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. 2001 Nov 1;15(21):2822-36.
doi: 10.1101/gad.912901.

Smad3 recruits the anaphase-promoting complex for ubiquitination and degradation of SnoN

S L Stroschein  1 , S Bonni, J L Wrana, K Luo
Affiliations

Smad3 recruits the anaphase-promoting complex for ubiquitination and degradation of SnoN

S L Stroschein et al. Genes Dev. .

Abstract

Smad proteins mediate transforming growth factor-beta (TGF-beta) signaling to regulate cell growth and differentiation. SnoN is an important negative regulator of TGF-beta signaling that functions to maintain the repressed state of TGF-beta target genes in the absence of ligand. On TGF-beta stimulation, Smad3 and Smad2 translocate into the nucleus and induce a rapid degradation of SnoN, allowing activation of TGF-beta target genes. We show that Smad2- or Smad3-induced degradation of SnoN requires the ubiquitin-dependent proteasome and can be mediated by the anaphase-promoting complex (APC) and the UbcH5 family of ubiquitin-conjugating enzymes. Smad3 and to a lesser extent, Smad2, interact with both the APC and SnoN, resulting in the recruitment of the APC to SnoN and subsequent ubiquitination of SnoN in a destruction box (D box)-dependent manner. In addition to the D box, efficient ubiquitination and degradation of SnoN also requires the Smad3 binding site in SnoN as well as key lysine residues necessary for ubiquitin attachment. Mutation of either the Smad3 binding site or lysine residues results in stabilization of SnoN and in enhanced antagonism of TGF-beta signaling. Our studies elucidate an important mechanism and pathway for the degradation of SnoN and more importantly, reveal a novel role of the APC in the regulation of TGF-beta signaling.

