Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M414027200 on April 6, 2005

J. Biol. Chem., Vol. 280, Issue 23, 22115-22123, June 10, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/23/22115    most recent
M414027200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Morén, A.
Right arrow Articles by Moustakas, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Morén, A.
Right arrow Articles by Moustakas, A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Degradation of the Tumor Suppressor Smad4 by WW and HECT Domain Ubiquitin Ligases*

Anita Morén{ddagger}, Takeshi Imamura§, Kohei Miyazono§, Carl-Henrik Heldin{ddagger}, and Aristidis Moustakas{ddagger}||

From the {ddagger}Ludwig Institute for Cancer Research, Box 595, Biomedical Center, Uppsala University, SE-751 24 Uppsala, Sweden, the §Department of Biochemistry, The Cancer Institute of Japanese Foundation for Cancer Research, 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 170-8455, Japan, and the Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Received for publication, December 14, 2004 , and in revised form, March 7, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Smad4 mediates signaling by the transforming growth factor-{beta} (TGF-{beta}) superfamily of cytokines. Smad signaling is negatively regulated by inhibitory (I) Smads and ubiquitin-mediated processes. Known mechanisms of proteasomal degradation of Smads depend on the direct interaction of specific E3 ligases with Smads. Alternatively, I-Smads elicit degradation of the TGF-{beta} receptor by recruiting the WW and HECT domain E3 ligases, Smurfs, WWP1, or NEDD4–2. We describe an equivalent mechanism of degradation of Smad4 by the above E3 ligases, via formation of ternary complexes between Smad4 and Smurfs, mediated by R-Smads (Smad2) or I-Smads (Smad6/7), acting as adaptors. Smurfs, which otherwise cannot directly bind to Smad4, mediated poly-ubiquitination of Smad4 in the presence of Smad6 or Smad7. Smad4 co-localized with Smad7 and Smurf1 primarily in the cytoplasm and in peripheral cell protrusions. Smad2 or Smad7 mutants defective in Smad4 interaction failed to induce Smurf1-mediated down-regulation of Smad4. A Smad4 mutant defective in Smad2 or Smad7 interaction could not be effectively down-regulated by Smurf1. We propose that Smad4 is targeted for degradation by multiple ubiquitin ligases that can simultaneously act on R-Smads and signaling receptors. Such mechanisms of down-regulation of TGF-{beta} signaling may be critical for proper physiological response to this pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Smad proteins mediate signaling by transforming growth factor-{beta} (TGF-{beta})1 superfamily members (1–3). Upon binding of TGF-{beta} to cell surface complexes of type I and type II receptor serine/threonine kinases, the type II receptor phosphorylates the type I receptor, which further phosphorylates the receptor-regulated (R-) Smads, Smad2 and Smad3 (3). Phosphorylated R-Smads oligomerize with the common mediator (Co) Smad4 and accumulate in the nucleus where they regulate gene expression. Alternatively, TGF-{beta} signals via different intracellular effectors, including protein or lipid kinases and small GTPases (1). A third class of Smad proteins, the inhibitory (I) Smads, Smad6 and Smad7, mediate negative control of TGF-{beta} pathways (2, 3). TGF-{beta} family members induce nuclear Smad complexes that activate transcription of the Smad6 and Smad7 genes, thus creating a negative regulatory feedback loop.

The best-documented mechanism of negative regulation of Smad proteins is via their proteasomal degradation after ubiquitination (2, 3). Such mechanisms either maintain low steady-state Smad levels or alternatively lead to shutdown of the activated Smad pathway after proper nuclear signaling. The bone morphogenetic protein-specific R-Smads, Smad1 and Smad5, are targeted by the WW and HECT domain E3 ligase Smad ubiquitination regulatory factor (Smurf) 1, whereas Smad1 can also be ubiquitinated via carboxyl terminus of Hsc70-interacting protein (CHIP) (4–8). The TGF-{beta}-activated R-Smad, Smad2, can be ubiquitinated by the second Smurf family member, Smurf2 (9, 10). A second WW-HECT domain E3 ligase, Tiul1/WWP1, forms complexes with Smad2 after TGF-{beta} stimulation via the Smad2-interacting transcriptional co-repressor TGF-{beta}-induced factor, and leads to Smad2 poly-ubiquitination and down-regulation of TGF-{beta} signaling (11). Similar to WWP1, another E3 ligase that contains WW and HECT domains, NEDD4–2, interacts with Smad2 and induces its poly-ubiquitination and degradation (12). In contrast, Smad3 is down-regulated by the Roc1·SCF ubiquitin ligase complex (13). In Drosophila, Smurf-mediated down-regulation of the Smad pathway has also been observed (14, 15). Emerging evidence suggests that ubiquitination of Smad proteins could also serve alternative functions. The E3 ligase Itch induces Smad2 poly-ubiquitination, which then positively modulates phosphorylation of Smad2 by the TGF-{beta} type I receptor (16). This example increases the complexity of physiological effects Smad ubiquitination can offer to TGF-{beta} superfamily signaling.

Wild type Smad4 can be proteasomally degraded after poly-ubiquitination by the E3 ligase complexes of Jab1, SCF{beta}-TrCP1, or CHIP (4, 17, 18). In addition, wild type Smad4 can be mono-ubiquitinated by yet unidentified E3 ligases or sumoylated by Ubc9 and PIAS family E3 ligases (19–26). In Drosophila, the E3 ligase Highwire down-regulates the Co-Smad, Medea (27). Both mono-ubiquitination and sumoylation of Smad4 lead to stabilization of the protein and enhanced TGF-{beta} signaling. However, Smad4 sumoylation can also lead to inactivation of nuclear Smad signaling in a gene-specific manner (24). Alternatively, Smad4 deletion or point mutants that accumulate in human cancers of the pancreas, colon, and certain other organs exhibit dramatic instability due to preferential poly-ubiquitination and proteasomal degradation by the SCFSkp2 E3 ligase complex (25, 28–32).

Ubiquitin-mediated regulation of TGF-{beta} pathways becomes even more complex when one encounters the function of I-Smads (reviewed in Refs. 3 and 33). During the inhibitory feedback mechanism, I-Smads recruit the E3 ligases Smurf1, Smurf2, WWP1/Tiul1, or NEDD4–2 and bind to TGF-{beta} receptor complexes leading to their degradation (11, 12, 34–37). In zebrafish, the E3 ligase Dapper2 mediates lysosomal degradation of nodal receptors, although the contribution of I-Smads to this mechanism remains unexplored (38). I-Smads can also recruit Smurf1 to bone morphogenetic protein receptor complexes on the one hand, and to R-Smads, like Smad1 and Smad5, on the other hand, leading to their ubiquitination and degradation (39). Thus, I-Smads play the role of adaptor proteins that bind directly to E3 ligases and mediate their association with substrates, i.e. receptors or R-Smads. The I-Smad level is not necessarily affected by the associating E3 ligase. However, under certain conditions, I-Smads can also be targeted for proteasomal degradation either by Smurfs themselves or by additional E3 ligases such as Arkadia or signalosome components such as Jab1/CSN5 (40–42).

In the present study, we examined the role of I-Smads as adaptors that mediate recruitment of Smurf E3 ligases to components of the TGF-{beta} pathway to regulate their protein levels. We also analyzed effects of two additional E3 ligases, structurally and functionally related to Smurfs, WWP1 and NEDD4–2. We concentrated on the Co-Smad, Smad4, because this protein is known to make complexes with R-Smads but cannot directly interact with Smurf members. The study originated by observing that ectopic expression of I-Smads or Smurf1 could lead to dramatic down-regulation of Smad4. We therefore establish the formation of ternary complexes between Smad4, I-Smads and Smurfs, which result in poly-ubiquitination and proteasomal degradation of Smad4. Thus, Smurf E3 ligases (but also WWP1 and NEDD4–2) can act at all levels of the TGF-{beta} signaling pathway, mediating degradation of receptors, R-Smads, I-Smads, and of the unique Co-Smad, as established for the first time here.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture, Adenoviruses, Plasmids, and Antibodies—Human embryonic kidney (HEK) 293 and HEK-293T cells were obtained and cultured according to protocols from the American Type Culture Collection. The human keratinocyte cell line HaCaT was a gift from N. Fusenig, Heidelberg.

