Deletion of the Carboxyl-Terminal Transactivation Domain of MGF-Stat5 Results in Sustained DNA Binding and a Dominant Negative Phenotype

Abstract

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...

Cooperative activity between HER oncogenes and the tumor suppressor IRF-1 results in apoptosis

“…A dominant negative version of STAT5a, STAT5aD750, together with a STAT sensitive reporter (Figure 8a) or the GAScontaining reporter (Figure 8b) was transiently transfected into NIH3T3 cells expressing IRF-1-hER and HER1. Figure 8a indicates that, as expected, the STAT5 dominant negative protein STAT5aD750 (Moriggl et al, 1996) inhibited the induction of the STAT responsive promoter construct after addition of EGF. In addition, the presence of STAT5aD750 abolished the synergistic activity of HER1 signaling and IRF-1 when the GAS-containing promoter construct (Figure 8b) or the ISRE-containing construct were used (Figure 8c).…”

“…Using the dominant negative mutant of STAT5, STAT5aD750 (Moriggl et al, 1996), we could show the STAT5 signaling is essential for this transcriptional synergy. Thus, STAT5 might synergize directly with IRF-1 on these promoters.…”

IL-3 dependent regulation of Bcl-xL gene expression by STAT5 in a bone marrow derived cell line

“…This dominant negative mutant contains a deletion of the last 45 amino acids that removes the transactivation domain but retains its DNA binding capacity (Moriggl et al, 1996). Ba/F3 cells were transfected with a pRSV/neo vector or a pRSVneo vector containing STAT5AD749, cDNA and stable G418 resistant cells were obtained.…”

“…STAT5AD749 has been shown to inhibit the transactivation of the b-casein gene promoter by STAT5A or STAT5B in transfected COS cells (Moriggl et al, 1996). We examined the dominant negative e ect of STAT5AD749 on the endogenous STAT5 activity in Ba/F3 cells.…”

“…All STAT members contain in their structure a SH2 domain which allows their selective recruitment to the receptor-kinase activated complex and the dimerization of the STATs (Heim et al, 1995;Shuai et al, 1994;Stahl et al, 1995). Functional domains involved in DNA binding and activation of transcription have been de®ned (Horvath et al, 1995;Schindler et al, 1995;Moriggl et al, 1996Moriggl et al, , 1997Schaefer et al, 1997).…”