Structures and biochemical characterization of bcr/abl proteins.
(A) Schematic of full-length and mutant bcr/abl proteins. The identical COOH-terminal abl portion of the chimeric protein (shaded bar) is present in all fusions. Some of the characterized domains are indicated on the left of wild-type P210. The NH2-terminal of the Abl portion is preceded by different truncated Bcr fragments to produce (1-63) bcr/abl, (1-210) bcr/abl, and Δ(1-63) bcr/abl. P210Tyr177Phe bcr/abl is derived from wild-type P210 with a Tyr177Phe substitution. (B) Kinase activity of wild-type P210 and bcr/abl mutants. Bosc cells were transiently transfected with the indicated constructs and labeled with 35S-methionine. Whole cell lysates were then immunoprecipitated with anti-Abl antibody 24-11. Equal amounts of each immunoprecipitated sample were subjected to SDS-PAGE to reveal the immunoprecipitated bcr/abl protein. Equal aliquots of each immunoprecipitated sample were subjected to an in vitro kinase assay. The figure shows both autophosphorylated bcr/abl and the phosphorylated exogenous substrate GST-Crk. To show specificity, Bosc cells were transfected with MigR1 or P210, immunoprecipitated with control mouse IgG or 24-11 and subjected to identical procedures (middle panel). To show that the immunoprecipitated bcr/abl specifically phosphorylated Crk on phosphotyrosine, the Crk212 mutant,20 which lacks the critical Tyr221 residue, was also used as an exogenous substrate. The α-abl mAb, 24-11, was used to immunoprecipitate the lysates of a v-Abl–expressing cell line N54 or P185-expressing cell line FL5.12 and identical assays were performed using 5 μg of either GST-Crk225 or GST-Crk212. As shown in the bottom section of panel B, Crk225 (lanes 2 and 4), but not Crk212 (lanes 1 and 3), was phosphorylated in this assay. Identical results were obtained in assays using P210 or P210Tyr177-expressing FL5.12 cells (data not shown). (C) P210 bcr/abl was in vitro translated and labeled with 35S-methionine alone or together with either the C-terminal His-tagged bcr polypeptide from P210's bcr portion or the C-terminal His-tagged Δ(1-63) bcr polypeptide from Δ(1-63) bcr/abl's Δ(1-63) bcr portion (lanes 1-3). The in vitro translated products were immunoprecipitated with an α-His mAb. The α-His mAb did not pull down P210 (lane 4), whereas it was able to pull down both the bcr and Δ(1-63) bcr polypeptides (lanes 5 and 6). P210 could only be coimmunoprecipitated with bcr polypeptides but not with the Δ(1-63) bcr polypeptide (lane 5 and 6). The correct P210 band was confirmed by immunoprecipitation with the α-abl mAb 24-11 (data not shown). (D) Grb2 fails to associate with bcr/abl mutants lacking Tyr177. Whole cell lysates of Bosc cells transfected with the indicated construct were coimmunoprecipitated with α-abl 24-11. The coimmunoprecipitated samples were immunoblotted with α-abl (upper panel) or α-grb2 antibody (middle panel). The upper panel shows that each bcr/abl protein was immunoprecipitated and the corresponding proteins are indicated on the right. The middle panel shows that only the wild-type P210 (lane 2), (1-210) bcr/abl (lane 5) and Δ(1-63) bcr/abl (lane 6) bound grb2. In the bottom panel, the whole cell lysates were immunoblotted with α-grb2 antibody as the internal control.
