|Budget Amount *help
¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1995 : ¥1,900,000 (Direct Cost : ¥1,900,000)
RAC-protein kinase has a regulatory domain and a protein-serine/threonine kinase catalytic domain at its amino-and carboxyl-terminal regions, respectively. The regulatory domain, that had been regarded to contain a SH2 domain at its amino-terminal end, was revealed to have a pleckstrin homology (PH) domain. Three types of rat RAC-protein kinase, designated alpha, beta, and gamma, were cloned from rat cDNA libraries, and Northern blot analysis showed that alpha and beta are distributed widely, whereas gamma was expressed abundantly in brain and testis. Biochemical studies indicated that the specific activities of RAC-protein kinase alpha, beta, and gamma purified from transfected COS-7 cells were similar when measured by using myelin basic protein as a phosphate acceptor. Analysis using fusion proteins of glutathione S-transferase revealed that the PH domain of the three types of RAC-protein kinase associates with protein kinase C subspecies. Binding studies using deletion mutants indicated that protein kinase C interacts with the beta-sheet structure in the amino-terminal portion of the PH domain. The betagamma subunits of G proteins, that had been reported to associate with the PH domain of beta-adreenergic receptor kinase, were shown to bind to the PH domain of three RAC-PKs. Immunoprecipitation assay using cell extracts co-expressing these two protein kinases indicated that RAC-protein kinase and protein kinase C associate in cells. These results suggest that the protein-protein interaction between RAC protein kinase and protein kinase C through the PH domain may be important for the regulation of these protein kinases, and the PH domain of RAC protein kinase family associates more than one protein to regulate the activity and/or intracellular distribution of this enzyme family by different ways.