The adenoviral vector expressing human Smurf1 epitope-tagged with FLAG at its N terminus (AdX-Smurf1) was constructed using the BD Clontech Ad-X kit after subcloning of the FLAG-tagged cDNA of human Smurf1 from pcDNA3 to the adenoviral backbone vector using the manufacturer's protocol. Adenovirus was produced in transfected HEK-293 cells, and it was amplified and titrated in the same cells as previously described (43). The adenoviral vectors expressing FLAG-tagged Smad7 and the control {beta}-galactosidase (Ad-LacZ) have been previously described (43).

The mammalian expression vectors pcDNA3 empty and pcDNA3 encoding HA-tagged constitutively active ALK5(TD), 6myc-Smad2 and 6myc-Smad4 were described before (44). pcDNA3 encoding FLAG-Smad4, FLAG-Smad4(K507R), the HA-tagged ubiquitin vector, pGEX4T-1 encoding GST, and GST-Smad4 have been described previously (25). pcDNA3 encoding 6myc-Smad4(K507R), was constructed by transferring the Smad4(K507R) mutant cDNA from pcDNA3-FLAG to pcDNA3–6myc. pcDNA3 encoding FLAG-Smad2, FLAG-Smad6, FLAG-Smad6N, FLAG-Smad6C, FLAG-Smurf1, 6myc-Smurf1(CS), and FLAG-Smad7 have been described before (35, 45). pcDNA3-FLAG-Smurf2 was created by subcloning the human wild type Smurf2 cDNA (EcoRI-HindIII fragment from pRK1M-Smurf2) into pcDNA3-FLAG. The pRK1M-Smurf2 construct was provided by R. Derynck (10). pcDNA3 expressing untagged Smad7 has been described before (46). pcDNA3 encoding FLAG-Smad7N and FLAG-Smad7C were created using PCR-based deletion mutagenesis. The encoded Smad7 fragments correspond to amino acids 1–261 (Smad7N) and 204–426 (Smad7C). GST-Smad7 in vector pGEX4T-1 and GFP-Smad7 and GFP-Smad4 in vector pEGFP-C1 were provided by E. Grönroos of our Institute (40). pcDNA3 encoding FLAG-tagged dominant-negative mutant of Smad2 (Smad2SA) was provided by S. Souchelnytskyi of our Institute (47).

Mouse monoclonal anti-FLAG (M2 and M5) antibodies were purchased from Sigma-Aldrich, mouse monoclonal anti-HA from Roche Applied Science, rabbit polyclonal anti-GFP from Molecular Probes, and mouse monoclonal anti-Smad4 from BD Transduction Laboratories. Mouse monoclonal anti-myc (9E10) and rabbit polyclonal anti-Smad7 (KAF (48)) antibodies were produced in-house.

Cell Transfections and Infections—Transient transfections of cells using calcium phosphate co-precipitation were performed as previously described (25). Cells were transfected directly on plastic tissue culture dishes for biochemical assays or on glass coverslips coated with collagen (Vitrogel, Cohesion) for immunofluorescence assays. Cell monolayers were infected with the multiplicity of infection specified in the figure legends as described before (43), and protein analysis was performed 24 h post-infection.

Analysis of Smad4 Protein Expression—Transiently transfected, infected, or intact cell monolayers were lysed in solubilization buffer (0.5% Triton X-100, 0.5% deoxycholate, 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 1% aprotinin, 1 mM phenylmethylsulfonyl fluoride). After Bradford analysis of total protein levels, the lysates were adjusted prior to subsequent immunoblotting or immunoprecipitation. Endogenous Smad4 was directly immunoblotted using the mouse anti-Smad4 antibody. Transfected FLAG-tagged Smad4 was detected by direct immunoblotting of cell lysates with anti-FLAG M2 antibody. All immunoblotted proteins were detected by ECL.

Ubiquitination Assays—HEK-293T cells were transfected with 6myc-Smad4 and HA-ubiquitin in the absence or presence of Smad6, Smad7, and Smurfs as explained in the figure legends. Forty-eight hours after transfection cells were lysed in solubilization buffer and sonicated for 5 s. Smad4 was immunoprecipitated with anti-myc (9E10); after four washes with solubilization buffer, the immunocomplexes were resolved by SDS-PAGE and immunoblotted with anti-HA antibody for ubiquitin. Alternatively, cell extracts were immunoprecipitated with anti-HA and immunoblotted with anti-myc (9E10). For Smad4 ubiquitination analysis after proteasomal inhibition, transfected HEK-293T cells were treated with 50 µM MG-132 inhibitor (Calbiochem) for 6 h. All control cell treatments included equal volumes of Me2SO, the solvent of MG-132.

Smad4/I-Smad/Smurf Complex Formation Assays—Association of transfected FLAG- or 6myc- or GFP-Smad4, with FLAG-Smad2 and its phosphorylation mutant, FLAG-Smad6 and its deletion mutants, or FLAG-Smad7 and its deletion mutants and 6myc-Smurf1(CS) mutant, was monitored by co-immunoprecipitation assays. Transfected HEK-293T cells were lysed and immunoprecipitated with anti-FLAG antibody as described above, prior to immunoblotting with anti-myc or anti-GFP antibodies and ECL detection.

GST Pull-down—GST fusion proteins (2.5 ng) were mixed with a normalized amount of FLAG-tagged protein expressing cell lysates from transfected HEK-293T cells in solubilization buffer with protease inhibitors. Before washing, the GST beads were transferred into clean tubes, and the pull-down was washed three times in solubilization buffer and once in high salt buffer (0.5 M NaCl, 20 mM Tris-HCl, pH 7.4, 1% Triton X-100). Bound proteins were resolved by SDS-PAGE and immunoblotted with anti-FLAG for Smad proteins.

Immunofluorescence Microscopy—Transiently transfected cell mono-layers at ~70% confluency were analyzed by immunofluorescence 48 h post-transfection. Cells were processed with anti-FLAG (M2) or anti-myc (9E10) primary antibodies, followed by anti-mouse-tetramethylrhodamine isothiocyanate (TRITC) secondary antibody (DAKO) and 4',6'-diamidino-2-phenylindole (DAPI) staining of nuclei, as described before (49).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Smad4 Is Down-regulated by Smurf1 in the Presence of I- or R-Smads—We screened a number of known E3 ligases for their ability to induce down-regulation of wild type Smad4 after ectopic expression in transfected HEK-293T cells.2 Among the E3 ligases examined were the two members of the Smurf family, Smurf1 and Smurf2 (Fig. 1A). Smurfs have been previously shown to interact directly with both R-Smads and I-Smads (8–10, 35, 36, 39). The interaction is mediated via specific proline-tyrosine (PPXY) motifs located in the linker domains of R-Smads and I-Smads. Such motifs are not present in the linker or other domains of Smad4 and thus preclude the direct interaction of Smad4 with the WW domains of Smurfs (35). However, because Smad4 forms heteromeric complexes with R-Smads after receptor phosphorylation, we reasoned that an R-Smad might mediate complex formation between Smurfs and Smad4. Alternatively, because the interactions between Smad proteins are mediated via their highly conserved C-terminal Mad homology (MH) 2 domains, we also reasoned that I-Smads, which also contain conserved MH2 domains, might mediate the Smurf-Smad4 interaction. Based on all these reasons, we co-transfected R-Smads or I-Smads together with Smurf1 or Smurf2 and scored for their effects on the protein levels of wild type Smad4. Fig. 1B shows that, although cotransfection of Smad7 alone did not alter the levels of wild type Smad4, and co-transfection of Smurf1 only weakly down-regulated wild type Smad4, the combination of these proteins induced a strong decrease in Smad4 levels. The same effect was observed when Smad2 was co-transfected with Smurf1, and the effect was even stronger when both Smad2 and Smad7 were co-expressed with Smurf1. Under these conditions of transient transfection we could always observe the previously established down-regulation of Smad7 or Smad2 by the co-expressed Smurf1 (Fig. 1B). Dose-response experiments showed that, with higher levels of Smad7 co-expressed with Smurf1, higher degrees of Smad4 down-regulation could be observed (Fig. 1C).



