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, 98 (6), 3203-7

A Transcriptional Corepressor of Stat1 With an Essential LXXLL Signature Motif

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A Transcriptional Corepressor of Stat1 With an Essential LXXLL Signature Motif

B Liu et al. Proc Natl Acad Sci U S A.

Abstract

Interferon (IFN) treatment induces tyrosine phosphorylation and nuclear translocation of Stat1 (signal transducer and activator of transcription) to activate or repress transcription. We report here that a member of the protein inhibitor of activated STAT family, PIASy, is a transcriptional corepressor of Stat1. IFN treatment triggers the in vivo interaction of Stat1 with PIASy, which represses Stat1-mediated gene activation without blocking the DNA binding activity of Stat1. An LXXLL coregulator signature motif located near the NH(2) terminus of PIASy, although not involved in the PIASy-Stat1 interaction, is required for the transrepression activity of PIASy. Our studies identify PIASy as a transcriptional corepressor of Stat1 and suggest that different PIAS proteins may repress STAT-mediated gene activation through distinct mechanisms.

Figures

Figure 1
Figure 1
PIASy is localized in the nucleus. Human 2fTGH cells were stained with anti-GST (A) or anti-PIASy (B). 2fTGH cells were transfected with an empty vector (C) or pCF-PIASy (D) and stained with anti-Flag.
Figure 2
Figure 2
The in vivo PIASy–Stat1 interaction is dependent on IFN stimulation. (A) Western blot analysis of protein extracts from 293T or U2OS cells with anti-pStat1 (New England Biolabs) or anti-Stat1. (B) Extracts from A were subjected to immunoprecipitation with anti-PIASy followed by Western blot analysis with anti-Stat1 (Upper). The same filter was reprobed with anti-PIASy (Lower). (C) Western blot analysis of protein extracts from 293T cells transfected with Flag-PIAS1 or Flag-PIASy. The filter was probed with anti-Flag, anti-PIAS1, or anti-PIASy as indicated.
Figure 3
Figure 3
PIASy represses Stat1-mediated gene activation. (A) Luciferase assays. Human U2OS cells were transfected with a Stat1 luciferase reporter construct (3xLy6E) with or without Stat1 or PIASy as indicated. Cells were treated with (black bar) or without (gray bar) IFN-γ (5 ng/ml) for 6 h before harvesting. Dual-luciferase assays were performed. (B) Same as A except HeLa cells were used. (C) Same as A except 293T cells were transfected by calcium precipitation method, and PIAS3 was included as a control as indicated. luc., luciferase.
Figure 4
Figure 4
PIASy does not block the DNA binding activity of Stat1. (A) Electrophoretic mobility-shift assay analysis. Nuclear extracts prepared from Daudi cells untreated or treated with IFN-α were mixed with a 32P-labeled Stat1 binding oligonucleotide (11) in the absence (lanes 1, 2, 10, and 11) or presence of various amounts of GST (lanes 3–5 and 12–14), GST-PIASy (lanes 6–9), or GST-PIAS1 (lanes 15–17) as indicated. (B) Same as A except purified tyrosine-phosphorylated Stat1 (lanes 1 and 7–13) or purified unphosphorylated Stat1 (lanes 2–6) was mixed with increasing concentrations of GST or GST-PIASy (30, 90, and 300 ng).
Figure 5
Figure 5
The LXXLL motif of PIASy is required for the transrepression activity of PIASy. (A) The region containing the α-helical LXXLL motif of PIASy is indicated. (B) In vitro GST-pull-down assays. GST or GST-tyrosine-phosphorylated Stat1 (GST-pStat1) immobilized on glutathione beads were mixed with in vitro translated, 35S-labeled luciferase, PIASy or PIASy-AA as indicated. Proteins bound were analyzed on an 8% SDS/PAGE followed by autoradiography. (C) Luciferase assays. 293T cells were transfected with a Stat1 luciferase reporter construct together with or without Flag-Stat1 or PIASy or PIASy-AA as indicated (Upper). Protein extracts from the same samples were analyzed by Western blot with anti-Flag antibody (Lower). luc., luciferase.

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