Ongoing work funded by this grant has focused on the role of Ca2+/calmodulin-dependent protein kinase II (CaMKII), a ubiquitous multifunctional serine/threonine kinase with unique structural and autoregulatory properties, in the control of vascular smooth muscle (VSM) contractile and growth responses. Our prior work in this area resulted in the biochemical and molecular characterization of CaMKII isozyme expression in VSM. Using this knowledge and experience, we have developed a number of specific immunochemical tools and molecular approaches that, for the first time, are capable of dissecting CaMKII activity and function in VSM. Recent studies have led to the general hypothesis that CaMKIIdelta2, the principal isoform expressed in cultured rat aortic VSM, has unique structural features that result in its coupling to nonreceptor tyrosine kinases and ERK1/2-dependent signaling pathways necessary for cell migration and growth. Biochemical and molecular approaches are proposed to:
(Aim 1) test the requirement for and specificity of CaMKIIdelta2 isozymes in regulating Ca2+depedent activation of ERK1/2, migration, and proliferation in cultured VSM cells.
(Aim 2) determine the functional significance of a novel CaMKIIdelta2/Fyn tyrosine kinase physical interaction with respect to CaMKll-dependent signaling and VSM cell migration and proliferation;
(Aim 3) determine the effect of autophosphorylation in regulating CaMKlIdelta2 scaffolding functions and cellular function with respect to Ca2+dependent signaling, migration, and proliferation. Based on published analyses of CaMKII gene expression in VSM, we propose that a change in expression from CaMKII gamma-isozymes, which have been associated with control of VSM contraction, to the delta2-isozyme is an important component of the response to vascular injury and enables Ca2+-dependent control of VSM cell migration and growth.
Aim 4 tests this hypothesis in the rat carotid artery injury and restenosis model. The proposed work includes the development of novel approaches and tools to address the function and regulation of specific CaMKII isozymes in arterial smooth muscle, focusing on cell migration and growth. Knowledge gained from these studies can potentially be applied to developing new therapeutic approaches for diseases that are known to involve vascular smooth muscle proliferation and migration such as atherosclerosis and restenosis.
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