Inactivation of calmodulin-dependent protein kinase IV by autophosphorylation of serine 332 within the putative calmodulin-binding domain

Abstract

When brain calmodulin-dependent protein kinase IV is incubated with calmodulin-dependent protein kinase IV kinase under the phosphorylation conditions in the presence of Ca 2+/calmodulin, rapid initial incorporation of 1 mol of phosphate into 1 mol of the enzyme by the action of the kinase kinase occurs, resulting in marked activation of the enzyme, and the subsequent incorporation of more than 3 mol of phosphate by autophosphorylation occurs, resulting in no significant change in the activity (Okuno, S., Kitani, T., and Fujisawa, H. (1994) J. Biochem. (Tokyo) 116, 923-930; Okuno, S., Kitani, T., and Fujisawa, H. (1995) J. Biochem. (Tokyo) 117, 686-690). After the maximal phosphorylation, the continued incubation in the presence of excess EGTA resulted in additional autophosphorylation of the enzyme, leading to a complete loss of the Ca 2+/calmodulin-dependent activity, while causing no significant change in the Ca 2+/calmodulin-independent activity. The amino acid sequence analysis revealed that the autophosphorylation after removal of Ca 2+ occurred on Ser 332, Ser 333, Ser 337, and Ser 341. Analysis by site-directed mutagenesis clearly showed that the autophosphorylation site responsible for the inactivation is Ser 332. Thus, calmodulin-dependent protein kinase IV activated by the kinase kinase may lose its Ca 2+/calmodulin-dependent activity by autophosphorylation on Ser 332 located within the putative calmodulin-binding domain in the absence of Ca 2+.