Shear Stress Stimulates Phosphorylation of Endothelial Nitric-oxide Synthase at Ser1179 by Akt-independent Mechanisms ROLE OF PROTEIN KINASE A

Abstract Recently, we have shown that shear stress stimulates NO⋅ production by the protein kinase B/Akt (Akt)-dependent mechanisms in bovine aortic endothelial cells (BAEC) (Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. Physiol. 91, 1574–1581). Akt has been believed to regulate shear-dependent production of NO⋅ by directly phosphorylating endothelial nitric-oxide synthase (eNOS) at the Ser1179 residue (eNOS-S1179), but a critical evaluation using specific inhibitors or dominant negative mutants (AktAA or AktAAA) has not been reported. In addition, other kinases, including protein kinase A (PKA) and AMP kinase have also shown to phosphorylate eNOS-S1179. Here, we show that shear-dependent phosphorylation of eNOS-S1179 is mediated by an Akt-independent, but a PKA-dependent, mechanism. Expression of AktAAor AktAAA in BAEC by using recombinant adenoviral constructs inhibited phosphorylation of eNOS-S1179 if cells were stimulated by vascular endothelial growth factor (VEGF), but not by shear stress. As shown before, expression of AktAAinhibited shear-dependent NO⋅ production, suggesting that Akt is still an important regulator in NO production. Further studies showed that a selective inhibitor of PKA, H89, inhibited shear-dependent phosphorylation of eNOS-S1179and NO⋅ production. In contrast, H89 did not inhibit phosphorylation of eNOS-S1179 induced by expressing a constitutively active Akt mutant (AktMyr) in BAEC, showing that the inhibitor did not affect the Akt pathway. 8-Bromo-cAMP alone phosphorylated eNOS-S1179 within 5 min without activating Akt, in an H89-sensitive manner. Collectively, these results demonstrate that shear stimulates phosphorylation of eNOS-S1179 in a PKA-dependent, but Aktindependent manner, whereas the NO⋅ production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction between Akt and PKA may be an important mechanism by which eNOS activity is regulated in response to physiological stimuli such as shear stress.