We have identified a cytoskeletal associated protein kinase, called death associated protein kinase (DAPK) with a major role in apoptosis signaling. DAPK is a muti-domain ser/thr protein kinase that can phosphorylate myosin II RLC in vivo to activate acto-myosin contractile force production. In vivo DAPK is found in a complex with a discrete set of cytoskeletal, cellular and apoptosis regulatory proteins. Depletion of DAPK expression by antisense strategies induces apoptosis in cells suggesting that DAPK is an essential protein with an important function in apoptosis. We hypothesize that the proteins associated with DAPK form a cytoskeletal associated regulatory complex that functions as a """"""""gate-keeper"""""""" to regulate entry into the apoptotic pathway and that the cytoprotective activities of DAPK are regulated by the interaction of DAPK with this complex of proteins. One novel protein that directly binds DAPK has been identified and is called DIP (DAPK Interacting Protein). DIP has been partially characterized and shown to have 3 RING fingers with E3 ubiquitin ligase activity. Expression of DIP antagonizes the survival function of DAPK, suggesting that these apoptosis regulators have an essential role in determining a balance between survival and death. In this competitive renewal application, we propose to further test this hypothesis by examining the molecular basis through which DAPK regulates apoptosis.
Specific aim 1 is to determine the physiological role of DAPK in vascular smooth muscle cells.
Specific aim 2 is to determine the mechanism by which DAPK promotes cell survival.
Specific aim 3 is to determine the interactions and roles of the kinase domain, death domain and carboxyl-terminal tail in the regulation of apoptosis regulation by DAPK.
Aim 4 is to understand the mechanism by which DIP, antagonizes the cytoprotective function of DAPK. The proposed studies could identify a new therapeutic target that would be important in vascular diseases and will contribute to the general understanding the chain of events that occurs when cells receive death-inducing stimuli.
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