USE OF A TEMPERATURE-SENSITIVE MUTANT TO DEFINE THE BIOLOGICAL EFFECTS OF THE P210(BCR-ABL) TYROSINE KINASE ON PROLIFERATION OF A FACTOR-DEPENDENT MURINE MYELOID CELL-LINE

The Philadelphia chromosome, detected in virtually all cases of chroni c myelogenous leukemia, is formed by a reciprocal translocation betwee n chromosomes 9 and 22 that fuses BCR encoded sequences upstream of ex on 2 of c-ABL. This oncogene produces a fusion protein, p210(BCR-ABL), in which the ABL tyrosine kinase activity is elevated. This elevated kinase activity is essential for transformation, but the mechanisms in volved are unknown. To investigate p210(BCR-ABL) function we construct ed a model system in which the tyrosine kinase activity of p210(BCR-AB L) was inducible. Two amino acid substitutions, Arg to His at amino ac id 457 and Tyr to His at amino acid 469 of c-abl, modeled on mutations known to render v-src temperature-sensitive for tyrosine kinase activ ity, were introduced into p210(BCR-ABL). This mutant was characterized in an IL-3 growth factor dependent murine myeloid cell line, 32Dc13. Cell lines expressing the temperature-sensitive mutant remained factor dependent at the non-permissive temperature, but at the permissive te mperature displayed a marked reduction in cell death in the absence of growth factor and an exaggerated proliferative response to low levels of IL-3. Both the kinase activity of the mutant and the levels of tyr osine phosphorylated proteins are increased in the temperature-sensiti ve mutant at the permissive temperature. Further, tyrosine phosphoryla tion of potential substrates of the p210(BCR-ABL) tyrosine kinase, p12 0 rasGAP and its associated proteins of p190 and p62, only occurs at t he permissive temperature in cells expressing the temperature-sensitiv e mutant.