Figure 3.

Brk binds to EGFR in both an EGFR kinase-dependent and a Brk kinase-dependent manner and competes with Cbl for binding to EGFR Y1045. (a) Brk binds to EGFR in an EGFR kinase-dependent manner. SUM102 cells were transiently transfected with wild-type Brk for 24 h and then exposed to 0.5 μM gefitinib or not for 16 h prior to 10 nM EGF in 0.5% FBS medium for 5 min as indicated. Brk immunoprecipitates (I.P. α Brk), EGFR immunoprecipitates (I.P. α EGFR), or whole cell lysates were subjected to Western blotting with the indicated antibodies. IgG H.C. = IgG heavy chain. (b) Brk binds to EGFR in a Brk kinase-dependent manner. CHO cells were transiently transfected with a control vector, cotransfected with wild-type EGFR and wild-type Brk, or cotransfected with wild-type EGFR and Brk-K219M for 24 h. The cells were then exposed to 10 nM EGF in 0.5% FBS medium for 5 min as indicated. Brk (I.P. α Brk) and EGFR immunoprecipitates (I.P. α EGFR) and whole cell lysates were subjected to Western blotting with the indicated antibodies. (c) Brk binds to EGFR and is activated by EGFR in an EGFR Y1045 phosphorylation-dependent manner. CHO cells were transiently cotransfected with wild-type Brk construct and one of neo vector, wild-type EGFR, or EGFR-Y1045F for 24 h. The cells were then exposed to 10 nM EGF in 0.5% FBS medium for 5 min as indicated. Brk (I.P. α Brk) and EGFR immunoprecipitates (I.P. α EGFR) and whole cell lysates were subjected to Western blotting with the indicated antibodies. (d) Brk competes with Cbl for binding to EGFR Y1045. CHO cells were transiently transfected with stepwise increasing DNA concentrations of Brk constructs for 24 h. The cells were stimulated with 10 nM EGF for 5 min. EGFR immunoprecipitates (I.P. α EGFR) and whole cell lysates were subjected to Western blotting with the indicated antibodies.