Caveolin-1 is phosphorylated on Tyr14 in response to both oxidative and hyperosmotic stress. In the present paper, we show that this phosphorylation requires activation of the Src family kinase Fyn. Stress-induced caveolin phosphorylation was abolished by three Src kinase inhibitors, SU6656, PP2 and PD180970, and was not observed in fibroblasts derived from a Src, Yes and Fyn triple-knockout mouse (SYF−/−). Using cell lines derived from single-kinase-knockout mice (Src−/−, Yes−/− and Fyn−/−), we show that expression of Fyn, but not Src or Yes, is required for stress-induced caveolin phosphorylation. Heterologous expression of Fyn in the SYF−/− and Fyn−/− cells was sufficient to reconstitute stress-induced caveolin phosphorylation, and overexpression of Fyn in wild-type cells induced hyperphosphorylation of caveolin. Fyn was autophosphorylated following oxidative stress, verifying activation of this kinase. Interestingly, there was a concomitant increase in the phosphorylation of Fyn on its Csk (C-terminal Src kinase) site, indicating feedback inhibition. Csk binds to phosphocaveolin [Cao, Courchesne and Mastick (2002) J. Biol. Chem. 277, 8771–8774] and should phosphorylate any co-localized Src-family kinases. Oxidative-stress-induced phosphorylation of caveolin-1 also requires expression of Abl [Sanguinetti and Mastick (2003) Cell Signal. 15, 289–298]. Using inhibitors and cells derived from knockout mice, we verified a requirement for both Abl and Fyn in stress-induced caveolin phosphorylation in a single cell type. Our data suggest a novel mechanism for attenuation of Src-kinase activity by Abl: stable tyrosine phosphorylation of a scaffolding protein, caveolin, and recruitment of Csk. Paxillin, a substrate of both Abl and Src, organizes a similar regulatory complex.
- oxidative stress
- stress-induced tyrosine phosphorylation
↵1 Present address: Department of Physiology, School of Medicine, University of Nevada, Reno, NV 89557, U.S.A.
↵2 Present address: Department of Nutrition, Harvard School of Public Health, Boston, MA 02115, U.S.A.
Abbreviations used: Abl, Abelson tyrosine kinase; MAPK, mitogen-activated protein kinase; BMK1, big MAPK 1; Csk, C-terminal Src kinase; ERK, extracellular-signal-regulated kinase; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; HF, human fibroblast; JAK, Janus kinase; JNK, c-Jun N-terminal kinase; SH2, Src homology 2; SYF−/−, Src, Yes and Fyn triple-knockout mouse; WT, wild-type.
- The Biochemical Society, London ©2003