title: Tyrosyl phosphorylation and DNA binding activity of signal transducers and activators of transcription (STAT) proteins in hematopoietic cell lines transformed by Bcr/Abl.
abstract: Bcr/Abl is a chimeric oncogene that can cause both acute and chronic human leukemias. Bcr/Abl-encoded proteins exhibit elevated kinase activity compared to c-Abl, but the mechanisms of transformation are largely unknown. Some of the biological effects of Bcr/Abl overlap with those of hematopoietic cytokines, particularly interleukin 3 (IL-3). Such effects include mitogenesis, enhanced survival, and enhanced basophilic differentiation. Therefore, it has been suggested that p210Bcr/Abl and the IL-3 receptor may activate some common signal transduction pathways. An important pathway for IL-3 signaling involves activation of the Janus family kinases (JAKs) and subsequent tyrosyl phosphorylation of STAT proteins (signal transducers and activators of transcription). This pathway directly links growth factor receptors to gene transcription. We analyzed JAK activation, STAT protein phosphorylation, and the formation of specific DNA-binding complexes containing STAT proteins, in a series of leukemia cell lines transformed by Bcr/Abl or other oncogenes. We also examined these events in cell lines transformed by a temperature sensitive (ts) mutant of Bcr/Abl, where the kinase activity of Abl could be regulated. STAT1 and STAT5 were found to be constitutively phosphorylated in 32D, Ba/F3, and TF-1 cells transformed by Bcr/Abl, but not in the untransformed parental cell lines in the absence of IL-3. Phosphorylation of STAT1 and STAT5 was also observed in the human leukemia cell lines K562 and BV173, which express the Bcr/Abl oncogene, but not in several Bcr/Abl-negative leukemia cell lines. Phosphorylation of STAT1 and STAT5 was directly due to the tyrosine kinase activity of Bcr/Abl since it could be activated or deactivated by temperature shifting of cells expressing the Bcr/Abl ts mutant. DNA-STAT complexes were detected in all Bcr/Abl-transformed cell lines and they were supershifted by antibodies against STAT1 and STAT5. DNA-STAT complexes in 32Dp210Bcr/Abl cells were similar, but not identical, to those formed after IL-3 stimulation. It is interesting to note that JAK kinases (JAK1, JAK2, JAK3, and Tyk2) were not consistently activated in Bcr/Abl-positive cells. These data suggest that STATs can be activated directly by Bcr/Abl, possibly bypassing JAK family kinase activation. Overall, our results suggest a novel mechanism that could contribute to some of the major biological effects of Bcr/Abl transformation.
authors.affiliation: Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
authors.forename: N
authors.initials: N
authors.lastname: Carlesso
authors.forename: D A
authors.initials: DA
authors.lastname: Frank
authors.forename: J D
authors.initials: JD
authors.lastname: Griffin
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism; D002471:Cell Transformation, Neoplastic; D016207:Cytokines / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D016044:Fusion Proteins, bcr-abl / Q000096:biosynthesis* / Q000378:metabolism; D018922:HL-60 Cells; D006412:Hematopoietic Stem Cells; D006801:Humans; D008894:Milk Proteins; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D050794:STAT1 Transcription Factor; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
chemical_list: D016207:Cytokines; D004268:DNA-Binding Proteins; D008894:Milk Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl