Activation of Src kinases p53/56(lyn) and p59(hck) by p210(bcr/abl) in myeloid cells

Chronic myeloid leukemia is characterized by the Philadelphia (Ph¹) translocation t(9;22) that generates a hybrid gene, bcr/abl, translated to a M(r)210,000 tyrosine kinase (p210(bcr/abl)) with transforming activity for hematopoietic cells. Hematopoietic cell transformation by p210(bcr/abl) seems to involve activation of the Ras signaling pathway by at least two different signaling intermediates, growth factor receptor-bound protein 2 and Src homology and collagen protein, but additional signaling proteins are likely to be required as well. In an effort to identify additional phosphoproteins activated by p210(bcr/abl), we studied the murine, interleukin 3-dependent, myeloid cell line, 32D, and a bcr/abl-transfected, factor-independent subline, 32Dp210. The analysis of whole-cell lysates of 32D and 32Dp210 cells showed that several proteins with a molecular weight of M(r)50,000-60,000 were phosphorylated on tyrosine residues in 32Dp210 cells. Because Src family kinases have an apparent molecular weight of M(r)50,000-60,000, we asked whether they could become activated by p210(bcr/abl). Two Src family kinases, p53/56(lyn) and p59(hck), showed a severalfold higher phosphokinase activity in 32Dp210 cells than in 32D cells. Coimmunoprecipitation experiments with anti-Lyn, anti-Hck, and anti-Abl antibodies demonstrated an intracellular association of p210(bcr/abl) with p53/56(lyn) and p59(hck). Moreover, the phosphokinase activity of p53/56(lyn) was higher in bcr/abl-positive myeloid cell lines (K562, BV173, and LAMA84) than in the bcr/abl-negative myeloid cell line JOSK-M. In conclusion, the results show that p210(bcr/abl) induces the activation of at least two Src family kinases, p53/56(lyn) and p59(hck) in myeloid cells. These findings extend the range of potential targets of p210(bcr/abl) that might mediate its transforming effects.