Characterization of P210^BCR-ABL mutants. (A) Schematic representation of wild-type P210^BCR-ABL and corresponding mutants with deletions or point mutations in critical functional domains. The C-C domain mediates protein oligomerization, Y177 binds GRB2, and Ph stands for pleckstrin homology. (B) Each construct was cloned into the pK1 vector that coexpresses the mutant of interest along with the puromycin resistance gene as a bicistronic message through an internal ribosomal entry site. Each construct was expressed in 32D cells and selected for growth in puromycin-treated medium in the presence of IL-3. A representative plot of 32D-cell proliferation is shown in which IL-3 was withdrawn from the media at day 0. All mutants and wild-type P210^BCR-ABL led to growth factor independence when expressed in this cell line. However, the Δ(1-63) mutant proliferated at a significantly lower rate (*), and cells expressing the control vector, pK1, rapidly died within 48 hours. Viability was determined by trypan blue exclusion; each sample was tested in triplicate. (C) Western blots of whole cell lysates from 32D cells expressing constructs shown in panel A were prepared using cells that had recently been transduced and selected (passage 3 or sooner after selection). Blots were assessed for BCR-ABL expression (anti-ABL), for whole cell phosphotyrosine (anti-PY), or for GRB2 expression as a loading control (anti-GRB2). The Δ(1-63) mutant shows reduced levels of phosphotyrosine, which may account for the lower proliferation rate seen in panel B.