Relevance of Phosphotyrosines in the Transactivation Domain of STAT5b: Implications for STAT5b in Breast Cancer

Signal transducers and activators of transcription (STATs) are mediators of cytokine and growth factor induced gene expression. With the ability to regulate transcription of genes involved in cell cycle progression, cell survival, and cell growth, it is not surprising that STATs, especially STAT3, 5a, and 5b, are activated in several cancers, including breast cancer. Phosphorylation of a single tyrosine is required for transcriptional activity, and for STAT5b this is tyrosine 699. Although additional tyrosine phosphorylation sites (Y725, Y740, Y743) have been identified, their implications for biological activity have not been elucidated. The significance of tyrosine phosphorylation of STAT5b by kinases known to be overexpressed in breast cancer, such as the epidermal growth factor receptor (EGFR), cSrc, and breast tumor kinase (Brk) was investigated. Through site-specific tyrosine to phenylalanine (Y→F) mutations, the biological importance of these tyrosines was examined in the EGFR/c-Src/Brk overexpressing human breast cancer cell line, SKBr3. Mutation of Y725 decreased basal and EGF-induced DNA synthesis. In contrast, mutation of Y740 and/or Y743 increased basal DNA synthesis, Y699 phosphorylation, and transcriptional activity. These data indicate that Y699 and Y725 are positive regulators, and Y740 and Y743 are negative regulators of STAT5b activity. The ability of EGFR, c-Src, and Brk to phosphorylate the four tyrosines was examined. While c-Src kinase mediated the phosphorylation of Y699 and Y725, the EGFR kinase mediated the phosphorylation of all four tyrosines. Additionally, anti-phospho-Y740/3 specific antibodies demonstrated that c-Src kinase activity inhibited EGF-induced phosphorylation of Y740 and Y743. Phosphorylation specific antibodies and mutational analysis demonstrated that Brk specifically mediated the phosphorylation of Y699, but 3 not Y725, Y740, or Y743. The establishment of how STAT5b activity is regulated, through tyrosine phosphorylation (both positively and negatively) and which kinases mediate this phosphorylation provides mechanistic insight into the function and regulation of the STAT5b protein. This knowledge sets the foundation for targeting the transactivation domain of this protein to modulate its molecular function in breast cancer. Elucidating the regulation of STAT5b activity will further aid in the development of therapies designed to prevent the initiation and progression of breast cancer.

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