Stat1 and Stat3 interact with c-Jun in vivo. Nuclear extracts (300 μg) from IL-6-treated (+) or untreated (−) HepG2 cells were immunoprecipitated (IP) with the antibodies (Ab) indicated at the top, and the immunoprecipitates were then subjected to SDS–10% PAGE followed by Western blotting with the antibodies indicated on the left. Rabbit immunoglobulin (rIgG) and mouse immunoglobulin (mIgG) (Santa Cruz) were used as controls for the Stat1 and -3 or c-Jun immunoprecipitations, respectively. α-c-Jun, anti-c-Jun; α-Stat3, anti-Stat3; αStat1, anti-Stat1.

Mapping of the regions in Stat1 and -3 that interact with in vitro-translated c-Jun by GST pull-down assays. (A) A schematic diagram of the structural domains of Stat3, and a summary of the interactions between c-Jun and various GST-Stat3 fusion fragments. −, no binding evident; +, weak binding exhibited; +++, strong binding demonstrated. (B) c-Jun interacts with GST-Stat3(107–377). (C) Mapping of the minimal c-Jun-interactive region in Stat3. Equivalent amounts of each GST-Stat3 fusion protein attached to glutathione-Sepharose beads were incubated with in vitro-translated full-length c-Jun labeled with [³⁵S]methionine. The bound proteins were analyzed by SDS–10% PAGE and radiography. (D) Endogenous c-Jun interacts with Stat3-GST fusion proteins. HepG2 cell extracts were incubated with GST-Stat3 fusion proteins bound to glutathione-Sepharose beads. The precipitates were analyzed by SDS–10% PAGE and blotted with an anti-c-Jun antibody (α-c-Jun).

Mapping of the Stat3-interactive region in c-Jun by GST pull-down assays. (A) Schematic diagram of the structural domains of c-Jun. The fragments of c-Jun that were in vitro translated were residues 1 to 104 and 105 to 334. (B) The fragment containing residues 105 to 334 of c-Jun is sufficient to bind to GST-Stat3(107–377). bZIP, basic leucine zipper.

Site-directed mutagenesis in regions 1 and 2 of the Stat3 molecule. (A) Sequence alignment of STAT proteins in regions 1 and 2. The five boxed residues in Stat3 were changed to alanine individually. The three boxed residues in region 2 were changed to alanines simultaneously. (B) Three Stat3 mutants showed decreased c-Jun binding. Mutants L₁₄₈A and V₁₅₁A (lanes 5 and 6) demonstrated weaker c-Jun binding. Mutant TKR (lane 12) in region 2 lost the c-Jun binding. WT, wild-type GST-Stat3(130–358).

Ribbon diagrams of regions 1 and 2, where site-directed mutagenesis was performed, and the corresponding mutated residues in the Stat1 molecule. (A) Two c-Jun-interactive regions in Stat3 are shown in a ribbon diagram of the Stat1 core dimer on DNA. Region 1 is shown in magenta, and region 2 is shown in purple. The coiled-coil domain is shown in green, the DNA binding domain is in red, the linker domain is in orange, and the SH2 domain is in cyan. The tail segments are shown in green and in magenta. (B) Four corresponding mutated residues in region 1 of Stat3 are shown in a ribbon diagram of the coiled-coil domain (green) and DNA binding domain (red) of the Stat1 monomer. M₁₃₅ in Stat1, the residue corresponding to V₁₃₇ in Stat3, is not included in the ribbon diagram. (C) Three corresponding mutated residues in region 2 of Stat3 are shown in a ribbon diagram of the DNA binding domain of the Stat1 monomer with DNA.

Requirement of Stat3–c-Jun interaction for maximal activation of an IL-6-inducible α₂-macroglobulin reporter gene containing both Stat3 and AP-1 binding sites. (A) Cotransfection of wild-type Stat3 and c-Jun boosted the IL-6-dependent response, while Stat1 and three noninteractive Stat3 mutants were ineffective with c-Jun at increasing the IL-6-dependent response. HepG2 cells were transfected with 0.5 μg of luciferase reporter, 0.2 μg of CMVβgal, 50 ng of Stat3, or 50 ng of c-Jun. Twenty-four hours after transfection, cells were left untreated (−) or were treated with 5 ng of IL-6 per ml for 6 h (+) prior to being harvested for luciferase and β-gal assays. Results shown are the means ± standard deviations of data from three experiments. The luciferase activity was normalized against the internal-control β-gal activity and calculated relative to the activity of cells transfected with the vector plasmid pRcCMV. (B) Stat1 was ineffective at cooperating with c-Jun to activate the IL-6-induced transcriptional response. HepG2 cells were cotransfected with 0.5 μg of α₂-macroglobulin luciferase reporter, 50 ng of c-Jun, and increasing amounts of either Stat3 or Stat1 as indicated. (C) Stat1 is functionally active on IFN-γ treatment in HepG2 cells. (Left panel) EMSA with ³²P-labeled α2MGAS probe. IL-6 treatment led to the activation of Stat1 and Stat3 while IFN-γ treatment led to the activation of Stat1 in HepG2 cells. −, no cytokines added. SIF A, Stat3 homodimer; SIF B, Stat3-Stat1 heterodimer; SIF C, Stat1 homodimer. (Right panel) IFN-γ induced activation of Stat1 with the reporter gene 3xLy6E, but not with the α₂-macroglobulin reporter gene (α₂M).

The noninteractive Stat3 mutants can bind DNA and activate IL-6-dependent transcription. (A) The DNA binding abilities of three noninteractive Stat3 mutants were examined by gel mobility shift analysis with ³²P-labeled M67 probe. 293T cells were transiently transfected with either wild-type (WT) Stat3 or mutant Stat3 cDNAs treated with IL-6 at a concentration of 5 ng/ml and recombinant human IL-6 soluble receptor at a concentration of 5 ng/ml for 30 min. Nuclear extracts were prepared from these cells, and 3 mg of extract was used in each EMSA. Rc, pRcCMV; α-Stat3, anti-Stat3; α-FLAG, anti-FLAG. (B) Phosphorylation on tyrosine and serine residues of the three Stat3 mutants was indistinguishable from that of wild-type Stat3. Nuclear extracts (75 μ) from transfected 293T cells were immunoprecipitated (IP) with anti-FLAG antibody, and the immunoprecipitates were then subjected to SDS–7% PAGE followed by Western blotting with the antibodies indicated. (C) The IL-6-dependent transcriptional activities of three Stat3 mutants were examined by using the 3xLy6E luciferase reporter. −, no IL-6 added; +, IL-6 present.