The N domain of Smad7 is essential for specific inhibition of transforming growth factor-beta signaling

Abstract

Inhibitory Smads (I-Smads) repress signaling by cytokines of the transforming growth factor-beta (TGF-beta) superfamily. I-Smads have conserved carboxy-terminal Mad homology 2 (MH2) domains, whereas the amino acid sequences of their amino-terminal regions (N domains) are highly divergent from those of other Smads. Of the two different I-Smads in mammals, Smad7 inhibited signaling by both TGF-beta and bone morphogenetic proteins (BMPs), whereas Smad6 was less effective in inhibiting TGF-beta signaling. Analyses using deletion mutants and chimeras of Smad6 and Smad7 revealed that the MH2 domains were responsible for the inhibition of both TGF-beta and BMP signaling by I-Smads, but the isolated MH2 domains of Smad6 and Smad7 were less potent than the full-length Smad7 in inhibiting TGF-beta signaling. The N domains of I-Smads determined the subcellular localization of these molecules. Chimeras containing the N domain of Smad7 interacted with the TGF-beta type I receptor (TbetaR-I) more efficiently, and were more potent in repressing TGF-beta signaling, than those containing the N domain of Smad6. The isolated N domain of Smad7 physically interacted with the MH2 domain of Smad7, and enhanced the inhibitory activity of the latter through facilitating interaction with TGF-beta receptors. The N domain of Smad7 thus plays an important role in the specific inhibition of TGF-beta signaling.

Figure 1.

Inhibition of TGF-β and BMP signaling by I-Smads. (A and B) Comparison of the inhibitory effects of Smad6 and Smad7 on transcription from p3TP–Lux (A) and AR3–Luc (B) induced byTβR-I(TD). In A–C, R mutant Mv1Lu cells or COS7 cells were transfected with the indicated plasmids, and luciferase activities were determined as described in the Materials and methods. + and ++ are 0.1 and 0.3 μg of DNA, respectively, transfected in R mutant or COS7 cells. 6 and 7 denote Smad6 and Smad7, respectively. For the analysis using AR3–Luc, FAST1/FoxH3 cDNA was cotransfected. (C) Comparison of the inhibitory effects of Smad6 and Smad7 on 3GC2–Lux transcription induced by a BMPR-I, ALK-6(QD). (D) Inhibitory effects of Smad6 and Smad7 on Tlx2–Lux transcription induced by ALK-6(QD) and BMPR-II. P19 embryonal carcinoma cells were transfected with the indicated plasmids, and luciferase activities were determined as described in the Materials and methods. + and ++ are 0.1 and 0.3 μg of DNA, respectively, transfected in P19 cells.

Figure 2.

Inhibition of TGF-β or BMP signaling by Smad6 and Smad7 mutants. (A) Structures of Smad6 and Smad7 and their deletion mutants and chimeras. The amino acid numbers of Smad6 and Smad7 are indicated. (B) Inhibitory effects of I-Smads and their deletion mutants and chimeras on p3TP–Lux transcription induced by TβR-I(TD). In B and C, R mutant Mv1Lu cells were transfected with the indicated plasmids, and luciferase activities were determined. + and ++ are 0.1 and 0.3 μg of DNA, respectively, transfected in R mutant cells. (C) Inhibitory effects of I-Smads and their deletion mutants and chimeras on 3GC2–Lux transcription induced by ALK-6(QD).

Figure 3.

Multiple regions in the Smad7 N domain are important for efficient inhibition of TGF-β signaling. (A) Structures of the Smad6 and Smad7 chimeras are shown. The amino acid numbers of Smad6 and Smad7 are indicated. (B and C) Inhibition of TGF-β signaling by the Smad6 and Smad7 chimeras. Effects of the chimeras on p3TP–Lux (B) and AR3–Luc (C) transcription induced by TβR-I(TD) were examined. + and ++ are 0.1 and 0.3 μg of DNA, respectively, transfected in R mutant cells. FAST1/FoxH3 cDNA was cotransfected in C.

Figure 4.

Subcellular localization of I-Smads. Subcellular localization of Smad6, Smad7, and their chimeras was determined in transfected COS7 (A) or HepG2 cells (B). FLAG-tagged I-Smads were transfected in cells and the subcellular localization of I-Smads was demonstrated by FITC. Nuclear staining was performed by 4,6-diamidino-2-phenylindole (PI).

Figure 4.

