Overexpression of 14-3-3ζ in cancer cells activates PI3K via binding the p85 regulatory subunit.

The ubiquitously expressed 14-3-3 proteins regulate many pathways involved in transformation. Previously, we found that 14-3-3ζ overexpression increased Akt phosphorylation in human mammary epithelial cells. Here, we investigated the clinical relevance and molecular mechanism of 14-3-3ζ-overexpression-mediated Akt phosphorylation, and its potential impact on breast cancer progression. We found that 14-3-3ζ overexpression was significantly (P=0.005) associated with increased Akt phosphorylation in human breast tumors. Additionally, 14-3-3ζ overexpression combined with strong Akt phosphorylation was significantly (P=0.01) associated with increased cancer recurrence in patients. In contrast, knockdown of 14-3-3ζ expression by small interfering RNA in cancer cell lines and tumor xenografts reduced Akt phosphorylation. Furthermore, 14-3-3ζ enhanced Akt phosphorylation through activation of phosphoinositide 3-kinase (PI3K). Mechanistically, 14-3-3ζ bound to the p85 regulatory subunit of PI3K and increased PI3K translocation to the cell membrane. A single 14-3-3-binding motif encompassing serine 83 on p85 is largely responsible for 14-3-3ζ-mediated p85 binding and PI3K/Akt activation. Mutation of serine 83 to alanine on p85 inhibited 14-3-3ζ binding to the p85 subunit of PI3K, reduced PI3K membrane localization and activation, impeded anchorage-independent growth and enhanced stress-induced apoptosis. These findings revealed a novel mechanism by which 14-3-3ζ overexpression activates PI3K, a key node in the mitogenic signaling network known to promote malignancies in many cell types.

Differential interactions of specific nuclear factor I isoforms with the glucocorticoid receptor and STAT5 in the cooperative regulation of WAP gene transcription.

The distal region (-830 to -720 bp) of the rat whey acidic protein (WAP) gene contains a composite response element (CoRE), which has been demonstrated previously to confer mammary gland-specific and hormonally regulated WAP gene expression. Point mutations in the binding sites for specific transcription factors present within this CoRE have demonstrated the importance of both nuclear factor I (NFI) and STAT5 as well as cooperative interactions with the glucocorticoid receptor (GR) in the regulation of WAP gene expression in the mammary gland of transgenic mice. This study reports the characterization of NFI gene expression during mammary gland development and the identification and cloning of specific NFI isoforms (NFI-A4, NFI-B2, and NFI-X1) from the mouse mammary gland during lactation. Some but not all of these NFI isoforms synergistically activate WAP gene transcription in cooperation with GR and STAT5, as determined using transient cotransfection assays in JEG-3 cells. On both the WAP CoRE and the mouse mammary tumor virus long terminal repeat promoter, the NFI-B isoform preferentially activated gene transcription in cooperation with STAT5A and GR. In contrast, the NFI-A isoform suppressed GR and STAT cooperativity at the WAP CoRE. Finally, unlike their interaction with the NFI consensus binding site in the adenovirus promoter, the DNA-binding specificities of the three NFI isoforms to the palindromic NFI site in the WAP CoRE were not identical, which may partially explain the failure of the NFI-A isoform to cooperate with GR and STAT5A.

Cooperative effects of STAT5 (signal transducer and activator of transcription 5) and C/EBPbeta (CCAAT/enhancer-binding protein-beta) on beta-casein gene transcription are mediated by the glucocorticoid receptor.

Beta-casein gene transcription is controlled primarily by a composite response element (CoRE) that integrates signaling from the lactogenic hormones, PRL, insulin, and hydrocortisone, in mammary epithelial cells. This CoRE contains binding sites for STAT5 (signal transducer and activator of transcription 5) and C/EBPbeta (CCAAT/enhancer-binding protein-beta) and several half-sites for glucocorticoid receptor (GR). To examine how interactions among these three transcription factors might regulate beta-casein gene transcription, a COS cell reconstitution system was employed. Cooperative transactivation was observed when all three factors were expressed, but unexpectedly was not seen between STAT5 and C/EBPbeta in the absence of full-length, transcriptionally active GR. Cooperativity required the amino-terminal transactivation domain of C/EBPbeta, and neither C/EBPalpha nor C/EBPdelta was able to substitute for C/EBPbeta when cotransfected with STAT5 and GR. Different GR determinants were needed for transcriptional cooperation between STAT5 and GR as compared with those required for all three transcription factors. These studies provide some new insights into the mechanisms responsible for high level, tissue-specific expression conferred by the beta-casein CoRE.

