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 cDNAs encoding the alpha and beta subunits of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) were ligated into the bacterial expression vector pET and expressed in Escherichia coli. The bacterially expressed alpha and beta subunits exhibited Ca2+/calmodulin-dependent activity and were easily purified to apparent homogeneity from cell extracts. To determine the minimum size required for catalytic activity and the properties of the calmodulin-binding domain, mutated CaM kinase II cDNAs were expressed in E. coli and the enzymatic property of expressed proteins was examined. The replacement of Thr-286 of the alpha subunit with the negatively charged amino acid Asp or that of Arg-283 with the neutral amino acid Gly induced the partially Ca2+ independent activity. The mutant enzymes alpha-I(delta 283-478) and alpha-II(delta 359-478), which truncated the C-terminal region of the alpha subunit, exhibited CaM kinase II activity and the activities of alpha-I(delta 283-478) and alpha-II(delta 359-478) were completely independent of and partially dependent on Ca2+ and calmodulin, respectively. However, the truncated protein alpha(delta 250-478), which was only 33 amino acids shorter than the alpha-I(delta 283-478) protein had no enzymatic activity, indicating that alpha-I(delta 283-478) was close to the minimum size of the active form. The mutant enzyme alpha(delta 291-315), which lacked the calmodulin-binding domain exhibited Ca2+ independent activity. The molecular mass was, however, smaller than that expected from the amino acid sequence. The mutant enzyme alpha(delta 304-315), which lacked the C-terminal half of the calmodulin-binding domain of the alpha subunit, however, exhibited Ca(2+)-independent activity without a reduction in molecular size, indicating that residues 304-315 of the alpha subunit constituted the core calmodulin-binding domain.
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PMID:Studies on the regulatory domain of Ca2+/calmodulin-dependent protein kinase II by expression of mutated cDNAs in Escherichia coli. 165 35

An endogenous 95 kDa chick embryo cytosolic protein (p95) was phosphorylated in the presence of [gamma-32P]ATP and the kinase activity for p95 was mostly associated with particulate fraction. Phosphorylation of p95 was prominent in embryos of early developmental stage. Hydrolysis of p95 phosphoprotein yielded phosphotyrosine in addition to phosphothreonine and phosphoserine. Native p95 was also tyrosine-phosphorylated. p95 phosphoprotein was purified by DEAE-Sephacel chromatography and immunoprecipitation with anti-phosphotyrosine antibody and the amino acid sequence was determined. The N-terminal sequence, Val-Asn-Phe-Thr-Val-Asp-Gln-Ile-Arg-Ala-Ile-Met-Asp- Lys-Lys-Ala-Asn-Ile-Arg-Asn-Met-, was found to be identical to those of elongation factor-2 (EF-2) of both rat and hamster. Our results suggest the presence of other EF-2 kinase in chick embryo cell than the previously reported Ca2+/calmodulin-dependent protein kinase III.
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PMID:Elongation factor-2 in chick embryo is phosphorylated on tyrosine as well as serine and threonine. 170 37

A purified bovine lung cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) was rapidly phosphorylated by purified bovine lung cGMP-dependent protein kinase (cGK). Within a physiological concentration range, cGK catalyzed phosphorylation of cG-BPDE at a rate approximately 10 times greater than did equimolar concentrations of purified catalytic subunit of cAMP-dependent protein kinase (cAK). cG-BPDE was a poor substrate for either purified protein kinase C or Ca2+/calmodulin-dependent protein kinase II. Binding of cGMP to the cG-BPDE binding site was required for phosphorylation since (a) phosphorylation of cG-BPDE by the catalytic subunit of cAK was cGMP-dependent, (b) phosphorylation of cG-BPDE in the presence of a cGMP analog specific for activation of cGK was cGMP-dependent, and (c) occupation of the cG-BPDE hydrolytic site with competitive inhibitors did not produce the cGMP-dependent effect. cGMP-dependent phosphorylation of cG-BPDE by both cGK and cAK occurred at serine. Proteolytic digestion of cG-BPDE phosphorylated by either cGK or cAK revealed the same phosphopeptide pattern, suggesting that phosphorylation by the two kinases occurred at the same or adjacent site(s). Tryptic digestion of cG-BPDE phosphorylated by cGK and [gamma-32P]ATP produced a single major phosphopeptide of approximately 2 kDa with the following amino-terminal sequence: Lys-Ile-Ser-Ala-Ser-Glu-Phe-Asp-Arg-Pro-Leu-Arg- Radioactivity was released during the third cycle of Edman degradation. cG-BPDE is one of few specific in vitro cGK substrates of known function to be identified. Elevation of intracellular cGMP may cause phosphorylation of cG-BPDE by modulating the substrate site availability as well as by activating cGK. Such regulation would greatly increase the selectivity of the phosphorylation of cG-BPDE and would represent a unique mechanism of action of a cyclic nucleotide or other second messenger.
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PMID:Substrate- and kinase-directed regulation of phosphorylation of a cGMP-binding phosphodiesterase by cGMP. 216 96

