The Stat (signal transducer and activator of transcription) factors transmit cytokine, growth factor, and hormone responses. Seven members of the Stat gene family are known. MGF-Stat5a has been discovered as a mediator of the prolactin response in mammary epithelial cells. Two closely related variants of Stat5, Stat5a and Stat5b, are encoded by distinct genes. We examined the functional properties of the carboxyl termini of these molecules. Wild-type Stat5a (794 amino acids) and the carboxyl-terminal deletion mutant Stat5a⌬772 supported prolactin-induced transcription of a ␤-casein promoter-reporter construct in COS7 cells; Stat5a⌬750 did not. Upon prolactin activation, tyrosine phosphorylation and the specificity of DNA binding were indistinguishable among the three Stat5a variants. Tyrosine dephosphorylation and the downregulation of the DNA-binding activity were delayed in the Stat5a⌬750 mutant. The carboxyl-terminal transactivation domain of Stat5a, amino acids 722 to 794, can be conferred to the DNA-binding domain of the yeast transcription factor GAL4. Coexpression of Stat5a or Stat5b and of the carboxyl-terminal deletion mutants resulted in the suppression of transcriptional induction in COS or Ba/F3 cells. We propose that Stat5a⌬750 and Stat5b⌬754 are lacking functional transactivation domains and exert their dominant negative effects by blocking the DNA-binding site in Stat5-responsive gene promoters.The Jak-Stat pathway relays cytokine and growth factor signals from the cell surface to the nucleus (19, 20, 36). Jak (Janus kinase) factors are a family of receptor-associated tyrosine kinases. Stat (signal transducer and activator of transcription) factors are transcription factors regulated by Jak-catalyzed phosphorylation events. The Stat gene family contains seven members which share structural similarities.First insights have been gained into the domain structure of the Stat factors. A distinctive feature of the Stat factors is an SH2 domain, a phosphotyrosine-binding domain. It mediates specific interactions of the Stat factors with the cytoplasmic region of cytokine receptors (14,16,45) and is required for Stat dimerization (43). Stat dimers translocate to the nucleus and bind to specific DNA sequences in the promoters of responsive genes. The use of chimeric Stat1-Stat3 or Stat1-Stat6 fusion molecules led to the identification of a DNA-binding domain. It is localized in a region between amino acid positions 400 and 500, distinct from the SH3 and SH2 domains (17, 38).The slightly different sequences of the Stat DNA-binding motifs, their arrangement in the promoter regions of individual genes, and the properties of the Stat dimers are determinants of the specificity of transcriptional regulation (23,38,42). Selective recruitment of Stats to the receptor, specificity of Stat DNA binding, and specific transactivation properties are mechanisms by which a specific cytokine response is elicited.Alignments show that sequence diversity among members of the Stat family is most pronounced in the carboxyl-termi...

Stat5 was discovered as a PRL-induced member of the Stat (signal transducer and activator of transcription) family. Its induction by many other cytokines and interleukins suggests that Stat5 plays a crucial role not only in mammary epithelial, but also in hematopoietic cells. Cell type- and promoter-specific functions of Stat5 are most likely modulated by the interaction with other transcription factors. We recently showed cross-talk between Stat5 and the glucocorticoid receptor. The activated glucocorticoid receptor forms a complex with Stat5 and enhances Stat5-mediated transcriptional induction. Conversely, Stat5 diminishes the induction of glucocorticoid-responsive genes. Here, we investigated the role of p300/CBP(CREB-binding protein), a transcriptional coactivator of several groups of transcription factors, in Stat5-mediated transactivation and in the cross-talk between Stat5 and the glucocorticoid receptor. p300/ CBP acts as a coactivator of Stat5. Its ectopic expression enhances PRL-induced Stat5-mediated transcriptional activation. Consistent with this observation, we find that the adenovirus E1A protein, which binds to p300/CBP, suppresses Stat5-induced transcriptional activation. This inhibition requires the Stat5 transactivation domain and the p300/CBP binding site of E1A. Coimmunoprecipitation and mammalian two-hybrid assays demonstrate a direct interaction between the carboxyl-terminal transactivation domain of Stat5 and p300/CBP. p300/CBP also positively interacts with the glucocorticoid receptor and enhances glucocorticoid receptor-dependent transcriptional activation of the mouse mammary tumor virus-long terminal repeat promoter. Overexpression of p300/CBP does not counteract the Stat5-mediated inhibition of glucocorticoid receptor-dependent transactivation, i.e. the repression of the glucocorticoid response by Stat5 is not a consequence of competition for limiting amounts of p300/CBP.

Deleted in liver cancer 1 (DLC1) is a Rho-GTPase-activating protein (GAP) that is downregulated in various tumor types. In vitro, DLC1 specifically inactivates the small GTPases RhoA, RhoB and RhoC through its GAP domain and this appears to contribute to its tumor suppressor function in vivo. Molecular mechanisms that control DLC1 activity have not so far been investigated. Here, we show that phorbol-ester-induced activation of protein kinase C and protein kinase D stimulates association of DLC1 with the phosphoserine/phosphothreonine-binding 14-3-3 adaptor proteins via recognition motifs that involve Ser327 and Ser431. Association with 14-3-3 proteins inhibits DLC1 GAP activity and facilitates signaling by active Rho. We further show that treatment of cells with phorbol ester or coexpression of 14-3-3 proteins, blocks DLC1 nucleocytoplasmic shuttling, probably by masking a previously unrecognized nuclear localization sequence. The binding to 14-3-3 proteins is thus a newly discovered mechanism by which DLC1 activity is regulated and compartmentalized.

Deleted in liver cancer (DLC) 1 and 2 are Rho GTPaseactivating proteins that are frequently down-regulated in various types of cancer. Ectopic expression in carcinoma cell lines lacking these proteins has been shown to inhibit cell migration and invasion. However, whether the loss of DLC1 or DLC2 is the cause of aberrant Rho signaling in transformed cells has not been investigated. Here, we have down-regulated DLC1 and DLC2 expression in breast cancer cells using a RNA interference approach. Silencing of DLC1 led to the stabilization of stress fibers and focal adhesions and enhanced cell motility in wound-healing as well as chemotactic Transwell assays. We provide evidence that enhanced migration of cells lacking DLC1 is dependent on the Rho effector protein Dia1 but does not require the activity of Rho kinase. By contrast, DLC2 knockdown failed to affect the migratory behavior of cells, suggesting that the two proteins have distinct functions. This is most likely due to their differential subcellular localizations, with DLC1 found in focal adhesions and DLC2 being mainly cytosolic. Collectively, our data show that DLC1 is critically involved in the control of Rho signaling and actin cytoskeleton remodeling and that its cellular loss is sufficient for the acquisition of a more migratory phenotype of breast cancer cells. [Cancer Res 2008;68(21):8743-51]

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