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Signal transducers and activators of transcription (STATs) are most notably known for their pivotal role in cytokine and growth factor signalling pathways. Activation of STAT proteins is mediated by phosphorylation on a conserved tyrosine residue. This tyrosine phosphorylation leads to dimerization and translocation to the nucleus resulting gene transcription. STAT3, 5a, and 5b regulate tran scription of genes involved in cell cycle progression, cell survival, and cell growth and are activated in several cancers, including breast cancer. Although phosphorylation of Y694 and Y699 of STAT5a and STAT5b (respectively) is required for transcriptional activity, additional tyrosine phosphorylation sites have been identified. In several human breast cancer cell lines STAT5b is expressed at higher levels than STAT5a, we examined the biological implications of additional STAT5b tyrosine phosphorylation in this context. In the SKBr3 human breast cancer cell line, EGF stimulation induced STAT5b tyrosine phosphorylation on Y699, Y725, Y740, and Y743. Mutation of Y740 and/or Y743 resulted in a basally active STAT5b, as measured by Y699 phosphorylation, transcriptional analysis, and DNA synthesis. In contrast, the mutation of Y725 decreased basal and EGF-induced DNA synthesis. Together, these data suggest that in addition to Y699, Y725 phosphorylation is a positive regulator of STAT5b activity, while Y740 and Y743 phosphorylation is a negative regulator. Serine 731 (S731), found previously to regulate STAT5b activity, lies between these positive and negative regulatory tyrosine sites in the STAT5b transactivation domain. We found that the Y740/743F STAT5b mutant increased S731 phosphorylation. Furthermore, mutation of S731 abrogated the increased basal transcriptional activity of the Y740/743F mutant. These results indicate that the enhanced basal activity of the Y740/743F mutant is dependent upon S731 phosphorylation. Finally, investigation of the kinases responsible for phosphorylation of each tyrosine site showed that c-Src mediated Y699 and Y725 phosphorylation, while EGF-induced activation of the EGFR mediated the phosphorylation of all four tyrosines. Understanding the molecular mechanism by which these tyrosines (Y740/Y743) modulate the activity of the carboxy terminal transactivation domain of STAT5b will provide insight into STAT5b mediated gene transcription in cancer cells, and these insights may lead to potential therapeutic targets for breast cancer.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]