Figure 4
Interaction of cortactin with FAK. (A) Diagram of the various cortactin mutants. Y represents the tyrosine residues at amino acid positions 421, 466, 475 and 482. F and E indicate Phe and Glu mutations, respectively. NTA, the N-terminal acidic region; Repeats, the cortactin tandem repeats; H, the helix region; Proline-rich, the proline-rich region; SH3, the Src-homology 3 domain. (B) Diagram of the various FAK mutants. P712/715A and P876/879A indicate Ala mutations at Pro712, 715 and Pro876, 879, respectively. FERM, the N-terminal FERM domain; Kinase, the central kinase domain; FAT, the C-terminal focal-adhesion targeting domain; FRNK, FAK-related non-kinase region; P, the proline-rich region. (C) GFP-tagged cortactin mutants expressed in COS7 cells were co-immunoprecipitated with anti-FAK antibody (α-FAK). Input, cell lysates; WB, western blot; IP, immunoprecipitation; α-GFP, anti-EGFP antibody. (D) The interaction of FAK with the cortactin phosphorylation mimic (Y/E) and phosphorylation-incompetent (Y/F) cortactin. (E) Interaction of SH3-deleted cortactin with FAK. GST fusion proteins immobilized on glutathione-beads were used to pull-down GFP-tagged FAK expressed in HEK293 cells. Coomassie, Coomassie blue staining. The arrow-heads indicate GST-cortactin and GST-SH3-deleted cortactin. The rectangle highlighted the low-exposed GFP-FAK bands. (F) The interaction of cortactin with FAK mutant (Y397F). GFP-tagged FAK or FAK mutant (Y397F) expressed in HEK293 cells was pulled-down with GST-cortactin. The arrowhead indicates GST-cortactin. (G) Interaction of cortactin with FAK proline-rich regions. GFP-tagged FAK mutants expressed in HEK293 cells were pulled down with GST-cortactin. (H) Interaction of cortactin with proline-rich region-mutated FRNK. FRNKm1, P712/715A FRNK; FRNKm2, P786/789A FRNK; FRNKm1/2, P712/715/786/789A FRNK.