Inactivation of Ca2+/calmodulin-dependent protein kinase II by Ca2+/calmodulin.
Incubation of calmodulin-dependent protein kinase II with Ca2+ and calmodulin resulted in a marked inactivation of the enzyme. Chelation of Ca2+ by EGTA or addition of calmodulin antagonists, W-7 or trifluoperazine, completely blocked this inactivation. The concentration required for the half-maximal inactivation, 127 microM, is three to four orders of magnitude higher than that for the half-maximal activation of the enzyme. The Ca2+/calmodulin-independent activity of the proteolytic fragment of the enzyme, whose calmodulin-binding site involved in the enzyme activation was deleted, was also decreased by incubation with Ca2+ and calmodulin. These results suggest that calmodulin-dependent protein kinase II possesses a second, low-affinity calmodulin-binding site, which is distinct from the calmodulin-binding site involved in the activation of the enzyme, and that the binding of calmodulin to the second binding site causes the inactivation of the enzyme. The inactivation by Ca2+/calmodulin was temperature-dependent. The addition of both 500 microM ADP and 10 mM MgCl2 markedly protected the enzyme against the inactivation, while such a marked protection was not observed after the addition of either of the two alone. The addition of 5 microM autocamtide-2, a synthetic substrate peptide containing the amino acid sequence of the autophosphorylation site (Thr286/Thr287 in alpha/beta, gamma, and delta isoforms) lying within the autoinhibitory domain, also protected the enzyme against the inactivation by Ca2+/calmodulin, while syntide-2, another synthetic substrate peptide corresponding to a phosphorylation site of glycogen synthase, did not protect it even at a concentration as high as 304 microM.
Ishida
A
A
Department of Biochemistry, Asahikawa Medical College, Hokkaido.
Kitani
T
T
Okuno
S
S
Fujisawa
H
H
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors*; D002147:Calmodulin / Q000494:pharmacology*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D036341:Intercellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins / Q000494:pharmacology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000037:antagonists & inhibitors; D010455:Peptides / Q000494:pharmacology; D051381:Rats; D013379:Substrate Specificity; D013552:Swine
D002147:Calmodulin; D036341:Intercellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins; D010446:Peptide Fragments; D010455:Peptides; C085903:autocamtide-2; C063391:syntide-2; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium