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. 2001 Aug 1;20(15):4132-42.
doi: 10.1093/emboj/20.15.4132.

Promoting bone morphogenetic protein signaling through negative regulation of inhibitory Smads

Affiliations

Promoting bone morphogenetic protein signaling through negative regulation of inhibitory Smads

F Itoh et al. EMBO J. .

Abstract

Inhibitory Smads, i.e. Smad6 and Smad7, are potent antagonists of the BMP-Smad pathway by interacting with activated bone morphogenetic protein (BMP) type I receptors and thereby preventing the activation of receptor-regulated Smads, or by competing with activated R-Smads for heteromeric complex formation with Smad4. The molecular mechanisms that underlie the regulation of I-Smad activity have remained elusive. Here we report the identification of a cytoplasmic protein, previously termed associated molecule with the SH3 domain of STAM (AMSH), as a direct binding partner for Smad6. AMSH interacts with Smad6, but not with R- and Co-Smads, upon BMP receptor activation in cultured cells. Consistent with this finding, stimulation of cells with BMP induces a co-localization of Smad6 with AMSH in the cytoplasm. Ectopic expression of AMSH prolongs BMP-induced Smad1 phosphorylation, and potentiates BMP-induced activation of transcriptional reporter activity, growth arrest and apoptosis. The data strongly suggest that the molecular mechanism by which AMSH exerts its action is by inhibiting the binding of Smad6 to activated type I receptors or activated R-Smads.

