NF-κB is a major gene regulator in immune responses and ribosomal protein S3 (RPS3) is an NF-κB subunit that directs specific gene transcription. However, it is unknown how RPS3 nuclear translocation is regulated. Here we report that IKKβ phosphorylation of serine 209 (S209) was crucial for RPS3 nuclear localization in response to activating stimuli. Moreover, the foodborne pathogen Escherichia coli O157:H7 virulence protein NleH1 specifically inhibited RPS3 S209 phosphorylation and blocked RPS3 function, thereby promoting bacterial colonization and diarrhea but decreasing mortality in a gnotobiotic piglet infection model. Thus, the IKKβ-dependent modification of a specific amino acid in RPS3 promotes specific NF-κB functions that underlie the molecular pathogenetic mechanisms of E. coli O157:H7.

17beta-Estradiol (E2) acts through the estrogen receptor alpha (ERalpha) to stimulate breast cancer proliferation. Here, we investigated the functional relationship between ERalpha and signal transducer and activator of transcription (STAT)5b activity in ER+ MCF-7 and T47D human breast cancer cells after specific knockdown of STAT5b. STAT5b small interfering RNA (siRNA) inhibited E2-induced bromodeoxyuridine (BrdU) incorporation in both cell lines, as well as the E2-induced increase in MCF-7 cell number, cyclin D1 and c-myc mRNA, and cyclin D1 protein expression, indicating that STAT5b is required for E2-stimulated breast cancer proliferation. E2 treatment stimulated STAT5b tyrosine phosphorylation at the activating tyrosine Y699, resulting in increased STAT5-mediated transcriptional activity, which was inhibited by a Y669F STAT5b mutant. E2-induced STAT5-mediated transcriptional activity was inhibited by overexpressing a kinase-defective epidermal growth factor receptor (EGFR), or the EGFR tyrosine kinase inhibitor tyrphostin AG1478, indicating a requirement for EGFR kinase activity. Both E2-induced STAT5b tyrosine phosphorylation and STAT5-mediated transcription were also inhibited by the ER antagonist ICI 182,780 and the c-Src inhibitor PP2, indicating additional requirements for the ER and c-Src kinase activity. EGFR and c-Src kinase activities were also required for E2-induced cyclin D1 and c-myc mRNA. Together, these studies demonstrate positive cross talk between ER, c-Src, EGFR, and STAT5b in ER+ breast cancer cells. Increased EGFR and c-Src signaling is associated with tamoxifen resistance in ER+ breast cancer cells. Here we show that constitutively active STAT5b not only increased basal DNA synthesis, but also conferred tamoxifen resistance. Because STAT5b plays an integral role in E2-stimulated proliferation and tamoxifen resistance, it may be an effective therapeutic target in ER+ breast tumors.

Introduction Signal transducers and activators of transcription (STATs) are mediators of cytokine and growth factor signaling. In recent years, STAT5b has emerged as a key regulator of tumorigenesis. STAT5b phosphorylation and activation is mediated by several kinases known to be overexpressed in breast cancer, such as epidermal growth factor receptor, HER2, and c-Src. Breast tumor kinase (Brk), also known as protein tyrosine kinase 6, is a nonreceptor tyrosine kinase expressed in more than 60% of breast cancers. Only a few substrates of the Brk tyrosine kinase have been identified, the most recent being STAT3. In the present article we investigate the potential role of Brk in the phosphorylation and activation STAT5b.

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.