Components of a calmodulin-dependent protein kinase cascade -: Molecular cloning, functional characterization and cellular localization of Ca2+/calmodulin-dependent protein kinase kinase β

Ca2+/calmodulin-dependent protein kinases I and IV (CaMKI and CaMKIV, respectively) require phosphorylation on an equivalent single Thr in the activation loop of subdomain MI for maximal activity. Two distinct CaMHI/IV kinases, CaMKK alpha and CaMKK beta, were purified from rat brain and partially sequenced (Edelman, A. M., Mitchelhill, K., Selbert, M. A., Anderson, K. A., Hook, S. S., Stapleton, D., Goldstein, E. G., Means, A. R., and Kemp, B. E. (1996) J. Biol. Chem. 271, 10806-10810). We report here the cloning and sequencing of cDNAs for human and rat CaMKK beta, tissue and regional brain localization of CaMKK beta protein, and mRNA and functional characterization of recombinant CaMKK beta in vitro and in Jurkat T cells. The sequences of human and rat CaMKK beta demonstrate 65% identity and 80% similarity with CaMKK alpha and 30-40% identity with CaMKI and CaMKIV themselves. CaMKK beta is broadly distributed among rat tissues with highest levels in CaMKIV-expressing tissues such as brain, thymus, spleen, and testis. In brain, CaMKK beta tracks more closely with CaMKIV than does CaMKK alpha. Bacterially expressed CaMKK beta undergoes intramolecular autophosphorylation, is regulated by Ca2+/CaM, and phosphorylates CaMKI and CaMKIV on Thr(177) and Thr(200), respectively. CaMKK beta activates both CaMKI and CaMKIV when coexpressed in Jurkat T cells as judged by phosphorylated cAMP response element-binding protein-dependent reporter gene expression. CaMKK beta activity is enhanced by elevation of intracellular Ca2+, although substantial activity is observed at the resting Ca2+ concentration. The strict Ca2+ requirement of CaMKIV-dependent phosphorylation of cAMP response element-binding protein, is therefore controlled at the level of CaMKIV rather than CaMKK.