EFFECT OF MUTATING THE REGULATORY PHOSPHOSERINE AND CONSERVED THREONINE ON THE ACTIVITY OF THE EXPRESSED CATALYTIC DOMAIN OF ACANTHAMOEBA MYOSIN-I HEAVY-CHAIN KINASE

Phosphorylation of Ser-627 is both necessary and sufficient for full a ctivity of the expressed 35-kDa catalytic domain of myosin I heavy cha in kinase (MIHCK). Ser-627 lies in the variable loop between highly co nserved residues DFG and APE at a position at which a phosphorylated S er/Thr also occurs in many other Ser/Thr protein kinases, The variable loop of MIHCK contains two other hydroxyamino acids: Thr-631, which i s conserved in almost all Ser/Thr kinases, and Thr-632, which is not c onserved. We determined the effects on the kinase activity of the expr essed catalytic domain of mutating Ser-627, Thr-631, and Thr-632 indiv idually to Ala, Asp, and Glu, The S627A mutant was substantially less active than wild type (wt), with a lower k(cat) and higher K-m for bot h peptide substrate and ATP, but was more active than unphosphorylated wt. The S627D and S627E mutants were also less active than phosphoryl ated wt, i.e., acidic amino acids cannot substitute for phospho-Ser-62 7. The activity of the T631A mutant was as low as that of the S627A mu tant, whereas the T632A mutant was as active as phosphorylated wt, ind icating that highly conserved Thr-631, although not phosphorylated, is essential for catalytic activity. Asp and Glu substitutions for Thr-6 31 and Thr-632 were inhibitory to various degrees. Molecular modeling indicated that Thr-631 can hydrogen bond with conserved residue Asp-59 1 in the catalytic loop and that similar interactions are possible for other kinases whose activities also are regulated by phosphorylation in the variable loop. Thus, this conserved Thr residue may be essentia l for the activities of other Ser/Thr protein kinases as well as for t he activity of MIHCK.