Myosin phosphorylation regulates the ATPase activity of permeable skeletal muscle fibers

Abstract

Reversible covalent phosphorylation is a mechanism which is widely used to regulate enzyme activity. The phosphorylation of one of the subunits of myosin, the P-light chain, has been found to occur in a variety of muscle and non-muscle cells (review [ 11). The phosphorylation is catalyzed by a CA 2+ activated kinase whose sole substrate appears to be the P-light chain of myosin. In skeletal muscle, myosin is phosphorylated in vivo during and after long tetanic contractions [2,3]. The time course of myosin phosphorylation is slower than the events of a muscle twitch and the maximum level of phosphorylation may occur several seconds after the contraction had ceased 141. These findings suggest that myosin phosphoryjation in skeletal muscle is not an obligatory event for the production of force, and thus any role which it does play must involve a modulation of the contractile interaction. In smooth muscle and in some non-muscle cells, myosin phosphorylation has been shown to regulate the actomyosin contractile interaction [5-S]. In contrast to these results phosphorylation of skeletal muscle myosin was found to have no effect on actomyosin ATPase [9,10]. In [l l] myosin phosphorylation correlated with a decrease in the isometric ATPase of living fibers; however, this effect was not found in [ 121. Here, we provide direct evidence that thiophosphorylation regulates the ATPase activity of isometric permeable skeletal fibers and that this effect occurs only in the organized array of the myofibril. A high degree of thiophosphorylation (50-90%) of the myosin light chain was achieved in situ in glycerinated rabbit psoas fibers by incubation with purified myosin light chain kinase. Thiophosphorylation decreased the ATPase activity of the fiber by a factor of -2. Either thiophosphorylation or phosphorylation caused a similar effect in myofibrils only when they were prevented from shortening by light cross-linking with glutaraldehyde. We conclude that myosin phosphorylation in skeletal fibers is a mechanism which modulates the rate of ATP hydrolysis and that the expression of this modulation requires an intact filament array.