Pin1 Regulates Endothelial Nitric Oxide Synthase

Endothelial nitric oxide synthase (eNOS), which generates the important vasodilating and vasculoprotective molecule, nitric oxide (NO), has a crucial role in controlling blood pressure and in protection from atherosclerotic lesion formation. Obtaining a detailed understanding of the structure, function, and regulation of the eNOS enzyme is thus clearly a worthy goal of scientific investigation. Posttranslational regulation of eNOS by the alternative mechanisms of 1) protein-protein interactions and 2) phosphorylation has been well-studied to date. A complete picture of either process, however, has not yet emerged. Moreover, virtually nothing is yet known regarding how these two different eNOS regulation processes are interrelated. eNOS regulation by phosphorylation is complex and 7 regulatory phosphorylation sites have been identified in eNOS at Y83, S116, T497, S617, S635, Y659, and S1179 (bovine sequence numbering). The eNOS protein-protein interactome is also very complex with many proteins that are known to interact with eNOS either directly or indirectly to influence eNOS activity or subcellular localization. Preliminary data presented in this proposal identify a new member of the eNOS interactome as the Pin1 prolyl isomerase. Pin1 isomerizes phospho-serine/threonine-proline [p(S/T)-P] motifs in target proteins which profoundly affects target protein conformation and function. Pin1 acts in concert with proline-directed protein kinases that phosphorylate serines or threonines immediately preceding proline in protein amino acid sequences. Our preliminary studies show that eNOS is subject under basal conditions in endothelial cells to proline-directed phosphorylation at S116 by the ERK 1/2 MAP kinase. Our data show further that Pin1 interacts with eNOS in a manner that depends on phosphorylation of S116 preceding P117 in the eNOS sequence. Most importantly, we show that the Pin1 interaction with eNOS suppresses the basal and agonist-stimulated activity of eNOS in endothelial cells. We therefore propose the following specific aims. We will test the hypothesis that basal phosphorylation of eNOS at S116 acts as an on-switch for eNOS-Pin1 binding and suppression of eNOS activity in endothelial cells and in blood vessels. We will elucidate the mechanism(s) by which Pin1 reduces eNOS activity in endothelial cells. (AHA Program: Grant-in-Aid)