title: Molecular basis for the recognition of phosphorylated STAT1 by importin alpha5.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Nardozzi
authors.forename: Nikola
authors.initials: N
authors.lastname: Wenta
authors.forename: Noriko
authors.initials: N
authors.lastname: Yasuhara
authors.forename: Uwe
authors.initials: U
authors.lastname: Vinkemeier
authors.forename: Gino
authors.initials: G
authors.lastname: Cingolani
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019913:Nuclear Localization Signals; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D050794:STAT1 Transcription Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D028901:alpha Karyopherins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: C423142:KPNA1 protein, human; D019913:Nuclear Localization Signals; D010446:Peptide Fragments; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D028901:alpha Karyopherins