Research Project
6513664
DESCRIPTION (provided by applicant): Changes in intracellular calcium ion concentrations ([Ca2+li) has been linked to the progression of the cell division cycle of mammalian cells. The mechanism by which this universal second messenger regulates such a complex process is unclear; however, many of its actions are most likely mediated by calcium-sensitive protein kinases and phosphatases. Recent work on the identification and characterization of calcium/calmodulin-dependent protein kinase 11 (CaM kinase 11) in rat aortic vascular smooth muscle (VSM) cells indicates that this enzyme is responsive to changes in [Ca2+]i and so, may play a fundamental role in processes such as cell division. It is well accepted that cellular signaling events are regulated by phosphorylation and dephosphorylation of critical cellular proteins. There is also compelling evidence that kinases and phosphatases are themselves targets of such phosphorylation/dephosphorylation events. Further, the complex activation/inactivation steps of CaM kinase 11 would uniquely lend itself to regulation by specific cellular phosphatases. An emerging paradigm of signal transduction networks is the sequestration of kinase/phosphatase complexes with committed downstream cellular function. This proposal seeks to identify and characterize phosphatases that regulate CaM kinase 11 activity in cultured VSM cells. This will be achieved by determining how CaM kinase 11 activity changes when endogenous phosphatases are inhibited through pharmacological and physiological manipulations. These phosphatases will be characterized by immunological and activity assays and, evidence for kinase/phosphatase signalling complexes will be sought through colocalization and cell fractionation studies. The applicant's preliminary work suggests that CaM kinase 11 plays a prominent role in the cell division process of VSM cells. Such a role takes on added significance when one considers that unregulated proliferation of VSM cells is a fundamental process in the development of atherosclerotic lesions and subsequent heart disease. The characterization of novel cellular processes would hasten the development of more selective pharmacological and genetic tools that may be used to better manage or reverse the disease process.
