Analysis of the regulatory phosphorylation site in Acanthamoeba myosin IC by using site-directed mutagenesis

The actin-activated ATPase activity of Acanthamoeba myosin IC is stimulated 15- to 20-fold by phosphorylation of Ser-329 in the heavy chain. In most m yosins, either glutamate or aspartate occupies this position, which lies wi thin a surface loop that forms part of the actomyosin interface. To investi gate the apparent need for a negative charge at this site, we mutated Ser-3 29 to alanine, asparagine, aspartate, or glutamate and coexpressed the Flag -tagged wild-type or mutant heavy chain and light chain in baculovirus-infe cted insect cells. Recombinant wild-type myosin IC was indistinguishable fr om myosin IC purified from Acanthamoeba as determined by (i) the dependence of its actin-activated ATPase activity on heavy-chain phosphorylation, (ii ) the unusual triphasic dependence of its ATPase activity on the concentrat ion of F-actin, (iii) its K-m for ATP, and (iv) its ability to translocate actin filaments. The Ala and Asn mutants had the same low actin-activated A TPase activity as unphosphorylated wild-type myosin IC. The Glu mutant, lik e the phosphorylated wild-type protein, was 16-fold more active than unphos phorylated wild type, and the Asp mutant was 8-fold more active. The wild-t ype and mutant proteins had the same K-m for ATP. Unphosphorylated wild-typ e protein and the Ala and Asn mutants were unable to translocate actin fila ments, whereas the Glu mutant translocated filaments at the same velocity, and the Asp mutant at 50% the velocity, as phosphorylated wild-type protein s. These results demonstrate that an acidic amino acid can supply the negat ive charge in the surface loop required for the actin-dependent activities of Acanthamoeba myosin IC in vitro and indicate that the length of the side chain that delivers this charge is important.