Figure 1

In vitro binding of paxillin and FAK mutants. A) The I937A and EKR mutations are indicated on the structure of the FAT domain. The four α-helices are labeled. The paxillin binding sites are at the interface of α-helices 1/4 and the interface of α-helices 2/3. B) Schematic representation of paxillin and paxillin GST-fusion proteins used for the binding studies. White boxes denote the LD motifs. The proteins are designated WT, LD4, LD2 and none to reflect functional LD motifs in the constructs. The previously published designation of each construct is in parentheses. C) CE cells transfected with empty RCAS vector (lane 1), wild type FAK (lanes 2–6), EKR (lanes 7–10), or I937A (lanes 11–14) were lysed and 1 mg of lysate was used for binding assays. The functional paxillin binding site in each of the constructs is indicated in parentheses. Lysates were incubated with GST alone (lane 2), the WT (lanes 3, 7, 11), the LD4 (lanes 4, 8, 12), the LD2 fusion proteins (lanes 5, 9, 13), or with the protein lacking both LD2 and LD4 sites (none; lanes 6, 10, 14). The protein complexes were washed and bound FAK detected by Western blotting. D) GST-fusion proteins used in C were analyzed in parallel by SDS-PAGE and Coomassie staining to ensure equal loading.