Volume 4, Issue 4, Pages 499-509 (October 1999) Interaction of the Ski Oncoprotein with Smad3 Regulates TGF-β Signaling  Yin Sun, Xuedong Liu, Elinor Ng Eaton, William S Lane, Harvey F Lodish, Robert A Weinberg  Molecular Cell  Volume 4, Issue 4, Pages 499-509 (October 1999) DOI: 10.1016/S1097-2765(00)80201-4

Figure 1 TGF-β-Dependent Binding of GST–Smad3D to an 80 kDa Protein [35S]Methionine-labeled lysates of mink lung epithelial cells with or without TGF-β treatment were bound to GST–Smad3D recombinant fusion proteins. The associated proteins were retrieved by glutathione beads, separated by SDS-PAGE, and visualized by fluorography. The arrow indicates an 80 kDa protein that displays TGF-β-dependent binding to the GST–Smad3D protein. Molecular Cell 1999 4, 499-509DOI: (10.1016/S1097-2765(00)80201-4)

Figure 2 TGF-β-Dependent In Vivo Association of Ski with Smad3 Protein (A) Mink lung epithelial cells that were stably doubly infected with the viral expression vectors expressing HA-Ski and Flag–Smad3 were treated with 200 pM of TGF-β for 30 min. Lysates prepared from untreated and TGF-β-treated cells were immunoprecipitated with anti-HA or anti-Flag antibody. The Smad3-associated Ski proteins as well as Ski-associated Smad3 proteins were collected on protein G beads, separated by SDS-PAGE, and detected by immunoblot with anti-HA or anti-Flag antibody, respectively. Identical amounts of lysates were used for the immunoprecipitation as revealed by the immunoblot of the input lysate with antibody against HA or Flag epitopes. (B) A mink lung epithelial cell clone (Ski13) stably expressing only HA-tagged Ski was treated with 200 pM of TGF-β for 30 min. Lysates prepared from untreated and TGF-β-treated cells were immunoprecipitated with anti-HA antibody. The Ski-associated Smad3 proteins were collected on protein G beads, separated by SDS-PAGE, and detected by immunoblot with anti-Smad3 antibody. Identical amounts of HA-Ski proteins were used for the immunoprecipitation as revealed by immunoblotting the anti-HA immunoprecipitate with the anti-HA antibody. (C) C2C12 cells that were stably infected with the viral expression vector expressing Flag–Smad3 were treated with 200 pM of TGF-β for 30 min. Lysates prepared from untreated and TGF-β-treated cells were immunoprecipitated with anti-Flag antibody. The Smad3-associated Ski proteins were collected on protein G beads, separated by SDS-PAGE, and detected by immunoblotting with a monoclonal antibody against Ski. Identical amounts of Smad3 proteins were used for the immunoprecipitation as revealed by the immunoblot of the Flag-tagged Smad3 proteins with anti-Smad3 antibody. In parallel (upper panel), total lysates from control or Flag-Smad3-expressing cells were subjected to SDS-PAGE and Western blotting with the anti-Smad3 antibody. The Flag–Smad3 protein was expressed at a level 1.5-fold that of the endogenous Smad protein. Molecular Cell 1999 4, 499-509DOI: (10.1016/S1097-2765(00)80201-4)

Figure 3 Transcriptional Repression by Ski of the 3TP, 4xSBE, pL800, and PE2 Promoters In all experiments, relative luciferase activities whose value is the average of the duplicate samples with standard error were plotted after normalization with the β-galactosidase, and representative results from multiple separate experiments are presented. The closed bar represents cells that had been treated with TGF-β, and the open bar represents cells that had not been treated. (A) Mink lung epithelial cells were transfected with the 3TP-luciferase construct together with 1 μg of Smad3 or Ski expression plasmids or both together. After incubation for 20 hr in the absence or presence of 100 pM TGF-β, luciferase as well as β-galactosidase activity was determined. (B) Hep G2 cells were transfected with the 4xSBE-luciferase construct promoter with 1 μg of Smad3 and 0.25 or 1 μg of Ski expression plasmids. Luciferase and β-galactosidase activities were determined as in (A). (C and D) Hep G2 cells were transfected with pL800 promoter (C) and PE2 promoter (D) luciferase construct (Hua et al. 1998) together with 1 μg of Smad3 or Ski expression plasmids. Luciferase and β-galactosidase activities were determined as in (A). (E) Transcriptional repression by Ski can be reversed by overexpression of the Smad3 protein. Hep G2 cells were transfected with 3TP promoter luciferase construct together with 2 μg of Ski and 1 or 10 μg of Smad3 expression plasmids. Luciferase and β-galactosidase activities were determined as in (A). Molecular Cell 1999 4, 499-509DOI: (10.1016/S1097-2765(00)80201-4)

