ACCELERATED PUBLICATION
Smurf1 Interacts with Transforming Growth Factor-beta Type I Receptor through Smad7 and Induces Receptor Degradation*

Takanori EbisawaDagger §, Minoru FukuchiDagger §, Gyo MurakamiDagger , Tomoki Chiba, Keiji Tanaka, Takeshi ImamuraDagger , and Kohei MiyazonoDagger ||**

From the Dagger  Department of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research, and Research for the Future Program, the Japan Society for the Promotion of Science, 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 170-8455,  The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, and || Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Received for publication, January 8, 2001, and in revised form, February 2, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 (Tbeta R-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 Tbeta R-I via Smad7, with subsequent enhancement of turnover of Tbeta R-I and Smad7. These results thus reveal a novel function of Smad7, i.e. induction of degradation of Tbeta R-I through recruitment of an E3 ligase to the receptor.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Members of the transforming growth factor-beta (TGF-beta )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 intracellular signals (2). Members of the TGF-beta 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) (3-5).

R-Smads and Co-Smads positively regulate signaling by the TGF-beta superfamily (3-5). 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-beta /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-beta 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-beta and BMPs, I-Smads inhibit TGF-beta 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-beta signaling pathways, R-Smads, e.g. Smad2 and Smad1/5, have recently been shown to be degraded by the ubiquitin-proteasome pathway. Smad2 activated by TGF-beta 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-beta superfamily signaling. Inhibitory Smad7 associates with Smurf1 in the nucleus and is exported to the cytoplasm. Smad7 thus recruits Smurf1 to Tbeta R-I, resulting in the degradation and rapid turnover of the Tbeta R-I protein.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 µM MG132 (Peptide Institute) or 10 µM 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-beta 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-FLAG or rabbit anti-Myc antibody followed by incubation with FITC-labeled 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 [35S]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.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 efficiently 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).


View larger version (48K):
[in this window]
[in a new window]
 
Fig. 1.   Smad6 and Smad7 interact with Smurf1 through the PY motif. A, amino acid sequence alignment of PY motif of Smads 1 through 8. B, interaction of I-Smads with Smurf1. Binding of a Smurf1 mutant, Smurf1(C710A) (Smurf1(CA)), to different Smads was analyzed in vivo. 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 Smad7Delta PY. The binding between Smurf1(WT) and Smad7(WT) was slightly facilitated by Tbeta 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 Tbeta R-II·Tbeta R-I complex. COS7 cells were transfected or not with FLAG-tagged Smurf1(CA) and HA-tagged Tbeta R-I and Tbeta R-II in the presence or absence of FLAG- or 6Myc-tagged Smad7. Cells were affinity-labeled with 125I-TGF-beta 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 Tbeta R-II and Tbeta R-I were similar in each lane (data not shown).

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-beta type I receptor, Tbeta 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 (Smad7Delta PY) did not bind to Smurf1.

Smurf1 Interacts with Tbeta R-I via Smad7-- Smad7 interacts with Tbeta R-I activated by Tbeta R-II, thereby competing with Smad2 and Smad3 for inhibition of TGF-beta signaling. We therefore examined in an affinity cross-linking assay whether Smad7 acts as an adapter molecule that links Tbeta R-I to the ubiquitin-proteasome pathway. Although Smurf1 alone did not efficiently bind to Tbeta R-I in transfected COS7 cells, Smad7 dramatically enhanced the interaction between Smurf1 and the Tbeta R-I·Tbeta R-II complex (Fig. 1D, lanes 4 and 5). Moreover, Smurf1 failed to interact with the receptor complex in the presence of Smad7Delta PY (Fig. 1D, lane 6). These results indicate that Smurf1 is recruited to Tbeta R-I through Smad7.

Smad7 Is Translocated to the Cytoplasm by Smurf1-- We next examined the effect of Smurf1 on the subcellular localization of Smad7. In the absence of Smurf1, both Smad7(WT) and Smad7Delta 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). Smad7Delta PY failed to accumulate in the cytoplasm even in the presence of Smurf1, although there is a little leakage of Smad7Delta 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 Smad7·Smurf1 complex (Fig. 2B).


