Fig. 7. Cortactin phosphorylation is not required for N-cadherin association but is necessary for adhesion strengthening. (A) N-cadherin immunoprecipitates from acceptor monolayers and DAM samples at 15, 60 and 180 minutes. Cells were treated with and without PP2. Immunoprecipitation of the cytoplasmic domain of N-cadherin shows that cortactin association with N-cadherin is not dependent on the phosphorylation status of cortactin. Untreated N-cadherin immunoprecipitates immunoblotted with phospho-cortactin (tyrosine residue 421) or PY-20 antibodies show general and residue-specific tyrosine phosphorylation of cadherin-associated cortactin. No cortactin phosphorylation was detected in PP2-treated samples but the association of cortactin with cadherin was unchanged. (B) Inhibition of cortactin phosphorylation using either genistein (100 µm) or PP2 (25 µm) strongly reduced adhesion strengthening. Wash-off shear assays of DAM after 30 minutes of adhesion (n=3 replicate samples). (C) Tyrosine mutated cortactin Myc-tagged construct (F-cort: phenylalanine substitution) was physically associated with N-cadherin adhesion complex on N-cadherin ligation stimulated by DAM. Verification of expression of Myc-tagged tyrosine-mutated and wild-type cortactin (WT-cort) constructs in ratfibroblasts by western blot (left panel). Immunoprecipitations were conducted using N-cadherin antibody and Myc monoclonal antibody in Myc F-cort-transfected fibroblasts. (D) Samples transfected with WT-cort or F-cort were incubated with recombinant N-cad-Fc-coated beads and jet washed in a logarithmic series following a 15-minute incubation period. Cells within a sample were divided into two groups based on Myc-tagged protein expression. The number of beads bound per cell was quantified. Representative images for samples in the fourth, eighth and sixteenth washes show the degree of bead binding to cells for transfected (F-cort) and control cells. Outline provided for untransfected controls. Bar, 20 µm.