Abstract 16025: Phosphorylation of Endothelial Nitric Oxide Synthase at Ser633 by a Novel Protein Kinase Pim1
Background: Endothelium-derived nitric oxide, which produced by endothelial Nitric Oxide synthase (eNOS), has been implicated in the regulation of a variety of vascular functions, including vessel relaxation, vascular inflammation, and thrombotic formation. The activity of eNOS in endothelia cells (EC) is largely regulated by posttranslational modifications, such as phosphorylation. Indeed, several phosphorylation sites, including Ser114, Thr495, Ser615, Ser633, and Ser1177, Tyr657 (in human), have been identified and phosphorylation of NOS at Ser633 has been implicated in the regulation of eNOS activity in ECs, in response to shear stress and VEGF stimulation. The protein kinase(s) responsible for eNOS phosphorylation at Ser633, however, remains largely unknown.
Methods and Results: In this study, we, for the first time, identified a protein kinase, Pim1, as a novel kinase that mediates eNOS phosphorylation at Ser633. The interaction between Pim1 and eNOS was determined by co-IP of Pim1 and eNOS in native ECs and Cos7 cells co-transfected with eNOS and Pim1 cDNAs. Indeed, contransfection of Pim1 with eNOS increased eNOS phosphorylation at Ser633. Furthermore, phosphorylates of eNOS at Ser633 was confirmed by an in vitro kinase assay, using recombinant eNOS and Pim1. Moreover, phosphorylation of eNOS by Pim1 substantially increased eNOS activity by 2.5-fold and the nitric oxide (NO) production. In vascular ECs, both VEGF and Hypoxia substantially increased the expression of Pim1, with a concomitant increased phosphorylation of eNOS at Ser633. Interestingly, VEGF stimulation induces a two-phase phosphorylation of eNOS, with a rapid and transient phosphorylation at Ser1177, which is mainly mediated by Akt, and a sustained phosphorylation of eNOS at Ser633, which largely depends on the activity of Pim1. Moreover, ectopic expression of Pim1 significant increases EC migration and tube formation, which is markedly attenuated by an eNOS inhibitor, L-NAME, further implicating eNOS as an important mediator for the Pim1 induced angiogenesis.
Conclusion: Our results suggest that Pim1 is a novel protein kinase responsible for eNOS phosphorylation at Ser633, which enhances nitric oxide production, ECs migration, and vascular angiogenesis.
- © 2013 by American Heart Association, Inc.