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 1.
I-Smads and Smurf1 lead to down-regulation of Smad4 protein levels. A, diagrammatic representation of the four HECT domain E3 ligases studied in this report. Three characteristic domains of all four proteins are shown: the N-terminal lipid-binding C2 domain, the protein-binding WW domains that vary in number and position among the proteins, and the C-terminal catalytic HECT domain. Numbers indicate the N- and C-terminal amino acids of each protein. B–E, steady-state levels of transfected Smad4 in HEK-293T cells in the absence or presence of the indicated co-expressed proteins, detected by anti-FLAG immunoblotting. In C increasing amounts of Smad7 were transfected as shown (in nanograms of transfected plasmid DNA). In all panels arrows indicate the specific protein bands.

 



View larger version (47K):
[in this window]
[in a new window]
  		FIG. 2.
Down-regulation of endogenous Smad4 by Smurf1 and Smad7. A, steady-state levels of endogenous Smad4 in HaCaT keratinocytes after transient infection with adenoviruses expressing Smurf1 and Smad7 at multiplicity of infection 200 each. B, steady-state levels of endogenous Smad4 in HEK-293T cells infected with the indicated multiplicity of infection of control LacZ adenovirus, wild type Smad7 and Smurf1 adenoviruses. The levels of transfected FLAG-Smad7 and FLAG-Smurf1 are shown below, {beta}-tubulin levels serve as protein loading controls, and the lane marked as "MDA" represents a negative control for antibody specificity derived from cell extract of MDA-MB-468 carcinoma cells that lack endogenous Smad4. In all panels arrows indicate the specific protein bands.

 
We then asked whether stimulation of TGF-{beta} signaling by co-expression of the point-mutated, constitutively active TGF-{beta} type I receptor (activin receptor-like kinase-5(TD), ALK5(TD)) could influence the fate of Smad4. We could not measure any appreciable effect of ALK5(TD) co-expression on Smad4 levels either in the absence or presence of co-transfected Smurf1 (Fig. 1D).

We also examined whether Smad6, the second I-Smad of the TGF-{beta} superfamily pathways, could play similar roles as Smad7 with respect to the protein fate of Smad4. Indeed, although coexpression of Smad4 with Smad6 or Smad7 led to weak down-regulation of Smad4, we observed a dramatic down-regulation of Smad4 when Smurf1 was also expressed in the cells (Fig. 1E). Under these transfection conditions, the effect of Smad6 was stronger compared with the effect of Smad7, and both I-Smads were completely degraded when co-expressed with Smurf1.

To confirm that the above effects of Smad7 and Smurf1 on transfected Smad4 could apply to the endogenous protein, we constructed an adenoviral vector expressing FLAG-tagged Smurf1 and also made use of another adenovirus expressing FLAG-tagged Smad7 (43). Infection of human HaCaT keratinocytes with each adenoviral vector and immunoblotting for endogenous Smad4 revealed that the combination of Smurf1 together with Smad7 led to measurable down-regulation of Smad4, which correlated with an equivalent down-regulation of Smurf1, presumably due to its established proteasomal degradation (Fig. 2A). Infection with Smurf1 or Smad7 viruses alone, led to weak but detectable down-regulation of endogenous Smad4. The same result was obtained after infection of HEK-293T cells with different doses of each adenoviral vector and immunoblotting for endogenous Smad4 (Fig. 2B). The negative effect on endogenous Smad4 protein levels was particularly obvious after combination of high dose Smurf1 together with high dose Smad7 in HEK-293T cells.

Smurf2, WWP1, and NEDD4–2 Mediate Smad4 Degradation—We tested whether the second Smurf member, Smurf2, could mediate Smad4 down-regulation. Smurf2 had a similar negative effect on Smad4 protein levels, especially when coexpressed together with I-Smad proteins (Fig. 3A). Smurf2 showed a similar capacity to down-regulate Smad4 when it was co-expressed with the R-Smad, Smad2 (Fig. 3A). Whereas activated type I receptor ALK5(TD) showed no modulation of Smad4 down-regulation by I-Smads (Fig. 1D), when Smurf2 was co-expressed with Smad2, the down-regulation of Smad4 was dramatically enhanced by the activated receptor ALK5(TD) (Fig. 3B). We therefore conclude that both I-Smads, and also the R-Smad, Smad2, can mediate Smad4 down-regulation when co-expressed with Smurf family members in mammalian cells. The latter mechanism is significantly enhanced by sustained stimulation of the TGF-{beta} pathway.



View larger version (37K):
[in this window]
[in a new window]
  		FIG. 3.
Smurf2, WWP1, and NEDD4–2 cooperate with Smad7 for Smad4 down-regulation. A–C, steady-state levels of transfected Smad4 in HEK-293T cells in the absence or presence of the indicated co-expressed proteins, detected by anti-FLAG immunoblotting of total cell extracts. Expression of Smurf2 and Smad2 (B) and of Smad7 (C) is shown by anti-myc immunoblotting and expression of ALK-5(TD) (B) by anti-HA immunoblotting. In all panels arrows indicate the specific protein bands.

 
It has been recently demonstrated that Smad2 and I-Smads can interact not only with WW-HECT domain ligases of the Smurf family, but also with novel related proteins, named WWP1/Tiul1 and NEDD4–2 (11, 12, 37). These E3 ligases are larger in size compared with Smurfs and contain more WW domains than Smurfs (Fig. 1A). Functionally, however, they play very similar roles to those of Smurfs in regulating TGF-{beta} signaling. It was therefore tempting to ask whether WWP1 or NEDD4–2 could mediate Smad4 down-regulation in a similar manner to that of Smurf proteins. For this reason, we coexpressed WWP1 or NEDD4–2 together with Smad4 and in the absence or presence of the I-Smad, Smad7 (Fig. 3C). Smad4 was weakly down-regulated by WWP1 or NEDD4–2. In contrast, when co-expressed together with Smad7, both of these E3 ligases led to significant down-regulation of Smad4, which correlated with the already established down-regulation of Smad7 by the same E3 ligases. We therefore conclude that I-Smads (and possibly Smad2, which was not tested here) can mediate down-regulation of Smad4 not only by the action of Smurf proteins, but also by other WW-HECT domain E3 ligases such as WWP1 and NEDD4–2.

Smurf1 Leads to Poly-ubiquitination of Wild Type Smad4— Because Smurf proteins are known E3 ubiquitin ligases, we reasoned that the observed Smad4 down-regulation was due to its proteasomal degradation that was following poly-ubiquitination of the protein. We therefore examined the mode of Smad4 ubiquitination after co-expression of I-Smads and Smurfs (Fig. 4). To enhance the detection ability of ubiquitinated Smad4 species, we co-transfected the HEK-293T cells with HA-tagged ubiquitin. Ubiquitin alone gave rise to a strong mono-ubiquitinated Smad4 species, as we have previously described (25). However, co-expression of Smurf1 led to a distinct pattern of high molecular weight bands that correspond to poly- or multiubiquitinated Smad4 species (Fig. 4A). Because the present analysis cannot distinguish between these two alternative forms of ubiquitination, in the rest of this report we will refer to either of these terms as poly-ubiquitination. The presence of Smad7 led to the same pattern of poly-ubiquitinated species, except that the intensity of these protein bands was decreased. This must reflect the more efficient Smad4 degradation when Smad7 is co-expressed. Repeating this experiment by immunoprecipitating first for Smad4, instead of ubiquitin, gave the same pattern of results, except that continuous poly-ubiquitinated Smad4 smears could be detected (Fig. 4B). Co-expression of Smad7 enhanced the degree of poly-ubiquitination of Smad4, which was more evident when the cells were treated with a proteasomal inhibitor, MG-132, prior to their lysis. Transfection of a catalytically inactive mutant Smurf1(CS) in place of wild type Smurf1 resulted only in back-ground poly-ubiquitination, confirming that the observed protein smears were due to the action of the wild type Smurf1.

We also compared side by side the efficiency of Smad4 poly-ubiquitination by Smurf1 versus Smurf2 and the effects of Smad6 versus Smad7 (Fig. 4C). In several experiments we observed that Smurf2 led to Smad4 poly-ubiquitination much like Smurf1. However, this effect was more exacerbated when Smad6 was present together with Smurf2 compared with Smad7 (Fig. 4C). The combined experiments of Figs. 1 and 2 suggest that I-Smads mediate poly-ubiquitination of Smad4 by Smurf E3 ligases in mammalian cells.

Smad4 Forms Ternary Complexes with I-Smads and Smurf1—The above results strongly suggested the possibility that Smad4 could form protein complexes together with I-Smads and Smurf proteins. If such a model was true, I-Smads should mediate the association of Smad4 to Smurfs, because Smad4 cannot directly interact with Smurf proteins (35). Furthermore, because both Smad4 and I-Smads contain conserved C-terminal MH2 domains, which are well established self-interacting protein modules, it was formally possible that Smad4 and I-Smads interacted via their MH2 domains. We tested these possibilities using co-immunoprecipitation and pull-down assays (Figs. 5 and 6). First we examined pair-wise interactions between Smad4 and I-Smads. After co-expression of FLAG-tagged Smad4 and untagged Smad7 in HEK-293T cells we could detect the co-immunoprecipitation of the two proteins (Fig. 5A). We then mapped the domain in Smad7 required for interaction with Smad4 (Fig. 5B). We could reproducibly observe that a C-terminal fragment of Smad7 encompassing all of the MH2 domain and the adjacent linker was sufficient for interaction with Smad4. In contrast, the corresponding N-terminal fragment together with the linker never scored significantly in these co-immunoprecipitation experiments (Fig. 5B). We also tested the co-precipitation of Smad4 with Smad6 and could detect reproducibly complex formation between these two Smad proteins (Fig. 5C). This interaction was dramatically enhanced when a Smad6 construct expressing the conserved C-terminal MH2 domain of Smad6 was tested, whereas the corresponding N-terminal domain of Smad6 showed much weaker and almost background association with Smad4, similar to the results on Smad7 domain mutants. This finding agrees with the theoretical prediction that all Smad-Smad interactions are mediated via their conserved C-terminal MH2 domain.



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 4.
Smurfs lead to poly-ubiquitination of Smad4. A–C, ubiquitination assays in HEK-293T cells. After co-transfection of the indicated expression plasmids, protein extracts were immunoprecipitated (IP) with anti-HA antibody and the resolved proteins were immunoblotted (IB) with anti-myc antibody (panels A and C). Alternatively, protein extracts were immunoprecipitated with anti-myc antibody and the resolved proteins were immunoblotted with anti-HA antibody (B). Anti-myc and anti-FLAG immunoblots of the total cell lysates (TCL) are shown at the bottom of the panels. In B, cells were treated with vehicle (Me2SO) or with the proteasomal inhibitor MG-132 for 6 h prior to cell lysis. In all panels, poly-ubiquitination ladders are indicated with brackets or arrowheads, arrows indicate the specific protein bands, a gray arrow indicates the expected position of core Smad4 protein in A, the heavy chain of immunoglobulins is marked as "IgH," and arrows marked as "ns" indicate nonspecific protein bands.

 
Complementary pull-down assays using GST-Smad7 or GST as bait revealed efficient association of Smad7 with Smad4 (Fig. 5D). GST-Smad4 also showed significant association with Smad7, which was almost 20% of that seen with the positive control used, Smad2 (Fig. 5E, long exposure). The empty GST resulted in weak background as revealed in the immunoblot of Fig. 5E (long exposure), which was distinct from the specific Smad proteins under investigation. Interestingly, when both Smad2 and Smad7 were co-expressed in the cells, an almost stoichiometric amount of the two proteins associated with GST-Smad4, suggesting that the three Smad proteins formed a stronger complex compared with Smad4 and Smad7 alone. This finding is compatible with the crystallographic data that propose a trimeric structure of Smad oligomers (50). Although the existing crystallographic data are limited to the complexes of MH2 domains of Smad2 with Smad4, and of Smad3 with Smad4, the conservation of MH2 domain amino acid sequence and structure, and the above data, permit the hypothesis that Smad7 could replace one of the R-Smad subunits in such complexes and thus possibly lead to formation of Smad2·Smad7· Smad4 trimers.

We then examined the incorporation of Smurf1 into the Smad4·I-Smad complexes (Fig. 6). When Smurf1 was co-transfected in the cells together with Smad4 and Smad7, immunoprecipitation of Smad4 led to detection of both Smurf1 and Smad7 in the same protein complexes (Fig. 6A). In these assays, the catalytically inactive Smurf1(CS) mutant was utilized instead of the wild type Smurf1, to avoid protein degradation and be able to focus on the ability of these proteins to form complexes. Immunoprecipitation of Smad7 also led to detection of Smurf1 and Smad4 in the same complexes. From such experiments it was evident that the Smurf1·Smad7 complex was stronger, because these two proteins interact directly, whereas the Smad4·Smad7 or the Smad4·Smad7·Smurf1 complexes were significantly weaker but reproducible and relatively easy to detect. Immunoprecipitation of Smurf1 led to efficient detection of Smad7 in the complex, but the level of co-precipitating Smad4 was extremely low (Fig. 6A), again attesting to the notion that these complexes constitute a small and specialized fraction among the more abundant Smad7·Smurf1 complexes. Smurf1 could also be found in protein complexes together with Smad6 and Smad4 as revealed by co-precipitation assays (Fig. 6B). Smurf1 could not form complexes with Smad4 alone, but in the presence of Smad6 it was efficiently co-precipitated together with Smad4. These data support the model whereby I-Smads recruit Smurf1 E3 ligase to Smad4 based on the inter-Smad-specific interaction supported by the Smad MH2 domains.

Smad4, Smad7, and Smurf1 Co-localize Primarily in the Cytoplasm—It is well established that Smad4 is a shuttling protein that resides in both the nucleus and the cytoplasm, but at steady state, most of Smad4 seems to localize in the cytoplasm due to a strong nuclear export mechanism (reviewed in Refs. 51 and 52). Upon stimulation of cells with TGF-{beta}, Smad4 is known to rapidly translocate to the nucleus in complex with R-Smads. On the other hand, I-Smads are also shuttling, but at steady state, they reside primarily in the nucleus, and upon ligand stimulation they translocate to the cytoplasm together with Smurfs to bind to signaling receptors (3, 51). It is therefore possible that the observed Smad4·I-Smad·Smurf complexes could form in the nucleus, the cytoplasm, or both. To begin addressing the question of subcellular localization, we performed immunofluorescence experiments in transfected HEK-293T cells under the same conditions as those used for the biochemical experiments described above. To facilitate detection of the three proteins, we made use of FLAG-tagged Smad4 or Smad7, but we also used GFP-tagged versions of Smad4 (Fig. 7) and GFP-Smad7.2 Furthermore, because Smurf1 is known to be self-degraded and lead to rapid degradation of its substrates, we employed the catalytically inactive mutant Smurf1(CS) for easier visualization, but also the wild type Smurf1. When expressed alone, Smad4 localized primarily in the cytoplasm and weakly in the nucleus, as predicted (Fig. 7A). Smad7 localized primarily in the nucleus and weakly in the cytoplasm. Smurf1(CS) and Smurf1(WT) localized in both nuclear and cytoplasmic compartments, including peripheral cell protrusions and filopodia. We could not observe any clear-cut differences in the subcellular distribution of wild type and catalytically inactive forms of Smurf1. However, cells transfected with wild type Smurf1 exhibited a larger number of cell appendages and longer filopodia, which is compatible with the newly established role of Smurf1 as an E3 ligase of the RhoA small GTPase that regulates actin dynamics and cell protrusions (53).



View larger version (50K):
[in this window]
[in a new window]
  		FIG. 5.
Smad4 forms protein complexes with I-Smads. A–C, protein extracts from HEK-293T cells transfected with the indicated expression plasmids were immunoprecipitated (IP) with anti-FLAG and immunoblotted with anti-Smad7, anti-GFP or anti-myc antibodies. Anti-FLAG, anti-myc, anti-GFP, and anti-Smad7 immunoblots (IB) of the total cell lysate (TCL) are shown at the bottom. D and E, in vitro interaction between bacterial GST-Smad7, GST-Smad4, or GST proteins and the indicated FLAG-tagged proteins expressed in HEK-293T cells. The input FLAG-tagged protein and the proteins bound to the affinity beads are shown. In E, a lighter (top) and a heavier (bottom) exposure of the same immunoblot are shown. In all panels arrows indicate the specific protein bands, and asterisks indicate nonspecific bands.

 



View larger version (37K):
[in this window]
[in a new window]
  		FIG. 6.
Smad4 forms protein complexes with I-Smads and Smurf1. A and B, protein extracts from HEK-293T cells transfected with the indicated expression plasmids were immunoprecipitated (IP) with anti-FLAG or anti-Smad7 antibodies and immunoblotted with anti-Smad7, anti-myc, or anti-FLAG antibodies. Anti-FLAG, anti-myc, and anti-Smad7 immunoblots (IB) of the total cell lysate (TCL) are shown at the bottom. Arrows indicate the specific protein bands.

 
Upon co-expression of the three proteins, most cells expressing the different protein combinations exhibited co-localization primarily in their cytoplasm (Fig. 7B). However, nuclear colocalization of the three proteins cannot be firmly excluded, because all three individual proteins could localize in nuclei (Fig. 7A). Furthermore, when Smurf1(CS) or Smurf1(WT) were co-expressed, we could additionally observe strong co-localization in peripheral compartments close to the plasma membrane and in filopodia (Fig. 7B). In summary, the cellular analysis suggests that Smad4 can form complexes with Smad7 and Smurf1 primarily in the cytoplasm and can even localize in specialized sub-membranous compartments.

Mutants That Disrupt Formation of Complexes between Smad4 and Smad2 or Smad7 Do Not Support Smad4 Down-regulation by Smurf1—To establish the significance of protein complexes between Smad4 and Smad2 or Smad7 in mediating recruitment of Smurf1, we employed various deletion and point mutants of each protein (Fig. 8). Thus, we measured the ability of such mutants to mediate Smad4 degradation by Smurf1. Comparison between the two fragments of Smad7 (N versus C), which both contain the Smurf1-binding region in the linker, clearly showed that the C-terminal MH2 domain could elicit Smurf1-mediated down-regulation of Smad4 (Fig. 8A). The N-terminal domain of Smad7 was not capable of enhancing the weak effect of Smurf1 alone on Smad4 levels (Fig. 8A), suggesting that interaction with Smad4 via the conserved MH2 domain is necessary for Smad4 degradation.



View larger version (46K):
[in this window]
[in a new window]
  		FIG. 7.
Subcellular distribution of Smad4, Smurf1, and Smad7. A and B, immunofluorescence microscopy of transfected HEK-293T cells with the indicated plasmids (white lettering) under conditions identical to those of the previous figures. Immunodetection using anti-FLAG and anti-myc antibodies followed by anti-mouse-TRITC secondary antibody (red lettering) and GFP autofluorescence (green lettering) were coupled to nuclear staining with DAPI (blue lettering). In B, each set of three photomicrographs arranged vertically represents the same microscopy field photographed under different fluorescence channels, and the bottom photomicrographs (merge; yellow lettering) show the combination of the above two fluorescence patterns. Bars represent 10 µm.

 
In a reciprocal experiment, a point mutant (K507R) in the L3 loop of the Smad4 MH2 domain that cannot interact with Smad2 was used (25, 54). Smad4(K507R) failed to co-precipitate with the MH2 domain of Smad7 (Fig. 8B). This result strongly implicates the L3 loop of the MH2 domain in the Smad4-Smad7 interaction and suggests that the same conserved amino acids of the MH2 domain that are critical for R-Smad·Smad4 complex formation may be responsible for Smad4·I-Smad complex formation. Smad4(K507R) exhibited resistance to degradation by Smurf1, when compared with wild type Smad4 (Fig. 8C), again supporting the role of oligomerization with Smad7 in the Smad4 degradation process.

Finally, we tested a Smad2 point mutant that cannot be phosphorylated by the TGF-{beta} receptor (Smad2SA) and thus cannot undergo the critical conformational change of its MH2 domain that is required for oligomerization with Smad4 (50, 55). Under the conditions of this experiment, the Smad2SA mutant failed to form complexes with Smad4, especially after stimulation of the TGF-{beta} pathway using the constitutively active type I receptor ALK5(TD) (Fig. 8D). When Smad4 was co-expressed with the mutant Smad2SA and Smurf1, no detectable Smad4 degradation could be observed (Fig. 8E). Even after activation of the TGF-{beta} pathway via ALK5(TD), both Smad2SA and wild type Smad4 showed a significant degree of resistance to degradation. Thus, the ability of the Smad4 MH2 domain to interact with a properly folded MH2 domain in activated Smad2 or in Smad7 appears to be very critical for the observed down-regulation of Smad4 by Smurf1. All the above experiments support the model whereby I- or R-Smads act as adaptors between WW-HECT domain ligases and a novel substrate, Smad4.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The complexity of events that lead to ubiquitination and proteasomal degradation of components of the TGF-{beta}/Smad pathway necessitates careful evaluation of several complementary mechanistic models. Here we addressed the role of I-Smads as adaptors that mediate recruitment of E3 ligases of the Smurf family to the common mediator of all TGF-{beta} super-family pathways, Smad4. Thus the results of this study are relevant to the physiological regulation of all signaling pathways in the TGF-{beta} superfamily, to the extent that Smad4 mediates such responses. The present findings and those of previous investigations demonstrate that Smurf E3 ligases are able to ubiquitinate essentially all members of the Smad family, R-Smads, I-Smads, and the Co-Smad, Smad4. In addition, Smurfs ubiquitinate the activated TGF-{beta} type I receptor (35, 36). Thus, Smurfs play central roles in regulating turnover of all Smad proteins, and as a result, their effects on overall TGF-{beta} signaling must be finely tuned. In the present study, both Smurf1 and Smurf2 were capable of mediating Smad4 poly-ubiquitination and down-regulation (Figs. 1, 2, 3, 4). In addition, Smad2 and both I-Smads, Smad6 and Smad7, were found capable of mediating the negative effect of Smurfs on Smad4 turnover (Figs. 1, 2, 3). After stimulation of cells with TGF-{beta}, Smad4 forms complexes not only with Smad2 but also with Smad3. Smad2 is known to be ubiquitinated by Smurfs (9, 10), whereas Smad3 utilizes a distinct ubiquitination machinery, that of the Roc1·SCF ubiquitin ligase (13). Whether Smad3 can mediate ubiquitination of Smad4 remains to be determined. Our data on WWP1 and NEDD4–2 (Fig. 3C) support a general involvement of several WW-HECT domain E3 ligases in the down-regulation of Smad4. Accordingly, Smad4 seems to follow the fate of Smad2, I-Smads, and of the TGF-{beta} type I receptor, all of which interact and become ubiquitinated by the same group of WW-HECT domain ligases.



