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Fig. 2. CrkII mediates the Dock1 interaction with Dab1 and requires Dab1 Y220 or Y232 for binding. (A) Dock1 (lanes 1-3, upper panel) and CrkII (lanes 4-6, upper panel) were detected by western blotting lysates from HEK293T cells transfected with Dock1 (lanes 2,3), and/or CrkII (lanes 1,3). Dock1 only associated with the immobilized tyrosine-phosphorylated GST-Dab1 in the presence of overexpressed CrkII (lane 3, lower panel) and not in its absence (lane 2, lower panel). In contrast, Crk II associated with GST-Dab1 in both the absence (lane 4, lower panel) and presence (lane 6, lower panel) of Dock1. (B) Expression of Dab1 wild-type (lanes 1,3, upper panel), the indicated Dab1 tyrosine to phenylalanine mutants (lanes 4-7, upper panel) and CrkII (lanes 9-14, upper panel) was detected in total cell lysates of transfected HEK293T cells, which were cotransfected with the activated Src527F mutant. Co-immunoprecipitation of Dab1 and CrkII was detected by immunoprecipitation with anti-CrkII (lanes 1-7, lower panel) or anti-Dab1 (lanes 8-14, lower panel) and western blotting with anti-Dab1 (lanes 1-7, lower panel) or anti-CrkII antibodies (lanes 8-14, lower panel). Wild-type Dab1 (lanes 3,10, lower panel) and single-site substituted Dab1 (lanes 4-6 and 11-13, lower panel), but not the Dab1 Y220F-Y232F double mutant (lanes 7,14, lower panel) supported co-immunoprecipitation with CrkII. Dab1 molecules that are phosphorylated at Y232 have reduced electrophoretic mobility as compared with unphosphorylated Dab1 or Dab1 with phenylalanine substitutions at this position (compare lanes 1 and 3-5 with 6 and 7, lower panel) (see also Howell et al., 2000). This figure shows representative results obtained in three independent experiments.

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