Phosphorylation of the regulatory light chain of myosin II by the Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) plays a critical role in regulation of myosin motor activities including muscle contraction, cellular migration and proliferation. Recently, novel forms of MLCK have been identified that act in unique signaling pathways and are regulated by different mechanisms. A cDNA representing a novel MLCK, called 208 kDa embryonic MLCK has been identified and linked to adhesion mediated signaling from the extracellular matrix to the cytoskeleton via its association with the cytoplasmic domain of beta-integrin subunits. This cDNA is identical to Death Associated Protein kinase, suggesting a role for 208 kDa MLCK in apoptosis. The overall goal of this proposal is to characterize its role in signaling and motogenic activity in smooth muscle (SM) and other cell types.
Aims 1 and 2 will test the hypothesis that the activity and localization of 208 kDa MLCK can be regulated by phosphorylation and/or auto phosphorylation.
Aim 1 will be to identify mechanisms regulating the association of 208 kDa MLCK with the cytoskeleton and integrins and to characterize the structural motifs involved.
Aim 2 will be to determine if association with integrins regulates the activity of the 208 kDa MLCK. Studies for Aims 3 and 4 will address the hypothesis that 208 kDa MLCK has a role in regulating cellular migration, proliferation and apoptosis. Studies for Aim 3 will be to elucidate the role of 208 kDa MLCK in regulation of cellular migration, proliferation, and apoptosis by examining the effects of either over expression or under expression of the 208 kDa MLCK in vitro in COS cells. This will involve over expression of wild-type vs. dominant negative and constitutively active forms of the 208 kDa MLCK as well as use of antisense approaches.
Aim 4 will be to investigate the physiological role of the two 208 kDa MLCK in vivo in transgenic mice. Transgenic mice will be generated in which expression of wild-type or dominant negative 208 kDa MLCK is targeted to vascular SMC using the SM22alpha promoter to direct expression. Migration, proliferation, and apoptosis in injured vascular SM and control tissues will be examined using histological analyses, measurement of [3H]thymidine incorporation, and nuclear degradation analyses.
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