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Identification of Src-Specific Phosphorylation Site on Focal Adhesion Kinase: Dissection of the Role of Src SH2 and Catalytic Functions and Their Consequences for Tumor Cell Behavior

¹ Beatson Institute for Cancer Research, Cancer Research UK Beatson Laboratories, Glasgow, United Kingdom and ² ARIAD Pharmaceuticals, Cambridge, Massachusetts

Requests for reprints: Valerie G. Brunton, Beatson Institute for Cancer Research, Cancer Research UK Beatson Laboratories, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK. Phone: 44-141-330-3956; Fax: 44-141-942-6521; E-mail: v.brunton{at}beatson.gla.ac.uk

Src tyrosine kinase expression and activity are elevated during colon cancer progression. How this contributes to the malignant phenotype is not fully understood. We show that in KM12C colon carcinoma cells, expression of kinase-deficient Src proteins (SrcMF and Src251) does not alter cell growth. Src kinase activity is required for turnover of cell-matrix adhesions and, in particular, the Src-dependent phosphorylation of focal adhesion kinase (FAK) is required for their disassembly. Surprisingly, we found that expression of SrcMF or Src251 resulted in increased tyrosine phosphorylation of FAK on Tyr⁴⁰⁷, Tyr⁵⁷⁶, Tyr⁵⁷⁷, and Tyr⁸⁶¹, which are considered to be Src kinase substrates. This Src kinase–independent phosphorylation of FAK required an intact Src SH2 domain that mediates association of Src and FAK at peripheral adhesions. Use of a novel highly potent and selective Src kinase inhibitor AP23464 combined with experiments in Src/Fyn/Yes–deficient fibroblasts showed that increased phosphorylation of FAK in cells expressing SrcMF did not require Src-like kinases. However, specific phosphorylation on Tyr⁹²⁵ of FAK was not evident in SrcMF- or Src251-expressing cells, and lack of Src kinase–dependent phosphorylation on this site was associated with impaired adhesion turnover. Our data show that Src kinase activity is required for adhesion turnover associated with cell migration in cancer cells and that, in addition to the catalytic activity, Src also acts as an adaptor to recruit other kinases that can phosphorylate key substrates including FAK. These studies have implications for tumor progression with respect to the use of Src kinase inhibitors.

Key Words: Src kinase • focal adhesion kinase • colon cancer • cell migration

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