Tyrosine Phosphorylation of Connexin 43 by v-Src Is Mediated by SH2 and SH3 Domain Interactions*
- From the ‡Molecular Biology and Virology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037 and §Molecular Carcinogenesis, Cancer Research Center of Hawaii, University of Hawaii at Manoa, Honolulu, Hawaii 96813
Abstract
Reduction of gap junctional communication in v-src transformed cells is accompanied by tyrosine phosphorylation of the gap junction protein, connexin 43 (Cx43). Cx43 is phosphorylated on tyrosine by v-Src. The Src homology 3 (SH3) and Src homology 2 (SH2) domains of v-Src mediate interactions with substrate proteins. SH3 domains interact with proline-rich peptide motifs. SH2 domains associate with short amino acid sequences containing phosphotyrosine. We present evidence that the SH3 and SH2 domains of v-Src bind to proline-rich motifs and a phosphorylated tyrosine residue in the C-terminal tail of Cx43. Cx43 bound to the SH3 domain of v-Src, but not c-Src, in vitro. Tyrosine-phosphorylated Cx43 bound to the SH2 domain of v-Src in vitro. v-Src coprecipitated with Cx43 from v-src-transformed Rat-1 fibroblasts. Mutations in the SH3 and SH2 domains of v-Src, and in the proline-rich region or tyrosine 265 of Cx43, reduced interactions between v-Src and Cx43 in vivo. Tyrosine phosphorylation of Cx43 was dependent on the association of v-Src and Cx43. These results provide further evidence for the direct involvement of v-Src in tyrosine phosphorylation of Cx43 and inhibition of gap junctional communication in v-src-transformed cells.
Footnotes
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↵* This work was supported by Research Grants CA09370, CA13884, CA14195 (to W. E.), and CA52098 (to A. F. L.) from the National Cancer Institute, National Institutes of Health, and ACS-PF-4275 (to M. Y. K.) from the American Cancer Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ Present address: Basic Sciences Div., Fred Hutchinson Cancer Research Center, 1100 Fairview North, Seattle, WA 98109.
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↵‖ To whom correspondence should be addressed. Tel.: 619-453-4100 (ext. 1386); Fax: 619-457-4765.
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↵1 The abbreviations used are: SH, Src homology; GJC, gap junctional communication; Cx, connexin; Tyr(P), phosphotyrosine; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PI, phosphatidylinositol; RT loop, arginine 95 and threonine 96 within the SH3 domain of c-Src.
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↵2 L. W. M. Loo, M. Y. Kanemitsu, and A. F. Lau, manuscript in preparation.
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- Received April 30, 1997.
- Revision received June 10, 1997.
