Myosin I heavy chain kinase: Cloning of the full-length gene and acidic lipid-dependent activation by Rac and Cdc42

Acanthamoeba myosin I heavy chain kinase (MIHCK) phosphorylates the heavy c hains of amoeba myosins I, increasing their actin-activated ATPase activiti es. The activity of MIHCK is increased by binding to acidic phospholipids o r membranes and by autophosphorylation at multiple sites. Phosphorylation a t a single site is necessary and sufficient for full activation of the expr essed catalytic domain, The rate of autophosphorylation of native MIHCK is controlled by a region N-terminal to the catalytic domain, By its substrate specificity and the sequence of its C-terminal catalytic domain, MIHCK was identified as a p21-activated kinase (PAK). We have now cloned the full-le ngth genomic DNA and cDNA of MIHCK and have shown it to contain the conserv ed p21-binding site common to many members of the PAK family, Like some mam malian PAKs, MIHCK is activated by Rac and Cdc42, and this activation is GT P-dependent and accompanied by autophosphorylation, In contrast to mammalia n PAKs, activation of MIHCK by Rac and Cdc42 requires the presence of acidi c lipids. Also unlike mammalian PAK, MIHCK is not activated by sphingosine or other non-negatively charged lipids, The acidic lipid-binding site is ne ar the N terminus followed by the p21-binding region. The N-terminal regula tory domain of MIHCK contains alternating strongly positive and strongly ne gative regions. and the extremely Pro-rich middle region of MIHCK has a str ongly acidic N-terminal segment and a strongly basic C-terminal segment. We propose that autophosphorylation activates MIHCK by neutralizing the basic segment of the Pro-rich region, thus unfolding the regulatory domain and a bolishing its inhibition of the catalytic domain.