Fig. 3. Overexpression of the Chat-Cas-Crk pathway member activates Rap1. (A) Overexpression effect of Chat and Cas on Rap1 activation was examined by a pull-down assay of GTP-bound Rap1 as in Fig. 1. Overexpression of Cas increases the amount of GTP-bound Rap1 like Chat. Co-expression of Cas with Chat enhanced the Rap1 activation (Chat+Cas), but the C-terminal portion of Cas inhibited it (Chat+Cas-C-half). (B) Rap1 activation by Chat, Cas or Crk overexpression. Overexpression of Crk showed the strongest Rap1 activation. Enhanced Rap1 activation by co-expression of Chat and Cas with Crk was hard to detect because of the robustness of the Crk-induced Rap1 activation.