Ca²⁺/calmodulin-dependent protein kinases (CaM-kinases) I and IV are activated upon phosphorylation of their Thr¹⁷⁷ and Thr¹⁹⁶, respectively, by the upstream Ca²⁺/calmodulin-dependent protein kinases CaM-kinase kinase α and β, and deactivated upon dephosphorylation by protein phosphatases such as CaM-kinase phosphatase. Recent studies demonstrated that the activity of CaM-kinase kinase α is decreased upon phosphorylation by cAMP-dependent protein kinase (PKA), and the relationship between the inhibition and phosphorylation of CaM-kinase kinase α by PKA has been studied. In the present study, we demonstrate that the activity of CaM-kinase kinase α toward PKIV peptide, which contains the sequence surrounding Thr¹⁹⁶ of CaM-kinase IV, is increased by incubation with PKA in the presence of Ca²⁺/calmodulin but decreased in its absence, while the activity toward CaM-kinase IV is decreased by incubation with PKA in both the presence and absence of Ca²⁺/calmodulin. Six phosphorylation sites on CaM-kinase kinase α, Ser²⁴ for autophosphorylation, and Ser⁵², Ser⁷⁴, Thr¹⁰⁸, Ser⁴⁵⁸, and Ser
for phosphorylation by PKA, were identified by amino acid sequence analysis of the phosphopeptides purified from the tryptic digest of the phosphorylated enzymes. The presence of Ca²⁺/calmodulin suppresses phosphorylation on Ser⁵², Ser⁷⁴, Thr¹⁰⁸, and Ser⁴⁵⁸ by PKA, but accelerates phosphorylation on Ser⁴⁷⁵. The changes in the activity of the enzyme upon phosphorylation appear to occur as a result of conformational changes induced by phosphorylation on several sites.