Molecular Basis for the Recognition of Phosphorylated STAT1 by Importin alpha 5

作者：Nardozzi Jonathan; Wenta Nikola; Yasuhara Noriko; Vinkemeier Uwe; Cingolani Gino^*

来源：Journal of Molecular Biology, 2010, 402(1): 83-100.

DOI：10.1016/j.jmb.2010.07.013

• nuclear import
• STAT1
• importin alpha 5
• importin beta
• NLS

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 alpha 1, STAT1 possesses a nonclassical NLS recognized by the isoform importin alpha 5. In the present study, we have analyzed the mechanisms by which importin alpha 5 binds phosphorylated STAT1 (pSTAT1). We found that a homodimer of pSTAT1 is recognized by one equivalent of importin alpha 5 with K(d) = 191 +/- 20 nM. Whereas tyrosine phosphorylation at position 701 is essential to assemble a pSTAT1 importin alpha 5 complex, the phosphate moiety is not a direct binding determinant for importin alpha 5. In contrast to classical NLS substrates, pSTAT1 binding to importin alpha 5 is not displaced by the N-terminal importin beta binding domain and requires the importin alpha 5 C-terminal acidic tail (505-EEDD-508). A local unfolding of importin alpha 5 Armadillo (ARM) repeat 10 accompanies high-affinity binding to pSTAT1. This unfolding is mediated by a single conserved tyrosine at position 476 of importin alpha 5, 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 alpha 1, 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 alpha 5 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 alpha 5 from pSTAT1, suggesting a possible role for DNA in releasing activated STAT1 in the cell nucleus.

• 出版日期2010-9-10