Figure 6. PKN2 mediates eNOS activation by direct phosphorylation at serine 1179 and by AKT-dependent phosphorylation at serine 1177.

(A) HUAECs kept untreated or exposed to flow were lysed, and PKN2 was immunoprecipitated with a specific antibody. IgG served as a negative control. Shown are immunoblots (IB) of the lysate and the immunoprecipitate using anti-eNOS and anti-PKN2 antibodies. (B and C) Purified eNOS was incubated in kinase buffer with or without recombinant purified PKN2, together with 50 μM [γ-³²P]-ATP (3000 Ci/mmol/L) (B) or ATP (C) for 30 minutes at 30°C. Thereafter, samples were separated by SDS-PAGE. Shown are an autoradiogram and a Coomassie stain of the gel (B). Alternatively, immunoblot analysis was performed using antibodies against eNOS and PKN2 proteins or eNOS S1177 and S1179, as well as the PKN2 T816 phosphosites (C). (D) Model of the role of PKN2 in flow-induced phosphorylation of eNOS.