Chk1 and Hsp90 cooperatively regulate phosphorylation of endothelial nitric oxide synthase at serine 1179

• Published in: Free radical biology & medicine 2011-12, Vol.51 (12), p.2217-2226
• Main Authors: Park, Jung-Hyun, Kim, Wuon-Shik, Kim, Jin Yi, Park, Min-Ha, Nam, Jae-Hwan, Yun, Cheol-Won, Kwon, Young-Guen, Jo, Inho
• Format: Article
• Language: English
• Subjects: Alkaloids - pharmacology; Animals; Cattle; Cells, Cultured; Checkpoint kinase; Checkpoint Kinase 1; Endothelial nitric oxide synthase; Free radicals; Heat shock protein; HSP90 Heat-Shock Proteins - metabolism; Nitric Oxide - biosynthesis; Nitric Oxide Synthase Type III - metabolism; Phosphorylation; Phosphorylation - drug effects; Protein Kinases - metabolism; Serine - metabolism; Ultraviolet Rays; UV irradiation
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2011.09.021

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 Thr497 or eNOS Ser116 were found. SB218078, a checkpoint kinase 1 (Chk1) inhibitor, inhibited UV-irradiation-stimulated eNOS-Ser1179 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-Ser1179 phosphorylation. Purified Chk1 directly phosphorylated eNOS Ser1179 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-Ser1179 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 Ser1179 in response to UV irradiation, which is dependent on Hsp90 interaction. ► UV irradiation increases NO production and eNOS activity by p-eNOS-Ser1179 in EC. ► Chk1 mediates p-eNOS-Ser1179 in response to UV irradiation. ► UV irradiation increases Hsp90/eNOS binding, but decreases its binding to Chk1. ► Increased NO by UV irradiation induces apoptosis of EC.