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2.   Cytoplasmic translocation of Smad7 by Smurf1. A, translocation of Smad7 from the nucleus to the cytoplasm by Smurf1. Subcellular localization of Smad7 in the presence or absence of Smurf1 was analyzed. Anti-Myc staining for Smad7(WT) or Smad7Delta PY (green) and nuclear staining by 4,6-diamidino-2-phenylindole (PI; red) were performed in transfected COS7 cells. B, proteasomal inhibitors MG132 and lactacystin facilitated the cytoplasmic staining of Smad7. Similar findings were obtained using Smurf1(C710A) in the absence of proteasomal inhibitors.

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 Tbeta 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 Smad7Delta PY or Smurf1(C710A) was used (Fig. 3B). We also tested the effect of Smad7 on Tbeta R-I ubiquitination by Smurf1 in 293T cells. Although Smurf1 alone ubiquitinated Tbeta R-I weakly, Smad7 enhanced the receptor ubiquitination by Smurf1 (Fig. 3C).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 3.   Ubiquitination of Smad7 and Tbeta R-I by Smurf1 in vivo. COS7 cells (A and B) and 293T cells (C) were transfected with the indicated plasmids and treated with 50 µM MG132 for 4 h before cell lysis. Lysates from cells were subjected to anti-FLAG immunoprecipitation followed by anti-HA immunoblotting. Polyubiquitination species of Smad7 (A and B; [HA-Ub]n-FLAG-Smad7) and those of Tbeta R-I (C; [HA-Ub]n-FLAG-Tbeta R-I(TD)) are indicated in the top panel.

Smurf1 Induces Degradation of Smad7 and Tbeta R-I-- To investigate whether Smurf1 regulates degradation of Smad7 and Tbeta 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 Tbeta R-I (Fig. 4C). Our results thus demonstrate that Smad7 accelerates turnover of the Tbeta R-I protein by recruitment of an E3 ubiquitin ligase, Smurf1.


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 4.   Smurf1 induces rapid turnover of Smad7 and Tbeta R-I and inhibits transcriptional activity induced by Tbeta R-I. A and B, degradation of Smad7 is enhanced by Smurf1(WT) (A) but not by Smurf1(C710A) (B). In panel B, turnover of Smurf1(WT) or Smurf1(CA) is also shown. COS7 cells were transfected with FLAG-tagged Smad7 with or without FLAG-tagged Smurf1(WT) or Smurf1(CA). Cell lysates were immunoprecipitated by FLAG antibody and analyzed by SDS-PAGE. C, degradation of Tbeta R-I is enhanced in the presence of both Smad7 and Smurf1. COS7 cells were transfected with HA-tagged Tbeta R-I with or without Smad7 and Smurf1. Cell lysates were immunoprecipitated by HA antibody and analyzed by SDS-PAGE. D, Smad7Delta PY is less potent than Smad7(WT) in inhibiting TGF-beta signaling. Effects of Smad7(WT) and Smad7Delta PY on the transcriptional activity of constitutively active Tbeta R-I (Tbeta R-I(TD)) were examined. R mutant Mv1Lu cells that lack functional Tbeta R-I were co-transfected with p3TP-lux, together with various combinations of Tbeta R-I(TD) and Smad7 cDNAs. E, Smurf1 enhances inhibitory activity of Smad7. R mutant Mv1Lu cells were co-transfected with p3TP-lux, together with various combinations of Tbeta R-I(TD), Smad7, Smurf1(WT), and Smurf1(CA) cDNAs.

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 Smad7Delta PY with that of Smad7(WT) using a TGF-beta -responsible promoter-reporter construct, p3TP-lux (Fig. 4D). Smad7Delta 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-beta 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-beta Receptors for Degradation via I-Smads-- I-Smads have been shown to regulate TGF-beta 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. 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-beta superfamily signaling by I-Smads. The present findings also suggest that E3 ligases of the Smurf family regulate TGF-beta 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-beta 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-beta 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.

    ACKNOWLEDGEMENT

We thank G. H. Thomsen for Smurf1 cDNA.