Regulation of milk protein gene expression.

Studies using both transgenic mice and transfected mammary epithelial cells have established that composite response elements containing multiple binding sites for several transcription factors mediate the hormonal and developmental regulation of milk protein gene expression. Activation of signal transduction pathways by lactogenic hormones and cell-substratum interactions activate transcription factors and change chromatin structure and milk protein gene expression. The casein promoters have binding sites for signal transducers and activators of transcription 5, Yin Yang 1, CCAAT/enhancer binding protein, and the glucocorticoid receptor. The whey protein gene promoters have binding sites for nuclear factor I, as well as the glucocorticoid receptor and the signal transducers and activators of transcription 5. The functional importance of some of these factors in mammary gland development and milk protein gene expression has been elucidated by studying mice in which some of these factors have been deleted.

Differential effects of prolactin and src/abl kinases on the nuclear translocation of STAT5B and STAT5A.

In this study, DNA binding and tyrosine phosphorylation of STAT5A and STAT5B were compared with their subcellular localization determined using indirect immunofluorescence microscopy. Following prolactin activation, both STAT5A and STAT5B were rapidly translocated into the nucleus and displayed a detergent-resistant, punctate nuclear staining pattern. Similar to prolactin induction, src activation resulted in tyrosine phosphorylation and DNA binding of both STAT5A and STAT5B. However, nuclear translocation of only STAT5B but not STAT5A was observed. This selective nuclear translocation appears to be mediated via the carboxyl-terminal sequences in STAT5B. Furthermore, overexpression of a dominant negative kinase-inactive mutant of JAK2 prevented prolactin-induced tyrosine phosphorylation and nuclear translocation of STAT5A and STAT5B but did not block src kinase activation and nuclear translocation of STAT5B. In co-transfection assays, prolactin-mediated activation but not src kinase-mediated activation of STAT5B resulted in the induction of a beta-casein promoter-driven reporter construct. These results suggest that STAT5 activation by src may occur by a mechanism distinct from that employed in cytokine activation of the JAK/STAT pathway, resulting in the selective nuclear translocation of STAT5B.

Glucocorticoid receptor/signal transducer and activator of transcription 5 (STAT5) interactions enhance STAT5 activation by prolonging STAT5 DNA binding and tyrosine phosphorylation.

The regulation of casein gene expression by both PRL and glucocorticoids has been a well studied paradigm for understanding how the signaling pathways regulated by these two hormones interact in the nucleus. Previous studies have demonstrated that the downstream effectors of these pathways, signal transducer and activator of transcription 5 (STAT5) and the glucocorticoid receptor (GR), are associated via protein-protein interactions and act synergistically to enhance beta-casein gene transcription. Indirect immunofluorescence microscopy was used to demonstrate that PRL-activated STAT5 can translocate GR into the nucleus, and that ligand-bound GR can translocate STAT5 into the nucleus. This provided further support of an interaction between the two proteins. To better understand the mechanism of transcriptional synergy between STAT5 and GR, experiments were performed in cells transiently transfected with STAT5 alone or with STAT5 and GR. GR cotransfection enhanced the DNA-binding activity of STAT5 without affecting STAT5 protein levels. The enhancement of STAT5 DNA binding by GR resulted in the formation of a complex that exhibited prolonged DNA binding after PRL treatment. This was correlated with increased STAT5 tyrosine phosphorylation, suggesting that GR enhances STAT5 DNA binding by modulating the rate of STAT5 dephosphorylation. In contrast, cotransfection of the estrogen receptor resulted in an overall decrease in STAT5 tyrosine phosphorylation, without changing the kinetics of dephosphorylation. Enhancement of STAT5 activity by GR is, therefore, one component of the transcriptional synergy exhibited by STAT5 and GR at the beta-casein promoter and is an example of how transcription factors at a composite response element may modulate each other's activity.