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Figures

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Fig. 1. BMP receptor activation induces interaction and co-localization of AMSH with I-Smads. (A) Interaction of AMSH with I-Smads. Flag-AMSH was co-transfected with 6×Myc-Smads with or without caALK6 in COS7 cells. Immunoprecipitations were performed with Flag M5 antibody, and co-immunoprecipitated Smads were detected by western blotting with Myc antibody (upper panel). The expression of Flag-AMSH and 6×Myc-Smads was measured by applying total cell lysate (1:50) on an SDS–polyacrylamide gel followed by western blotting with Flag M5 (middle panel) or Myc antibody (lower panel). (B) I-Smads and AMSH are localized in the cytoplasm upon BMP-7 stimulation. C2C12 cells were transiently transfected with Myc-tagged AMSH, and Flag-tagged Smad6L or Flag-tagged Smad7. Then, the cells were stimulated for 2 h with BMP-7. Myc-AMSH was visualized with rabbit polyclonal Myc antibody followed by Texas red-conjugated goat anti-rabbit IgG (red), and Flag-I-Smads were detected with mouse monoclonal Flag M5 antibody followed by FITC-conjugated goat anti-mouse IgG (green). Co-localization of AMSH and I-Smads appears as yellow.
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Fig. 1. BMP receptor activation induces interaction and co-localization of AMSH with I-Smads. (A) Interaction of AMSH with I-Smads. Flag-AMSH was co-transfected with 6×Myc-Smads with or without caALK6 in COS7 cells. Immunoprecipitations were performed with Flag M5 antibody, and co-immunoprecipitated Smads were detected by western blotting with Myc antibody (upper panel). The expression of Flag-AMSH and 6×Myc-Smads was measured by applying total cell lysate (1:50) on an SDS–polyacrylamide gel followed by western blotting with Flag M5 (middle panel) or Myc antibody (lower panel). (B) I-Smads and AMSH are localized in the cytoplasm upon BMP-7 stimulation. C2C12 cells were transiently transfected with Myc-tagged AMSH, and Flag-tagged Smad6L or Flag-tagged Smad7. Then, the cells were stimulated for 2 h with BMP-7. Myc-AMSH was visualized with rabbit polyclonal Myc antibody followed by Texas red-conjugated goat anti-rabbit IgG (red), and Flag-I-Smads were detected with mouse monoclonal Flag M5 antibody followed by FITC-conjugated goat anti-mouse IgG (green). Co-localization of AMSH and I-Smads appears as yellow.
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Fig. 2. AMSH potentiates the BMP/Smad-induced transcriptional response. (A) AMSH stimulates BMP/Smad1-induced transcription. HepG2 cells were transfected with (SBE)4-luc, AMSH and Smads in the absence (solid bars) or presence (open bars) of 100 ng/ml BMP-7. (B) AMSH requires Smad4 to potentiate BMP-7-induced transcription. MDA-MB468 cells were transfected with (SBE)4-luc, AMSH and Smad4 without (solid bars) or with (open bars) BMP-7.
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Fig. 3. AMSH enhances the sensitivity to BMP-7 in HS-72 plasma cells. (A) Expression of AMSH in HS-72 transformants. Total lysate from each transformant (2 × 105 cells) was applied on the SDS–polyacrylamide gel, and the expression of AMSH was detected by western blotting using anti-Myc antibody. (B) AMSH potentiates BMP-7-mediated growth inhibition. The potentiation of growth inhibition by AMSH in three independent clones (AM101, AM103 and AM111) is shown. As a control, the effect of BMP-7 on parental HS-72 cells is shown. The relative growth compared with control is plotted against the concentration of BMP-7. All data shown are means ± SD. The percentage viability was calculated as follows: percentage viability = 100 × (A570–620 nm with BMP-7/A570–620 nm without BMP-7). (C) AMSH induces apoptosis in HS-72 cells in the presence of BMP-7. The cells were left unstimulated (solid bars) or stimulated with 500 ng/ml BMP-7 for 24 h (open bars), then the apoptotic cells in 1 × 104 cells analyzed were counted by FACScan. (D) AMSH increases the number of G1-arrested cells in the presence of BMP-7. Twenty-four hours after the treatment of each transformant with 500 ng/ml BMP-7, the cells were fixed and analyzed by FACScan. The data show the percentages of G1, S and G2/M phases in 1 × 104 cells. The experiments were performed at least three times, and the results from representative experiments are shown.
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Fig. 3. AMSH enhances the sensitivity to BMP-7 in HS-72 plasma cells. (A) Expression of AMSH in HS-72 transformants. Total lysate from each transformant (2 × 105 cells) was applied on the SDS–polyacrylamide gel, and the expression of AMSH was detected by western blotting using anti-Myc antibody. (B) AMSH potentiates BMP-7-mediated growth inhibition. The potentiation of growth inhibition by AMSH in three independent clones (AM101, AM103 and AM111) is shown. As a control, the effect of BMP-7 on parental HS-72 cells is shown. The relative growth compared with control is plotted against the concentration of BMP-7. All data shown are means ± SD. The percentage viability was calculated as follows: percentage viability = 100 × (A570–620 nm with BMP-7/A570–620 nm without BMP-7). (C) AMSH induces apoptosis in HS-72 cells in the presence of BMP-7. The cells were left unstimulated (solid bars) or stimulated with 500 ng/ml BMP-7 for 24 h (open bars), then the apoptotic cells in 1 × 104 cells analyzed were counted by FACScan. (D) AMSH increases the number of G1-arrested cells in the presence of BMP-7. Twenty-four hours after the treatment of each transformant with 500 ng/ml BMP-7, the cells were fixed and analyzed by FACScan. The data show the percentages of G1, S and G2/M phases in 1 × 104 cells. The experiments were performed at least three times, and the results from representative experiments are shown.
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Fig. 4. AMSH prolongs the BMP-7-induced Smad phosphorylation. HS-72 and AM111 cells were stimulated with 500 ng/ml BMP-7 for 0, 1, 4, 8 and 12 h. Total lysates from the cells (2 × 105 cells) were blotted and stained with anti-pS1 (upper panel) or anti-Smad1/5 (lower panel) antibody. Similar results were obtained in three independent experiments.
