title: B56δ subunit of protein phosphatase 2A decreases phosphorylation of endothelial nitric oxide synthase at serine 116: Mechanism underlying aphidicolin-stimulated NO production.
abstract: DNA damage is significant in endothelial cells (EC), particularly in anticancer chemotherapy. Here, we explored whether and how aphidicolin, a DNA-damaging chemical with a promising anticancer activity, alters NO production in bovine aortic endothelial cells (BAEC). In addition to increasing eNOS-Ser1179phosphorylation, aphidicolin decreased eNOS-Ser116phosphorylation with a concomitant increase in NO production in a time-dependent manner. The amino acid sequence around the eNOS-Ser116residue was identified as the substrate site of the regulatory subunit B56δ of protein phosphatase 2A (PP2A). As expected, okadaic acid, a specific PP2A inhibitor, reversed aphidicolin-induced eNOS-Ser116dephosphorylation in a dose-dependent manner. Aphidicolin also increased B56δ-Ser566phosphorylation, although expression of neither the catalytic subunit Cα (PP2A Cα) nor B56δ was altered. Ectopic expression of dominant negative (dn)-B56δ reversed all of the observed effects of aphidicolin with respect to phosphorylation of eNOS-Ser116and B56δ-Ser566. Lastly, aphidicolin-stimulated NO production was also partially attenuated by ectopic expression of dn-B56δ. Taken together, our results are the first to demonstrate that aphidicolin decreases phosphorylation of eNOS-Ser116, at least in part by activating PP2A B56δ, resulting in NO release in BAEC.
authors.affiliation: Department of Molecular Medicine, Ewha Womans University School of Medicine, Yangcheon-gu, Seoul 158-710, Republic of Korea.
authors.forename: Jung-Hyun
authors.initials: JH
authors.lastname: Park
authors.affiliation: Department of Pharmacology, School of Medicine, Eulji University, Jung-gu, Daejeon 301-746, Republic of Korea.
authors.forename: Du-Hyong
authors.initials: DH
authors.lastname: Cho
authors.affiliation: Department of Molecular Medicine, Ewha Womans University School of Medicine, Yangcheon-gu, Seoul 158-710, Republic of Korea.
authors.forename: Jee Young
authors.initials: JY
authors.lastname: Lee
authors.affiliation: Department of Molecular Medicine, Ewha Womans University School of Medicine, Yangcheon-gu, Seoul 158-710, Republic of Korea.
authors.forename: Hyeon-Ju
authors.initials: HJ
authors.lastname: Lee
authors.affiliation: Department of Biochemistry, Ewha Womans University School of Medicine, Yangcheon-gu, Seoul 158-710, Republic of Korea.
authors.forename: Yena
authors.initials: Y
authors.lastname: Ha
authors.affiliation: Department of Biochemistry, Ewha Womans University School of Medicine, Yangcheon-gu, Seoul 158-710, Republic of Korea. Electronic address: ahnj@ewha.ac.kr.
authors.forename: Jung-Hyuck
authors.initials: JH
authors.lastname: Ahn
authors.affiliation: Department of Molecular Medicine, Ewha Womans University School of Medicine, Yangcheon-gu, Seoul 158-710, Republic of Korea. Electronic address: inhojo@ewha.ac.kr.
authors.forename: Inho
authors.initials: I
authors.lastname: Jo
keywords: Aphidicolin; Endothelial nitric oxide synthase; PP2A B56δ subunit; Phosphorylation; Protein phosphatase 2A