Figure 5

Cell adhesion induced cortactin tyrosine phosphorylation requires FAK autophosphorylation at Tyr³⁹⁷. (A) Adhesion induced FAK and p130Cas tyrosine phosphorylation. 10, 30 and 120 min indicate the time elapsed after the HEK293 cells were plated onto fibronectin-coated dishes. 0 min, suspended cells; Ad, cells cultured in dishes for 24 hours. α-FAK, α-pFAK, α-p130Cas and α-pp130 indicate western blots with anti-FAK, anti-phosphotyrosine³⁹⁷ FAK, anti-p130Cas and anti-phosphotyrosine⁴¹⁰ p130Cas antibodies, respectively (B) Adhesion-induced cortactin tyrosine phosphorylation. Cortactin was immunoprecipitated (IP) from suspended HEK293 cells (0 min) and adhering cells (10, 30 and 120 min) with anti-cortactin antibody (α-Cttn). Cortactin tyrosine phosphorylation was detected with an anti-phosphotyrosine antibody (α-PY). (C) Co-immunoprecipitation of cortactin and FAK. Cortactin was immunoprecipitated from adherent HEK293 cells with anti-cortactin antibody (α-Cttn) or control IgG (IgG). The immunoprecipitated samples were analyzed by western blotting. (D) Immunofluorescence staining of FAK and cortactin during cell adhesion. Actin was stained with phalloidin. Bar, 10 μm. (E) Immunofluorescence staining of Tyr³⁹⁷-phosphorylated FAK and Tyr⁴²¹-phosphorylated cortactin during cell adhesion. Bar, 10 μm. (F) FAK expression and autophosphorylation in cortactin knockdown cells. Cortactin was knocked down by RNA interference in COS7 cells. Cortactin, FAK and Tyr³⁹⁷-autophosphorylated FAK were detected by western blotting. α-Tubulin was used as the loading control. (G) Cortactin expression and tyrosine phosphorylation in FAK knockdown cells. FAK was knocked down by RNA interference in COS7 cells. siRNA, small interfering RNA; Luci, luciferase siRNA; FAK, FAK siRNA. FAK and cortactin proteins were detected by western blotting. Cortactin was immunoprecipitated with anti-cortactin antibody (α-Cttn) and its tyrosine phosphorylation was detected using an anti-phosphotyrosine antibody (α-PY). (H) Immunofluorescence staining of tyrosine phosphorylated cortactin in FAK knockdown cells. FAK RNAi (FAK Ri) and control RNAi (Luci Ri) cells were stained for FAK (FAK) and Tyr⁴²¹-phosphorylated cortactin (pCttn). Bar, 50 μm. (I) Expression and phosphorylation of GFP-tagged FAK mutant (Y397F). The lysates of cells expressing GFP-tagged FAK or mutated FAK (Y397F) were blotted with anti-EGFP (α-GFP), anti-FAK (α-FAK), anti-phosphotyrosine³⁹⁷ FAK (α-pFAK) or anti-phosphotyrosine⁵⁷⁶ FAK (α-pFAK⁵⁷⁶) antibodies. (J) Cortactin tyrosine phosphorylation in cells expressing the GFP-tagged FAK mutant (Y397F). Cortactin in cells expressing the GFP-tagged or mutated (Y397F) FAK was immunoprecipitated with an anti-cortactin antibody and blotted with an anti-cortactin (α-Cttn), anti-phosphotyrosine (α-PY) or anti-EGFP (α-GFP) antibody.