Pim1 Is a Novel Kinase That Phosphorylates eNOS at Ser‐633 and Enhances Angiogenesis in Diabetic Mice

NO is produced by endothelial NO synthase (eNOS) whose function is modulated by posttranslational modifications such as phosphorylation and sumoylation. In this study, we identified Pim1 as a novel kinase that mediates eNOS phosphorylation at Ser633. The interaction between Pim1 and eNOS was demonstrated by co‐immunoprecipitation studies in native human endothelial cells and Cos7 cells transiently transfected with eNOS and Pim1 cDNAs. In a vitro kinase assay using recombinant eNOS and Pim1, we demonstrate that Pim1 primarily phosphorylates eNOS at Ser633. Furthermore, co‐transfection of Pim1 with eNOS markedly increased eNOS phosphorylation at Ser633 and substantially increased eNOS activity as determined by NO producton. Intriguingly, in response to VEGF stimulation, phosphorylation of eNOS at Ser633 in vascular ECs exhibits two distinct phases, including a transient phosphorylation occurring between 0 and 60 min and a sustained phosphorylation occurring between 2 h and 24 h, which are mediated by the PKA and Pim1 respectively. Inhibition of Pim1 by either pharmacological inhibitor SMI‐4a or the dominant negative form of Pim1 markedly inhibits VEGF‐induced tube formation, while ectopic expression of Pim1 significantly increases endothelial cell tube formation and EC migration in a NO dependent manner. Strikingly, Pim1 expression and eNOS phosphorylation at Ser633 were substantially decreased in ECs treated with high glucose as well as in the aorta of db/db diabetic mice. Overexpression of Pim1 ameliorates impaired vascular angiogenesis in diabetic mice, as determined by an ex vivo aortic ring assay. Together, these data suggest that Pim1 could potentially serve as a novel therapeutic target for revascularization strategies in diabetic patients.