The crystal structure of the DNA complex of a STAT-1 homodimer has been determined at 2.9 A resolution. STAT-1 utilizes a DNA-binding domain with an immunoglobulin fold, similar to that of NFkappaB and the p53 tumor suppressor protein. The STAT-1 dimer forms a contiguous C-shaped clamp around DNA that is stabilized by reciprocal and highly specific interactions between the SH2 domain of one monomer and the C-terminal segment, phosphorylated on tyrosine, of the other. The phosphotyrosine-binding site of the SH2 domain in each monomer is coupled structurally to the DNA-binding domain, suggesting a potential role for the SH2-phosphotyrosine interaction in the stabilization of DNA interacting elements.

Interferon ␥ (IFN-␥) induces rapid tyrosine phosphorylation of the latent cytoplasmic transcription factor, Stat1, which then forms homodimers, translocates to the nucleus and participates in IFN-␥-induced transcription. However, little is known of the interactions between Stat1 and the general transcription machinery during transcriptional activation. We show here that Stat1 can directly interact with the CREB-binding protein (CBP)͞p300 family of transcriptional coactivators. Specifically, two interaction regions were identified: the amino-terminal region of Stat1 interacts with the CREB-binding domain of CBP͞p300 and the carboxylterminal region of Stat1 interacts with the domain of CBP͞ p300 that binds adenovirus E1A protein. Transfection experiments suggest a role for these interactions in IFN-␥-induced transcription. Because CBP͞p300-binding is required for the adenovirus E1A protein to regulate transcription of many genes during viral replication and cellular transformation, it is possible that the anti-viral effect of IFN-␥ is based at least in part on direct competition by nuclear Stat1 with E1A for CBP͞p300 binding.

Stat1 alpha, Stat1 beta and a proteolytically defined truncated Stat1 (132–713, Stat1tc) have been prepared from recombinant sources. All three proteins were specifically phosphorylated on Tyr701 in vitro and the phosphoprotein purified to homogeneity. This was achieved by employing a new isolation scheme that does not include DNA affinity steps and readily allows for the isolation of tens of milligrams of activated Stat protein. The purified phosphoprotein was free of traces of unphosphorylated polypeptide as detected by mass spectrometry. The phosphorylated Stat1 preparations bound to various DNA recognition sites with the same Keq of approximately 1 × 10(‐9) M; distinction between ‘weak’ and ‘strong’ binding sites is determined by the very rapid dissociation (< 30 s, t1/2) from ‘weak’ sites compared with ‘strong’ sites (approximately 3 min, t1/2). Reports of ‘weak’ tandem binding sites in a natural gene caused us to examine binding to tandem sites leading to the finding that the Stat1 alpha or beta (38 amino acids shorter on the C terminus) bound to two tandem sites (but not two head‐to‐head sites) with a higher stability than to a single recognition site. The N‐terminally truncated protein Stat1tc did not show this cooperative binding, thus implicating the N‐terminal domain in promoting Stat1‐Stat1 dimer interaction.

STAT1 functions as both a constitutive transcriptional regulator and, in response to cytokine stimulation of cells, as an inducible tyrosine-phosphorylated transcription factor. Here, we identify and characterize a non-transferable nuclear targeting sequence in the STAT1 DNA-binding domain. This conserved signal is critical for the interferon-g (IFN-g)-induced nuclear import of phosphorylated STAT1 dimers and requires adjacent positively charged and hydrophobic residues for functioning. Additionally, the constitutive nucleocytoplasmic shuttling of STAT1 in the absence of IFN-g stimulation is revealed. Nuclear import and export of unphosphorylated STAT1 are demonstrated to be sensitive towards wheat germ agglutinin and to occur independently of the import receptor p97. Lossof-function mutations of the dimer-speci®c import signal block nuclear entry of tyrosine-phosphorylated STAT1, which in turn also prevents induction of cytokine-inducible target genes. Nevertheless, nuclear import of unphosphorylated STAT1 continues and the STAT1-dependent constitutive expression of caspases and the tumor necrosis factor-a-mediated induction of apoptosis proceed unaltered. Thus, tyrosine-phosphorylated and unphosphorylated STAT1 molecules shuttle via independent pathways to distinct sets of target genes.