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Fig. 5. AMSH inhibits interactions of Smad6L with activated BMP type I receptor and Smad4. (A) AMSH inhibits the association between Smad6L and ALK6. Myc-AMSH was co-transfected with Flag-Smad6L and HA-caALK6 in COS7 cells. Immunoprecipitations were performed with HA 12C5 (Boehringer Mannheim) antibody, and co-immuno precipitated Smad6L was detected by western blotting with Flag M5 antibody (upper panel). The expression of HA-caALK6, Flag-Smad6L and Myc-AMSH was measured by applying total cell lysate (1:50) on an SDS–polyacrylamide gel followed by western blotting with HA (second panel), Flag M5 (third panel) or Myc antibody (lower panel). (B) The interaction between Smad6L and activated Smad1 upon BMP type I receptor activation is inhibited by AMSH. Myc-AMSH was co-transfected with 6×Myc-Smad1 and Flag-Smad6L with or without caALK6 in COS7 cells. Immunoprecipitations were performed with Flag M5 antibody, and co-immunoprecipitated Smad6L was detected by western blotting with Myc antibody (upper panel). The expression of Flag-Smad6L, 6×Myc-Smad1 and Myc-AMSH was measured by applying total cell lysate (1:50) on an SDS–polyacrylamide gel followed by western blotting with Flag M5 (middle panel) or Myc antibody (lower panel).
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Fig. 6. BMP type I receptor-induced AMSH phosphorylation. (A) SB203580 inhibits the phosphorylation of AMSH by caALK6. Flag-AMSH was transfected with or without caALK6. Then, PD98059 or SB203580 was added to culture medium 1 h before the addition of [32P]orthophosphate. Immunoprecipitations were performed with Flag M5 antibody (upper panel). The expression of Flag-AMSH in COS7 cells was visualized by western blotting using Flag M5 antibody (lower panel). (B) Dominant-negative forms of JNK2 and p38 block the phosphorylation of AMSH by caALK6. COS7 cells were transfected with the combinations of Flag-AMSH, HA-JNK2(VPF), HA-p38(AGF) and caALK6 and then metabolically labeled with [32P]orthophosphate. Immunoprecipitations were performed with Flag M5 antibody (upper panel). The expression of Flag-AMSH, HA-JNK2(VPF) and HA-p38(AGF) in COS7 cells was visualized by western blotting using Flag M5 (middle panel) or HA antibody (lower panel).
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Fig. 6. BMP type I receptor-induced AMSH phosphorylation. (A) SB203580 inhibits the phosphorylation of AMSH by caALK6. Flag-AMSH was transfected with or without caALK6. Then, PD98059 or SB203580 was added to culture medium 1 h before the addition of [32P]orthophosphate. Immunoprecipitations were performed with Flag M5 antibody (upper panel). The expression of Flag-AMSH in COS7 cells was visualized by western blotting using Flag M5 antibody (lower panel). (B) Dominant-negative forms of JNK2 and p38 block the phosphorylation of AMSH by caALK6. COS7 cells were transfected with the combinations of Flag-AMSH, HA-JNK2(VPF), HA-p38(AGF) and caALK6 and then metabolically labeled with [32P]orthophosphate. Immunoprecipitations were performed with Flag M5 antibody (upper panel). The expression of Flag-AMSH, HA-JNK2(VPF) and HA-p38(AGF) in COS7 cells was visualized by western blotting using Flag M5 (middle panel) or HA antibody (lower panel).
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Fig. 7. Phosphopeptide mapping in AMSH. Flag-AMSH or its mutants were co-transfected in the absence or presence of caALK6 in COS cells. After metabolic labeling with [32P]orthophosphate, proteins were immunoprecipitated with Flag M5 antibody. After trypsinization, two-dimensional electrophoresis was carried out. Four major spots, surrounded with broken lines, are shown as a–d.
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Fig. 8. AMSH (S243A,S245A,S247A) mutant is more active than wild-type AMSH. (A) Interaction of the AMSH (S243A,S245A,S247A) mutant with Smad6L upon BMP type I receptor activation. Immuno precipitations were performed with Flag M5 antibody, and co-immunoprecipitated Smad6L was detected by western blotting with Myc antibody (upper panel). The expression of Flag-AMSH or Flag-AMSH (S243A,S245A,S247A), and 6×Myc-Smad6L was measured by applying total cell lysate (1:50) on an SDS–polyacrylamide gel followed by western blotting with Flag M5 (middle panel) or Myc antibody (lower panel). (B) AMSH (S243A,S245A,S247A) is more active than wild-type AMSH in potentiating BMP-7-induced transcriptional responses in HepG2 cells. Experiments were performed as described in the legend to Figure 2A. (C) Dominant-negative p38 and JNK augment the interaction of AMSH with Smad6L upon BMP type I receptor activation. Immunoprecipitations were performed with Flag M5 antibody, and co-immunoprecipitated Smad6L was detected by western blotting with Myc antibody (upper panel). The expression of Flag-AMSH, 6×Myc-Smad6L and HA-JNK2(VPF) or HA-p38(AGF) was measured by applying total cell lysate (1:50) on an SDS–polyacrylamide gel followed by western blotting with Flag M5 (second panel), Myc (third panel) and HA antibodies (lower panel), respectively.
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Fig. 8. AMSH (S243A,S245A,S247A) mutant is more active than wild-type AMSH. (A) Interaction of the AMSH (S243A,S245A,S247A) mutant with Smad6L upon BMP type I receptor activation. Immuno precipitations were performed with Flag M5 antibody, and co-immunoprecipitated Smad6L was detected by western blotting with Myc antibody (upper panel). The expression of Flag-AMSH or Flag-AMSH (S243A,S245A,S247A), and 6×Myc-Smad6L was measured by applying total cell lysate (1:50) on an SDS–polyacrylamide gel followed by western blotting with Flag M5 (middle panel) or Myc antibody (lower panel). (B) AMSH (S243A,S245A,S247A) is more active than wild-type AMSH in potentiating BMP-7-induced transcriptional responses in HepG2 cells. Experiments were performed as described in the legend to Figure 2A. (C) Dominant-negative p38 and JNK augment the interaction of AMSH with Smad6L upon BMP type I receptor activation. Immunoprecipitations were performed with Flag M5 antibody, and co-immunoprecipitated Smad6L was detected by western blotting with Myc antibody (upper panel). The expression of Flag-AMSH, 6×Myc-Smad6L and HA-JNK2(VPF) or HA-p38(AGF) was measured by applying total cell lysate (1:50) on an SDS–polyacrylamide gel followed by western blotting with Flag M5 (second panel), Myc (third panel) and HA antibodies (lower panel), respectively.

