Molecular Characterization of Calmodulin Trapping by Calcium/Calmodulin-dependent Protein Kinase II

Sheela I. Singla, Andy Hudmon, Jonathan M. Goldberg, Janet L. Smith, Howard Schulman

Research output: Contribution to journalArticle

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Abstract

Autophosphorylation of α-Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) at Thr286 results in calmodulin (CaM) trapping, a > 10,000-fold decrease in the dissociation rate of CaM from the enzyme. Here we present the first site-directed mutagenesis study on the dissociation of the high affinity complex between CaM and full-length CaM kinase II. We measured dissociation kinetics of CaM and CaM kinase II proteins by using a fluorescently modified CaM that is sensitive to binding to target proteins. In low [Ca2+], the phosphorylated mutant kinase F293A and the CaM mutant E120A/M124A exhibited deficient trapping compared with wild-type. In high [Ca2+], the CaM mutations E120A, M124A, and E120A/M124A and the CaM kinase II mutations F293A, F293E, N294A, N294P, and R297E increased dissociation rate constants by factors ranging from 2.3 to 116. We have also identified residues in CaM and CaM kinase II that interact in the trapped state by mutant cycle-based analysis, which suggests that interactions between Phe293 in the kinase and Glu120 and Met124 in CaM specifically stabilize the trapped CaM-CaM kinase II complex. Our studies further show that Phe293 and Asn294 in CaM kinase II play dual roles, because they likely destabilize the low affinity state of CaM complexed to unphosphorylated kinase but stabilize the trapped state of CaM bound to phosphorylated kinase.

Original languageEnglish (US)
Pages (from-to)29353-29360
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number31
DOIs
StatePublished - Aug 3 2001
Externally publishedYes

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Calcium-Calmodulin-Dependent Protein Kinase Type 2
Calcium-Calmodulin-Dependent Protein Kinases
Calmodulin
Phosphotransferases
Mutagenesis
Mutation
Site-Directed Mutagenesis
Rate constants
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Molecular Characterization of Calmodulin Trapping by Calcium/Calmodulin-dependent Protein Kinase II. / Singla, Sheela I.; Hudmon, Andy; Goldberg, Jonathan M.; Smith, Janet L.; Schulman, Howard.

In: Journal of Biological Chemistry, Vol. 276, No. 31, 03.08.2001, p. 29353-29360.

Research output: Contribution to journalArticle

Singla, Sheela I. ; Hudmon, Andy ; Goldberg, Jonathan M. ; Smith, Janet L. ; Schulman, Howard. / Molecular Characterization of Calmodulin Trapping by Calcium/Calmodulin-dependent Protein Kinase II. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 31. pp. 29353-29360.
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