Use of a temperature-sensitive mutant to define the biological effects of the p210BCR-ABL tyrosine kinase on proliferation of a factor-dependent murine myeloid cell line.
The Philadelphia chromosome, detected in virtually all cases of chronic myelogenous leukemia, is formed by a reciprocal translocation between chromosomes 9 and 22 that fuses BCR encoded sequences upstream of exon 2 of c-ABL. This oncogene produces a fusion protein, p210BCR-ABL, in which the ABL tyrosine kinase activity is elevated. This elevated kinase activity is essential for transformation, but the mechanisms involved are unknown. To investigate p210BCR-ABL function we constructed a model system in which the tyrosine kinase activity of p210BCR-ABL was inducible. Two amino acid substitutions, Arg to His at amino acid 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 activity, were introduced into p210BCR-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 temperature 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 tyrosine phosphorylated proteins are increased in the temperature-sensitive mutant at the permissive temperature. Further, tyrosine phosphorylation of potential substrates of the p210BCR-ABL tyrosine kinase, p120 rasGAP and its associated proteins of p190 and p62, only occurs at the permissive temperature in cells expressing the temperature-sensitive mutant.
Carlesso
N
N
Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, MA.
Griffin
J D
JD
Druker
B J
BJ
D000818:Animals; D001483:Base Sequence; D002455:Cell Division; D002460:Cell Line; D016044:Fusion Proteins, bcr-abl / Q000502:physiology*; D007377:Interleukin-3 / Q000494:pharmacology; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics / Q000473:pathology; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D016315:Proto-Oncogene Proteins c-abl / Q000502:physiology; D013696:Temperature
D007377:Interleukin-3; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl; D016315:Proto-Oncogene Proteins c-abl