Regulation of Ca^/Calmodulin-Dependent Protein Kinase Kinase a by cAMP-Dependent Protein Kinase: I. Biochemical Analysis1

Ca2Vcalmodulin-dependent protein kinases (CaM-kinases) I and IV are activated upon phosphorylation of their Thr177 and Thr186, respectively, by the upstream Ca2+/calmodu-lin-dependent protein kinases CaM-kinase kinase a and p, and deactivated upon dephosphorylation by protein phosphatases such as CaM-kinase phosphatase. Recent studies demonstrated that the activity of CaM-kinase kinase a is decreased upon phos-phorylation by cAMP-dependent protein kinase (PKA), and the relationship between the inhibition and phosphorylation of CaM-kinase kinase a by PKA has been studied. In the present study, we demonstrate that the activity of CaM-kinase kinase a toward PKTV peptide, which contains the sequence surrounding Thr198 of CaM-kinase IV, is increased by incubation with PKA in the presence of Ca27calmodulin 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 Ca27calmodulin. Six phosphorylation sites on CaM-kinase kinase a, Ser24 for autophosphorylation, and Ser42, Ser74, Thr108, Ser458, and Ser476 for phosphorylation by PKA, were identified by amino acid sequence analysis of the phos-phopeptides purified from the tryptic digest of the phosphorylated enzymes. The pres