Characterization of the interaction between endothelial nitric oxide synthase and heat shock protein 90: Implications foreNOS activation and nitric oxide release

Nitric oxide derived from the endothelium is a critical mediator of a variety of functions within the cardiovascular system. Nitric oxide is a potent vasodilator and anti-inflammatory autocoid that has profound effects on basic cellular functions including gene expression, cell migration, proliferation, and differentiation. Physiologically, endothelial cells manufacture nitric oxide via the enzyme endothelial nitric oxide synthase (eNOS). eNOS is a peripheral membrane protein that within endothelial cells targets to the Golgi and plasma membrane micro domains, termed Caveolae. This compartmentalization of eNOS is optimal for nitric oxide synthesis providing the rationale for the discovery of additional protein regulators of eNOS function such as calmodulin, caveolin, and Akt. Recent studies have demonstrated the importance of a stimulus dependent interaction of eNOS with heat shock protein 90. Based on this finding my thesis examines the role of hsp90 as component of stimulation dependent signal transduction leading activation of eNOS. My objectives were to characterize the localization of hsp90 and eNOS within endothelial cells, and identify the molecular mechanism for stimulus dependent eNOS activation by hsp90. The data presented herein describes direct and definitive evidence for the importance of the interaction between hsp90 and eNOS. We demonstrate the co-localization of hsp90 and eNOS within endothelial cells and blood vessels and provide further evidence for the involvement of hsp90 in eNOS function in vivo through a model of cell permeability. We present further support by demonstrating hsp90 can facilitate the calmodulin dependent displacement of eNOS from caveolin and can serve as a molecular scaffold to regulate Akt dependent phosphorylation of eNOS. Finally, we characterized the domains of hsp90 required to bind eNOS, using yeast 2-hybrid, cell based co-precipitation experiments and GST-fusion proteins, revealing that the M region of hsp90 (amino acids 442–600) interacts with the amino terminus of eNOS (amino acids 300–400). In conclusion, these data provide direct evidence for that hsp90 is indeed the critical link between the various pathways leading to eNOS activation and nitric oxide release.