STATs (signal transducers and activators of transcription) are a family of transcription factors that are specifically activated to regulate gene transcription when cells encounter cytokines and growth factors. The crystal structure of an NH2-terminal conserved domain (N-domain) comprising the first 123 residues of STAT-4 was determined at 1.45 angstroms. The domain consists of eight helices that are assembled into a hook-like structure. The N-domain has been implicated in several protein-protein interactions affecting transcription, and it enables dimerized STAT molecules to polymerize and to bind DNA cooperatively. The structure shows that N-domains can interact through an extensive interface formed by polar interactions across one face of the hook. Mutagenesis of an invariant tryptophan residue at the heart of this interface abolished cooperative DNA binding by the full-length protein in vitro and reduced the transcriptional response after cytokine stimulation in vivo.

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.

Interferon stimulation of cells leads to the tyrosine phosphorylation of latent Stat1 and subsequent transient accumulation in the nucleus that requires canonical transport factors. However, the mechanisms that control the predominantly cytoplasmic localization in unstimulated cells have not been resolved. We uncovered that constitutive energy- and transport factor-independent nucleocytoplasmic shuttling is a property of unphosphorylated Stat1, Stat3, and Stat5. The NH2- and COOH-terminal Stat domains are generally dispensable, whereas alkylation of a single cysteine residue blocked cytokine-independent nuclear translocation and thus implicated the linker domain into the cycling of Stat1. It is revealed that constitutive nucleocytoplasmic shuttling of Stat1 is mediated by direct interactions with the FG repeat regions of nucleoporin 153 and nucleoporin 214 of the nuclear pore. Concurrent active nuclear export by CRM1 created a nucleocytoplasmic Stat1 concentration gradient that is significantly reduced by the blocking of energy-requiring translocation mechanisms or the specific inactivation of CRM1. Thus, we propose that two independent translocation pathways cooperate to determine the steady-state distribution of Stat1.

The activation/inactivation cycle of STAT transcription factors entails their transition between different dimer conformations. Unphosphorylated STATs can dimerize in an antiparallel conformation via extended interfaces of the globular N-domains, whereas STAT activation triggers a parallel dimer conformation with mutual phosphortyrosine:SH2 domain interactions, resulting in DNA-binding and nuclear retention. However, despite the crucial role of STAT tyrosine phosphorylation in cytokine signaling, it has not been determined how this modification affects the stability and the conformational flexibility of STAT dimers. Here, we use analytical ultracentrifugation and electrophoretic mobility shift assay (EMSA) to study the association of STAT1 in solution before and after tyrosine phosphorylation. It is revealed that STAT1 formed high-affinity dimers (K d of Ϸ50 nM) with estimated half-lives of 20 -40 min irrespective of the phosphorylation status. Our results demonstrate that parallel and antiparallel conformations of STAT1 were present simultaneously, supported by mutually exclusive interfaces; and the transition between conformations occurred through affinity-driven dissociation/association reactions. Therefore, tyrosine phosphorylation was dispensable for DNA binding, but the phosphorylation enforced preformed SH2 domainmediated dimers, thus enhancing the DNA-binding activity of STAT1 >200-fold. Moreover, upon STAT1 activation the N-domains adopted an open conformation and engaged in interdimer interactions, as demonstrated by their participation in tetramerization instead of dimerization. Yet, homotypic N-domain interactions are not conserved in the STAT family, because the N-domain dissociation constants of STAT1, STAT3, and STAT4 differed by more than three orders of magnitude. In conclusion, STAT1 constantly oscillated between different dimer conformations, whereby the abundance of the dimerization interfaces was determined by tyrosine phosphorylation.analytical ultracentrifugation ͉ dimerization ͉ SH2 domain ͉ STAT transcription factors I nterferons and other cytokines are extracellular signaling molecules that exert their effects after binding to specific cell surface expressed receptors (1). The binding of IFN-␥ activates receptor-associated Jak kinases, which catalyze a series of tyrosine phosphorylation reactions leading to the activation of the transcription factor STAT1. The STATs, of which seven different genes are expressed in mammals, all consist of three independent structural subunits (2, 3). At the N terminus there is a proteolytically separable N-domain of 130 residues, followed by a large contiguous core fragment of almost 600 residues containing a coiled-coil domain, a DNA-binding domain, and an SH2 domain. A transferable transactivation domain of variable length and sequence that frequently is subject to alternative splicing is located at the C terminus (3). The STATs are best known for their cytokine-dependent transcriptional activities, and STAT activation was recognized as the central eve...