Regulation of Nonmuscle Myosin II by Tropomyosin Isoforms

Abstract

Tropomyosin (Tm) regulates actin myosin interactions in eukaryotic cells ranging from yeast to mammalian muscle and nonmuscle cells. Tropomyosin is a α-helical coiled-coil actin binding protein that associates end-to-end to form continuous strands along both sides of the actin filament. Tropomyosins can inhibit or activate actomyosin MgATPase activity and motility depending on the myosin and Tm isoforms. In this study, we have attempted to determine whether activation or inhibition is specified by the Tm or the myosin isoform. We carried out in vitro motility assays with four myosin isoforms (skeletal muscle myosin, and nonmuscle IIA, IIB and IIC HMMs) in the presence of five Tm isoforms (skeletal muscle αTm, and nonmuscle isoforms, Tm2, Tm5a, Tm5NM1 and Tm4) and skeletal muscle actin. With skeletal muscle myosin, actin-Tm filament velocities were inhibited by αTm (∼60%) but activated by Tm5a and Tm5NM1 (30-60%) relative to actin alone, whereas Tm2 and Tm4 had little or no effect. In the case of nonmuscle IIA and IIC HMMs, all nonmuscle Tms activated filament velocities, whereas αTm had no effect, relative to actin alone. None of the Tm isoforms affected filament velocities with nonmuscle IIB HMM. Therefore, the primary determinant of the effect of Tm on actin filament velocities on myosin is the myosin isoform. The actin-activated MgATPase activities of IIA, IIB and IIC HMMs were measured to determine the mechanism of activation by a nonmuscle Tm, Tm5NM1. Tm5NM1 increased the Vmax of both IIA (26%), and IIC (19%), with a much smaller effect on IIB (12%) compared to actin alone thus supporting the motility data. Tm5NM1 also decreased the KATPase of IIA and IIB. Therefore, Tm5NM1 activates the MgATPase activity of IIA and IIB by increasing the Vmax and decreasing KATPase. Supported by NIH.