptide(645-661)] on Ser-655. Ca2+/calmodulin-dependent protein kinase II phosphorylated ADAP peptide (645-661) on Thr-654 and Ser-655. This peptide was virtually ineffective as a substrate for cAMP-dependent protein kinase, cGMP-dependent protein kinase, casein kinase II, or insulin receptor protein-tyrosine kinase. When a homogenate of rat cerebral cortex was used as the source of protein kinase, phosphorylation of ADAP peptide(645-661) was stimulated by calcium/phosphatidylserine/diolein to a level 4.6-fold above the basal level of phosphorylation, consistent with a prominent stimulation by protein kinase C. Using rat cerebral cortex synaptosomes prelabeled with 32Pi, a 32P-labeled phosphoprotein of approximately equal to 135 kDa was immunoprecipitated by using antisera prepared against ADAP peptide(597-624), consistent with the possibility that the holoform of ADAP in rat brain is a phosphoprotein. Based on analogy with the effect of phosphorylation by protein kinase C of juxtamembrane residues in the cytoplasmic domain of the epidermal growth factor receptor and the interleukin 2 receptor, phosphorylation of ADAP may target it for internalization.
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PMID:Phosphorylation of Alzheimer disease amyloid precursor peptide by protein kinase C and Ca2+/calmodulin-dependent protein kinase II. 313 67

The phosphorylation of synthetic peptides derived from the NH2-terminal sequence of smooth-muscle myosin was studied with purified protein kinase C. The protein kinase C phosphorylation domain included both serine residues and threonine residues in the sequence SSKRAKAKTTKKR(G), denoted myosin light chain (1-13) (MLC(1-13)). Kinetic analysis of MLC(1-13) and truncated peptides derived from the parent peptide established that removal of the serine residues had little effect on protein kinase C reactivity. MLC(1-13) had a V/K of 2.4 min-1.mg-1, whereas the V/K of MLC(3-13) was 3.0 min-1.mg-1. Removal of Lys-3 resulted in a 50% decrease in V/K which was attributable to a 50% decrease in apparent Vmax.Arg-4 was established as a significant protein kinase C specificity determinant, since the apparent Km increased 7-fold and the Vmax decreased 3-fold when the parent peptide was truncated at that residue. All peptides studied required calcium and lipid effectors for full activity with protein kinase C, indicating that they are Class C substrates as defined by Bazzi and Nelsestuen (Biochemistry 26 (1987) 5002) for protein kinase C. Other protein kinases, including cyclic AMP- and cyclic GMP-dependent protein kinase, S6/H4 kinase, myosin light-chain kinase and calcium/calmodulin-dependent kinase II, had little or no activity with these peptides. In studies on the purification of lymphosarcoma protein kinase C by several chromatographic procedures, the results showed that the myosin light-chain peptides can provide convenient and well-characterized substrates for purification and mechanistic studies of protein kinase C biochemistry.
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PMID:Synthetic peptides derived from the nonmuscle myosin light chains are highly specific substrates for protein kinase C. 317 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

Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) autophosphorylated under limiting conditions (7 microM [gamma-32P]ATP, 500 microM magnesium acetate, 4 degrees C) was analyzed by CNBr cleavage and peptide mapping to determine the site of autophosphorylation that brings about transition of the kinase to the Ca2+-independent form. Reverse phase high performance liquid chromatography (HPLC) (C3) revealed one major CN-Br 32P-peptide (CB1) that eluted at about 6% propanol. This peptide contained [32P]threonine, but almost no [32P]serine, and migrated as a single band (Mr = 3000-3500) in polyacrylamide gels run in the presence of urea and sodium dodecyl sulfate. The properties of CB1 were compared to the properties of a 26-residue synthetic peptide containing the CaM-binding and inhibitory domains as well as a consensus phosphorylation sequence (-Arg-Gln-Glu-Thr-) of rat brain CaM-kinase II (residues 282-307 and 283-308 of the alpha and beta subunits, respectively). CB1 and the synthetic peptide comigrated in urea/sodium dodecyl sulfate gels, co-eluted from reverse phase HPLC (C3 and C18) and from Sephadex G-50, and exhibited Ca2+-dependent calmodulin-binding properties. When the two peptides were subjected to automated Edman sequence analysis, both exhibited a burst of 32P release at cycle 5, which is consistent with the expected amino-terminal sequence of the two peptides, i.e. His-Arg-Gln-Glu-Thr(PO4)-. These findings indicate that autophosphorylation of Thr286 (alpha subunit) and Thr287 (beta subunit) is responsible for transition of CaM-kinase II to the Ca2+-independent form.
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PMID:Ca2+/calmodulin-dependent protein kinase II. Identification of a regulatory autophosphorylation site adjacent to the inhibitory and calmodulin-binding domains. 341 68