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Figures

Figure 1
Figure 1
Smad3- and TGF-β-induced degradation of SnoN is mediated by the ubiquitin-dependent proteasome. (A) Pulse-chase analysis of SnoN degradation by various Smad proteins. 293T cells cotransfected with HA–SnoN and the indicated Flag-tagged Smads were subjected to pulse-chase assays as described in Materials and Methods. Immunoprecipitation was carried out with anti-Flag to purify Smad-associated SnoN or with anti-HA to purify total cellular HA–SnoN. The half-lives of the two populations were similar. As a control, SnoN from singly transfected cells was isolated by immunoprecipitation with anti-HA. Abbreviations used: S3, Smad3; S3C, MH2 domain of Smad3; S3N, MH1 domain of Smad3; S2, Smad2; S2C, MH2 domain of Smad2; S2NL, MH1 and linker domains of Smad2; S4, Smad4. (B) Proteasome inhibitors block Smad3-induced degradation of SnoN. Transfected 293T cells were pretreated with DMSO (control) or various proteolytic inhibitors for 45 min followed by a pulse-chase assay as described in Materials and Methods. Inhibitors were present throughout the duration of the pulse-chase. Quantification of 35S-labeled SnoN was carried out with the Bio-Rad Molecular Imager FX system and is shown below the gels. (C) Lactacystin blocks TGF-β-induced degradation of endogenous SnoN. Hep3B cells were pretreated with DMSO (lanes 1–3) or lactacystin (lanes 4–6) for 4 h followed by stimulation with 200 pM TGF-β1 for the indicated times. Endogenous SnoN was isolated by immunoprecipitation with an anti-SnoN anti-serum and analyzed by immunoblotting with anti-SnoN. (D) Smad2 and Smad3 induce ubiquitination of SnoN. HA–Smads or Flag–SnoN were transfected either alone or together into 293T cells and isolated by immunoprecipitation with either anti-HA (lanes 1–3) or anti-Flag (lanes 4–7). Polyubiquitination of SnoN was detected by immunoblotting of the immunoprecipitates with an anti-ubiquitin antibody (top). The amounts of SnoN (middle) and Smads present in the immunoprecipitates were shown on the two lower panels. (E) The MH2 domain of Smad3 induces SnoN ubiquitination. The experiments were carried out as described in D.
Figure 1
Figure 1
Smad3- and TGF-β-induced degradation of SnoN is mediated by the ubiquitin-dependent proteasome. (A) Pulse-chase analysis of SnoN degradation by various Smad proteins. 293T cells cotransfected with HA–SnoN and the indicated Flag-tagged Smads were subjected to pulse-chase assays as described in Materials and Methods. Immunoprecipitation was carried out with anti-Flag to purify Smad-associated SnoN or with anti-HA to purify total cellular HA–SnoN. The half-lives of the two populations were similar. As a control, SnoN from singly transfected cells was isolated by immunoprecipitation with anti-HA. Abbreviations used: S3, Smad3; S3C, MH2 domain of Smad3; S3N, MH1 domain of Smad3; S2, Smad2; S2C, MH2 domain of Smad2; S2NL, MH1 and linker domains of Smad2; S4, Smad4. (B) Proteasome inhibitors block Smad3-induced degradation of SnoN. Transfected 293T cells were pretreated with DMSO (control) or various proteolytic inhibitors for 45 min followed by a pulse-chase assay as described in Materials and Methods. Inhibitors were present throughout the duration of the pulse-chase. Quantification of 35S-labeled SnoN was carried out with the Bio-Rad Molecular Imager FX system and is shown below the gels. (C) Lactacystin blocks TGF-β-induced degradation of endogenous SnoN. Hep3B cells were pretreated with DMSO (lanes 1–3) or lactacystin (lanes 4–6) for 4 h followed by stimulation with 200 pM TGF-β1 for the indicated times. Endogenous SnoN was isolated by immunoprecipitation with an anti-SnoN anti-serum and analyzed by immunoblotting with anti-SnoN. (D) Smad2 and Smad3 induce ubiquitination of SnoN. HA–Smads or Flag–SnoN were transfected either alone or together into 293T cells and isolated by immunoprecipitation with either anti-HA (lanes 1–3) or anti-Flag (lanes 4–7). Polyubiquitination of SnoN was detected by immunoblotting of the immunoprecipitates with an anti-ubiquitin antibody (top). The amounts of SnoN (middle) and Smads present in the immunoprecipitates were shown on the two lower panels. (E) The MH2 domain of Smad3 induces SnoN ubiquitination. The experiments were carried out as described in D.
Figure 2
Figure 2
A direct interaction between Smad3 and SnoN is necessary but not sufficient for degradation of SnoN. (A) Residues 230–289 of Smad3 are required for interaction with SnoN. Flag-tagged full-length or truncated Smad3 was cotransfected into 293T cells together with HA–SnoN and isolated by immunoprecipitation with anti-Flag agarose. The immunoprecipitates were subjected to immunoblotting with an anti-HA antibody to detect SnoN that bound to Smad3 (top) or with anti-Flag to control for Smad levels (middle). Cell lysates were blotted with anti-HA to control for SnoN expression (bottom). (B) Residues 279–318 of Smad2 are required for interaction with SnoN. Flag-tagged Smad1, Smad2, or Smad1/Smad2 chimeras were cotransfected together with HA–SnoN and analyzed as described in A. (C) Residues 237–276 of Smad3 are necessary but not sufficient for degradation of SnoN. (Left) Binding assay. Flag–SnoN was transfected either alone or together with HA-tagged Smads and isolated by immunoprecipitation with anti-Flag antibodies followed by Western blotting with anti-HA to detect Smads that bound to SnoN (top) or with anti-Flag to detect SnoN (middle). Cell lysates were blotted with anti-HA to control for Smad expression (bottom). (Right) Pulse-chase assay. 293T cells cotransfected with HA–SnoN and Flag–Smad were subjected to pulse-chase assay. (S3/S1/S3) Smad3 residues 1–236 and 277–425, and Smad1 residues 276–317. (S1/S3/S1) Smad1 residues 1–275 and 318–465, and Smad3 residues 237–276.
