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Fig. 5. Identification of the principal phosphorylation site of Asef. (A) Domain structures of Asef and Asef2. ABR, APC-binding region; SH3, Src-homology 3; DH, Dbl-homology; PH, pleckstrin homology. (B) Alignment of amino-terminal sequences of Asef and Asef2. Tyr94 and Tyr104 are shown in bold, and the ABR and SH3 domains are underlined and boxed, respectively. The NcoI recognition site used to construct Asef2-Asef1 and Asef1-Asef2 mutants (Asef2/1 and Asef/2, respectively) is shown. (C,D) HA-tagged proteins were expressed transiently in A431 cells and immunoprecipitated with anti-HA antibody before or after 25 ng/mL EGF stimulation for 2 minutes. Immunoprecipitates and total-cell lysates were resolved by SDS-PAGE and probed with anti-phosphotyrosine (pTyr) or anti-HA antibody. (E) A431 cells expressing HA-tagged Asef were stimulated with or without 100 ng/mL EGF for 2 minutes. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the antibodies indicated above each lane. In the case of anti-phosphorylated-Y94Asef ( ${alpha}$ -pY94Asef), phenylphosphate was included in the reaction buffer to reduce the signals of non-specific anti-phosphotyrosine antibody. (F) HA-tagged constructs were expressed in A431 cells and immunoprecipitated with anti-HA antibody as in C. Immunoprecipitates and total cell lysates were analyzed using the antibodies as indicated.

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