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

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 major substrate for Ca2+/calmodulin-dependent protein kinase III in mammalian cells is a species of Mr 100,000 that has a primarily cytoplasmic localization. This substrate has now been identified as elongation factor-2 (EF-2), a protein that catalyzes the translocation of peptidyl-tRNA on the ribosome. The amino acid sequence of 18 residues from the N-terminal of the Mr 100,000 CaM-dependent protein kinase III substrate purified from rat pancreas was found to be identical to the N-terminal sequence of authentic rat EF-2 as previously deduced from nucleic acid sequencing of a cDNA (Kohno, K., Uchida, T., Ohkubo, H., Nakanishi, S., Nakanishi, T., Fukui, T., Ohtsuka, E., Ikehara, M., and Okada, Y. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4978-4982). CaM-dependent protein kinase III phosphorylated EF-2 in vitro with a stoichiometry of approximately 1 mol/mol on a threonine residue. Amino acid sequencing of the purified tryptic phosphopeptide revealed that this threonine residue lies within the sequence: Ala-Gly-Glu-Thr-Arg-Phe-Thr-Asp-Thr-Arg (residues 51-60 of EF-2). The Mr 100,000 protein was stoichiometrically ADP-ribosylated in vitro by the addition of diphtheria toxin and NAD. The Mr 100,000 protein was photoaffinity labeled with a GTP analog and the protein had an endogenous GTPase activity that could be stimulated by the addition of salt-washed ribosomes. These properties are all characteristic of EF-2. Dephospho-EF-2 could support poly(U)-directed polyphenylalanine synthesis in a reconstituted elongation system when combined with EF-1. In the same system, phospho-EF-2 was virtually inactive in supporting polypeptide synthesis; this effect could be reversed by dephosphorylation of phospho-EF-2. These results suggest that intracellular Ca2+ inhibits protein synthesis in mammalian cells via CaM-dependent protein kinase III-catalyzed phosphorylation of EF-2.
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PMID:Identification of the major Mr 100,000 substrate for calmodulin-dependent protein kinase III in mammalian cells as elongation factor-2. 369 53

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

We assessed hippocampal-dependent memory in mice with a Ca(2+)-independent form of CaMKII generated by the introduction of an aspartate at amino acid 286. The CaMKII-Asp-286 mice show normal LTP at high frequency stimulation, but in the 5-10 Hz range, they show a shift in the frequency-response curve favoring LTD. This range of frequencies is similar to the theta rhythm, which is associated with exploration in rodents. Using the Barnes maze to assess spatial memory, we found the transgenic mice could not learn to navigate to a specific location using spatial cues. In contrast, one line of transgenic mice performed normally in contextual fear conditioning, a task that is also hippocampal dependent. This dissociation between spatial and contextual memory suggests that even though both require the hippocampus, they may be mediated by different synaptic mechanisms.
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PMID:Impairment of spatial but not contextual memory in CaMKII mutant mice with a selective loss of hippocampal LTP in the range of the theta frequency. 778 Oct 67

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

The substrate recognition determinants of Ca2+-calmodulin-dependent protein kinase (CaMK) IV and CaMKIIalpha were investigated using peptide substrates modeled on the amino acid sequence encompassing Ser-9 of synapsin I. For both kinases, hydrophobic residues (Leu or Phe) at the -5 position, are well tolerated, whereas non-hydrophobic residues (Arg, Ala, or Asp) decrease Vmax/Km by 55- to >4000-fold. At the -3 position, substitution of Ala for Arg leads to decreases of 99- and 343- fold in Vmax/Km for CaMKIV and CaMKIIalpha, respectively. For both kinases, the nature of the residues occupying the -4, -1, and + 4 positions exerts relatively little influence on phosphorylation kinetics. CaMKIV and CaMKIIalpha respond differently to substitutions at the -2 and +1 positions. Substitution of Arg at the -2 position with non-basic residues (Gln or Ala) leads to 6-fold decreases in Vmax/Km for CaMKIV, but 17-28-fold increases for CaMKIIalpha. Additionally, peptides containing Leu, Asp, or Ala at the +1 position are phosphorylated with similar efficiencies by CaMKIV, whereas the Leu-substituted peptide is preferred by CaMKIIalpha (by a factor of 5.8-9.7-fold). Thus, CaMKIV and CaMKIIalpha preferentially phosphorylate substrates with the motifs: Hyd-X-Arg-X-X-Ser*/Thr*, and Hyd-X-Arg-NB-X-Ser*/Thr*-Hyd, respectively, where Hyd represents a hydrophobic, X any, and NB a non-basic amino acid residue. The different specificities of the two kinases may contribute to their targeting to distinct physiological substrates during Ca2+-dependent cellular events.
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PMID:Definition of optimal substrate recognition motifs of Ca2+-calmodulin-dependent protein kinases IV and II reveals shared and distinctive features. 945 27


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