The Significance of Tetramerization in Promoter Recruitment by Stat5

Abstract

Stat5a and Stat5b are rapidly activated by a wide range of cytokines and growth factors, including interleukin-2 (IL-2). We have previously shown that these signal transducers and activators of transcription (STAT proteins) are key regulatory proteins that bind to two tandem gamma interferon-activated site (GAS) motifs within an IL-2 response element (positive regulatory region III [PRRIII]) in the human IL-2R␣ promoter. In this study, we demonstrate cooperative binding of Stat5 to PRRIII and explore the molecular basis underlying this cooperativity. We demonstrate that formation of a tetrameric Stat5 complex is essential for the IL-2-inducible activation of PRRIII. Stable tetramer formation of Stat5 is mediated through protein-protein interactions involving a tryptophan residue conserved in all STATs and a lysine residue in the Stat5 N-terminal domain (N domain). The functional importance of tetramer formation is shown by the decreased levels of transcriptional activation associated with mutations in these residues. Moreover, the requirement for STAT protein-protein interactions for gene activation from a promoter with tandemly linked GAS motifs can be relieved by strengthening the avidity of protein-DNA interactions for the individual binding sites. Taken together, these studies demonstrate that a dimeric but tetramerization-deficient Stat5 protein can activate only a subset of target sites. For functional activity on a wider range of potential recognition sites, N-domainmediated oligomerization is essential.

IL-2 and long-term T cell activation induce physical and functional interaction between STAT5 and ETS transcription factors in human T cells

“…To characterize the composition of the IL-2-inducible complexes, EMSAs were performed with three probes corresponding to human GASd/EBSd, FcgRI GAS and mouse site I (Table 1). In contrast to previous reports (John et al, 1999;Meyer et al, 1997), the three probes revealed both complexes in human T cell nuclear extracts, evidencing that they were not a singularity of the human GASd/EBSd element ( Figure 1b). Supershift and inhibition of binding assays were performed with speci®c antisera.…”

“…This argues against an IL-2 inducible cooperative binding on both sites but does not preclude their functional interaction. Moreover, we con®rmed our previous report (Lecine et al, 1996) and observed e cient binding of two GAS-speci®c complexes on monomeric GAS-speci®c motif using both tumoral and primary human T cell extracts, whereas John and collaborators (John et al, 1999) did not report any signi®cant Stat5 binding with a monomeric GASc probe (almost identical of our GASd/EBSd probe). We did not elucidate this discrepancy but we suspect some di erences in EMSA protocols.…”

“…Two recent studies have underscored the requirement for Stat5 tetramerization mediated by the two PRRIII GAS motifs for e cient promoter recruitment and transcriptional activation (John et al, 1999;Meyer et al, 1997). Although the experiments presented here were not designed to address the respective role of the two GAS elements present within human PRRIII, some of our results di er signi®cantly from those recently reported.…”

“…To analyse this putative synergy and to avoid the complex interactions observed within the native IL-2rE (John et al, 1996(John et al, , 1999Meyer et al, 1997), wild-type and mutant GASd/EBSd trimers were inserted upstream of HSV TK minimal promoter ( Figure 4a). The design of these arti®cial targets was validated by the binding properties of WT and mutants GASd/EBSd motifs ( Figure 1a).…”

Potential role of signal transducer and activator of transcription (STAT)3 signaling pathway in inflammation, survival, proliferation and invasion of hepatocellular carcinoma

“…The N-terminal domain is involved in STAT dimerization and also in tertramerization interactions. The tetramerization of STATs contributes to stability of the STAT-DNA binding by means of the interaction with randomly arranged low-affinity STAT binding sites, thus increasing transcriptional activity [83]. Several studies have implicated the N-domain in various protein-protein interactions affecting transcription and it has been suggested that this domain enables dimerized STAT molecules to polymerize and to bind multiple DNA sites that are involved in oncogenic growth signaling pathways [84].…”