Functional analysis of tail domains of Acanthamoeba myosin IC by characterization of truncation and deletion mutants

Acanthamoeba myosin IC has a single 129-kDa heavy chain and a single l7-kDa light chain. The heavy chain comprises a 75-kDa catalytic head domain with an ATP-sensitive F-actin-binding site, a 3-kDa neck domain, which binds a single 17-kDa light chain, and a 50-kDa tail domain, which binds F-actin in the presence or absence of ATP. The actin-activated MgATPase activity of m yosin IC exhibits triphasic actin dependence, apparently as a consequence o f the two actin-binding sites, and is regulated by phosphorylation of Ser-3 29 in the head. The 50-kDa tail consists of a basic domain, a glycine/proli ne/alanine-rich (GPA) domain, and a Src homology 3 (SH3) domain, often refe rred to as tail homology (TH)-1, -2, and -3 domains, respectively. The SH3 domain divides the TH-3 domain into GPA-1 and GPA-2. To define the function s of the tail domains more precisely, we determined the properties of expre ssed wild type and six mutant myosins, an SH3 deletion mutant and five muta nts truncated at the C terminus of the SH3, GPA-2, TH-1, neck and head doma ins, respectively. We found that both the TH-1 and GPA-2 domains bind F-act in in the presence of ATP, Only the mutants that retained an actin-binding site in the tail exhibited triphasic actin-dependent MgATPase activity, in agreement with the F-actin-cross-linking model, but truncation reduced the MgATPase activity at both low and high actin concentrations. Deletion of th e SH3 domain had no effect. Also, none of the tail domains, including the S H3 domain, affected either the K-m or V-max for the phosphorylation of Ser- 329 by myosin I heavy chain kinase.