Complex formation of Grb/F16 and Bcr-Abl.

(A) Grb4/F16 and Bcr-Abl form a complex in vitro. Bcr-Abl mutants with internal deletions of Bcr resulting in varying lengths of Bcr as indicated were in vitro translated and S35 labeled. We allowed 50 μL of the translation mix to autophosphorylate in vitro and then incubated it with 5 μg purified GST or GST-Grb4/F16. Bound fractions were separated by SDS-PAGE, and Bcr-Abl mutants were visualized by autoradiography. (B) The association of Grb4/F16 and Bcr-Abl is kinase-dependent. We transiently transfected 1 × 10⁶ 293 cells with a ts mutant of Bcr-Abl and grew these at either 32° (permissive temperature) or 39° C (restrictive temperature) for 8 hours before harvest. Cell lysates were incubated with 5 μg of GST or GST-Grb4/F16, and bound fractions were resolved on SDS-PAGE. Western blotting was performed with the anti-Abl antibody, 8E9. (C) Grb4/F16 binds to v-Abl. We transiently transfected 1 × 10⁶ 293 cells with v-Abl. Cells were harvested 48 hours after transfection, and lysates were incubated with 5 μg of GST or GST-Grb4/F16. Bound fractions were run out on SDS-PAGE. Western blotting was performed with the anti-Abl antibody 8E9. (D) Grb4/F16 and Bcr-Abl coimmunoprecipitate in vivo. In this experiment, 1 × 10⁶ 293 cells were co-transfected with either WT Bcr-Abl or the ts variant and Grb4/F16. Cells transfected with the ts mutant were shifted to the permissive temperature, 32°, or the restrictive temperature, 39°, for 8 hours before harvest. A coimmunoprecipitation experiment (IP) was performed on cell lysates with the use of an anti-Xpress antibody to detect tagged Grb4 (lanes 5-7) or a rabbit antimouse (RαM) antibody as control (lanes 1-4). Bound fractions were resolved on SDS-PAGE and immunoblotted with a phosphotyrosine antibody (δ-PY) (4G10) (upper 2 left panels), an anti-Xpress antibody to detect Grb4 (lowest left panel), or an anti-Abl antibody (8E9) (right panel).