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 Soderling

Research output: Contribution to journalArticle

64 Citations (Scopus)

Abstract

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
Volume269
Issue number46
StatePublished - Nov 18 1994
Externally publishedYes

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Calcium-Calmodulin-Dependent Protein Kinases
Calmodulin
Catalytic Domain
Phosphotransferases
Adenosine Triphosphate
Sf9 Cells
Molecular Models
Baculoviridae
Peptides

ASJC Scopus subject areas

  • Biochemistry

Cite this

Brickey, D. A., Bann, J. G., Fong, Y. L., Perrino, L., Brennan, R. G., & Soderling, T. (1994). Mutational analysis of the autoinhibitory domain of calmodulin kinase II. Journal of Biological Chemistry, 269(46), 29047-29054.

Mutational analysis of the autoinhibitory domain of calmodulin kinase II. / Brickey, Debra A.; Bann, James G.; Fong, Yiu Lian; Perrino, Lilly; Brennan, Richard G.; Soderling, Thomas.

In: Journal of Biological Chemistry, Vol. 269, No. 46, 18.11.1994, p. 29047-29054.

Research output: Contribution to journalArticle

Brickey, DA, Bann, JG, Fong, YL, Perrino, L, Brennan, RG & Soderling, T 1994, 'Mutational analysis of the autoinhibitory domain of calmodulin kinase II', Journal of Biological Chemistry, vol. 269, no. 46, pp. 29047-29054.
Brickey DA, Bann JG, Fong YL, Perrino L, Brennan RG, Soderling T. Mutational analysis of the autoinhibitory domain of calmodulin kinase II. Journal of Biological Chemistry. 1994 Nov 18;269(46):29047-29054.
Brickey, Debra A. ; Bann, James G. ; Fong, Yiu Lian ; Perrino, Lilly ; Brennan, Richard G. ; Soderling, Thomas. / Mutational analysis of the autoinhibitory domain of calmodulin kinase II. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 46. pp. 29047-29054.
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