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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.

SUBMITTER: Nardozzi J

PROVIDER: S-EPMC2930068 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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

Nardozzi Jonathan J Wenta Nikola N Yasuhara Noriko N Vinkemeier Uwe U Cingolani Gino G

Journal of molecular biology 20100717 1

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 alp ...[more]

PMID: 20643137

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Project description:Introduction: The mechanisms governing synaptonuclear transport, and the transcriptional pathways that are at the core of neuronal disorders are poorly understood. The importin family of nuclear import factors has pivotal roles in such pathways, due to their involvement in intracellular transport from both synapse to soma and cytoplasm to nucleus. Mammals express six different isoforms of importin α, which bind an Importin β to form transport complexes for specific cargoes. Methods: We addressed the roles of importin α isoforms in mammalian CNS by evaluation of a systematic series of importin α knockout mice. A comprehensive battery of behavioral tests was used to investigate spontaneous and novelty-induced locomotion, basal ganglia function, motor coordination, neuromuscular integration, anxiety-related behaviors and memory. Subsequent analyses using acute-brain slice electrophysiology, RNA-seq, bioinformatics, imaging and pharmacology was used in order to delineate the contribution of genes and signaling pathways involved in the identified phenotype and screen for new therapeutic approaches. Results and Discussion: During the course of comprehensive behavioral profiling of importin α mouse mutants, we identified an importin α knockout (KO) mouse with significantly reduced anxiety levels. Electrophysiological recordings in acute hippocampal slices showed a reduced short-term and presynaptic plasticity (paired-pulse facilitation deficit, lower PTP), while LTP was not affected in these mutants. We then performed transcriptional profiling (RNA-seq) from hippocampal extracts of wild type versus mutant animals after exposure to an anxiogenic drug. Computational analysis revealed differences in the expression of genes and the involvement of transcription factors. Drugs that activate LXR or the Sphingosine 1 phosphate receptor have robust anxiolytic effects, while a Sphk1 blocker reverses anxiolysis in the importin α5 knockout mouse. Thus, importin α5 influences anxiety by regulating nuclear import in neurons.

Project description:Padavannil et al. 2019 show that Importin-9 (Imp9) transports Histones H2A-H2B from the cytoplasm to the nucleus using a non-canonical mechanism whereby binding of a GTP-bound Ran GTPase (RanGTP) fails to evict the H2A-H2B cargo. Instead, a stable complex forms, comprised of equimolar RanGTP, Imp9, and H2A-H2B. Unlike the binary Imp9•H2A-H2B complex, this RanGTP•Imp9•H2A-H2B ternary complex can release H2A-H2B to an assembling nucleosome. Here, we define the molecular basis for this RanGTP-activated nucleosome assembly by Imp9. We use hydrogen-deuterium exchange coupled with mass spectrometry and compare the dynamics and interfaces of the RanGTP•Imp9•H2A-H2B ternary complex to those in the Imp9•H2A-H2B or Imp9•RanGTP binary complexes. Our data are consistent with the Imp9•H2A-H2B structure by Padavannil et al. 2019 showing that Imp9 HEAT repeats 4-5 and 18-19 contact H2A-H2B, as well as many homologous importin•RanGTP structures showing that importin HEAT repeats 1 and 3, and the h8 loop, contact RanGTP. We show that Imp9 stabilizes H2A-H2B beyond the direct binding site, similar to other histone chaperones. Importantly, we reveal that binding of RanGTP releases H2A-H2B interaction at Imp9 HEAT repeats 4-5, but not 18-19. This exposes DNA- and histone-binding surfaces of H2A-H2B, thereby facilitating nucleosome assembly. We also reveal that RanGTP has a weaker affinity for Imp9 when H2A-H2B is bound. This may ensure that H2A-H2B is only released in high RanGTP concentrations near chromatin. We delineate the molecular link between the nuclear import of H2A-H2B and its deposition into chromatin by Imp9.SignificanceImp9 is the primary importin for shuttling H2A-H2B from the cytoplasm to the nucleus. It employs an unusual mechanism where the binding of RanGTP alone is insufficient to release H2A-H2B. The resulting stable RanGTP•Imp9•H2A-H2B complex gains nucleosome assembly activity as H2A-H2B can be deposited onto an assembling nucleosome. We show that H2A-H2B is allosterically stabilized via interactions with both N- and C-terminal portions of Imp9, reinforcing its chaperone-like behavior. RanGTP binding causes H2A-H2B release from the N-terminal portion of Imp9 only. The newly-exposed H2A-H2B surfaces can interact with DNA or H3-H4 in nucleosome assembly. Imp9 thus plays a multi-faceted role in histone import, storage, and deposition regulated by RanGTP, controlling histone supply in the nucleus and to chromatin.

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

Publications

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

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