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J. Biochem, 1997, Vol. 121, No. 4 804-810
© 1997 Japanese Biochemical Society

research-article

Studies on the Site of Phosphorylation of Ca2+/Calmodulin-Dependent Protein Kinase (CaM-Kinase) IV by CaM-Kinase Kinase1

Takako Kitani, Sachiko Okuno and Hitoshi Fujisawa

Department of Biochemistry, Asahikawa Medical College Asahikawa, Hokkaido 078
2Laboratory for Radioactive Isotope Research, Asahikawa Medical College.

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 Lys71 (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 Thr196. The recombinant mutant CaM-kinase IV in which Thr196 or Thr200 was replaced with nonphosphorylatable alanine showed little activity in the presence and absence of the kinase kinase. The mutant enzyme in which Thr196 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 Thr200 was replaced with aspartic acid showed little enzyme activity. Thus, it may be concluded that the phosphorylation of Thr196 in CaM-kinase IV by CaM-kinase kinase is necessary for the subsequent autophosphorylation and activation of CaM-kinase IV.

1This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, the Byotai Taisha Research Foundation, the Uehara Memorial Foundation, the Smoking Research Foundation, the Brain Science Foundation, and the Mitsubishi Foundation.


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