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References

    1. Afrakhte M., Morén,A., Jossan,S., Itoh,S., Sampath,K., Westermark,B., Heldin,C.-H., Heldin,N.-E. and ten Dijke,P. (1998) Induction of inhibitory Smad6 and Smad7 mRNA by TGF-β family members. Biochem. Biophys. Res. Commun., 249, 505–511. - PubMed
    1. Atfi A., Djelloul,S., Chastre,E., Davis,R. and Gespach,C. (1997) Evidence for a role of Rho-like GTPases and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in transforming growth factor β-mediated signaling. J. Biol. Chem., 272, 1429–1432. - PubMed
    1. Bitzer M., von Gersdorff,G., Liang,D., Dominguez-Rosales,A., Beg,A.A., Rojkind,M. and Böttinger,E.P. (2000) A mechanism of suppression of TGF-β/SMAD signaling by NF-κB/RelA. Genes Dev., 14, 187–197. - PMC - PubMed
    1. Boyle W.J., van der Geer,P. and Hunter,T. (1991) Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol., 201, 110–149. - PubMed
    1. de Caestecker M.P., Hemmati,P., Larisch-Bloch,S., Ajmera,R., Roberts,A.B. and Lechleider,R.J. (1997) Characterization of functional domains within Smad4/DPC4. J. Biol. Chem., 272, 13690–13696. - PubMed

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