PIAS1 inhibits Stat1-mediated gene activation. (A) Luciferase reporter assays. Human 293 cells were transiently transfected with [(3x)Ly6] luciferase reporter construct together with empty expression vector, FLAG–Stat1 or various amounts of FLAG–PIAS1 vectors, alone or in combination as indicated. Cells were either treated or untreated with IFN-γ for 6 h. Shown is the fold increase after IFN-γ treatment. (B) Western blot analysis. Equal amounts of protein extracts from A were analyzed by immunoblot with anti-FLAG (Sigma).

Specificity of PIAS–STAT interaction. (A) PIAS1 interacts with Stat1 in vivo. Protein extracts from Daudi cells untreated (−) or treated (+) with IFN-α for 15 min were prepared and used for immunoprecipitation with anti-PIAS1n. Immunoprecipitates were then analyzed on SDS/PAGE and the blot was probed anti-Stat1. The filter was washed and reprobed with anti-PIAS1 (Lower). (B) PIAS1 does not interact with Stat2. Daudi cells were treated with IFN-α for the times indicated. Whole-cell extracts were prepared and one-half of these extracts were used for immnunoprecipitation with anti-PIAS1n (Left). Immunoprecipitation was carried out as described in A and the filter was probed with anti-Stat1 and anti-Stat2. The same filter was washed and reprobed with anti-PIAS1n (Lower). The other half of protein extracts were subjected to immunoprecipitation with anti-Stat1 (Right). The filter was probed with a mixture of anti-Stat1 and anti-Stat2 antibodies. p-Stat1, phosphorylated Stat1. (C) Western blot analysis. HepG2 cells were untreated or treated with IL-6 or IFN-γ for 15 min, and protein extracts were prepared and analyzed by immunoblot with anti-Stat1, anti-pStat1 (New England Biolabs), anti-Stat3 (Santa Cruz Biothechnology), or anti-pStat3 (New England Biolabs) as indicated. (D) PIAS1 interacts with Stat1 but not Stat3. Protein extracts from C were immunoprecipitated with anti-PIAS1n. The precipitates were subjected to electrophoresis, and the filter was blotted with anti-Stat1 (Left). The same filter was washed and reprobed with anti-Stat3 (Left) or reprobed with anti-PIAS1n (Lower). (E) Same as D except that anti-PIAS3c was used for immunoprecipitation. The filter was probed with anti-Stat3 (Left) or anti-Stat1 (Right) or PIAS3c (Lower).

Sequence comparison of the PIAS family of proteins. The predicted amino acid sequences of PIAS1, PIAS3, PIASxα, PIASxβ, and PIASy are shown. h, Human; m: mouse. Cysteine and histidine residues that are predicted to form a zinc finger are shaded. The conserved acidic region is boxed. Dots indicate amino acid identity.

PIAS1 inhibits the DNA binding activity of Stat1. EMSA analysis was performed with nuclear extracts prepared from Daudi cells with (+) or without (−) IFN-α treatment in the absence or presence of GST, GST–PIASxα, GST–PIAS3, or GST–PIAS1 proteins (15–75 ng) as indicated. The probe used is a high-affinity Stat1–DNA binding site (10). The concentrations of GST fusion proteins were estimated on 7% SDS/PAGE with various dilutions of BSA as the standard.

Phosphorylation on Tyr-701 of Stat1 is required for PIAS1–Stat1 interaction. U3A cells complemented with Stat1 or Stat1(Tyr-701 → Phe) mutant protein were untreated or treated with IFN-γ for 15 min, and protein extracts were prepared. (A) Immunoprecipitation was performed with anti-Stat1 followed by blotting with a specific phosphotyrosine antiserum [anti-pTyr (Transduction Laboratories, Lexington, KY)]. The same filter was washed and reprobed with anti-Stat1 (Lower). (B) Immunoprecipitation was performed with anti-PIAS1n antiserum and blotted with anti-Stat1. The filter was washed and reprobed with anti-PIAS1 (Lower).