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Fig. 4. Deletion effect of Cas domain structure on Cas-induced Rap1 activation. (A) Schematic representation of Cas variants used for the evaluation of Rap1 activation competence. (B) Overexpression effect of Cas and its deletion mutants on Rap1 activity was examined as in Fig. 1. Cas overexpression increases the amount of GTP-bound Rap1 compared with control pCAX transfectant. Cas {Delta}SD failed to activate Rap1, whereas Cas {Delta}SCB substantially retained the activity. (C) Tyrosine phosphorylation and Crk interaction of Cas mutants. 293T cells were transfected with a Cas derivative expression plasmid together with Crk. After immunoprecipitation with anti-HA antibody, the tyrosine phosphorylation levels of Cas proteins and co-immunoprecipitated Crk in the immunoprecipitates were analyzed.