We are interested in the molecular bases of the regulation of the actin-activated ATPase activities of class-I and class-II myosins, the biological roles of these myosins and the regulation of their biological activities. One study investigated the role of the cardiomyopathy (CM)-loop on the biochemical and biological properties of Dictyostelium myosin II. The CM-loop of the heavy chain of all class-II myosins begins with an Arg residue (whose mutation in human ?-cardiac myosin II results in familial hypertrophic cardiomyopathy. The CM-loop of Dictyostelium myosin II (Arg397-Gln407) is essential for its biological functions and biochemical activities, i.e. the CM-loop deletion mutant is biochemically and biologically inactive. We found that the CM-loop of smooth muscle myosin II substitutes partially and the CM-loop of ?-cardiac myosin II much less well in supporting growth in suspension culture, capping of concanavalin A surface receptors and development of mounds to fruiting bodies in vivo and the actin-activated MgATPase and in vitro motility activities of purified myosins. The abilities of the smooth and cardiac muscle chimeras and 19 CM-loop point mutants to support growth and development correlate well with each other but not with the ability to support capping of concanavalin A receptors. There is little overall correlation between the biochemical and biological activities of the myosins but only the five mutant constructs (of 21 constructs tested) that support essentially full biological function have kcat/Kactin values, a measure of catalytic efficiency, equivalent to wild-type myosin. Biological and biochemical functions are particularly dependent on Ala at position 400, where almost all other class-II myosins have Val. The three point mutations of Arg397 equivalent to those that result in hypertrophic cardiomyopathy in humans had minimal biological effects and different biochemical effects. We are now studying S1 constructs to determine which step(s) in the actomyosin ATPase cycle is affected by the Ala/Val mutation. The tail domain of Acanthamoeba myosin IC consists of a basic region (BR) and two Gly/Pro/Ala-rich regions (GPA1 and GPA2) separated by a Src homology (SH3) region. Reconstructed cryo-electron microscopic images F-actin decorated with WT and truncated tail mutants show that the BR forms a ~40 angstrom-long oval diverging obliquely from the head domain and that the GPA1 and GPA2 regions are folded back on the actin-proximal side of the BR region. These studies, which imply interactions between the N-terminal and C-terminal halves of the tail, are being followed up by NMR of N15 and C13 labeled tail constructs to determine the nature of these interactions. Ongoing studies on the affect of blebbistatin on the biology of Dictyostelium are consistent with it being a specific inhibitor of myosin II, as published by others, but also show that the enzymatically inactivated myosin II can affect cellular processes that do not directly involve myosin II. These results have significant implications for interpreting the effects of blebbistatin on cells in which the biological roles of myosin II are not as clearly defined as they are in Dictyostelium. As an extension of studies of Acanthamoeba PAK (myosin I heavy chain kinase), we found that expression of constitutively active Rac in HeLa cells increases the level of phosphorylation of the regulatory light chain of myosin II substantially, but not exclusively, by activation of PAK, as shown by the use of kinase-specific inhibitors. This is the first evidence that Rac-activation of myosin II may be one of the ways that Rac affects the cytoskeleton,
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