title: Pim1 kinase promotes angiogenesis through phosphorylation of endothelial nitric oxide synthase at Ser-633.
abstract: OBJECTIVE
  Posttranslational modification, such as phosphorylation, plays an essential role in regulating activation of endothelial NO synthase (eNOS). In the present study, we aim to determine whether eNOS could be phosphorylated and regulated by a novel serine/threonine-protein kinase Pim1 in vascular endothelial cells (ECs).
  
  
  RESULTS
  Using immunoprecipitation and protein kinase assays, we demonstrated that Pim1 specifically interacts with eNOS, which leads to a marked phosphorylation of eNOS at Ser-633 and increased production of nitric oxide (NO). Intriguingly, in response to VEGF stimulation, eNOS phosphorylation at Ser-633 exhibits two distinct phases: transient phosphorylation occurring between 0 and 60 min and sustained phosphorylation occurring between 2 and 24 h, which are mediated by the protein kinase A (PKA) and Pim1, respectively. Inhibiting Pim1 by either pharmacological inhibitor SMI-4a or the dominant-negative form of Pim1 markedly attenuates VEGF-induced tube formation, while Pim1 overexpression significantly increases EC tube formation and migration in an NO-dependent manner. Importantly, Pim1 expression and eNOS phosphorylation at Ser-633 were substantially decreased in high glucose-treated ECs and in the aorta of db/db diabetic mice. Increased Pim1 expression ameliorates impaired vascular angiogenesis in diabetic mice, as determined by an ex vivo aortic ring assay.
  
  
  CONCLUSIONS
  Our findings demonstrate Pim1 as a novel kinase that is responsible for the phosphorylation of eNOS at Ser-633 and enhances EC sprouting of aortic rings from diabetic mice, suggesting that Pim1 could potentially serve as a novel therapeutic target for revascularization strategies.
authors.affiliation: Center for Translational Medicine, Thomas Jefferson University, 1020 Locust Street, Room 286G, Philadelphia, PA 19107, USA.
authors.forename: Ming
authors.initials: M
authors.lastname: Chen
authors.affiliation: Center for Translational Medicine, Thomas Jefferson University, 1020 Locust Street, Room 286G, Philadelphia, PA 19107, USA.
authors.forename: Bing
authors.initials: B
authors.lastname: Yi
authors.affiliation: Center for Translational Medicine, Thomas Jefferson University, 1020 Locust Street, Room 286G, Philadelphia, PA 19107, USA.
authors.forename: Ni
authors.initials: N
authors.lastname: Zhu
authors.affiliation: Center for Translational Medicine, Thomas Jefferson University, 1020 Locust Street, Room 286G, Philadelphia, PA 19107, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Wei
authors.affiliation: Center for Translational Medicine, Thomas Jefferson University, 1020 Locust Street, Room 286G, Philadelphia, PA 19107, USA The Institute of Cardiothoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
authors.forename: Guan-Xin
authors.initials: GX
authors.lastname: Zhang
authors.affiliation: The Institute of Cardiothoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
authors.forename: Shengdong
authors.initials: S
authors.lastname: Huang
authors.affiliation: Center for Translational Medicine, Thomas Jefferson University, 1020 Locust Street, Room 286G, Philadelphia, PA 19107, USA The Institute of Cardiothoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai 200433, China jianxin.sun@jefferson.edu.
authors.forename: Jianxin
authors.initials: J
authors.lastname: Sun
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000502:physiology; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018919:Neovascularization, Physiologic; D009569:Nitric Oxide / Q000502:physiology; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation; D051573:Proto-Oncogene Proteins c-pim-1 / Q000502:physiology*; D012694:Serine / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
chemical_list: D042461:Vascular Endothelial Growth Factor A; D009569:Nitric Oxide; D012694:Serine; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; C496636:Pim1 protein, mouse; D051573:Proto-Oncogene Proteins c-pim-1
keywords: Angiogenesis; Diabetes; Phosphorylation; Pim1; eNOS