Subcellular localization of I-Smads. Subcellular localization of Smad6, Smad7, and their chimeras was determined in transfected COS7 (A) or HepG2 cells (B). FLAG-tagged I-Smads were transfected in cells and the subcellular localization of I-Smads was demonstrated by FITC. Nuclear staining was performed by 4,6-diamidino-2-phenylindole (PI).

Figure 8.

Physical interaction of Smad7N leads to nuclear export and enhancement of the inhibitory activity of Smad7C. (A) Enhancement of the inhibitory activity of Smad7C by Smad7N. Effects of Smad6N and Smad7N were examined in the presence of their MH2 domains (Smad6C and Smad7C) by a p3TP–Lux transcription assay induced by TβR-I(TD). + and ++ are 0.1 and 0.3 μg of DNA, respectively, transfected in R mutant cells. (B) Physical interaction between Smad7N and the MH2 domains of Smad7 (7C), Smad3 (3C), and Smad1 (1C). Interaction of Smad7N with the full-length Smad7 (7) was also tested. Interaction was examined by FLAG immunoprecipitation of FLAG–Smads, followed by Myc immunoblotting of Smad7N using transfected COS7 cells (top). Expression levels of transfected proteins were determined and are shown in the lower two panels. (C and D) Subcellular localization of Smad7C in the presence and absence of Smad7N was examined in transfected HepG2 (C) or COS7 cells (D) using confocal laser scanning microscopy. FLAG–Smad7C was stained by anti-FLAG antibody in the absence and presence of Smad7N and Smurf1(CA), and its subcellular localization was demonstrated by FITC. In HepG2 cells, 6Myc–Smad7N was stained by rhodamine isothiocyanate (RITC; C, bottom). (E) Affinity cross-linking of ¹²⁵I–TGF-β1 was performed using transfected COS7 cells followed by FLAG immunoprecipitation of FLAG–I-Smads (upper). The lower two panels demonstrate the levels of expression of transfected proteins.

Figure 8.

Physical interaction of Smad7N leads to nuclear export and enhancement of the inhibitory activity of Smad7C. (A) Enhancement of the inhibitory activity of Smad7C by Smad7N. Effects of Smad6N and Smad7N were examined in the presence of their MH2 domains (Smad6C and Smad7C) by a p3TP–Lux transcription assay induced by TβR-I(TD). + and ++ are 0.1 and 0.3 μg of DNA, respectively, transfected in R mutant cells. (B) Physical interaction between Smad7N and the MH2 domains of Smad7 (7C), Smad3 (3C), and Smad1 (1C). Interaction of Smad7N with the full-length Smad7 (7) was also tested. Interaction was examined by FLAG immunoprecipitation of FLAG–Smads, followed by Myc immunoblotting of Smad7N using transfected COS7 cells (top). Expression levels of transfected proteins were determined and are shown in the lower two panels. (C and D) Subcellular localization of Smad7C in the presence and absence of Smad7N was examined in transfected HepG2 (C) or COS7 cells (D) using confocal laser scanning microscopy. FLAG–Smad7C was stained by anti-FLAG antibody in the absence and presence of Smad7N and Smurf1(CA), and its subcellular localization was demonstrated by FITC. In HepG2 cells, 6Myc–Smad7N was stained by rhodamine isothiocyanate (RITC; C, bottom). (E) Affinity cross-linking of ¹²⁵I–TGF-β1 was performed using transfected COS7 cells followed by FLAG immunoprecipitation of FLAG–I-Smads (upper). The lower two panels demonstrate the levels of expression of transfected proteins.

Figure 8.

Physical interaction of Smad7N leads to nuclear export and enhancement of the inhibitory activity of Smad7C. (A) Enhancement of the inhibitory activity of Smad7C by Smad7N. Effects of Smad6N and Smad7N were examined in the presence of their MH2 domains (Smad6C and Smad7C) by a p3TP–Lux transcription assay induced by TβR-I(TD). + and ++ are 0.1 and 0.3 μg of DNA, respectively, transfected in R mutant cells. (B) Physical interaction between Smad7N and the MH2 domains of Smad7 (7C), Smad3 (3C), and Smad1 (1C). Interaction of Smad7N with the full-length Smad7 (7) was also tested. Interaction was examined by FLAG immunoprecipitation of FLAG–Smads, followed by Myc immunoblotting of Smad7N using transfected COS7 cells (top). Expression levels of transfected proteins were determined and are shown in the lower two panels. (C and D) Subcellular localization of Smad7C in the presence and absence of Smad7N was examined in transfected HepG2 (C) or COS7 cells (D) using confocal laser scanning microscopy. FLAG–Smad7C was stained by anti-FLAG antibody in the absence and presence of Smad7N and Smurf1(CA), and its subcellular localization was demonstrated by FITC. In HepG2 cells, 6Myc–Smad7N was stained by rhodamine isothiocyanate (RITC; C, bottom). (E) Affinity cross-linking of ¹²⁵I–TGF-β1 was performed using transfected COS7 cells followed by FLAG immunoprecipitation of FLAG–I-Smads (upper). The lower two panels demonstrate the levels of expression of transfected proteins.