Figure 3
Figure 3
Smad2- or Smad3-binding site in SnoN is required for the ubiquitination and degradation of SnoN. (A) SnoN mutants. (B) Ubiquitination of SnoN requires binding to Smad2 or Smad3. HA–Smad3 or Flag–SnoN was transfected either individually or together into 293T cells. Flag–SnoN was isolated by immunoprecipitation with anti-Flag (lanes 2–7) antibody followed by Western blotting with anti-ubiquitin (top), anti-Flag (second panel), or anti-HA (third panel). As a control, HA–Smad3 was isolated from singly transfected cells by immunoprecipitation with anti-HA (lane 1). Total Smad levels were detected by immunoblotting of the cell lysates with anti-HA (bottom). (C) Pulse-chase assay. 293T cells transfected with HA–SnoN and Flag–Smad3 were subjected to the pulse-chase assay as described in Materials and Methods. SnoN was isolated from the transfected cells by immunoprecipitation with the anti-HA antibody. (D) TGF-β-induced degradation of SnoN is mediated by the R-Smads. Hep3B cells were transiently transfected with Flag-tagged wild-type or mutant SnoN constructs. Cells were treated with TGF-β for 30 min, and SnoN was isolated by immunoprecipitation with anti-Flag agarose and detected by Western blotting with anti-Flag antiserum.
Figure 4
Figure 4
Lysines 440, 446, and 449 of SnoN are required for ubiquitination. (A) SnoN deletions and point mutations. Lysine residues were mutated to arginine and numbered as follows: 1, K383; 2, K407; 3, K423; 4, K427; 5, K432; 6, K440; 7, K446; and 8, K449. (B) Pulse-chase assay of the SnoN deletion mutants. 293T cells were transfected with HA–SnoN and Flag–Smad2C and subjected to the pulse-chase assay. SnoN associated with Smad2C was isolated by immunoprecipitation with the anti-Flag antibody and detected by autoradiography. (C) Mutation of lysines 440, 446, and 449 disrupts Smad3-induced ubiquitination of SnoN. Flag–SnoN was cotransfected together with HA–Smad3 and isolated by immunoprecipitation with anti-Flag (lanes 2–15) followed by Western blotting with anti-ubiquitin (top), anti-Flag (middle) or anti-HA antibodies (bottom). As a control, HA–Smad3 was isolated from singly transfected cells by immunoprecipitation with anti-HA (lane 1). (D) Mutation of lysines 440, 446, and 449 blocks TGF-β-induced degradation of SnoN. Hep3B cells were transiently transfected with the indicated Flag-tagged SnoN constructs. Cells were treated with TGF-β for 30 min, and SnoN was isolated by immunoprecipitation with anti-Flag agarose and detected by Western blotting with anti-Flag antiserum.
Figure 5
Figure 5
Ubiquitination pathways and components involved in TGF-β-induced degradation of SnoN. (A) The UbcH5 family of E2 ubiquitin-conjugating enzymes are required for SnoN degradation. Dominant negative forms of various E2 enzymes were cotransfected into 293T cells together with Smad3 and SnoN. The ability of Smad3 to induce degradation of SnoN was analyzed by pulse-chase assay as described in Materials and Methods. (B) Smad3-induced ubiquitination and degradation of SnoN does not require the PY motif of Smad3. (Top) Pulse-chase assay. 293T cells were transfected with SnoN and the Smad3 constructs as indicated and subjected to pulse-chase assays. (Bottom) Ubiquitination assay. HA–Smad3 or Flag–SnoN constructs were transfected either individually or together into 293T cells and isolated by immunoprecipitation with anti-HA (lanes 1,2) or anti-Flag (lanes 3–5) antibodies followed by Western blotting with anti-ubiquitin (top), anti-Flag (middle), or anti-HA (bottom). (C) Smad3-induced ubiquitination and degradation of SnoN requires the D box sequence. (Top) Alignment of various D box sequences. (Middle) Pulse-chase assay. (Bottom) Ubiquitination assay. The experiments were carried out as in B.
Figure 6
Figure 6
Smad3 and Smad2 interact with the APC to induce ubiquitination of SnoN. (A) Smad3 and Smad2 interact with the APC components. The indicated Flag-tagged full-length or mutant Smads were transfected into 293T cells and isolated from nuclear extracts by immunoprecipitation with anti-Flag agarose. The immunoprecipitates were subjected to immunoblotting with an anti-Cdc27 antibody (top) or anti-Cdc16 (middle) to detect associated APC components, or with anti-Flag to control for Smad levels (bottom). (B) SnoN associates with CDH1 in a D box-dependent manner. Flag-tagged wild-type or mutant SnoN or Smads were cotransfected with HA–CDH1 into 293T cells and isolated by immunoprecipitation with anti-Flag agarose. The immunoprecipitates were subjected to immunoblotting with anti-HA to detect CDH1 bound to SnoN (top) and anti-Flag to control for Smad and SnoN levels (middle). Total CDH1 levels were detected by immunoblotting