title: NADPH Oxidase 1 Activity and ROS Generation Are Regulated by Grb2/Cbl-Mediated Proteasomal Degradation of NoxO1 in Colon Cancer Cells.
abstract: The generation of reactive oxygen species (ROS) is required for proper cell signaling, but must be tightly regulated to minimize deleterious oxidizing effects. Activation of the NADPH oxidases (Nox) triggers ROS production and, thus, regulatory mechanisms exist to properly control Nox activity. In this study, we report a novel mechanism in which Nox1 activity is regulated through the proteasomal degradation of Nox organizer 1 (NoxO1). We found that through the interaction between NoxO1 and growth receptor-bound protein 2 (Grb2), the Casitas B-lineage lymphoma (Cbl) E3 ligase was recruited, leading to decreased NoxO1 stability and a subsequent reduction in ROS generation upon epidermal growth factor (EGF) stimulation. Additionally, we show that EGF-mediated phosphorylation of NoxO1 induced its release from Grb2 and facilitated its association with Nox activator 1 (NoxA1) to stimulate ROS production. Consistently, overexpression of Grb2 resulted in decreased Nox1 activity, whereas knockdown of Grb2 led to increased Nox1 activity in response to EGF. CRISPR/Cas9-mediated NoxO1 knockout in human colon cancer cells abrogated anchorage-independent growth on soft agar and tumor-forming ability in athymic nude mice. Moreover, the expression and stability of NoxO1 were significantly increased in human colon cancer tissues compared with normal colon. Taken together, these results support a model whereby Nox1 activity and ROS generation are regulated by Grb2/Cbl-mediated proteolysis of NoxO1 in response to EGF, providing new insight into the processes by which excessive ROS production may promote oncogenic signaling to drive colorectal tumorigenesis.
authors.affiliation: Department of Life Science, Ewha Womans University, Seoul, Korea.
authors.forename: Jung Hee
authors.initials: JH
authors.lastname: Joo
authors.affiliation: Department of Life Science, Ewha Womans University, Seoul, Korea.
authors.forename: Hyunjin
authors.initials: H
authors.lastname: Oh
authors.affiliation: School of Biological Sciences, Seoul National University, Seoul, Korea.
authors.forename: Myungjin
authors.initials: M
authors.lastname: Kim
authors.affiliation: Department of Life Science, Ewha Womans University, Seoul, Korea.
authors.forename: Eun Jung
authors.initials: EJ
authors.lastname: An
authors.affiliation: Department of Life Science, Research Institute for Natural Sciences, Hanyang University, Seoul, Korea.
authors.forename: Rae-Kwon
authors.initials: RK
authors.lastname: Kim
authors.affiliation: Department of Life Science, Ewha Womans University, Seoul, Korea.
authors.forename: So-Young
authors.initials: SY
authors.lastname: Lee
authors.affiliation: Department of Life Science, Ewha Womans University, Seoul, Korea.
authors.forename: Dong Hoon
authors.initials: DH
authors.lastname: Kang
authors.affiliation: Department of Life Science, Ewha Womans University, Seoul, Korea.
authors.forename: Sang Won
authors.initials: SW
authors.lastname: Kang
authors.affiliation: Department of Pathology, Yonsei University College of Medicine, Seoul, Korea.
authors.forename: Cheol
authors.initials: C
authors.lastname: Keun Park
authors.affiliation: Department of Pathology, Yonsei University College of Medicine, Seoul, Korea.
authors.forename: Hoguen
authors.initials: H
authors.lastname: Kim
authors.affiliation: Department of Life Science, Research Institute for Natural Sciences, Hanyang University, Seoul, Korea.
authors.forename: Su-Jae
authors.initials: SJ
authors.lastname: Lee
authors.affiliation: Department of Life Science, Ewha Womans University, Seoul, Korea. baeys@ewha.ac.kr jhseol@snu.ac.kr daekee@ewha.ac.kr.
authors.forename: Daekee
authors.initials: D
authors.lastname: Lee
authors.affiliation: School of Biological Sciences, Seoul National University, Seoul, Korea. baeys@ewha.ac.kr jhseol@snu.ac.kr daekee@ewha.ac.kr.
authors.forename: Jae Hong
authors.initials: JH
authors.lastname: Seol
authors.affiliation: Department of Life Science, Ewha Womans University, Seoul, Korea. baeys@ewha.ac.kr jhseol@snu.ac.kr daekee@ewha.ac.kr.
authors.forename: Yun Soo
authors.initials: YS
authors.lastname: Bae
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics* / Q000378:metabolism; D051380:GRB2 Adaptor Protein / Q000235:genetics*; D006801:Humans; D051379:Mice; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000074624:NADPH Oxidase 1; D017382:Reactive Oxygen Species; D015398:Signal Transduction; D014162:Transfection
chemical_list: D051380:GRB2 Adaptor Protein; D017382:Reactive Oxygen Species; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; C000614711:NOX1 protein, mouse