(Verbatim from applicant's application): A long-term goal of this laboratory has been to elucidate the function of specific protein kinases in the control of vascular smooth muscle (VSM) contractile and growth responses. Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is a ubiquitous multifunctional serine/threonine kinase with unique structural and autoregulatory properties. The general goal of this proposal is to understand to what extent CaM Kinase II medicates the diverse consequences of intracellular Ca2+ signals in VASM. Combined pharmacological and molecular approach employing recombinant replication defective adenoviruses to infect cultured VSM cells with specific active CaM kinase II subunits of inactive mutants will be used to the test the following hypothesis:
(AIM 1) CaM kinase II is an intermediate in the Ca2+-dependent activation of non-receptor tyrosine kinases and the MAP kinase signaling pathway in VSM cells.
(AIM 2) CaM kinase II isozymes are involved in the Ca2+-dependent regulation of VSM migration and integrin-dependent signaling;
(AIM 3) CaM Kinase II is involved in the regulation of tonic contractile function and protein tyrosine phosphorylation responses in intact arterial smooth muscle. Detailed structural information pertaining to CaM kinase II subunit and holoenzyme has also raised interesting questions regarding to the relationships between CaM kinase II isozyme structure and its unique autoregulatory properties or subcellular localization.
AIM 4 proposes experiments to define specific amino acid residues that are (auto) phosphorylated within CaM kinase II following activation of VSM cells by physiological stimuli, and the relationships between phosphorylation of these sites and modulation of CaM kinase II activity. Finally, in AIM 5, we propose to determine the structural basis for specific subcellular targeting of CaM kinase II isozymes in VSM and to identify binding or targeting proteins involved in modulating CaM kinase II localization or activity. The proposed research addresses questions relating to one mechanism (activation of CaM kinase II) through which intracellular Ca2+ signals may regulate fundamental cellular processes such as gene transcription, cell motility, and proliferation. Specifically, the research will lead to a more complete understanding of the mechanisms for controlling VSM cell migration and contraction, processes that are involved in pervasive vascular diseases including atherosclerosis, restenosis, hypertension and vasospasm.
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