Mutational analysis of the autoinhibitory domain of calmodulin kinase II

Debra A. Brickey, James G. Bann, Yiu Lian Fong, Lilly Perrino, Richard G. Brennan, Thomas R. Soderling

Research output: Contribution to journalArticlepeer-review

65 Scopus citations


Calmodulin (CaM)-kinase II is inactive in the absence of Ca2+/CaM due to interaction of its autoinhibitory domain with its catalytic domain. Previous studies using synthetic autoinhibitory domain peptides (residues 281-302) identified several residues as important for inhibitory potency and suggested that His282 may interact with the ATP-binding motif of the catalytic domain. To further examine the autoinhibitory domain, site-specific mutants were expressed using the baculovirus/Sf9 cell system. The purified mutants had many biochemical properties identical to wild-type kinase, but mutants H282Q, H282R, R283E, and T286D had 10-20% constitutive Ca2+-independent activities, indicating that these residues are involved in the autoinhibitory interaction. The Ca2+-independent activities of the H282Q, H282R, and R283E mutants exhibited 10-fold lower K(m) values for ATP than the wild-type kinase. Wild-type and mutant kinases, except T286A and T286D, generated Ca2+ independence upon autophosphorylation in the presence of Ca2+/CaM, and those mutants having constitutive Ca2+ independence also exhibited enhanced Ca2+/CaM-independent autophosphorylation. This Ca2+-independent autophosphorylation resulted in a decrease in total kinase activity, but there was little increase in Ca2+-independent activity, consistent with autophosphorylation of predominantly Thr306 rather than Thr286. These results are consistent with an inhibitory interaction of His282 and possibly Arg283 with the ATP-binding motif of the catalytic domain, and they indicate that constitutively active CaM-kinase II cannot autophosphorylate on Thr286 in the absence of bound Ca2+/CaM. Based on these and other biochemical characterizations, we propose a molecular model for the interaction of a bisubstrate autoinhibitory domain with the catalytic domain of CaM-kinase II.

Original languageEnglish (US)
Pages (from-to)29047-29054
Number of pages8
JournalJournal of Biological Chemistry
Issue number46
StatePublished - Nov 18 1994
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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