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 partly through the activation of endothelial nitric-oxide synthase (eNOS). Association with heat shock protein 90 (hsp90) and phosphorylation by Akt were recently shown to separately activate eNOS upon VEGF stimulation in endothelial cells. Here, we examined the interplay between these different mechanisms in VEGF-exposed endothelial cells. We documented that hsp90 binding to eNOS is, in fact, the crucial event triggering the transition from the Ca(2+)-dependent activation of eNOS to the phosphorylation-mediated potentiation of its activity by VEGF. Accordingly, we showed that early VEGF stimulation first leads to the Ca(2+)/calmodulin disruption of the caveolin-eNOS complex 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 expressing either wild-type or S1177A mutant eNOS led us to identify the serine 1177 as the critical residue for the hsp90-dependent Akt-mediated activation of eNOS. Finally, we documented that although the VEGF-induced phosphorylation of eNOS leads to a sustained production of NO independently of a maintained increase in [Ca(2+)](i), this late stage of eNOS activation is strictly conditional on the initial VEGF-induced Ca(2+)-dependent stimulation of the enzyme. These data establish the critical temporal sequence of events leading to the sustained activation of eNOS by VEGF and suggest new ways of regulating the production of NO in response to this cytokine through the ubiquitous chaperone protein, hsp90.
Brouet
A
A
Department of Medicine, University of Louvain Medical School, UCL-FATH 5349, Unit of Pharmacology and Therapeutics, Avenue E. Mounier 53, B-1200 Brussels, Belgium.
Sonveaux
P
P
Dessy
C
C
Balligand
J L
JL
Feron
O
O
D000818:Animals; D000831:Animals, Newborn; D019556:COS Cells; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D016228:Endothelial Growth Factors / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation; D018841:HSP90 Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D008222:Lymphokines / Q000494:pharmacology*; D051379:Mice; D019001:Nitric Oxide Synthase / Q000302:isolation & purification / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D009573:Nitrites / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014471:Umbilical Veins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
C495540:CAV1 protein, human; C495541:Cav1 protein, mouse; D051242:Caveolin 1; D022461:Caveolins; D016228:Endothelial Growth Factors; D018841:HSP90 Heat-Shock Proteins; D007328:Insulin; D008222:Lymphokines; D009573:Nitrites; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D000001:Calcimycin; D004533:Egtazic Acid; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D011505:Protein-Tyrosine Kinases; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium