Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degradation.

Smad7 is an inhibitory Smad that acts as a negative regulator of signaling by the transforming growth factor-beta (TGF-beta) superfamily proteins. Smad7 is induced by TGF-beta, stably interacts with activated TGF-beta type I receptor (TbetaR-I), and interferes with the phosphorylation of receptor-regulated Smads. Here we show that Smurf1, an E3 ubiquitin ligase for bone morphogenetic protein-specific Smads, also interacts with Smad7 and induces Smad7 ubiquitination and translocation into the cytoplasm. In addition, Smurf1 associates with TbetaR-I via Smad7, with subsequent enhancement of turnover of TbetaR-I and Smad7. These results thus reveal a novel function of Smad7, i.e. induction of degradation of TbetaR-I through recruitment of an E3 ligase to the receptor.


Smad7 is an inhibitory Smad that acts as a negative regulator of signaling by the transforming growth factor-␤ (TGF-␤) superfamily proteins. Smad7 is induced by TGF-␤, stably interacts with activated TGF-␤ type I receptor (T␤R-I), and interferes with the phosphorylation of receptor-regulated Smads.
Here we show that Smurf1, an E3 ubiquitin ligase for bone morphogenetic proteinspecific Smads, also interacts with Smad7 and induces Smad7 ubiquitination and translocation into the cytoplasm. In addition, Smurf1 associates with T␤R-I via Smad7, with subsequent enhancement of turnover of T␤R-I and Smad7. These results thus reveal a novel function of Smad7, i.e. induction of degradation of T␤R-I through recruitment of an E3 ligase to the receptor.
Members of the transforming growth factor-␤ (TGF-␤) 1 superfamily initiate cellular responses (1) by binding to two different types of serine/threonine kinase receptors, termed type I and type II. Type I receptor is activated by type II receptor upon ligand binding and mediates specific intracel-lular signals (2). Members of the TGF-␤ superfamily transduce intracellular signals by Smad proteins. Eight different Smad proteins have been identified in mammals and are classified into three subgroups, i.e. receptor-regulated Smads (R-Smads), common-partner Smads (Co-Smads), and inhibitory Smads (I-Smads) (3)(4)(5).
R-Smads and Co-Smads positively regulate signaling by the TGF-␤ superfamily (3)(4)(5). R-Smads directly interact with type I receptors and become activated through phosphorylation of the C-terminal SSXS motif. R-Smads then form heteromeric complexes with Co-Smads (Smad4) and translocate into the nucleus. Nuclear Smad complexes bind to transcriptional coactivators or corepressors and regulate transcription of target genes. Smad2 and Smad3 act in the TGF-␤/activin pathway, whereas Smad1, Smad5, and Smad8 are thought to act as bone morphogenetic protein (BMP)-specific Smads.
The third class of Smads are I-Smads, which include Smad6 and Smad7 in mammals (6 -8). I-Smads associate with activated TGF-␤ superfamily type I receptors, thereby preventing phosphorylation of R-Smads. In addition, Smad6 has been demonstrated to interact with phosphorylated Smad1 to prevent complex formation between Smad1 and Smad4 (9). Smad6 was also reported to interact with Hoxc-8 and function as a transcriptional corepressor for inhibition of BMP signaling (10). Because expression of Smad6 and Smad7 is induced by TGF-␤ and BMPs, I-Smads inhibit TGF-␤ superfamily signaling by a negative feedback system (11).
Ubiquitin-dependent protein degradation plays a key role in various biological processes, including signal transduction, cell cycle progression, and transcriptional regulation (12). In the TGF-␤ signaling pathways, R-Smads, e.g. Smad2 and Smad1/5, have recently been shown to be degraded by the ubiquitinproteasome pathway. Smad2 activated by TGF-␤ is degraded by the ubiquitin-proteasome pathway after translocation into the nucleus (13). Smurf1, a member of the HECT family of E3 ubiquitin ligases, ligand-independently induces the ubiquitination and degradation of BMP-specific Smads 1 and 5 through binding to a PY motif in the linker regions (14).
Here we demonstrate a novel function of Smurf1 in receptor degradation in TGF-␤ superfamily signaling. Inhibitory Smad7 associates with Smurf1 in the nucleus and is exported to the cytoplasm. Smad7 thus recruits Smurf1 to T␤R-I, resulting in the degradation and rapid turnover of the T␤R-I protein.

EXPERIMENTAL PROCEDURES
Transfection, Immunoprecipitation, and Immunoblotting-COS7 cells or 293T cells were transiently transfected using FuGENE6 (Roche Molecular Biochemicals). Immunoprecipitation and immunoblotting were performed as described (15). For inhibition of proteasomal degradation, cells were incubated with 50 M MG132 (Peptide Institute) or 10 M lactacystin (Calbiochem) for 4 h. Each experiment has been repeated at least three times with essentially similar results.
Affinity Cross-linking and Immunoprecipitation-Recombinant TGF-␤1 (R & D Systems) was iodinated using the chloramine T method. Cross-linking was performed on ice to avoid degradation of the receptors and other proteins. Subsequent immunoprecipitation and analysis by SDS polyacrylamide gel electrophoresis (PAGE) were performed as described (15).
Immunofluorescence Labeling-Immunohistochemical staining of 6Myc-Smad7 in transfected COS7 cells was performed using mouse anti-Myc antibody followed by incubation with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG as described (15). For double staining of Smad7 and Smurf1, immunohistochemical staining of FLAG-Smad7 and 6Myc-Smurf1 was performed using mouse anti-* This research was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan and by special coordination funds for promoting science and technology from the Science and Technology Agency of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
FLAG or rabbit anti-Myc antibody followed by incubation with FITClabeled goat anti-mouse IgG or rhodamine isothiocyanate (RITC)-labeled goat anti-rabbit IgG, respectively. Nuclei of the cells were stained by 4,6-diamidino-2-phenylindole. Intracellular localization was determined by confocal laser scanning microscopy.
Pulse-Chase Analysis-Cells were labeled for 10 min at 37°C with 50 mCi/ml [ 35 S]methionine and cysteine (Amersham Pharmacia Biotech) in methionine-and cysteine-free Dulbecco's modified Eagle's medium and chased as described (13). Cells were then lysed and subjected to immunoprecipitation.
Luciferase Assay-R mutant mink lung epithelial cells were transiently transfected with an appropriate combination of a p3TP-lux promoter-reporter construct, expression plasmids, and pcDNA3. Total amounts of transfected DNAs were the same in each experiment, and values were normalized using Renilla luciferase activity.

Smurf1
Interacts with Smad6 and Smad7-Smurf1 has been identified as an E3 ubiquitin ligase for BMP-specific Smads (14). Smurf1 has two WW domains that facilitate protein-protein interactions by binding to the PPXY sequence (PY motif) on partner proteins. Of eight different Smads, not only R-Smads including Smad1 and Smad5 but also I-Smads have a PY motif in their linker regions (Fig. 1A). We therefore examined whether Smurf1 binds to I-Smads. We first analyzed the interaction of Smurf1 with different Smads in transfected COS7 cells. A Smurf1 mutant, Smurf1(C710A), which has a mutation in the HECT domain and fails to recruit ligase activity, was used for this study. Of Smads 1 through 8, Smad6 and Smad7 strongly interacted with Smurf1(C710A) (Fig. 1B). Smurf1(C710A) interacted with Smad1 and Smad5 less effi-ciently than with Smad6 and Smad7. In contrast, it bound to Smad3 only weakly and failed to bind to Smads 2 and 4. Because Smad8 lacks the PY motif, Smurf1(C710A) did not bind to Smad8 either (Fig. 1B).
The mode of interaction between Smad7 and Smurf1 was further studied. In COS7 cells, weak interaction of wild-type Smad7 (Smad7(WT)) with wild-type Smurf1 (Smurf1(WT)) was detected, and it was slightly facilitated in the presence of the constitutively active TGF-␤ type I receptor, T␤R-I(TD) (Fig.  1C). Moreover, the interaction between Smurf1(WT) and Smad7 was enhanced by the proteasome inhibitor lactacystin. In contrast, a Smad7 deletion mutant that lacks the PY motif (amino acids 207-211) in the linker region (Smad7⌬PY) did not bind to Smurf1.
Smurf1 Interacts with T␤R-I via Smad7-Smad7 interacts with T␤R-I activated by T␤R-II, thereby competing with Smad2 and Smad3 for inhibition of TGF-␤ signaling. We therefore examined in an affinity cross-linking assay whether Smad7 acts as an adapter molecule that links T␤R-I to the ubiquitin-proteasome pathway. Although Smurf1 alone did not efficiently bind to T␤R-I in transfected COS7 cells, Smad7 dramatically enhanced the interaction between Smurf1 and the T␤R-I⅐T␤R-II complex (Fig. 1D, lanes 4 and 5). Moreover, Smurf1 failed to interact with the receptor complex in the presence of Smad7⌬PY (Fig. 1D, lane 6). These results indicate that Smurf1 is recruited to T␤R-I through Smad7.
Smad7 Is Translocated to the Cytoplasm by Smurf1-We next examined the effect of Smurf1 on the subcellular localiza- Transfected COS7 cells were subjected to FLAG immunoprecipitation (IP) followed by Myc immunoblotting (Blot). The top panel shows the interaction, and the lower two panels the expression of each protein as indicated. C, binding of Smurf1(WT) to Smad7(WT) but not to Smad7⌬PY. The binding between Smurf1(WT) and Smad7(WT) was slightly facilitated by T␤R-I(TD), which was further enhanced by the presence of a proteasome inhibitor lactacystin. The top panel shows the interaction of Smurf1 and Smad7. FLAG-Smad7 was immunoprecipitated in lanes 2-7 from the left, whereas FLAG-Smurf1 was precipitated in lanes 8 -11. Note that the expression levels of Smurf1 were lower than those of Smad7; thus the degradation of Smad7 was not remarkable in this figure. D, binding of Smurf1 to the T␤R-II⅐T␤R-I complex. COS7 cells were transfected or not with FLAG-tagged Smurf1(CA) and HA-tagged T␤R-I and T␤R-II in the presence or absence of FLAG-or 6Myc-tagged Smad7. Cells were affinity-labeled with 125 I-TGF-␤1, and lysates were immunoprecipitated (IP) with anti-FLAG M2 antibody. Immune complexes were subjected to SDS-PAGE and analysis using a Fuji BAS 2500 bio-imaging analyzer (Fuji Photo Film). FLAG-Smurf1 was immunoprecipitated in lanes 4 -6 from the left. As controls, FLAG-Smad7 was precipitated in lanes 1-3. Cross-linking analysis revealed that the expression levels of T␤R-II and T␤R-I were similar in each lane (data not shown).

Smurf1 Induces Degradation of T␤R-I by Smad7 12478
tion of Smad7. In the absence of Smurf1, both Smad7(WT) and Smad7⌬PY were predominantly located in the nucleus, although weak staining in the cytoplasm was also detected ( Fig.  2A). When transfected alone, Smurf1 was detected in the cytoplasm (data not shown). In the presence of Smurf1, Smad7(WT) was mainly observed in the cytoplasm. The cytoplasmic staining of Smad7 was further enhanced by the presence of proteasomal inhibitor MG132 or lactacystin (Fig. 2B). Smad7⌬PY failed to accumulate in the cytoplasm even in the presence of Smurf1, although there is a little leakage of Smad7⌬PY out of the nucleus (Fig. 2A); these results strongly suggest that interaction of Smurf1 with Smad7 is required for the cytoplasmic localization of Smad7. Consistent with this, Smurf1 and Smad7 colocalized in the cytoplasm (Fig. 2B). Interestingly, similar findings were obtained using Smurf1(C710A), suggesting that recruitment of ligase activity is not required for cytoplasmic translocation of the Smad7⅐Smurf1 complex (Fig. 2B).
An E3 ubiquitin ligase, MDM2, has been reported to promote ubiquitin-dependent degradation and nuclear export of p53 (16,17). In this case, a mutation within the MDM2 RING-finger domain that cannot induce p53 ubiquitination also lacks the ability to promote the p53 nuclear export. Thus, both Smurf1 and MDM2 promote not only ubiquitin-dependent degradation but also nuclear export of the substrates, although the mechanisms of nuclear export appear to differ between them. Itoh et al. (18) reported that Smad7 is predominantly located in the nucleus and that it is exported to the cytoplasm after ligand stimulation. It is possible that Smurf1 functions as a carrier protein for Smad7 for nuclear export, although it is currently not known whether ligand stimulation triggers the nuclear export of Smad7 by Smurf1.
Smurf1 Induces Ubiquitination of Smad7 and T␤R-I-To determine whether Smurf1 acts as an E3 ubiquitin ligase for Smad7, ubiquitination of Smad7 by Smurf1 was investigated in vivo. Smad7 was transfected into COS7 cells, together with Smurf1 and HA-tagged ubiquitin. Smurf1 efficiently induced the ubiquitination of Smad7 (Fig. 3A). Notably, Smad7 ubiquitination occurred more efficiently than that of Smad1 or Smad4. Polyubiquitination of Smad7 was not observed when Smad7⌬PY or Smurf1(C710A) was used (Fig. 3B). We also tested the effect of Smad7 on T␤R-I ubiquitination by Smurf1 in 293T cells. Although Smurf1 alone ubiquitinated T␤R-I weakly, Smad7 enhanced the receptor ubiquitination by Smurf1 (Fig. 3C).
Smurf1 Induces Degradation of Smad7 and T␤R-I-To investigate whether Smurf1 regulates degradation of Smad7 and T␤R-I, we analyzed turnover of these proteins by pulse-chase experiments. Smurf1(WT), but not Smurf1(C710A), enhanced the degradation of Smad7 (Fig. 4, A and B), suggesting that Smurf1-induced Smad7 degradation is dependent on the HECT catalytic activity and through the proteasome. Smurf1(WT), but not Smurf1(C710A), was also rapidly degraded (Fig. 4B). Moreover, Smad7 and Smurf1 induced the degradation of T␤R-I (Fig. 4C). Our results thus demonstrate that Smad7 accelerates turnover of the T␤R-I protein by recruitment of an E3 ubiquitin ligase, Smurf1.
Smurf1 Enhances the Inhibitory Activity of Smad7-To examine the effect of Smurf1 on the inhibitory activity of Smad7, we first compared the effect of Smad7⌬PY with that of Smad7(WT) using a TGF-␤-responsible promoter-reporter construct, p3TP-lux (Fig. 4D). Smad7⌬PY suppressed activation of the reporter gene in a dose-dependent manner, but its inhibitory effect was less potent than that of Smad7(WT), suggesting that the interaction of Smad7 with Smurf-like molecules is important for efficient inhibition of TGF-␤ signaling by Smad7. Next, we tested the effect of Smurf1 on the inhibitory activity of Smad7 using p3TP-lux (Fig. 4E). Smurf1(WT), but not Smurf1(C710A), enhanced the inhibitory activity of Smad7. These data indicate that E3 ligase activity of Smurf1 is crucial for its effect on the inhibitory activity of Smad7.
Smurf-like Molecules Target TGF-␤ Receptors for Degradation via I-Smads-I-Smads have been shown to regulate TGF-␤ superfamily signaling through multiple mechanisms, e.g. competition with R-Smads for type I receptor interaction, inhibition of complex formation between R-Smads and Co-Smads, and transcriptional repression by interaction with transcription factors, such as Hoxc-8 (6 -10). Our present findings revealed a novel mechanism for the inhibitory activity of Smad7.

Smurf1 Induces Degradation of T␤R-I by Smad7 12479
Although degradation of receptor complexes by Smurf1 may not be absolutely required for the action of I-Smads, it may play an important role in the negative regulation of TGF-␤ superfamily signaling by I-Smads. The present findings also suggest that E3 ligases of the Smurf family regulate TGF-␤ superfamily signaling through dual mechanisms. (i) By interaction with and degradation of R-Smads, Smurf1 negatively regulates BMP signaling. (ii) Smurf1 also interacts with Smad7 and inhibits TGF-␤ signaling by receptor degradation. Recently, another Smurf, Smurf2, has been suggested to exhibit similar dual specificities. Lin et al. (19) reported that Smurf2 interacts with Smad2, as well as other R-Smads, and induces the degradation of Smad2. Moreover, Kavsak et al. (20) reported that Smurf2 binds to TGF-␤ receptor complex via Smad7 and causes degradation of receptors and Smad7. It will be important to determine in the future whether there are some functional differences between Smurf1 and Smurf2 in vivo, especially in the interaction with I-Smads or receptors.