Ca2+/calmodulin-dependent protein kinase II contains two types of subunit, alpha (Mr 50,000) and beta (Mr 60,000/58,000), both of which undergo Ca2+/calmodulin-dependent autophosphorylation. Autophosphorylation is known to convert the enzyme to a Ca2+/calmodulin-independent form. In the present study, we have characterized the autophosphorylation sites on rat forebrain Ca2+/calmodulin-dependent protein kinase II that are most likely to be responsible for the generation of Ca2+/calmodulin-independence. Under conditions (0 degree C, low concentrations of ATP) sufficient to generate close to maximal Ca2+/calmodulin-independence, only a few of the phosphorylatable sites on the enzyme became phosphorylated. These autophosphorylation sites were examined by phospho amino acid analysis, two-dimensional thermolytic phosphopeptide mapping, and high-performance liquid chromatography. The time course of phosphorylation of threonine in both alpha and beta subunits was similar to the time course of the generation of Ca2+/calmodulin-independence. Moreover, the time course of phosphorylation of one set of peptides, referred to as peptide 1/1', present in both alpha and beta subunits was similar to the time course of the generation of Ca2+/calmodulin-independence. Threonine was the only amino acid phosphorylated in peptide 1/1'. An additional peptide, referred to as peptide 2, was phosphorylated in the beta subunit. The time course of phosphorylation of peptide 2, which also contained only phosphothreonine, did not parallel the time course of the generation of Ca2+/calmodulin-independence. It is likely that the phosphorylation of a threonine residue on peptide 1/1' is responsible for the generation of Ca2+/calmodulin-independence of Ca2+/calmodulin-dependent protein kinase II.
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PMID:Ca2+/calmodulin-dependent protein kinase II: identification of autophosphorylation sites responsible for generation of Ca2+/calmodulin-independence. 347 99

We have used oligonucleotide probes, based on a portion of the p60v-src autophosphorylation sequence, Glu-Asp-Asn-Glu-Tyr-Thr, to identify and characterize a cDNA from the human T-leukemia cell line, JURKAT. The JURKAT cDNA (designated ptk-JURKAT) was homologous to but distinct from the src, yes and fgr oncogenes, which encode protein-tyrosine kinases (ATP:protein phosphotransferase, EC 2.7.1.37). The ptk-JURKAT cDNA hybridized with a 2.2 kb RNA transcript from JURKAT cells and the human T-cell lymphoma line, MOLT-4, but failed to identify any transcript in two human B-cell lymphoma lines or a human erythroid-myeloid leukemia line, K562. Recently the nucleotide sequence has been established for the murine lymphocyte protein tyrosine kinase, p56LSTRA. The ptk-JURKAT cDNA appears to encode the human homolog of p56LSTRA.
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PMID:Human T lymphocytes express a protein-tyrosine kinase homologous to p56LSTRA. 348 86

The phosphorylation state of six cytoplasmic proteins is increased following treatment of isolated rat hepatocytes with hormones that elevate free intracellular Ca2+ levels (Garrison, J. C. and Wagner, J. D. (1982) J. Biol. Chem. 257, 13135-13143). Tryptic 32P-phosphopeptide maps of two of the substrates, pyruvate kinase and a 49,000-dalton protein, the major 32P-labeled protein in hepatocytes, were prepared following stimulation of cells with vasopressin, a Ca2+-linked hormone. Peptide maps of the 49,000-dalton protein phosphorylated in vitro with the recently identified multifunctional Ca2+/calmodulin-dependent protein kinase contained phosphopeptides identical to those observed in the intact cell, suggesting that this kinase is activated in response to Ca2+-mobilizing hormones. Similar in vitro phosphorylation experiments with pyruvate kinase suggested that the Ca2+/calmodulin-dependent protein kinase can phosphorylate not only the serine residues observed following vasopressin stimulation of the intact cell but also additional threonine residues. Both pyruvate kinase and the 49,000-dalton protein are also phosphorylated in the hepatocyte in response to glucagon and in vitro by the cAMP-dependent protein kinase. Both vasopressin and glucagon appear to stimulate the phosphorylation of identical serine residues in pyruvate kinase but only vasopressin enhances the phosphorylation of certain sites in the 49,000-dalton protein. Comparison of the tryptic phosphopeptide maps of these substrates phosphorylated in vitro with either the Ca2+/calmodulin-dependent protein kinase or the cAMP-dependent protein kinase suggests that the Ca2+-dependent kinase can phosphorylate unique sites in both substrates. It appears to share specificity at other sites with the cAMP-dependent protein kinase. Overall, the results suggest that the multifunctional Ca2+/calmodulin-dependent protein kinase plays an important role in the response of the hepatocyte to a Ca2+ signal.
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PMID:Evidence for the activation of the multifunctional Ca2+/calmodulin-dependent protein kinase in response to hormones that increase intracellular Ca2+. 361 Oct 57


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