title: Recruitment of Nox4 to a plasma membrane scaffold is required for localized reactive oxygen species generation and sustained Src activation in response to insulin-like growth factor-I.
abstract: Nox4-derived ROS is increased in response to hyperglycemia and is required for IGF-I-stimulated Src activation. This study was undertaken to determine the mechanism by which Nox4 mediates sustained Src activation. IGF-I stimulated sustained Src activation, which occurred primarily on the SHPS-1 scaffold protein. In vitro oxidation experiments indicated that Nox4-derived ROS was able to oxidize Src when they are in close proximity, and Src oxidation leads to its activation. Therefore we hypothesized that Nox4 recruitment to the plasma membrane scaffold SHPS-1 allowed localized ROS generation to mediate sustained Src oxidation and activation. To determine the mechanism of Nox4 recruitment, we analyzed the role of Grb2, a component of the SHPS-1 signaling complex. We determined that Nox4 Tyr-491 was phosphorylated after IGF-I stimulation and was responsible for Nox4 binding to the SH2 domain of Grb2. Overexpression of a Nox4 mutant, Y491F, prevented Nox4/Grb2 association. Importantly, it also prevented Nox4 recruitment to SHPS-1. The role of Grb2 was confirmed using a Pyk2 Y881F mutant, which blocked Grb2 recruitment to SHPS-1. Cells expressing this mutant had impaired Nox4 recruitment to SHPS-1. IGF-I-stimulated downstream signaling and biological actions were also significantly impaired in Nox4 Y491F-overexpressing cells. Disruption of Nox4 recruitment to SHPS-1 in aorta from diabetic mice inhibited IGF-I-stimulated Src oxidation and activation as well as cell proliferation. These findings provide insight into the mechanism by which localized Nox4-derived ROS regulates the sustained activity of a tyrosine kinase that is critical for mediating signal transduction and biological actions.
authors.affiliation: Department of Medicine, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Xi
authors.forename: Xin-Chun
authors.initials: XC
authors.lastname: Shen
authors.forename: Christine
authors.initials: C
authors.lastname: Wai
authors.forename: David R
authors.initials: DR
authors.lastname: Clemmons
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism; D001011:Aorta / Q000378:metabolism / Q000473:pathology; D021961:Cell Membrane Structures / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D003921:Diabetes Mellitus, Experimental / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D004789:Enzyme Activation / Q000235:genetics; D051380:GRB2 Adaptor Protein / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D020125:Mutation, Missense; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D013552:Swine; D018909:src Homology Domains; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D000943:Antigens, Differentiation; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495949:Grb2 protein, mouse; C501922:Sirpa protein, mouse; D017382:Reactive Oxygen Species; D011971:Receptors, Immunologic; C501920:SIRPA protein, human; D007334:Insulin-Like Growth Factor I; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; C422943:Nox4 protein, mouse; D019061:src-Family Kinases
keywords: Atherosclerosis; Diabetes; Insulin-like Growth Factor (IGF); NADPH Oxidase; Reactive Oxygen Species (ROS); Signal Transduction; Src