FIG. 5.

Phosphorylation of plakoglobin by Fer and Fyn kinases decreases plakoglobin-desmoplakin interaction and increases plakoglobin-α-catenin association. (A and D) GST or GST-plakoglobin fusion proteins (5 pmol) were phosphorylated with Fer purified from transfected RWP1 cells (A) or Fyn kinase immunoprecipitated from transfected RWP1 cells (D). Pull-down assays were then performed by incubating the GST proteins with 100 μg of total cell extracts from SW480. The amounts of the associated proteins were determined by using specific MAbs. (B and E) GST or GST-plakoglobin (1.2 pmol) (control and Fer phosphorylated [B] or Fyn phosphorylated [E]) was incubated with 0.4 or 0.8 pmol of α-catenin in a final volume of 200 μl. α-Catenin (0.08 pmol) was included as a reference (St). The numbers below the lanes indicate the amounts of bound α-catenin. (C and G) GST or GST-plakoglobin fusion proteins (5 pmol) were phosphorylated with Fer (C) or Fyn kinases (G), and pull-down assays were then performed by incubating the GST proteins with 100 or 200 μg of total cell extracts from SW480. The amount of associated desmoplakin was determined by using a specific MAb. (F) GST or GST-Tcf-4(1-80) (1.2 pmol) was incubated with 0.5 or 1 pmol of plakoglobin incubated or not with Fyn in a final volume of 200 μl. Plakoglobin (0.03 pmol) was included as a reference (St). The numbers below the lanes indicate the amounts of bound plakoglobin. WB, Western blotting; +, present; −, absent.