TYROSYL PHOSPHORYLATION AND DNA-BINDING ACTIVITY OF SIGNAL TRANSDUCERS AND ACTIVATORS OF TRANSCRIPTION (STAT) PROTEINS IN HEMATOPOIETIC-CELL LINES TRANSFORMED BY BCR ABL/

Bcr/Abl is a chimeric oncogene that can cause both acute and chronic h uman leukemias. Bcr/Abl-encoded proteins exhibit elevated kinase activ ity compared to c-Abl, but the mechanisms of transformation are largel y unknown. Some of the biological effects of Bcr/Abl overlap with thos e of hematopoietic cytokines, particularly interleukin 3 (IL-3). Such effects include mitogenesis, enhanced survival, and enhanced basophili c differentiation. Therefore, it has been suggested that p210Bcr/Abl a nd the IL-3 receptor may activate some common signal transduction path ways. An important pathway for IL-3 signaling involves activation of t he 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 transcrip tion. We analyzed JAK activation, STAT protein phosphorylation, and th e formation of specific DNA-binding complexes containing STAT proteins , in a series of leukemia cell lines transformed by Bcr/Abl or other o ncogenes. We also examined these events in cell lines transformed by a temperature sensitive (ts) mutant of Bcr/Abl, where the kinase activi ty of Abl could be regulated. STAT1 and STAT5 were found to be constit utively phosphorylated in 32D, Ba/F3, and TF-1 cells transformed by Bc r/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 on cogene, but not in several Bcr/Abl-negative leukemia cell lines. Phosp horylation of STAT1 and STAT5 was directly due to the tyrosine kinase activity of Bcr/Abl since it could be activated or deactivated by temp erature shifting of cells expressing the Bcr/Abl tr mutant. DNA-STAT c omplexes were detected in all Bcr/Abl-transformed cell lines and they were supershifted by antibodies against STAT1 and STAT5. DNA-STAT comp lexes in 32Dp210Bcr/Abl cells were similar, but not identical, to thos e formed after IL-3 stimulation. It is interesting to note that JAK ki nases (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.