title: TGFβ1 rapidly activates Src through a non-canonical redox mechanism.
abstract: Transforming growth factor-β (TGF-β1 is involved in multiple cellular processes including epithelial-mesenchymal transition (EMT), and many of them are mediated through activating Src kinase, a non-receptor tyrosine kinase. How TGF-β1activates Src remains largely unknown. In the classic pathway Src activation is initiated by dephosphorylation of pTyr530 followed by a conformational change that permits autophosphorylation at Tyr419 with full activation. As Src is activated by various oxidative stimuli and TGF-β reportedly stimulates H2O2 production, we hypothesize that TGF-β activates Src through a redox dependent mechanism. We found that TGF-β exposure increased Src phosphorylation at Tyr419 (pTyr419 Src), but also at Tyr530 (pTyr530 Src), which is inconsistent with the canonical activation mechanism. The non-specific tyrosine phosphatase inhibitor vanadate alone increased pTyr530 Src, but had little effect on TGF-β-mediated Src activation. TGF-β increased extracellular H2O2 transiently with a peak at about 10min. GSH-ester and catalase increased basal Src activity but abrogated TGF-β-mediated Src activation. The potential involvement of any cysteine residues in Src activation by TGF-β1was further examined by site-directed mutation of cysteine to alanine using a FLAG-tagged human Src construct. Mutation of 248C/A, 277C/A, 490C/A, or 501C/A did not change Src stability but abrogated the activation of Src by TGF-β. Taken together our data demonstrate that TGF-β-mediated Src activation is through a redox dependent mechanism, and mutation of cysteines 248, 277, 490, and 501 all inhibit Src activation.
authors.affiliation: Andrus Gerontology Center, Davis School of Gerontology, University of Southern California.
authors.forename: Hongqiao
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Andrus Gerontology Center, Davis School of Gerontology, University of Southern California; School of Natural Science, University of California, Merced.
authors.forename: Henry Jay
authors.initials: HJ
authors.lastname: Forman