Molecular characterization of calmodulin trapping by calcium/calmodulin-dependent protein kinase II

Autophosphorylation of alpha -Ca2+/calmodulin-dependent protein kinase II ( CaM kinase II) at Thr(286) results in cal. modulin (CaM) trapping, a > 10,0 00-fold decrease in the dissociation rate of CaM from the enzyme. Here we p resent the first site-directed mutagenesis study on the dissociation of the high affinity complex between CaM and full-length CaM kinase IEL We measur ed dissociation kinetics of CaM and CaM kinase II proteins by using a fluor escently modified CaM that is sensitive to binding to target proteins. In l ow [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 muta tions F293A, F293E, N294A, N294P, and R297E increased dissociation rate con stants 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 cycl e-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 farther show that Phe(293) and Asn(2 94) in CaM kinase II play dual roles, because they likely destabilize the l ow affinity state of CaM complexed to unphosphorylated kinase but stabilize the trapped state of CaM bound to phosphorylated kinase.