Modulation of signal transducer and activator of transcription 5b activity in breast cancer cells by mutation of tyrosines within the transactivation domain

The signal transducer and activator of transcription (STAT) proteins are latent transcription factors activated by a variety of cytokines and growth factors. Activation leads to phosphorylation on a conserved tyrosine residue. Although phosphorylation of STAT5b on Y699 is required for activation, it was previously shown that in epidermal growth factor receptor (EGFR)-overexpressing cell lines, three tyrosines (Y725, Y740, and Y743) in the STAT5b transactivation domain are also phosphorylated upon epidermal growth factor stimulation. The significance of these additional tyrosine phosphorylation sites was analyzed in the context of the human breast cancer cell line SKBr3, which overexpresses the EGFR and c-Src. When compared with wild-type STAT5b, mutation of Y725 decreased basal and epidermal growth factor-induced DNA synthesis. In contrast, mutation of Y740 and/or Y743 enhanced basal STAT5b Y699 phosphorylation, basal transcriptional activity, and basal DNA synthesis compared with wtSTAT5b. This indicates that Y699 and Y725 are positive regulators and Y740 and Y743 are negative regulators for STAT5b activity. Anti-phospho-Y740/743-specific antibodies demonstrated that the c-Src tyrosine kinase inhibits the phosphorylation of these two sites. Furthermore, Y740 and Y743 were not detectably phosphorylated in breast cancer cells overexpressing c-Src, but the Y740/743F mutant increased basal activity suggesting that the conformation of the transactivation domain is important in regulating STAT5b activity. Mechanistic insight into the inhibitory action of Y740 and Y743 may lead to the development of therapeutics that specifically modulate the activity of STAT5b in breast cancer and potentially other EGFR/c-Src-overexpressing cancers.