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 8.
Mutant forms of Smad2 and Smad7, which cannot form complexes with Smad4, do not support efficient down-regulation of Smad4 by Smurf1. A, C, and E, steady-state levels of transfected Smad4 in HEK-293T cells in the absence or presence of the indicated co-expressed proteins, detected by anti-FLAG immunoblotting of total cell extracts. B and D, protein extracts from HEK-293T cells transfected with the indicated expression plasmids were immunoprecipitated (IP) with anti-FLAG antibody and immunoblotted with anti-myc or anti-GFP antibodies. Anti-FLAG, anti-myc, and anti-GFP immunoblots (IB) of the total cell lysate (TCL) are shown at the bottom. In all panels arrows indicate the specific protein bands.

 
The Smad·Smurf protein complexes identified in this study could have been partially predicted theoretically based on the well established mechanisms of interaction between Smurf proteins and I-Smads or R-Smads and between Smad4 and R-Smads. However, no previous attempt has evaluated the possible formation of complexes between Smad4 and I-Smads. The findings of Figs. 5 and 6 demonstrate the existence of such complexes in mammalian cells and extend the paradigm of Smurf action as E3 ligases toward substrates that they do not contact directly, i.e. Smad4 or TGF-{beta} type I receptor, but rather contact via intermediate adaptor proteins, such as I- or R-Smads. The ability of I-Smads and Smad4 to associate is compatible with the fact that all these proteins contain highly conserved C-terminal MH2 domains, which upon crystallization tend to form trimers (50, 56). The experiments of Fig. 8 using point mutants of Smad4 or Smad2 corroborate the above statement and implicate the Smad4 MH2 domain L3 loop (54) and the established conformational change of the Smad2 MH2 domain that is induced by receptor phosphorylation (50, 55), in the formation of complexes with Smad4 that recruit Smurf1 and lead to degradation of Smad4. It is therefore tempting to speculate that such Smad heterotrimers could accommodate, in addition to R-Smads and Smad4, the I-Smads, Smad6 or Smad7. Within such trimers, WW-HECT domain E3 ligases bound to the PPXY motifs in the linker domains of R-Smads or I-Smads could then ubiquitinate any of the three different subunits of the trimer. The existence of such heterotrimers deserves future structural analysis. Alternatively, we can envision formation of I-Smad/Smad4 oligomers in the absence of an activated R-Smad.

Under all conditions tested, we observed efficient poly-ubiquitination of Smad4 by Smurf proteins (Fig. 4). This is in contrast to the efficient mono-ubiquitination of Smad4 established by our previous work (25). So far we have no evidence supporting a role for Smurf proteins on mono-ubiquitination of Smad4. Neither siRNA-mediated knock-down approaches nor expression of catalytically inactive forms of Smurf1 or Smurf2 have resulted in inhibition of Smad4 mono-ubiquitination. Thus, there must be additional E3 ligases that mediate Smad4 mono-ubiquitination, a modification that leads to Smad4 stabilization and enhanced TGF-{beta} signaling.

Smurfs seem to mediate turnover of wild type Smad4. Recently, the SCFSkp2 ubiquitin ligase complex was shown to ubiquitinate selectively point mutants of Smad4 (29) that others and we previously showed to be preferentially poly-ubiquitinated and proteasomally degraded (25, 28, 30–32). Our unpublished experiments2 have shown that Smurf1 or Smurf2 do not appreciably enhance the already high levels of poly-ubiquitination of such cancer-derived point mutants of Smad4. This is in agreement with the role of the SCFSkp2 ubiquitin ligase on Smad4 cancer mutant degradation and thus suggests that Smurfs may be responsible primarily for the turnover of Smad4 under normal conditions.

Smurf-mediated turnover of Smad4 most probably occurs at later stages post-TGF-{beta} signaling at which point Smurf gene expression is activated by TGF-{beta}; at this stage down-regulation of signaling as a means of pathway shut-off makes sense (2, 3, 57). Activation of TGF-{beta} signaling did not show appreciable effects of Smurf1 on Smad4 turnover (Fig. 1D). This probably reflects the fact that the primary physiological effect of the TGF-{beta} pathway is to induce Smurf gene expression and accumulation of Smurf protein above sub-critical levels. Once the Smurf levels are sufficient in the cell, the E3 ligase can make complexes with various components of the Smad family. In contrast, activation of the same pathway significantly enhances Smad4 down-regulation by the Smad2/Smurf combination (Figs. 3B and 8E), presumably because receptor activation enhances formation of both Smad2·Smad4 complexes and of Smad2·Smurf complexes. Under such conditions, the critical event triggered by TGF-{beta} signaling must be phosphorylation of the C-terminal di-serine motif of Smad2, leading to the characteristic conformational change of the MH2 domain.

The above model could suggest that Smurfs might form complexes with Smads in cellular compartments where their abundance is highest. Smurf1 was found to be localized primarily in the nucleus and in peripheral cell protrusions (Fig. 7), as previously reported (53, 58). It is tempting to suggest that Smad4 could first associate with I-Smads and Smurfs in the nucleus, presumably after translocation induced by TGF-{beta} signaling. However, at steady state, the majority of Smad4 colocalized with Smurf1 and Smad7 in the cytoplasm (Fig. 7B). In addition, the clear co-localization at cell protrusions, suggests that Smad4 may also form complexes with I-Smads and Smurfs at locations under the plasma membrane, possible sites enriched in signaling TGF-{beta} receptor complexes.

In conclusion, the present data expand the roles of Smurf family ubiquitin ligases to the Co-Smad that is ubiquitously utilized by all TGF-{beta} superfamily pathways and establish even stronger than previously appreciated the mechanisms by which Smurfs and their related WW-HECT domain proteins regulate TGF-{beta} superfamily signaling components.


   FOOTNOTES
 
* This work was supported by grants from the Human Frontier Science Program (to A. Moustakas) and the Swedish Foundation for International Cooperation in Research and High Education (to C.-H. H.). 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. Back

|| To whom correspondence should be addressed. Tel.: 46-18-160-414; Fax: 46-18-160-420; E-mail: aris.moustakas{at}licr.uu.se.

1 The abbreviations used are: TGF-{beta}, transforming growth factor-{beta}; ALK, activin-like receptor kinase; DAPI, 4',6'-diamidino-2-phenylindole; HEK, human embryonic kidney; MH, Mad homology; Smurf, Smad ubiquitination regulatory factor; TCL, total cell lysate; TRITC, tetramethylrhodamine isothiocyanate; I-Smad, inhibitory Smad; R-Smad, receptor-regulated Smad; Co-Smad, common mediator Smad; E3, ubiquitin-protein isopeptide ligase; HA, hemagglutinin; GST, glutathione S-transferase; GFP, green fluorescent protein; Roc1, regulator of cullin 1 protein; SCF, Skp1·cullin·F-box protein complex. Back

