Molecular Mechanisms of Nonmuscle Myosin II Regulation

DESCRIPTION (provided by applicant): Late in mitosis, actin and myosin-II transiently accumulate at the equator of the cell to form a contractile ring that divides the cell in two. Although myosin-II is known to be required for the fidelity of the cytokinetic process, the molecular signals and underlying mechanisms that mediate its recruitment to the contractile ring and stimulate its motor activity have not been elucidated. During metaphase the regulatory light chain (RLC) of myosin-II is phosphorylated on residues inhibitory for activity; however as mitosis progresses through anaphase, the RLC is dephosphorylated on these sites and phosphorylated on residues that activate the motor. The observation that RLC phosphorylation is cell cycle dependent demonstrates that myosin-II activity is under temporal regulation during mitosis and suggests that the assembly and activity of the contractile ring depends upon the modulation of myosin-II function via phosphorylation. Importantly, the kinases that mediate mitosis-specific phosphorylation of the RLC have not been identified. We have initiated a multidisciplinary approach involving quantitative in vitro biochemical analyses and in vivo studies to identify the kinases that phosphorylate the regulatory light chain (RLC) of myosin-II during mitosis in vertebrate nonmuscle cells. These studies will provide the basis for a model describing the signaling pathways that regulate myosin-II activity and which contribute to the assembly of the contractile ring. Our objectives are: (1) quantify the effects of phosphorylation on the inhibitory sites of the RLC on myosin-II activity, (2) identify the mitotic kinase that phosphorylates the inhibitory sites on the myosin-II RLC, (3) test the requirement for MLCK in proper contractile ring assembly and the fidelity of the cytokinetic process, (4) identify the mechanisms that down regulate MLCK activity in early mitosis and (5) identify the molecular determinants required for targeting MLCK to the contractile ring.