Hsp90 ensures the transition from the early Ca2+-dependent to the late phosphorylation-dependent activation of the endothelial nitric-oxide synthase in vascular endothelial growth factor-exposed endothelial cells

Vascular endothelial growth factor (VEGF) exerts its angiogenic effects par tly through the activation of endothelial nitric-oxide synthase (eNOS). Ass ociation with heat shock protein 90 (hsp90) and phosphorylation by Akt were recently shown to separately activate eNOS upon VEGF stimulation in endoth elial cells. Here, we examined the interplay between these different mechan isms in VEGF-exposed endothelial cells. We documented that hsp90 binding to eNOS is, in fact, the crucial event triggering the transition from the Ca2 +-dependent activation of eNOS to the phosphorylation-mediated potentiation of its activity by VEGF. Accordingly, we showed that early VEGF stimulatio n first leads to the Ca2+/calmodulin disruption of the caveolin-eNOS comple x and promotes the association between eNOS and hsp90. eNOS-bound hsp90 can then recruit VEGF-activated (phosphorylated) Akt to the complex, which in turn can phosphorylate eNOS. Further experiments in transfected COS cells e xpressing either wild-type or S1177A mutant eNOS led us to identify the ser ine 1177 as the critical residue for the hsp90-dependent Akt-mediated activ ation of eNOS. Finally, we documented that although the V-EGF-induced phosp horylation of eNOS leads to a sustained production of NO independently of a maintained increase in [Ca2+](i), this late stage of eNOS activation is st rictly conditional on the initial VEGF-induced Ca2+-dependent stimulation o f the enzyme. These data establish the critical temporal sequence of events leading to the sustained activation of eNOS by VEGF and suggest new ways o f regulating the production of NO in response to this cytokine through the ubiquitous chaperone protein, hsp90.