of the cell lysates with anti-HA (bottom). (C) Smad3 or Smad2 recruits the APC to SnoN in response to activation of TGF-β signaling. Flag–SnoN and HA–Smad were cotransfected into 293T cells in the absence or presence of a constitutive active (T204D)TβRI (TβRI*). The APC was isolated from the nuclear extract by immunoprecipitation with anti-Cdc27 and anti-Cdc16 antisera, and the associated SnoN or Smad3 was detected by immunoblotting with anti-Flag or anti-HA as labeled (two top panels). Total APC levels were detected by immunoblotting with anti-Cdc27 and anti-Cdc16 (third and fourth panels from the top). Total SnoN and Smad3 levels were detected by immunoblotting of the cell lysates with anti-Flag and anti-HA (two bottom panels). (D) TGF-β induces formation of an endogenous Smad3/SnoN/APC complex. Nuclear extracts were prepared from Ba/F3 cells that were stimulated with TGF-β for 20 min. Smad3 and the APC associated with SnoN was isolated from these nuclear extracts by immunoprecipitation with anti-SnoN and detected by Western blotting with anti-Smad3, anti-Cdc27, or anti-Cdc16 antibodies. Controls for the levels of total Smad3, SnoN, and APC in the nuclear extracts are shown. (E) In vitro reconstituted ubiquitination reactions. In vitro ubiquitin reactions were carried out with the purified Flag–SnoN, Flag–Smad3 or Flag–Smad2, GST–ubiquitin, and the indicated ubiquitin enzymes as described in Materials and Methods. Ubiquitinated SnoN was detected by Western blotting with anti-SnoN and anti-Flag antisera (top). The membrane was reblotted with anti-Flag (middle) to measure the level of SnoN and Smad3 in each reaction, and with anti-Cdc27 and anti-Cdc16 to determine the amounts of APC in each reaction (two bottom panels). (*) A nonspecific band recognized by anti-SnoN antibody.
Figure 6
Figure 6
Smad3 and Smad2 interact with the APC to induce ubiquitination of SnoN. (A) Smad3 and Smad2 interact with the APC components. The indicated Flag-tagged full-length or mutant Smads were transfected into 293T cells and isolated from nuclear extracts by immunoprecipitation with anti-Flag agarose. The immunoprecipitates were subjected to immunoblotting with an anti-Cdc27 antibody (top) or anti-Cdc16 (middle) to detect associated APC components, or with anti-Flag to control for Smad levels (bottom). (B) SnoN associates with CDH1 in a D box-dependent manner. Flag-tagged wild-type or mutant SnoN or Smads were cotransfected with HA–CDH1 into 293T cells and isolated by immunoprecipitation with anti-Flag agarose. The immunoprecipitates were subjected to immunoblotting with anti-HA to detect CDH1 bound to SnoN (top) and anti-Flag to control for Smad and SnoN levels (middle). Total CDH1 levels were detected by immunoblotting of the cell lysates with anti-HA (bottom). (C) Smad3 or Smad2 recruits the APC to SnoN in response to activation of TGF-β signaling. Flag–SnoN and HA–Smad were cotransfected into 293T cells in the absence or presence of a constitutive active (T204D)TβRI (TβRI*). The APC was isolated from the nuclear extract by immunoprecipitation with anti-Cdc27 and anti-Cdc16 antisera, and the associated SnoN or Smad3 was detected by immunoblotting with anti-Flag or anti-HA as labeled (two top panels). Total APC levels were detected by immunoblotting with anti-Cdc27 and anti-Cdc16 (third and fourth panels from the top). Total SnoN and Smad3 levels were detected by immunoblotting of the cell lysates with anti-Flag and anti-HA (two bottom panels). (D) TGF-β induces formation of an endogenous Smad3/SnoN/APC complex. Nuclear extracts were prepared from Ba/F3 cells that were stimulated with TGF-β for 20 min. Smad3 and the APC associated with SnoN was isolated from these nuclear extracts by immunoprecipitation with anti-SnoN and detected by Western blotting with anti-Smad3, anti-Cdc27, or anti-Cdc16 antibodies. Controls for the levels of total Smad3, SnoN, and APC in the nuclear extracts are shown. (E) In vitro reconstituted ubiquitination reactions. In vitro ubiquitin reactions were carried out with the purified Flag–SnoN, Flag–Smad3 or Flag–Smad2, GST–ubiquitin, and the indicated ubiquitin enzymes as described in Materials and Methods. Ubiquitinated SnoN was detected by Western blotting with anti-SnoN and anti-Flag antisera (top). The membrane was reblotted with anti-Flag (middle) to measure the level of SnoN and Smad3 in each reaction, and with anti-Cdc27 and anti-Cdc16 to determine the amounts of APC in each reaction (two bottom panels). (*) A nonspecific band recognized by anti-SnoN antibody.
Figure 7
Figure 7
Stabilization of SnoN enhances its ability to block TGF-β signaling. (A) Ba/F3 cells stably expressing wild-type SnoN, SnoN 1–366, or SnoN K6–8R were stimulated with (lanes 3,5,7) or without (lanes 1,2,4,6) TGF-β1 for 30 min. Flag–SnoN was isolated by immunoprecipitation with anti-Flag antiserum followed by immunoblotting with anti-Flag. (B) Growth inhibition assay. The same stable Ba/F3 cell lines shown in A were incubated for 4 d with various concentrations of TGF-β1 as indicated. The growth of cells was quantified by cell counting and compared with the growth of unstimulated cells.
Figure 8
Figure 8
Model of ubiquitination and degradation of SnoN.
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