THE CATALYTIC DOMAIN OF ACANTHAMOEBA MYOSIN-I HEAVY-CHAIN KINASE .2. EXPRESSION OF ACTIVE CATALYTIC DOMAIN AND SEQUENCE HOMOLOGY TO P21-ACTIVATED KINASE (PAK)

Acanthamoeba myosin I heavy chain (MIHC) kinase is a monomeric 97-kDa protein that is activated by binding to acidic phospholipids or by aut ophosphorylation. Activation by phospholipids is inhibited by Ca2+-cal modulin. In the accompanying paper (Brzeska, H., Martin, B., and Korn, E. D. (1996) J. Biol. Chem. 271, 27049-27055), we identified the cata lytic domain as the COOH-terminal 35 kDa produced by trypsin digestion of phosphorylated MIHC kinase, In this paper, we report the cloning a nd sequencing of the corresponding cDNA and expression of fully active catalytic domain. The expressed catalytic domain has substrate specif icity similar to that of native kinase and resistance to trypsin simil ar to that of fully phosphorylated MIHC kinase, MIHC kinase catalytic domain has only 25% sequence identity to the catalytic domain of prote in kinase A and similarly low sequence identity to the catalytic domai ns of protein kinase C- and calmodulin-dependent kinases, but 50% sequ ence identity and 70% similarity to the p21-activated kinase (PAK) and STE20 family of kinases. This suggests that MIHC kinase is (at least) evolutionarily related to the PAK family, whose activities are regula ted by small GTP-binding proteins, The homology includes the presence of a potential MIHC kinase autophosphorylation site as well as conserv ed Tyr and Ser/Thr residues in the region corresponding to the P+1 loo p of protein kinase A. A synthetic peptide corresponding to this regio n of MIHC kinase is phosphorylated by both the expressed catalytic dom ain and native MIHC kinase.