2 A. Morén, T. Imamura, K. Miyazono, C.-H. Heldin, and A. Moustakas, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank R. Derynck, N. Fusenig, E. Grönroos, and S. Souchelnytskyi for reagents and M. Kowanetz, P. Lönn, and K. Pardali from our laboratory for useful discussions and sharing of unpublished data.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Derynck, R., and Zhang, Y. E. (2003) Nature 425, 577–584[CrossRef][Medline] [Order article via Infotrieve]
  2. Miyazono, K., Suzuki, H., and Imamura, T. (2003) Cancer Sci. 94, 230–234[CrossRef][Medline] [Order article via Infotrieve]
  3. Shi, Y., and Massagué, J. (2003) Cell 113, 685–700[CrossRef][Medline] [Order article via Infotrieve]
  4. Li, L., Xin, H., Xu, X., Huang, M., Zhang, X., Chen, Y., Zhang, S., Fu, X. Y., and Chang, Z. (2004) Mol. Cell. Biol. 24, 856–864[Abstract/Free Full Text]
  5. Shi, W., Chen, H., Sun, J., Chen, C., Zhao, J., Wang, Y. L., Anderson, K. D., and Warburton, D. (2004) Am. J. Physiol. 286, L293–L300[CrossRef]
  6. Ying, S. X., Hussain, Z. J., and Zhang, Y. E. (2003) J. Biol. Chem. 278, 39029–39036[Abstract/Free Full Text]
  7. Zhao, M., Qiao, M., Harris, S. E., Oyajobi, B. O., Mundy, G. R., and Chen, D. (2004) J. Biol. Chem. 279, 12854–12859[Abstract/Free Full Text]
  8. Zhu, H., Kavsak, P., Abdollah, S., Wrana, J. L., and Thomsen, G. H. (1999) Nature 400, 687–693[CrossRef][Medline] [Order article via Infotrieve]
  9. Lin, X., Liang, M., and Feng, X.-H. (2000) J. Biol. Chem. 275, 36818–36822[Abstract/Free Full Text]
  10. Zhang, Y., Chang, C., Gehling, D. J., Hemmati-Brivanlou, A., and Derynck, R. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 974–979[Abstract/Free Full Text]
  11. Seo, S. R., Lallemand, F., Ferrand, N., Pessah, M., L'Hoste, S., Camonis, J., and Atfi, A. (2004) EMBO J. 23, 3780–3792[CrossRef][Medline] [Order article via Infotrieve]
  12. Kuratomi, G., Komuro, A., Goto, K., Shinozaki, M., Miyazawa, K., Miyazono, K., and Imamura, T. (2005) Biochem. J. 386, 461–470[CrossRef][Medline] [Order article via Infotrieve]
  13. Fukuchi, M., Imamura, T., Chiba, T., Ebisawa, T., Kawabata, M., Tanaka, K., and Miyazono, K. (2001) Mol. Biol. Cell 12, 1431–1443[Abstract/Free Full Text]
  14. Liang, Y. Y., Lin, X., Liang, M., Brunicardi, F. C., ten Dijke, P., Chen, Z., Choi, K. W., and Feng, X.-H. (2003) J. Biol. Chem. 278, 26307–26310[Abstract/Free Full Text]
  15. Podos, S. D., Hanson, K. K., Wang, Y. C., and Ferguson, E. L. (2001) Dev. Cell 1, 567–578[CrossRef][Medline] [Order article via Infotrieve]
  16. Bai, Y., Yang, C., Hu, K., Elly, C., and Liu, Y.-C. (2004) Mol. Cell 15, 825–831[CrossRef][Medline] [Order article via Infotrieve]
  17. Wan, M., Cao, X., Wu, Y., Bai, S., Wu, L., Shi, X., and Wang, N. (2002) EMBO Rep. 3, 171–176[CrossRef][Medline] [Order article via Infotrieve]
  18. Wan, M., Tang, Y., Tytler, E. M., Lu, C., Jin, B., Vickers, S. M., Yang, L., Shi, X., and Cao, X. (2004) J. Biol. Chem. 279, 14484–14487[Abstract/Free Full Text]
  19. Lee, P. S., Chang, C., Liu, D., and Derynck, R. (2003) J. Biol. Chem. 278, 27853–27863[Abstract/Free Full Text]
  20. Liang, M., Melchior, F., Feng, X.-H., and Lin, X. (2004) J. Biol. Chem. 279, 22857–22865[Abstract/Free Full Text]
  21. Lin, X., Liang, M., Liang, Y. Y., Brunicardi, F. C., and Feng, X.-H. (2003) J. Biol. Chem. 278, 31043–31048[Abstract/Free Full Text]
  22. Lin, X., Liang, M., Liang, Y. Y., Brunicardi, F. C., Melchior, F., and Feng, X.-H. (2003) J. Biol. Chem. 278, 18714–18719[Abstract/Free Full Text]
  23. Long, J., Matsuura, I., He, D., Wang, G., Shuai, K., and Liu, F. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 9791–9796[Abstract/Free Full Text]
  24. Long, J., Wang, G., He, D., and Liu, F. (2004) Biochem. J. 379, 23–29[CrossRef][Medline] [Order article via Infotrieve]
  25. Morén, A., Hellman, U., Inada, Y., Imamura, T., Heldin, C.-H., and Moustakas, A. (2003) J. Biol. Chem. 278, 33571–33582[Abstract/Free Full Text]
  26. Ohshima, T., and Shimotohno, K. (2003) J. Biol. Chem. 278, 50833–50842[Abstract/Free Full Text]
  27. McCabe, B. D., Hom, S., Aberle, H., Fetter, R. D., Marques, G., Haerry, T. E., Wan, H., O'Connor, M. B., Goodman, C. S., and Haghighi, A. P. (2004) Neuron 41, 891–905[CrossRef][Medline] [Order article via Infotrieve]
  28. De Bosscher, K., Hill, C. S., and Nicolas, F. J. (2004) Biochem. J. 379, 209–216[CrossRef][Medline] [Order article via Infotrieve]
  29. Liang, M., Liang, Y. Y., Wrighton, K., Ungermannova, D., Wang, X. P., Brunicardi, F. C., Liu, X., Feng, X.-H., and Lin, X. (2004) Mol. Cell. Biol. 24, 7524–7537[Abstract/Free Full Text]
  30. Maurice, D., Pierreux, C. E., Howell, M., Wilentz, R. E., Owen, M. J., and Hill, C. S. (2001) J. Biol. Chem. 276, 43175–43181[Abstract/Free Full Text]
  31. Morén, A., Itoh, S., Moustakas, A., ten Dijke, P., and Heldin, C.-H. (2000) Oncogene 19, 4396–4404[CrossRef][Medline] [Order article via Infotrieve]
  32. Xu, J., and Attisano, L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4820–4825[Abstract/Free Full Text]
  33. Moustakas, A., Souchelnytskyi, S., and Heldin, C.-H. (2001) J. Cell Sci. 114, 4359–4369[Medline] [Order article via Infotrieve]
  34. Di Guglielmo, G. M., Le Roy, C., Goodfellow, A. F., and Wrana, J. L. (2003) Nat. Cell Biol. 5, 410–421[CrossRef][Medline] [Order article via Infotrieve]
  35. Ebisawa, T., Fukuchi, M., Murakami, G., Chiba, T., Tanaka, K., Imamura, T., and Miyazono, K. (2001) J. Biol. Chem. 276, 12477–12480[Abstract/Free Full Text]
  36. Kavsak, P., Rasmussen, R. K., Causing, C. G., Bonni, S., Zhu, H., Thomsen, G. H., and Wrana, J. L. (2000) Mol. Cell 6, 1365–1375[CrossRef][Medline] [Order article via Infotrieve]
  37. Komuro, A., Imamura, T., Saitoh, M., Yoshida, Y., Yamori, T., Miyazono, K., and Miyazawa, K. (2004) Oncogene 23, 6914–6923[CrossRef][Medline] [Order article via Infotrieve]
  38. Zhang, L., Zhou, H., Su, Y., Sun, Z., Zhang, H., Zhang, Y., Ning, Y., Chen, Y. G., and Meng, A. (2004) Science 306, 114–117[Abstract/Free Full Text]
  39. Murakami, G., Watabe, T., Takaoka, K., Miyazono, K., and Imamura, T. (2003) Mol. Biol. Cell 14, 2809–2817[Abstract/Free Full Text]
  40. Grönroos, E., Hellman, U., Heldin, C.-H., and Ericsson, J. (2002) Mol. Cell 10, 483–493[CrossRef][Medline] [Order article via Infotrieve]
  41. Kim, B.-C., Lee, H.-J., Park, S. H., Lee, S. R., Karpova, T. S., McNally, J. G., Felici, A., Lee, D. K., and Kim, S.-J. (2004) Mol. Cell. Biol. 24, 2251–2262[Abstract/Free Full Text]
  42. Koinuma, D., Shinozaki, M., Komuro, A., Goto, K., Saitoh, M., Hanyu, A., Ebina, M., Nukiwa, T., Miyazawa, K., Imamura, T., and Miyazono, K. (2003) EMBO J. 22, 6458–6470[CrossRef][Medline] [Order article via Infotrieve]
  43. Kurisaki, K., Kurisaki, A., Valcourt, U., Terentiev, A. A., Pardali, K., ten Dijke, P., Heldin, C.-H., Ericsson, J., and Moustakas, A. (2003) Mol. Cell. Biol. 23, 4494–4510[Abstract/Free Full Text]
  44. Pardali, K., Kurisaki, A., Morén, A., ten Dijke, P., Kardassis, D., and Moustakas, A. (2000) J. Biol. Chem. 275, 29244–29256[Abstract/Free Full Text]
  45. Imamura, T., Takase, M., Nishihara, A., Oeda, E., Hanai, J., Kawabata, M., and Miyazono, K. (1997) Nature 389, 622–626[CrossRef][Medline] [Order article via Infotrieve]
  46. Nakao, A., Afrakhte, M., Morén, A., Nakayama, T., Christian, J. L., Heuchel, R., Itoh, S., Kawabata, M., Heldin, N. E., Heldin, C.-H., and ten Dijke, P. (1997) Nature 389, 631–635[CrossRef][Medline] [Order article via Infotrieve]
  47. Souchelnytskyi, S., Tamaki, K., Engström, U., Wernstedt, C., ten Dijke, P., and Heldin, C.-H. (1997) J. Biol. Chem. 272, 28107–28115[Abstract/Free Full Text]
  48. Brodin, G., ten Dijke, P., Funa, K., Heldin, C.-H., and Landström, M. (1999) Cancer Res. 59, 2731–2738[Abstract/Free Full Text]
  49. Kowanetz, M., Valcourt, U., Bergström, R., Heldin, C.-H., and Moustakas, A. (2004) Mol. Cell. Biol. 24, 4241–4254[Abstract/Free Full Text]
  50. Chacko, B. M., Qin, B. Y., Tiwari, A., Shi, G., Lam, S., Hayward, L. J., De Caestecker, M., and Lin, K. (2004) Mol. Cell 15, 813–823[CrossRef][Medline] [Order article via Infotrieve]
  51. Reguly, T., and Wrana, J. L. (2003) Trends Cell Biol. 13, 216–220[CrossRef][Medline] [Order article via Infotrieve]
  52. ten Dijke, P., and Hill, C. S. (2004) Trends Biochem. Sci. 29, 265–273[CrossRef][Medline] [Order article via Infotrieve]
  53. Wang, H. R., Zhang, Y., Ozdamar, B., Ogunjimi, A. A., Alexandrova, E., Thomsen, G. H., and Wrana, J. L. (2003) Science 302, 1775–1779[Abstract/Free Full Text]
  54. Chacko, B. M., Qin, B., Correia, J. J., Lam, S. S., de Caestecker, M. P., and Lin, K. (2001) Nat. Struct. Biol. 8, 248–253[CrossRef][Medline] [Order article via Infotrieve]
  55. Wu, J. W., Hu, M., Chai, J., Seoane, J., Huse, M., Li, C., Rigotti, D. J., Kyin, S., Muir, T. W., Fairman, R., Massagué, J., and Shi, Y. (2001) Mol. Cell 8, 1277–1289[CrossRef][Medline] [Order article via Infotrieve]
  56. Shi, Y., Hata, A., Lo, R. S., Massagué, J., and Pavletich, N. P. (1997) Nature 388, 87–93[CrossRef][Medline] [Order article via Infotrieve]
  57. Siegel, P. M., and Massagué, J. (2003) Nat. Rev. Cancer 3, 807–820[CrossRef][Medline] [Order article via Infotrieve]
  58. Tajima, Y., Goto, K., Yoshida, M., Shinomiya, K., Sekimoto, T., Yoneda, Y., Miyazono, K., and Imamura, T. (2003) J. Biol. Chem. 278, 10716–10721[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol. Biol. CellHome page
T. Kalkan, Y. Iwasaki, C. Y. Park, and G. H. Thomsen
Tumor Necrosis Factor-Receptor-associated Factor-4 Is a Positive Regulator of Transforming Growth Factor-{beta} Signaling That Affects Neural Crest Formation
Mol. Biol. Cell, July 15, 2009; 20(14): 3436 - 3450.
[Abstract] [Full Text] [PDF]