    FOOTNOTES

* 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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Contributed equally to this work.

** To whom correspondence should be addressed: Dept. of Biochemistry, The Cancer Inst. of the Japanese Foundation for Cancer Research, 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 170-8455, Japan. Tel.: 81-3-5394-3866; Fax: 81-3-3918-0342; E-mail: miyazono-ind@ umin.ac.jp.

Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.C100008200

    ABBREVIATIONS

The abbreviations used are: TGF-beta , transforming growth factor-beta ; R-Smad(s), receptor-regulated Smad(s); Co-Smad(s), common-partner Smad(s); I-Smad(s), inhibitory Smad(s); BMP(s), bone morphogenetic protein(s); PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; RITC, rhodamine isothiocyanate; HA, hemagglutinin; WT, wild type.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Roberts, A. B., and Sporn, M. B. (1990) in Pepide Growth Factors and Their Receptors, Part I (Sporn, M. B. , and Roberts, A. B., eds) , pp. 419-472, Springer-Verlag, Heidelberg
2. Massagué, J. (1998) Annu. Rev. Biochem. 67, 753-791[CrossRef][Medline] [Order article via Infotrieve]
3. Heldin, C.-H., Miyazono, K., and ten Dijke, P. (1997) Nature 390, 465-471[CrossRef][Medline] [Order article via Infotrieve]
4. Derynck, R., Zhang, Y., and Feng, X.-H. (1998) Cell 95, 737-740[Medline] [Order article via Infotrieve]
5. Attisano, L., and Wrana, J. L. (2000) Curr. Opin. Cell Biol. 12, 235-243[CrossRef][Medline] [Order article via Infotrieve]
6. 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]
7. Hayashi, H., Abdollah, S., Qiu, Y., Cai, J., Xu, Y. Y., Grinnell, B. W., Richardson, M. A., Topper, J. N., Gimbrone, M. A., Jr., Wrana, J. L., and Falb, D. (1997) Cell 89, 1165-1173[Medline] [Order article via Infotrieve]
8. Nakao, A., Afrakhte, M., Moren, 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]
9. Hata, A., Lagna, G., Massagué, J., and Hemmati-Brivanlou, A. (1998) Genes Dev. 12, 186-197[Abstract/Free Full Text]
10. Bai, S., Shi, X., Yang, X., and Cao, X. (2000) J. Biol. Chem. 275, 8267-8270[Abstract/Free Full Text]
11. Miyazono, K. (2000) J. Cell Sci. 113, 1101-1109[Abstract/Free Full Text]
12. Hershko, A., and Ciechanover, A. (1998) Annu. Rev. Biochem. 67, 425-479[CrossRef][Medline] [Order article via Infotrieve]
13. Lo, R. S., and Massagué, J. (1999) Nat. Cell Biol. 1, 472-478[CrossRef][Medline] [Order article via Infotrieve]
14. Zhu, H., Kavsak, P., Abdollah, S., Wrana, J. L., and Thomsen, G. H. (1999) Nature 400, 687-693[CrossRef][Medline] [Order article via Infotrieve]
15. Ebisawa, T., Tada, K., Kitajima, I., Tojo, K., Sampath, T. K., Kawabata, M., Miyazono, K., and Imamura, T. (1999) J. Cell Sci. 112, 3519-3527[Abstract/Free Full Text]
16. Boyd, S. D., Tsai, K. Y., and Jacks, T. (2000) Nat. Cell Biol. 2, 563-568[CrossRef][Medline] [Order article via Infotrieve]
17. Geyer, R. K., Yu, Z. K., and Maki, C. G. (2000) Nat. Cell Biol. 2, 569-573[CrossRef][Medline] [Order article via Infotrieve]
18. Itoh, S., Landstrom, M., Hermansson, A., Itoh, F., Heldin, C.-H., Heldin, N.-E., and ten Dijke, P. (1998) J. Biol. Chem. 273, 29195-29201[Abstract/Free Full Text]
19. Lin, X., Liang, M., and Feng, X.-H. (2000) J. Biol. Chem. 275, 36818-36822[Abstract/Free Full Text]
20. 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[Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.