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

• Published in: The Journal of biological chemistry Vol. 276; no. 31; pp. 29353 - 29360
• Main Authors: Singla, S I, Hudmon, A, Goldberg, J M, Smith, J L, Schulman, H
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 03.08.2001
• Subjects: Adenosine Triphosphate - metabolism; Amino Acid Sequence; Amino Acid Substitution; Animals; Binding Sites; Calcium - metabolism; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases - chemistry; Calcium-Calmodulin-Dependent Protein Kinases - metabolism; Calmodulin - chemistry; Calmodulin - genetics; Calmodulin - metabolism; Cloning, Molecular; Crystallography, X-Ray; Escherichia coli; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Phosphorylation; Protein Conformation; Rats; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Sea Urchins
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M101744200

Autophosphorylation of α-Ca 2+ /calmodulin-dependent protein kinase II (CaM kinase II) at Thr 286 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 [Ca 2+ ], the phosphorylated mutant kinase F293A and the CaM mutant E120A/M124A exhibited deficient trapping compared with wild-type. In high [Ca 2+ ], 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 Phe 293 in the kinase and Glu 120 and Met 124 in CaM specifically stabilize the trapped CaM-CaM kinase II complex. Our studies further show that Phe 293 and Asn 294 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.