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First published online 14 February 2006
doi: 10.1242/jcs.02794

Journal of Cell Science 119, 866-875 (2006)
Published by The Company of Biologists 2006
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Research Article

Molecular mapping of tyrosine-phosphorylated proteins in focal adhesions using fluorescence resonance energy transfer

Christoph Ballestrem, Noam Erez, Joachim Kirchner, Zvi Kam, Alexander Bershadsky and Benjamin Geiger*

Department of Molecular Cell Biology, The Weizmann Institute of Science, 76100 Rehovot, Israel

* Author for correspondence (e-mail: benny.geiger{at}weizmann.ac.il)

Accepted 14 November 2005

Microscopy-based fluorescence resonance energy transfer (FRET) provides an opportunity to monitor molecular processes in the natural environment in live cells. Here we studied molecular interactions and tyrosine phosphorylation of paxillin, Crk-associated substrate (CAS), and focal adhesion kinase (FAK) in focal adhesions. For that purpose, these focal adhesion phosphoproteins, fused to cyan or yellow fluorescent proteins (CFP or YFP) were expressed in cultured fibroblasts. To assess the dynamics of tyrosine phosphorylation we used YFP- or CFP-tagged SH2 domain of pp60src (dSH2), which specifically binds to phosphotyrosine residues. FRET measurements, combined with immunolabeling with phosphospecific antibodies revealed that FAK, CAS and paxillin are tyrosine phosphorylated in early matrix adhesions and that FAK is in FRET proximity to CAS and paxillin in focal complexes and focal adhesions. Data suggest that paxillin incorporation into nascent focal complexes precedes its tyrosine phosphorylation, which then gradually increases. In cells treated with Rho-kinase inhibitors or expressing constitutively active Rac, focal complexes showed similar levels of paxillin tyrosine phosphorylation as seen in mature focal adhesions. Dynamic FRET-based examination indicated that paxillin phosphorylation occurs in specific areas (hotspots) within focal adhesions, whereas FAK phosphorylation is broadly distributed.

Key words: Focal adhesions, FRET, Paxillin, FAK, CAS, Phosphorylation




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