title: Chk1 and Hsp90 cooperatively regulate phosphorylation of endothelial nitric oxide synthase at serine 1179.
abstract: The effects of DNA damage on NO production have not been completely elucidated. Using ultraviolet (UV) irradiation as a DNA-damaging agent, we studied its effect on NO production in bovine aortic endothelial cells (BAEC). UV irradiation acutely increased NO production, the phosphorylation of endothelial NO synthase (eNOS) at serine 1179, and eNOS activity. No alterations in eNOS expression nor phosphorylation at eNOS Thr(497) or eNOS Ser(116) were found. SB218078, a checkpoint kinase 1 (Chk1) inhibitor, inhibited UV-irradiation-stimulated eNOS-Ser(1179) phosphorylation and NO production. Similarly, ectopic expression of small interference RNA for Chk1 or a dominant-negative Chk1 repressed the UV-irradiation stimulatory effect, whereas wild-type Chk1 increased basal eNOS-Ser(1179) phosphorylation. Purified Chk1 directly phosphorylated eNOS Ser(1179) in vitro. Confocal microscopy and coimmunoprecipitation studies revealed a colocalization of eNOS and Chk1. In basal BAEC, heat shock protein 90 (Hsp90) predominantly interacted with Chk1. This interaction, which decreased significantly in response to UV irradiation, was accompanied by increased interaction of Hsp90 with eNOS. The Hsp90 inhibitor geldanamycin attenuated UV-irradiation-stimulated eNOS-Ser(1179) phosphorylation by dissociating Hsp90 from eNOS. UV irradiation and geldanamycin did not alter the interaction between eNOS and Chk1. Overall, this is the first study demonstrating that Chk1 directly phosphorylates eNOS Ser(1179) in response to UV irradiation, which is dependent on Hsp90 interaction.
authors: Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710, Korea.
authors: Jung-Hyun
authors: JH
authors: Park
authors: Wuon-Shik
authors: WS
authors: Kim
authors: Jin Yi
authors: JY
authors: Kim
authors: Min-Ha
authors: MH
authors: Park
authors: Jae-Hwan
authors: JH
authors: Nam
authors: Cheol-Won
authors: CW
authors: Yun
authors: Young-Guen
authors: YG
authors: Kwon
authors: Inho
authors: I
authors: Jo
mesh_terms: D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D000071877:Checkpoint Kinase 1; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D014466:Ultraviolet Rays
chemical_list: D000470:Alkaloids; D018841:HSP90 Heat-Shock Proteins; D009569:Nitric Oxide; D012694:Serine; D052250:Nitric Oxide Synthase Type III; D011494:Protein Kinases; D000071877:Checkpoint Kinase 1; C404329:SB 218078