Figure 4 The Ski:Smad3 Protein Complex Binds to the 2xSBE and a Natural Promoter, PE2 (A and B) Lysates prepared from the BOSC cells that were transfected with the Flag–Smad3, HA-Ski, and TβRI T204D (constitutively active type I TGF-β receptor) were exposed to the end-labeled 2xSBE oligonucleotide (A) or PE2 sequence (B). The resulting mixtures were separated on a native polyacrylamide gel, dried, and visualized by autoradiography. The arrow heads indicate the Smad3 DNA-binding complexes while the arrow indicates Ski:Smad3 DNA-binding complexes. In lane 8 of (A) and lane (7) of B, the lysate was first incubated with anti-HA antibody before it was mixed with the labeled oligonucleotides; in lane 9 of (A) and lane 8 of (B), the lysate was first incubated with anti-Flag antibody before it was mixed with the labeled oligonucleotides; and in lane 10 of (A) and lane 9 of (B), the lysate was first incubated with both anti-HA and anti-Flag antibodies before it was mixed with the labeled oligonucleotides. The arrows indicate the Smad3:Ski DNA-binding complex. (C) Nuclear lysates were prepared from TGF-β-treated or nontreated mink lung cell clone expressing Ski (Ski13) and were mixed with end-labeled 2xSBE oligonucleotide. The resulting mixtures were processed as in (A) and (B). The lysate was first incubated with anti-HA antibody (lane 3), anti-Smad3 antibody (lane 4), or both (lane 5) before it was mixed with the labeled oligonucleotides. Notice the complete disappearance of the discrete protein:DNA complex after the mixture was incubated with anti-Smad3 antibody and correspondingly increasing amounts of well-retained labeled oligonucleotides, suggesting that this complex contained Smad3 and Ski protein simultaneously. Molecular Cell 1999 4, 499-509DOI: (10.1016/S1097-2765(00)80201-4)

Figure 5 Growth Regulation of Mink Lung Epithelial Cells by Ectopic Expression of Ski and Smad3 (A) Percentage reduction of [3H]thymidine incorporation of mink lung epithelial cell stably infected with the cDNA encoding Ski protein or vector alone. Triplicate cultures of wild-type, vector-infected, or Ski-expressing mink lung epithelial cells were treated with various concentrations of TGF-β for 24 hr followed by 3 hr incubation with 1 μCi of [3H]thymidine, and the acid-insoluble [3H]thymidine was counted and compared to the incorporation from cells that had not been exposed to TGF-β treatment. The insert in the graph is a representative immunoblot probed with anti-HA antibody demonstrating the expression of the Ski protein. Lane 1, wild-type L20 cells; lane 2, Ski13 clone. (B) Reduction in cell number of various mink epithelial cells in a 5-day culture period in the presence or absence of 20 pM TGF-β. L20, wild-type mink lung epithelial cells; B1, a cell clone transfected with vector alone; Ski13, a stable transfectant expressing HA-tagged Ski protein. (C) Growth resistance to TGF-β in Ski-overexpressing cells can be overcome by overexpression of Smad3 proteins. Populations of mink lung epithelial cells infected with vector alone or vectors expressing Ski, Smad3, and Smad3A in various combinations were generated. Triplicate cultures of those cells were treated with TGF-β and were processed as in (A). The acid-insoluble [3H]thymidine was counted and compared to the incorporation from cells that had not been exposed to TGF-β treatment. Molecular Cell 1999 4, 499-509DOI: (10.1016/S1097-2765(00)80201-4)

Figure 6 Effect of Ectopic Expression of Ski in Mink Lung Epithelial Cells on p15INK4B and myc Transcript Levels (A) RNase protection assay of various derivatives of mink lung epithelial cells in response to 5, 10, and 20 pM TGF-β. Total RNA was prepared from cells that were treated with TGF-β for 12 hr at various concentrations. Total RNA (10 μg) was used for analysis of p15INK4B transcript; 50 μg of total RNA was used for the analysis of myc transcript. Actin transcripts were analyzed in each lane simultaneously as internal control. The antisense-labeled RNA transcripts were prepared from portions of mink myc, p15INK4B, and mouse actin cDNA. (B) The quantitation of the myc transcript level normalized against the actin transcript level. The myc transcript level of cells without TGF-β treatment was used as one unit, and all the other normalized myc levels were then plotted against that unit. (C) The quantitation of p15INK4B transcript. The fold induction of p15INK4B in response to TGF-β was plotted. p15INK4B RNA levels were normalized against actin RNA levels present in the same preparations. Molecular Cell 1999 4, 499-509DOI: (10.1016/S1097-2765(00)80201-4)