Ski interacts with the MH2 domains of Smad2, Smad3, and Smad4. (A) Cell lysates were prepared from control 293T cells or 293T cells stably expressing Flag–Smad4C or Flag–Smad4N, and Smad4-associated cellular proteins were isolated by affinity purification using anti-Flag agarose followed by elution with the Flag peptide as described in Materials and Methods. The purified Smad4 complexes were visualized by silver staining. The 97-kD protein indicated by an arrow was identified by microsequencing as the human c-Ski protein and the 80-kD protein as the human c-SnoN. (B) Flag-tagged full-length or truncated Smad proteins were cotransfected into 293T cells together with HA-Ski as indicated and isolated by immunoprecipitation with an anti-Flag M2 mAb. The Smad-bound Ski was visualized by Western blotting with an anti-HA mAb as indicated. Cell lysates were blotted directly with an anti-HA mAb as a control for HA–Ski expression (bottom).

Ski represses TGF-β-induced and Smad-dependent transcriptional activation. (A). Ski represses TGF-β-induced transcriptional activation. Hep3B cells were transfected with 0.5 μg p3TP–lux and increasing amounts of ski as indicated. Luciferase activity was measured 16 hr after stimulation with 50 pm TGFβ1. (B) Hep3B cells were cotransfected with the Smad proteins and Ski as indicated. Smad-mediated transcriptional activation of the p3TP–lux in the absence − or presence + of Ski was analyzed.

Smad proteins interact with the amino-terminal region of Ski. (A) Schematic drawings of Ski truncation mutants. The domain in c-Ski that is conserved with v-Ski (residues 24–441) is shaded. This domain contains transforming activity and also mediates binding to N-CoR. (B) Binding of the Ski mutants to Smad3 and Smad4. Flag–Smad3 or Flag–Smad4 were cotransfected with HA-tagged c-Ski and mutants. Association of Smads with various Ski proteins was analyzed by blotting of the Flag immunoprecipitates with an anti-HA antiserum. Cell lysates were blotted directly with an anti-HA antibody as a control for the expression of various Ski mutants. (*) A nonspecific background band. (C) Interaction of Smad3 and Smad4 with Ski(241–323). To detect the smallest Ski truncation mutant, 293T cells transfected with Flag–Smads and HA–Ski(214–323) were labeled with ³⁵S-Express, and Smad-bound Ski(241–323) was isolated by immunoprecipitation with anti-Flag agarose and visualized by autoradiography. Parallel immunoprecipitation with an anti-HA antiserum was carried out to control for the expression of the Ski mutants. Ski(197–441) was used as a positive control for this experiment. (D) Hep3B cells were transfected with p3TP–lux and various Ski mutants. After transfection (30 hr), cells were stimulated with 50 pm TGFβ1 for 16 hr and luciferase activity was measured.

Ski binds to the SBE through interaction with Smad4. ³²P-labeled SBE oligonucleotide (2 × 10⁴ cpm) was incubated with HA–Smad4, GST-Ski, Flag–Ski (F-Ski), or purified Ski/Smad4 complex in EMSA reactions. DNA-protein complexes were resolved by nondenaturing PAGE. Proteins used in the EMSA reactions: (Lane 1) 0.5 μg HA–Smad4; (lane 2) 0.8 μg GST–Ski; (lane 3) 0.8 μg Flag-Ski purified from 293T cells transfected with Flag–Ski alone; (lanes 4–7) Flag–Ski/HA–Smad4 complex purified using anti-Flag agarose from cotransfected 293T cells. Antibodies used in supershift reactions: (lane 5) NR (nonrelevant antibody); (lane 6) F (anti-Flag); (lane 7) S4 (anti-Smad4).

Smad2, Smad3, and Smad4 complex with N-CoR through Ski. 293T cells were transfected with Flag–Smad2, Flag–Smad3, or Flag–Smad4 either alone (lanes 1–3) or together with HA–Ski (lanes 4–6). Endogenous N-CoR complexed with the Smads was isolated by immunoprecipitation with an anti-Flag mAb and detected by Western blotting with an anti-N-CoR antibody. Cell lysates were also blotted directly with anti-Flag and anti-HA antibodies for control of Smads and Ski expression. As a positive control, N-CoR associated with HA–Ski was isolated by immunoprecipitation with an anti-HA mAb and blotted with an anti-N-CoR antibody (lane 7). (Lane 8) HA-tagged mutant Ski(241–323) isolated from transfected 293T cells by immunoprecipitation with an anti-HA antibody. (Lane 9) Anti-Flag immunoprecipitate prepared from cells cotransfected with Flag–Smad3 and HA–Ski(241–323).

Overexpression of Ski greatly attenuates TGFβ-induced growth inhibition and activation of JunB expression. (A) Ba/F3 cell lines stably expressing Flag–Ski were generated by retroviral infection. Flag–Ski was isolated by immunoprecipitation with anti-Flag agarose from uninfected Ba/F3 cells or from two stable pools (B/ski-8 and B/ski-12) and analyzed by Western blotting with an anti-Flag mAb. (B) B/ski-8 cells (2 × 10⁸) were stimulated with 200 pm TGFβ1 for 15 or 30 min as indicated. Endogenous Smad proteins associated with Flag–Ski were isolated by immunoprecipitation with anti-Flag agarose and detected by Western blotting with anti-Smad2, anti-Smad3, or anti-Smad4 antibodies. (C) Nuclear extracts were prepared from B/ski-8 cells that had been stimulated with 200 pm TGFβ for 30 min and incubated with ³²P-labeled SBE in an EMSA assay. Antibodies used in the supershift assay: (S2/3) anti-Smad2/3; (S4) anti-Smad4; (F) anti-Flag; (NR) nonrelevant control antibody. (*) Non-specific background bands. (D) Growth inhibition assay. Uninfected Ba/F3 cells, B/ski-8 or B/ski-12 cells were incubated for 5 days with various concentrations of TGFβ1 as indicated. The growth of cells was quantified by cell counting and compared to unstimulated cells. The growth rate of B/ski-8 or B/ski-12 cells in the absence of TGFβ1 is similar to that of uninfected Ba/F3 cells. (E) Activation of JunB expression in uninfected Ba/F3 or B/ski-8 cells by Northern blotting. Cells were serum starved for 16 hr and stimulated with 100 pm TGFβ1 for various periods of time as indicated. An analysis of the JunB and human CAC1 RNA is shown. CAC1 was used as a control for equal loading. A quantification of the Northern blot was carried out using the Bio-Rad Molecular Imager FX system and folds of induction of JunB expression are shown in the graph.