Figure 5.

Smurf1 induces nuclear export of Smad6 and Smad7. (A–C) The nuclear export of Smad6, Smad7, and their deletion mutants (Smad6C and Smad7C) induced by Smurf1(CA) was examined in COS7 (A) or HepG2 cells (B and C) as in Fig. 4. (D) The effects of Smad7 amino-terminal deletion mutants on p3TP–Lux transcription induced by TβR-I(TD) were examined. + and ++ are 0.3 and 1.0 μg of DNA, respectively, transfected in R mutant cells. PPPY represents the PY motif.

Figure 5.

Smurf1 induces nuclear export of Smad6 and Smad7. (A–C) The nuclear export of Smad6, Smad7, and their deletion mutants (Smad6C and Smad7C) induced by Smurf1(CA) was examined in COS7 (A) or HepG2 cells (B and C) as in Fig. 4. (D) The effects of Smad7 amino-terminal deletion mutants on p3TP–Lux transcription induced by TβR-I(TD) were examined. + and ++ are 0.3 and 1.0 μg of DNA, respectively, transfected in R mutant cells. PPPY represents the PY motif.

Figure 6.

Repression of Smad2 phosphorylation by I-Smads, their deletion mutants, and chimeras. COS7 cells were transfected with the indicated plasmids, and phosphorylation of Smad2 was determined by FLAG immunoprecipitation of Smad2, followed by phosphoserine immunoblotting (top). Levels of expression for each protein were determined by immunoblotting using FLAG, HA, or Myc antibodies (lower). + and ++ are 0.1 and 0.3 μg of DNA, respectively, transfected in COS7 cells.

Figure 7.

Physical interaction of Smads with TGF-β receptors. (A) Affinity cross-linking using ¹²⁵I–TGF-β1 followed by immunoprecipitation by anti-FLAG antibody was performed using COS7 cells transfected with the indicated plasmids (top). Levels of expression of I-Smads and their mutants and chimeras were examined by immunoblotting using anti-FLAG antibody (bottom). (B) Interaction of I-Smads with TβR-I(TD) was examined by FLAG immunoprecipitation of I-Smads, followed by HA-immunoblotting of TβR-I(TD) using transfected COS7 cells (top). The Smad6 and Smad7 chimeras shown in Fig. 3 A were used. 6(32), S6(31/32)S7; 6(91), S6(171/91)S7; 6(158), S6(225/158); 6(211), S6(279/211)S7. Note that HA-tagged TβR-I(TD) was comigrated with IgG. Lower panels demonstrate the levels of expression of I-Smads (middle) and TβR-I(TD) (bottom).

Figure 7.

Physical interaction of Smads with TGF-β receptors. (A) Affinity cross-linking using ¹²⁵I–TGF-β1 followed by immunoprecipitation by anti-FLAG antibody was performed using COS7 cells transfected with the indicated plasmids (top). Levels of expression of I-Smads and their mutants and chimeras were examined by immunoblotting using anti-FLAG antibody (bottom). (B) Interaction of I-Smads with TβR-I(TD) was examined by FLAG immunoprecipitation of I-Smads, followed by HA-immunoblotting of TβR-I(TD) using transfected COS7 cells (top). The Smad6 and Smad7 chimeras shown in Fig. 3 A were used. 6(32), S6(31/32)S7; 6(91), S6(171/91)S7; 6(158), S6(225/158); 6(211), S6(279/211)S7. Note that HA-tagged TβR-I(TD) was comigrated with IgG. Lower panels demonstrate the levels of expression of I-Smads (middle) and TβR-I(TD) (bottom).

Figure 9.

Modes of interaction between the amino-terminal regions and the MH2 domains of R-Smads and I-Smads. The amino-terminal regions and the MH2 domains physically interact with each other in both R-Smads and I-Smads. The amino-terminal MH1 domains and the MH2 domains of R-Smads repress each other's activity, and this physical interaction is released upon receptor activation. In contrast, the N domain of Smad7 enhances the inhibitory activity of its MH2 domain through physical interaction.