Studies on the site of phosphorylation of Ca2+/calmodulin-dependent protein kinase (CaM-kinase) IV by CaM-kinase kinase

The phosphorylation site(s) involved in the activation of CaM-kinase IV by CaM-kinase kinase alpha was studied using a mutant CaM-kinase IV (K71R) in which Lys(71) (ATP-binding site) was replaced with Arg, because the autophosphorylation of CaM-kinase IV occurring at multiple sites made it difficult to study phosphorylation of the enzyme by CaM-kinase kinase, Sequence analysis of the phosphopeptide from the trypsin digest of CaM-kinase IV (K71R) phosphorylated by CaM-kinase kinase alpha suggested that the phosphorylation of CaM-kinase IV by CaM-kinase kinase only occurred at Thr(196). The recombinant mutant CaM-kinase IV in which Thr(196) Or Thr(200) was replaced with nonphosphorylatable alanine showed little activity in the presence and absence of the kinase kinase, The mutant enzyme in which Thr(196) was replaced with negatively charged aspartic acid showed almost 25 times as high activity as the wild-type enzyme in the absence of the kinase kinase, and no more activation was observed in its presence, In contrast, the enzyme in which Thr(200) was replaced with aspartic acid showed little enzyme activity, Thus, it may be concluded that the phosphorylation of Thr(196) in CaM-kinase IV by CaM-kinase kinase is necessary for the subsequent autophosphorylation and activation of CaM-kinase IV.