THE CATALYTIC DOMAIN OF ACANTHAMOEBA MYOSIN-I HEAVY-CHAIN KINASE .1. IDENTIFICATION AND CHARACTERIZATION FOLLOWING TRYPTIC CLEAVAGE OF THE NATIVE ENZYME

The actin-activated Mg2+-ATPase activities of the myosin I isoenzymes from Acanthamoeba castellanii are greatly increased by phosphorylation catalyzed by myosin I heavy chain kinase (MIHC kinase), a monomeric 9 7-kDa protein whose activity is greatly enhanced by acidic phospholipi ds and by autophosphorylation of multiple sites, In this paper, we sho w that the 35-kDa COOH-terminal fragment obtained by trypsin cleavage of maximally activated, autophosphorylated kinase retains the full act ivity and two to three of the autophosphorylation sites of the native enzyme, Other autophosphorylation sites occur in the middle third of t he native enzyme, A trypsin cleavage site within the 35-kDa region is protected in phosphorylated kinase but is readily cleaved in unphospho rylated kinase producing catalytically inactive 25 and 11-kDa fragment s from the NH2- and COOH-terminal ends, respectively, of the 35-kDa pe ptide, This implies that the conformation around the ''25/11'' cleavag e site changes upon phosphorylation of the native enzyme, The position of this site corresponds to the activation loop of protein kinase A ( see the accompanying paper: Brzeska, H., Szczepanowska, J., Hoey, J., and Korn, E. D. (1996) J. Biol. Chem. 271, 27056-27062), Exogenously a dded MIHC kinase phosphorylates the 11-kDa fragment, but not the 25-kD a fragment, indicating that the phosphorylation sites of the 35-kDa ca talytic fragment are located within the COOH-terminal 11 kDa, The acco mpanying paper describes the cloning, sequencing, and expression of a fully active 35-kDa catalytic domain.