Evidence against a role for ß-arrestin1 in STAT1 dephosphorylation and the inhibition of interferon-γ signaling.

Signal transducer and activator of transcription 1 (STAT1) is activated by tyrosine phosphorylation upon interferon-γ (IFNγ) stimulation, which results in the expression of genes with antiproliferative and immunomodulatory functions. The inactivation of STAT1 occurs through tyrosine dephosphorylation by the tyrosine phosphatase TC45. It was proposed that recruitment of TC45 required the direct interaction of STAT1 with the scaffold protein ß-arrestin1, making ß-arrestin1 an essential negative regulator of STAT1 and IFNγ signaling (Mo et al., 2008). We tested the relevance of ß-arrestin1 for STAT1 activity. Our results do not confirm ß-arrestin1 as a STAT1-interacting protein. The STAT1 phosphorylation/dephosphorylation cycle was found to be unaffected by both the overexpression and the genetic deletion of ß-arrestin1. Accordingly, ß-arrestin1 did not inhibit STAT1 transcriptional activity or the induction of IFNγ target genes in response to IFNγ. Our data indicate that ß-arrestin1 is dispensable for STAT1 dephosphorylation and the termination of IFNγ signaling.

Characterization of STAT self-association by analytical ultracentrifugation.

Multiple experimental tools have demonstrated that cytokine-induced STAT activation entails the transition of dimer conformations rather than de novo dimerization. In this chapter, we describe the utilization of analytical ultracentrifugation (AUC) as a powerful technique for the quantitative analysis of hydro- and thermodynamic properties of STAT proteins in solution. These studies provided a quantitative understanding of dimer stability and conformational transitions associated with the activation of STAT1.

STAT1:DNA sequence-dependent binding modulation by phosphorylation, protein:protein interactions and small-molecule inhibition.

The DNA-binding specificity and affinity of the dimeric human transcription factor (TF) STAT1, were assessed by total internal reflectance fluorescence protein-binding microarrays (TIRF-PBM) to evaluate the effects of protein phosphorylation, higher-order polymerization and small-molecule inhibition. Active, phosphorylated STAT1 showed binding preferences consistent with prior characterization, whereas unphosphorylated STAT1 showed a weak-binding preference for one-half of the GAS consensus site, consistent with recent models of STAT1 structure and function in response to phosphorylation. This altered-binding preference was further tested by use of the inhibitor LLL3, which we show to disrupt STAT1 binding in a sequence-dependent fashion. To determine if this sequence-dependence is specific to STAT1 and not a general feature of human TF biology, the TF Myc/Max was analysed and tested with the inhibitor Mycro3. Myc/Max inhibition by Mycro3 is sequence independent, suggesting that the sequence-dependent inhibition of STAT1 may be specific to this system and a useful target for future inhibitor design.

Molecular basis for the recognition of phosphorylated STAT1 by importin alpha5.

Interferon-gamma stimulation triggers tyrosine phosphorylation of the transcription factor STAT1 at position 701, which is associated with switching from carrier-independent nucleocytoplasmic shuttling to carrier-mediated nuclear import. Unlike most substrates that carry a classical nuclear localization signal (NLS) and bind to importin alpha1, STAT1 possesses a nonclassical NLS recognized by the isoform importin alpha5. In the present study, we have analyzed the mechanisms by which importin alpha5 binds phosphorylated STAT1 (pSTAT1). We found that a homodimer of pSTAT1 is recognized by one equivalent of importin alpha5 with K(d)=191+/-20 nM. Whereas tyrosine phosphorylation at position 701 is essential to assemble a pSTAT1-importin alpha5 complex, the phosphate moiety is not a direct binding determinant for importin alpha5. In contrast to classical NLS substrates, pSTAT1 binding to importin alpha5 is not displaced by the N-terminal importin beta binding domain and requires the importin alpha5 C-terminal acidic tail (505-EEDD-508). A local unfolding of importin alpha5 Armadillo (ARM) repeat 10 accompanies high-affinity binding to pSTAT1. This unfolding is mediated by a single conserved tyrosine at position 476 of importin alpha5, which is inserted between ARM repeat 10 helices H1-H2-H3, thereby preventing intramolecular helical stacking essential to stabilize the folding conformation of ARM 10. Introducing a glycine at this position, as in importin alpha1, disrupts high-affinity binding to pSTAT1, suggesting that pSTAT1 recognition is dependent on the intrinsic flexibility of ARM 10. Using the quantitative stoichiometry and binding data presented in this article, together with mutational information available in the literature, we propose that importin alpha5 binds between two STAT1 monomers, with two major binding determinants in the SH2 and DNA binding domains. In vitro, this model is supported by the observation that a 38-mer DNA oligonucleotide containing two tandem cfosM67 promoters can displace importin alpha5 from pSTAT1, suggesting a possible role for DNA in releasing activated STAT1 in the cell nucleus.

Tyrosine phosphorylation regulates the partitioning of STAT1 between different dimer conformations.

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 approximately 50 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 domain-mediated 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.