MLC Dephosphorylation in Smooth Muscle Cell Apoptosis

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The cytoskeleton undergoes dramatic changes during programmed cell death (apoptosis) and actin has been implicated in mediating these cytoskeletal changes. Although actin and myosin II work in concert, little is known about the role of myosin II and its regulation by myosin light chain phosphorylation in the mechanism of smooth muscle cell apoptosis. We have made three observations that impact the regulation of cytoskeletal dynamics during apoptosis. First, myosin light chains are dephosphorylated during apoptosis; Second, the activity of myosin light chain kinase (MLCK), the enzyme that phosphorylates myosin II, is decreased during apoptosis; First, myosin light chains are dephosphorylated during apoptosis. Second, the activity of myosin light chain kinase (MLCK), the enzyme that phosphorylates myosin II, is decreased during apoptosis; Second, the activity of myosin light chain kinase (MLCK), the enzyme that phosphorylates myosin II, is decreased during apoptosis; Third, prolonged inhibition of MLCK activity using a pharmacological agent results in nuclear fragmentation and genome digestion. Based on these data, I propose the hypothesis that myosin light chain dephosphorylation is necessary for and perhaps initiates the cytoskeletal changes characteristic of programmed cell death. Specifically, I will address the role of MLCK dephosphorylation in vascular smooth muscle cell death and determine how this may be regulated at the molecular level. The specific aims of this proposal are to: 1. Establish the relation between myosin light chain dephosphorylation and apoptosis of smooth muscle cells. 2. Determine the function of "non-apoptotic MLCK dephosphorylation" in sensitizing the smooth muscle cells to undergo apoptosis following exposure to the pro-inflammatory cytokines TNF-alpha, IL-1beta and IFN-gamma. The information derived from these studies will increase our understanding of cytoskeletal dynamics during apoptosis.