Bradykinin-regulated interactions of the mitogen-activated protein kinase pathway with the endothelial nitric-oxide synthase

Activation of the bradykinin B2 receptor in endothelial cells initiates a c omplex array of cellular responses mediated by diverse signaling pathways, including stimulation of the mitogen-activated protein (MAP) kinase cascade and activation of the endothelial isoform of nitric-oxide synthase (eNOS). Several protein kinases have been implicated in eNOS regulation, but the r ole of MAP kinases remains less well understood. We explored the interactio ns between eNOS and components of the MAP kinase pathway in bovine aortic e ndothelial cells (BAEC), Using co-immunoprecipitation experiments, we isola ted eNOS in a complex with the MAP kinases extracellular signal-regulated k inases 1 and 2 (ERK1/2) as well as the protein kinases Raf-1 and Akt. Withi n minutes of adding bradykinin to BAEC, the eNOS-Raf-1-ERK-Akt heteromeric complex dissociated, and it subsequently reassociated following more prolon ged agonist stimulation. Bradykinin treatment of BAEC led to the activation of ERK, associated with an increase in phosphorylation of eNOS; phosphoryl ation of eNOS by ERK in vitro significantly reduced eNOS enzyme activity. E vidence for the direct phosphorylation of eNOS by MAP kinase in BAEC came f rom "back-phosphorylation" experiments using [gamma-P-32]ATP and ERK in vit ro to phosphorylate eNOS isolated from cells previously treated with bradyk inin or the MAP kinase inhibitor PD98059. The ERK-catalyzed in vitro P-32 p hosphorylation of eNOS isolated from BAEC treated with bradykinin was signi ficantly attenuated compared with untreated cells, indicating that bradykin in treatment led to the phosphorylation of ERK-sensitive sites in cells. Co nversely, eNOS isolated from endothelial cells pretreated with the MAP kina se inhibitor PD98059 showed increased ERK-promoted phosphorylation in vitro . Taken together, our results suggest that bradykinin-induced activation of ERK leads to eNOS phosphorylation and enzyme inhibition, a process influen ced by the reversible associations of members of the MAP kinase pathway wit h eNOS.