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Fig. 1. Tyrosine phosphorylation of focal adhesion components. A construct consisting of two consecutive SH2 domains (dSH2) fused to CFP or YFP was used as a reporter for PY residues. For FRET measurements between PY and vinculin or paxillin, dSH2 (fused to CFP or YFP) was co-transfected with paxillin or vinculin, fused to the complementary fluorophore. (A) Single-color images, ratio images and FRET images are displayed for the indicated pairs. Note high CFP-paxillin/YFP-dSH2 (pax-dSH2) and low FRET for CFP-vinculin/YFP-dSH2 (vin-dSH2). Bar, 2 µm. (B) Quantification of FI measurements with the indicated FRET pairs. The data are presented as mean ± standard error of FI in focal adhesions of 15 to 32 cells. (C) Western blots showing, on the left, immunoprecipitation of YFP and YFP-dSH2 from cells expressing these constructs. The blot on the upper right shows that a large number of tyrosine phosphorylated proteins co-immunoprecipitate with the YFP-dSH2 domain expressed in NIH 3T3 cells. One of the proteins that co-precipitated is paxillin (lower right blot). No tyrosine-phosphorylated proteins co-precipitated from control lysates from NIH 3T3 cells expressing YFP only. (D) Quantification of FRET measurements with the indicated FRET pairs. Note that FRET between dSH2 and a truncated form of paxillin devoid of major tyrosine phosphorylation sites is reduced by half compared with FI values between dSH2 and full-length paxillin, but still significantly higher than between dSH2 and vinculin.
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