title: TGFβ1 rapidly activates Src through a non-canonical redox signaling mechanism.
abstract: Transforming growth factor-β1 (TGF-β) is involved in multiple cellular processes through Src activation. In the canonical pathway, Src activation is initiated by pTyr530 dephosphorylation followed by a conformational change allowing Tyr419 auto-phosphorylation. A non-canonical pathway in which oxidation of cysteine allows bypassing of pTyr530 dephosphorylation has been reported. Here, we examined how TGF-β activates Src in H358 cells, a small cell lung carcinoma cell line. TGF-β increased Src Tyr419 phosphorylation, but surprisingly, Tyr530 phosphorylation was increased rather than decreased. Vanadate, a protein tyrosine phosphatase inhibitor, stimulated Src activation itself, but rather than inhibiting Src activation by TGF-β, activation by vanadate was additive with TGF-β showing that pTyr530 dephosphorylation was not required. Thus, the involvement of the non-canonical oxidative activation was suspected. TGF-β increased extracellular H2O2 transiently while GSH-ester and catalase abrogated Src activation by TGF-β. Apocynin, a NADPH oxidase inhibitor, inhibited TGF-β-stimulated H2O2 production. Furthermore, mutation of cysteines to alanine, 248C/A, 277C/A, or 501C/A abrogated, while 490C/A significantly reduced, TGF-β-mediated Src activation. Taken together, the results indicate that TGF-β-mediated Src activation operates largely through a redox dependent mechanism, resulting from enhanced H2O2 production through an NADPH oxidase and that cysteines 248, 277, 490, and 501 are critical for this activation.
authors.affiliation: Ethel Percy Andrus Gerontology Center, Leonard Davis School of Gerontology, The University of Southern California, United States.
authors.forename: Hongqiao
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Ethel Percy Andrus Gerontology Center, Leonard Davis School of Gerontology, The University of Southern California, United States; Division of Molecular & Computational Biology, Department of Biological Sciences, Dornsife College of Letters, Arts, and Sciences, The University of Southern California, United States.
authors.forename: Kelvin J A
authors.initials: KJ
authors.lastname: Davies
authors.affiliation: Ethel Percy Andrus Gerontology Center, Leonard Davis School of Gerontology, The University of Southern California, United States; School of Natural Science, The University of California, Merced, United States. Electronic address: peroxideman@gmail.com.
authors.forename: Henry Jay
authors.initials: HJ
authors.lastname: Forman
mesh_terms: D045744:Cell Line, Tumor; D004789:Enzyme Activation; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism; D010084:Oxidation-Reduction; D010766:Phosphorylation; D017354:Point Mutation; D015398:Signal Transduction; D055752:Small Cell Lung Carcinoma / Q000235:genetics / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: C508960:TGFB1 protein, human; D053773:Transforming Growth Factor beta1; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; D019061:src-Family Kinases
keywords: Hydrogen peroxide; Oxidative modification; Redox signaling; Src; TGF-β