Home page
 Anticancer ResHome page
P. B. AZMI and A. K. SETH
The RING Finger Protein11 Binds to Smad4 and Enhances Smad4-dependant TGF-{beta} Signalling
Anticancer Res, June 1, 2009; 29(6): 2253 - 2263.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
S. W. Eastman, M. Yassaee, and P. D. Bieniasz
A role for ubiquitin ligases and Spartin/SPG20 in lipid droplet turnover
J. Cell Biol., March 23, 2009; 184(6): 881 - 894.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Carra, J. F. Brunsting, H. Lambert, J. Landry, and H. H. Kampinga
HspB8 Participates in Protein Quality Control by a Non-chaperone-like Mechanism That Requires eIF2{alpha} Phosphorylation
J. Biol. Chem., February 27, 2009; 284(9): 5523 - 5532.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
S.-M. Feng, R. S. Muraoka-Cook, D. Hunter, M. A. Sandahl, L. S. Caskey, K. Miyazawa, A. Atfi, and H. S. Earp III
The E3 Ubiquitin Ligase WWP1 Selectively Targets HER4 and Its Proteolytically Derived Signaling Isoforms for Degradation
Mol. Cell. Biol., February 1, 2009; 29(3): 892 - 906.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
E. C. Osmundson, D. Ray, F. E. Moore, Q. Gao, G. H. Thomsen, and H. Kiyokawa
The HECT E3 ligase Smurf2 is required for Mad2-dependent spindle assembly checkpoint
J. Cell Biol., October 20, 2008; 183(2): 267 - 277.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
M. Kowanetz, P. Lonn, M. Vanlandewijck, K. Kowanetz, C.-H. Heldin, and A. Moustakas
TGF{beta} induces SIK to negatively regulate type I receptor kinase signaling
J. Cell Biol., August 26, 2008; 182(4): 655 - 662.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
E. Glasgow and L. Mishra
Transforming growth factor-{beta} signaling and ubiquitinators in cancer
Endocr. Relat. Cancer, March 1, 2008; 15(1): 59 - 72.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
L. Levy, M. Howell, D. Das, S. Harkin, V. Episkopou, and C. S. Hill
Arkadia Activates Smad3/Smad4-Dependent Transcription by Triggering Signal-Induced SnoN Degradation
Mol. Cell. Biol., September 1, 2007; 27(17): 6068 - 6083.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
C. Chen, A. K. Seth, and A. E. Aplin
Genetic and Expression Aberrations of E3 Ubiquitin Ligases in Human Breast Cancer
Mol. Cancer Res., October 1, 2006; 4(10): 695 - 707.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
M. C Fleisch, C. A Maxwell, and M.-H. Barcellos-Hoff
The pleiotropic roles of transforming growth factor beta in homeostasis and carcinogenesis of endocrine organs.
Endocr. Relat. Cancer, June 1, 2006; 13(2): 379 - 400.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. Chen, X. Sun, P. Guo, X.-Y. Dong, P. Sethi, X. Cheng, J. Zhou, J. Ling, J. W. Simons, J. B. Lingrel, et al.
Human Kruppel-like Factor 5 Is a Target of the E3 Ubiquitin Ligase WWP1 for Proteolysis in Epithelial Cells
J. Biol. Chem., December 16, 2005; 280(50): 41553 - 41561.
[Abstract] [Full Text] [PDF]

Home page
 Genes Dev.Home page
J. Massague, J. Seoane, and D. Wotton
Smad transcription factors
Genes & Dev., December 1, 2005; 19(23): 2783 - 2810.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/23/22115    most recent
M414027200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Morén, A.
Right arrow Articles by Moustakas, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Morén, A.
Right arrow Articles by Moustakas, A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement