CHARACTERIZATION OF A CA2+ CALMODULIN-DEPENDENT PROTEIN-KINASE CASCADE - MOLECULAR-CLONING AND EXPRESSION OF CALCIUM/CALMODULIN-DEPENDENT PROTEIN-KINASE KINASE/

Recent studies have demonstrated that Ca2+/calmodulin dependent protei n kinase IV (CaM-kinase IV) can mediate Ca2+-dependent regulation of g ene expression through the phosphorylation of transcriptional activati ng proteins, We have previously identified and purified a 68-kDa rat b rain CaM-kinase kinase that phosphorylates and increases total and Ca2 +-independent activities of CaM-kinase IV (Tokumitsu, H., Brickey, D. A., Gold, J., Hidaka, H., Sikela, J., and Soderling, T. R. (1994) J. B iol. Chem, 269, 28640-28647), Using a partial amino acid sequence of t he purified brain kinase, a CaM-kinase kinase cDNA was cloned from a r at brain cDNA library, Northern blot analysis showed that CaM-kinase k inase mRNA (3.4 kilobases) was expressed in rat brain, thymus, and spl een. Sequence analyses revealed that the cDNA encoded a 505-amino acid protein, which contained consensus protein kinase motifs and was 30-4 0% homologous with members of the CaM-kinase family, Expression of the cDNA in COS-7 cells yielded an apparent 68-kDa CaM-binding protein, w hich catalyzed in vitro activation in the presence of Mg2+/ATP and Ca2 +/CaM of CaM-kinases I and IV but not of CaM-kinase II. Co-expression of CaM-kinase kinase with CaM-kinase IV gave a 14-fold enhancement of cAMP-response element-binding protein-dependent gene expression compar ed with CaM-kinase IV alone. These results are consistent with the hyp othesis that CaM-kinases I and IV are regulated through a unique signa l transduction cascade involving CaM-kinase kinase.