The Akt kinase signals directly to endothelial nitric oxide synthase

Endothelial nitric oxide synthase (eNOS) is an important modulator of angio genesis and vascular tone [1]. It is stimulated by treatment of endothelial cells in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent fashion b y insulin-like growth factor-1 (IGF-1) and vascular endothelial growth fact or (VEGF) [2,3] and is activated by phosphorylation at Ser1177 in the seque nce RIRTQS(1177)f (in the single-letter amino acid code) [4]. The protein k inase Akt is an important downstream target of PI 3-kinase [5,6], regulatin g VEGF stimulated endothelial cell survival [7], Akt phosphorylates substra tes within a defined motif [8], which is present in the sequence surroundin g Ser1177 in eNOS, Both Akt [5,6] and eNOS [9] are localized to, and activa ted at, the plasma membrane. We found that purified Akt phosphorylated card iac eNOS at Ser1177, resulting in activation of eNOS, Phosphorylation at th is site was stimulated by treatment of bovine aortic endothelial cells (BAE Cs) with VEGF or IGF-1, and Akt was activated in parallel. Preincubation wi th wortmannin, an inhibitor of Akt signalling, reduced VEGF- or IGF-1-induc ed Akt activity and eNOS phosphorylation, Akt was detected in immunoprecipi tates of eNOS from BAECs, and eNOS in immunoprecipitates of Akt, indicating that the two enzymes associate in vivo. It is thus apparent that Akt direc tly activates eNOS in endothelial cells. These results strongly suggest tha t Akt has an important role in the regulation of normal angiogenesis and ra ise the possibility that the enhanced activity of this kinase that occurs i n carcinomas may contribute to tumor vascularization and survival.