STATs Dimerize in the Absence of Phosphorylation

Abstract

Upon activation by tyrosine kinases, members of the STAT family of transcription factors form stable dimers that are able to rapidly translocate to the nucleus and bind DNA. Although crystal structures of activated, near full-length, Stat1 and Stat3 illustrate how STATs bind to DNA, they provide little insight into the dynamic regulation of STAT activity. To explore the unique structural changes Stat1 and Stat3 undergo when they become activated, full-length inactive recombinant proteins were prepared. To our surprise, even though these proteins are unable to bind DNA, our studies demonstrate that they exist as stable homodimers. Similarly, the Stat1 and Stat3 found in the cytoplasm of unstimulated cells also exhibit a dimeric structure. These observations indicate that Stat1 and Stat3 exist as stable homodimers prior to activation.Cytokines are important regulators of intercellular communication. They mediate pleiotropic cellular responses by binding to specific transmembrane spanning receptors (reviewed in Refs. 1 and 2). These receptors in turn activate intracellular signaling pathways, resulting in the induction of gene expression. The STAT 1 (signal transducers and activators of transcription) family of transcription factors transmits signals in response to cytokines. This family consists of seven members, Stat1, Stat2, Stat3, Stat4, Stat5a, Stat5b, and Stat6. Structural and functional studies have led to the identification of six conserved STAT domains (3-5): 1) the amino-terminal domain (ϳamino acids 1-125), which has been implicated in cooperativity of DNA binding to tandem gamma activation site (GAS) elements, nuclear translocation, and in receptor association (2); 2) the coiled-coil domain (ϳamino acids 125-325), which has been shown to mediate interactions with several other proteins (2); 3) the DNA binding domain (ϳamino acids 325-475); 4) the linker domain (ϳamino acids 475-575); 5) the SH2 domain ϩ tyrosine activation motif (ϳamino acids 575-710), which is essential for STAT recruitment to the receptor, STAT activation, and dimerization (2); and 6) the carboxyl-terminal transcriptional activation domain.The general outline for the STAT signaling paradigm elucidated a decade ago is now widely accepted (1, 2, 6). In this model, a specific interaction between a cytokine and its cognate receptor brings the cytoplasmic domains of this receptor into apposition, thereby promoting the transphosphorylation of receptor associated tyrosine kinases from the Janus kinase family. These activated Janus kinases in turn phosphorylate specific tyrosine motifs found in receptor endodomains, which then serve to mediate recruitment of the specific monomeric STATs to the receptor complex. Once at the receptor, the STATs become activated by a single tyrosine phosphorylation event. The activated STATs are then released, where upon they dimerize through the interaction between the SH2 domain of one STAT and the tyrosine-phosphorylated tail segment of the other STAT. This renders them competent for both rapid translocation ...

Interleukin 4 Regulates Phosphorylation of Serine 756 in the Transactivation Domain of Stat6

“…These results suggest that unlike Tyr 641 (27,28), Ser 756 phosphorylation is not essential for nuclear localization or Stat6 DNA binding (lane h). Recent work by Braunstein et al (12) has shown that Stat1 and Stat3 exist as preformed dimers in the absence of tyrosine phosphorylation yet cannot bind DNA (12). Here we show that, although IL4 can induce Stat6 serine phosphorylation in the absence of tyrosine phosphorylation, phosphorylation of Ser 756 alone is not sufficient to promote DNA binding (Fig.…”

“…Stat6 is one of seven Stat family members that is postulated to be recruited to newly phosphorylated tyrosine residues within activated receptors in the cytoplasm via their Src homology 2 domains, subsequently becoming tyrosine phosphorylated by Janus kinase or Src enzymes on a single and positionally conserved residue that is believed critical for Stat dimerization, nuclear localization, and gene transcription (10,11). However, recent evidence questions this model by proffering that Stats exist as preformed dimers in the absence of tyrosine phosphorylation (12).…”

Dietary compounds as potent inhibitors of the signal transducers and activators of transcription (STAT) 3 regulatory network

“…Areas in purple represent the different sites of action of specific inhibitors of STAT3 that have been recently identified. b Schematic representation of the structural domains of JAK proteins, adapted from Braunstein et al (2003). The N-terminal region of the JAK protein contains the JH3-JH7 domains (shown in purple) that are involved in protein binding with the receptor.…”

“…Activation of STATs is usually transient and highly regulated. STAT proteins contain seven conserved structural and functional domains ( First, the amino terminal NH 2 domain is involved in the dimerization of STAT proteins and in the stabilization of the interaction established between these dimers and DNA response elements (Braunstein et al 2003;Mertens et al 2006). The coiled-coil domain is responsible for controlling the process of import and export of proteins into and from the nucleus (Schindler et al 2007).…”

Implications of an antiparallel dimeric structure of nonphosphorylated STAT1 for the activation–inactivation cycle

“…These new structural data provide a basis for the often reported occurrence of nonphosphorylated STAT dimers (including STAT1) in cell extracts and with pure protein in solution (6)(7)(8)(9)(10). Biophysical assays show that, at moderate concentration (2 mg͞ml or less), the nonphosphorylated full-length STAT1 or STAT1 (1-683) used in the crystal structure study were mainly dimeric in solution (9).…”