Activation of type II calcium/calmodulin-dependent protein kinase by Ca^(2+)/calmodulin is inhibited by autophosphorylation of threonine within the calmodulin-binding domain

It is now well established that autophosphorylation of a threonine residue located next to each calmodulin-binding domain in the subunits of type II Ca^(2+)/calmodulin-dependent protein kinase causes the kinase to remain active, although at a reduced rate, after Ca^(2+) is removed from the reaction. This autophosphorylated form of the kinase is still sensitive to Ca2+/calmodulin, which is required for a maximum catalytic rate. After removal of Ca^(2+), new sites are autophosphorylated by the partially active kinase. Autophosphorylation of these sites abolishes sensitivity of the kinase to Ca^(2+)/calmodulin (Hashimoto, Y., Schworer, C. M., Colbran, R. J., and Soderling, T. R. (1987) J. Biol. Chem. 262, 8051-8055). We have identified two pairs of homologous residues, Thr^(305) and Ser^(314) in the alpha subunit and Thr^(306) and Ser^(315) in the beta subunit, that are autophosphorylated only after removal of Ca^(2+) from an autophosphorylation reaction. The sites were identified by direct sequencing of labeled tryptic phosphopeptides isolated by reverse-phase high pressure liquid chromatography. Thr^(305-306) is rapidly dephosphorylated by purified protein phosphatases 1 and 2A, whereas Ser^(314-315) is resistant to dephosphorylation. We have shown by selective dephosphorylation that the presence of phosphate on Thr^(305-306) blocks sensitivity of the kinase to Ca^(2+)/calmodulin. In contrast, the presence of phosphate on Ser^(314-315) is associated with an increase in the Kact for Ca^(2+)/calmodulin of only about 2-fold, producing a relatively small decrease in sensitivity to Ca^(2+)/calmodulin.