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

• Published in: The Journal of biological chemistry Vol. 288; no. 22; pp. 15641 - 15653
• Main Authors: Xi, Gang, Shen, Xin-Chun, Wai, Christine, Clemmons, David R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 31.05.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Substitution; Animals; Antigens, Differentiation - genetics; Antigens, Differentiation - metabolism; Aorta - metabolism; Aorta - pathology; Atherosclerosis; Cell Membrane Structures - genetics; Cell Membrane Structures - metabolism; Cell Membrane Structures - pathology; Cells, Cultured; Diabetes; Diabetes Mellitus, Experimental - genetics; Diabetes Mellitus, Experimental - metabolism; Diabetes Mellitus, Experimental - pathology; Enzyme Activation - genetics; GRB2 Adaptor Protein - genetics; GRB2 Adaptor Protein - metabolism; Humans; Insulin-like Growth Factor (IGF); Insulin-Like Growth Factor I - genetics; Insulin-Like Growth Factor I - metabolism; Mice; Mutation, Missense; NADPH Oxidase; NADPH Oxidase 4; NADPH Oxidases - genetics; NADPH Oxidases - metabolism; Oxidation-Reduction; Protein Binding; Reactive Oxygen Species (ROS); Reactive Oxygen Species - metabolism; Receptors, Immunologic - genetics; Receptors, Immunologic - metabolism; Signal Transduction; Signal Transduction - genetics; Src; src Homology Domains; src-Family Kinases - genetics; src-Family Kinases - metabolism; Swine; Reactive Oxygen Species (ROS); Signal Transduction; Src; Atherosclerosis; Diabetes; Insulin-like Growth Factor (IGF); NADPH Oxidase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.456046

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. Background: Nox4-derived ROS is required for Src oxidation and activation. Results: Grb2 recruits Nox4 to the scaffold protein SHPS-1, and Nox4 colocalization with Src on SHPS-1 allows Nox4 to activate Src. Conclusion: Nox4 recruitment to SHPS-1 is essential for sustained Src activation and cell proliferation. Significance: This is the first report that localized ROS generation mediates growth factor signaling in vitro and in vivo.