Molecular Basis for the Recognition of Phosphorylated STAT1 by Importin α5

• Published in: Journal of molecular biology 2010-09, Vol.402 (1), p.83-100
• Main Authors: Nardozzi, Jonathan, Wenta, Nikola, Yasuhara, Noriko, Vinkemeier, Uwe, Cingolani, Gino
• Format: Article
• Language: English
• Subjects: importin α5; importin β; NLS; nuclear import; STAT1
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2010.07.013

Interferon-γ 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 α1, STAT1 possesses a nonclassical NLS recognized by the isoform importin α5. In the present study, we have analyzed the mechanisms by which importin α5 binds phosphorylated STAT1 (pSTAT1). We found that a homodimer of pSTAT1 is recognized by one equivalent of importin α5 with K d = 191 ± 20 nM. Whereas tyrosine phosphorylation at position 701 is essential to assemble a pSTAT1–importin α5 complex, the phosphate moiety is not a direct binding determinant for importin α5. In contrast to classical NLS substrates, pSTAT1 binding to importin α5 is not displaced by the N-terminal importin β binding domain and requires the importin α5 C-terminal acidic tail (505-EEDD-508). A local unfolding of importin α5 Armadillo (ARM) repeat 10 accompanies high-affinity binding to pSTAT1. This unfolding is mediated by a single conserved tyrosine at position 476 of importin α5, 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 α1, 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 α5 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 α5 from pSTAT1, suggesting a possible role for DNA in releasing activated STAT1 in the cell nucleus.