Nitric oxide-dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition.
Endothelial nitric oxide synthase (eNOS)-mediated NO production plays a critical role in the regulation of vascular function and pathophysiology. Caveolin-1 (Cav-1) binding to eNOS holds eNOS in an inactive conformation; however, the mechanism of Cav-1-mediated inhibition of activated eNOS is unclear. Here the role of Src-dependent Cav-1 phosphorylation in eNOS negative feedback regulation is investigated. Using fluorescence resonance energy transfer (FRET) and coimmunoprecipitation analyses, we observed increased interaction between eNOS and Cav-1 following stimulation of endothelial cells with thrombin, vascular endothelial growth factor, and Ca(2+) ionophore A23187, which is corroborated in isolated perfused mouse lung. The eNOS/Cav-1 interaction is blocked by eNOS inhibitor L-N(G)-nitroarginine methyl ester (hydrochloride) and Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3, 4-d] pyrimidine. We also observe increased binding of phosphomimicking Y14D-Cav-1 mutant transduced in human embryonic kidney cells overexpressing eNOS and reduced Ca(2+)-induced NO production compared to cells expressing the phosphodefective Y14F-Cav-1 mutant. Finally, Src FRET biosensor, eNOS small interfering RNA, and NO donor studies demonstrate NO-induced Src activation and Cav-1 phosphorylation at Tyr-14, resulting in increased eNOS/Cav-1 interaction and inhibition of eNOS activity. Taken together, these data suggest that activation of eNOS promotes Src-dependent Cav-1-Tyr-14 phosphorylation and eNOS/Cav-1 binding, that is, eNOS feedback inhibition.
Department of Pharmacology, University of Illinois, Chicago, IL 60612, USA.
Zhenlong
Z
Chen
Farnaz R
FR
Bakhshi
Ayesha N
AN
Shajahan
Tiffany
T
Sharma
Mao
M
Mao
Andy
A
Trane
Pascal
P
Bernatchez
Geerten P
GP
van Nieuw Amerongen
Marcelo G
MG
Bonini
Randal A
RA
Skidgel
Asrar B
AB
Malik
Richard D
RD
Minshall
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D000001:Calcimycin / Q000494:pharmacology; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D025461:Feedback, Physiological; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007700:Kinetics; D008168:Lung / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008954:Models, Biological; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
C495540:CAV1 protein, human; C495541:Cav1 protein, mouse; D051242:Caveolin 1; D004791:Enzyme Inhibitors; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D009569:Nitric Oxide; D000001:Calcimycin; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D019061:src-Family Kinases; D019331:NG-Nitroarginine Methyl Ester