Regulation of eNOS by the cell division cycle homolog, cdc37

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DESCRIPTION (provided by applicant): Endothelial nitric oxide synthase (eNOS) via the production of nitric oxide (NO) in the vascular endothelium plays a key role in cardiovascular biology. NO is known to regulate vasodilation, platelet aggregation, platelet and leukocyte adhesion to the endothelium, endothelin-1 generation, and vascular smooth muscle proliferation thereby playing a role in the broader issues of blood pressure regulation and atherosclerosis. In addition, eNOS uncoupling in which the balance of NO production and superoxide production is shifted in favor of superoxide is associated with the endothelial dysfunction that occurs in various pathological conditions including hypertension and diabetes. Precise regulation of eNOS function is of vital importance to maintain vascular homeostasis. eNOS is regulated by co- and posttranslational mechanisms, phosphorylation, and protein-protein interactions. Understanding the relationship between these regulatory mechanisms is an important area of investigation. In our preliminary data, we have identified a novel eNOS interacting protein which may play an important role in mediating the eNOS protein complex formation as well as subsequent eNOS phosphorylation and activation. Using co-immunoprecipitation and in vitro binding experiments, we have determined that the cell division cycle 37 homolog (cdc37) interacts directly with eNOS and in the absence of other proteins asserts an inhibitory influence on eNOS activity. Cdc37 is critical component of the protein complex between heat shock protein 90 (Hs90) and Akt, two key eNOS regulatory proteins. However, no previous studies have examined the potential interaction between cdc37 and eNOS. Based on these observations and our preliminary work, we will use purified proteins in in vitro binding and kinase experiments as well as coimmunoprecipitation and adenovirus mediated gene transfer in endothelial cell cultures and isolated vessels to explore these interactions. The overall goals of this project are to investigate the effects of cdcd37 on eNOS function by examining cdc37's role in coordinating the interaction of eNOS with Hsp90 and Akt as well as subsequent phosphorylation and activation. The specific aims and hypotheses of this study are: Aim 1 - To test the hypothesis that specific domains in cdc37 and eNOS can be identified which govern the direct interaction between the two proteins, Aim 2 - To test the hypothesis that cdc37 mediates the eNOS complex formation with Hsp90 and Akt subsequently altering eNOS phosphorylation, and Aim 3 - To test the hypothesis that cdc37 modifies eNOS function both in vitro and in vivo. The data gathered will provide new insight into the mechanisms by which blood vessels are regulated and help us understand the underlying mechanisms of vascular dysfunction associated with diseases such as hypertension, diabetes, and atherosclerosis.