Smurf1 Regulates the Inhibitory Activity of Smad7 by Targeting Smad7 to the Plasma Membrane
2002; Elsevier BV; Volume: 277; Issue: 42 Linguagem: Inglês
10.1074/jbc.m201901200
ISSN1083-351X
AutoresChie Suzuki, Gyo Murakami, Minoru Fukuchi, Tomomasa Shimanuki, Yuko Shikauchi, Takeshi Imamura, Kohei Miyazono,
Tópico(s)Renal and related cancers
ResumoSmad ubiquitin regulatory factor 1 (Smurf1), a HECT-type E3 ubiquitin ligase, interacts with inhibitory Smad7 and induces cytoplasmic localization of Smad7. Smurf1 then associates with transforming growth factor-β type I receptor (TβR-I) and enhances the turnover of this receptor. However, the mechanisms of the nuclear export and plasma membrane localization of the Smurf1·Smad7 complex have not been elucidated. We show here that Smurf1 targets Smad7 to the plasma membrane through its N-terminal conserved 2 (C2) domain. Both wild-type Smurf1 (Smurf1(WT)) and Smurf1 lacking the C2 domain (Smurf1(ΔC2)) bound to Smad7 and translocated nuclear Smad7 to the cytoplasm. However, unlike Smurf1(WT), Smurf1(ΔC2) did not move to the plasma membrane and failed to recruit Smad7 to the cell surface TβR-II·TβR-I complex. Moreover, although Smurf1(ΔC2) induced ubiquitination of Smad7, it failed to induce the ubiquitination and degradation of TβR-I and did not enhance the inhibitory activity of Smad7. Thus, these results suggest that the plasma membrane localization of Smad7 by Smurf1 requires the C2 domain of Smurf1 and is essential for the inhibitory effect of Smad7 in the transforming growth factor-β signaling pathway. Smad ubiquitin regulatory factor 1 (Smurf1), a HECT-type E3 ubiquitin ligase, interacts with inhibitory Smad7 and induces cytoplasmic localization of Smad7. Smurf1 then associates with transforming growth factor-β type I receptor (TβR-I) and enhances the turnover of this receptor. However, the mechanisms of the nuclear export and plasma membrane localization of the Smurf1·Smad7 complex have not been elucidated. We show here that Smurf1 targets Smad7 to the plasma membrane through its N-terminal conserved 2 (C2) domain. Both wild-type Smurf1 (Smurf1(WT)) and Smurf1 lacking the C2 domain (Smurf1(ΔC2)) bound to Smad7 and translocated nuclear Smad7 to the cytoplasm. However, unlike Smurf1(WT), Smurf1(ΔC2) did not move to the plasma membrane and failed to recruit Smad7 to the cell surface TβR-II·TβR-I complex. Moreover, although Smurf1(ΔC2) induced ubiquitination of Smad7, it failed to induce the ubiquitination and degradation of TβR-I and did not enhance the inhibitory activity of Smad7. Thus, these results suggest that the plasma membrane localization of Smad7 by Smurf1 requires the C2 domain of Smurf1 and is essential for the inhibitory effect of Smad7 in the transforming growth factor-β signaling pathway. transforming growth factor-β TGF-β type I receptor constitutively active TβR-I receptor-regulated Smad common-partner Smad inhibitory Smad bone morphogenetic protein neuronal precursor cell-expressed developmentally down-regulated 4 Smad ubiquitin regulatory factor protein kinase C conserved 2 domain Smurf1 lacking the C2 domain hemagglutinin wild-type sulfosuccinimidyl 6-(biotinamide) hexanoate Members of the transforming growth factor-β (TGF-β)1 superfamily are multifunctional proteins that regulate a wide spectrum of cellular responses including growth, differentiation, apoptosis, and morphogenesis (1Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Peptide Growth Factors and Their Receptors, Part I. Springer-Verlag New York Inc., New York1990: 419-472Google Scholar). TGF-β and related proteins initiate cellular responses 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 (2Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar). Members of the TGF-β 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) (3Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3316) Google Scholar, 4Derynck R. Zhang Y. Feng X.-H. Cell. 1998; 95: 737-740Abstract Full Text Full Text PDF PubMed Scopus (945) Google Scholar, 5Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (475) Google Scholar). R-Smads and Co-Smad, Smad4, positively regulate signaling by the TGF-β superfamily (3Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3316) Google Scholar). Among R-Smads, Smad2 and Smad3 act in the TGF-β/activin pathway, whereas Smad1, Smad5, and Smad8 function in bone morphogenetic protein (BMP) and anti-Müllerian hormone pathways. In contrast to R-Smads and Co-Smad, I-Smads including Smad6 and Smad7 bind to type I receptors and compete with R-Smads for activation by the type I receptors, resulting in the inhibition of TGF-β superfamily signaling (6Imamura T. Takase M. Nishihara A. Oeda E. Hanai J. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar, 7Hayashi H. Abdollah S. Qiu Y. Cai J., Xu, Y.Y. Grinnell B.W. Richardson M.A. Topper J.N. Gimbrone Jr., M.A. Wrana J.L. Falb D. Cell. 1997; 89: 1165-1173Abstract Full Text Full Text PDF PubMed Scopus (1149) Google Scholar, 8Nakao A. Afrakhte M. Moren A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.-E. Heldin C.-H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1546) Google Scholar). Smad6 also inhibits BMP signaling by forming a complex with Smad1 and by interfering with complex formation between Smad1 and Smad4 (9Hata A. Lagna G. Massagué J. Hemmati-Brivanlou A. Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (577) Google Scholar).Ubiquitin-dependent protein degradation plays key roles in various biological processes including signal transduction, cell cycle progression, and transcriptional regulation (10Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6793) Google Scholar, 11Jesenberger V. Jentsch S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 112-121Crossref PubMed Scopus (308) Google Scholar). Ubiquitination of proteins is induced by an E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases. In the ubiquitin-proteasome pathway, E3 ligases play a crucial role in the recognition of target proteins and subsequent protein degradation by 26 S proteasomes (12Laney J.D. Hochstrasser M. Cell. 1999; 97: 427-430Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). Of the E3 ubiquitin ligases, the RING-type and HECT-type ligases have been well characterized in mammals. Many proteins containing RING finger domains have been found to function as E3 ligases, and some of those are involved in signaling pathways (12Laney J.D. Hochstrasser M. Cell. 1999; 97: 427-430Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). For example, ROC1·Skp1·Cullin1·F-box protein complex containing Fbw1a (also termed βTrCP1) is an E3 ligase for IκB and β-catenin, which participate in the NFκB- and Wnt-signaling pathways, respectively. We have recently shown that the ROC1·Skp1·Cullin1·F-box protein complex containing Fbw1a induced ubiquitination and degradation of activated Smad3 (13Fukuchi M. Imamura T. Chiba T. Ebisawa T. Kawabata M. Tanaka K. Miyazono K. Mol. Biol. Cell. 2001; 12: 1431-1443Crossref PubMed Scopus (176) Google Scholar). Neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) and Smad ubiquitin regulatory factor (Smurf) family proteins represent the HECT-type subclass of E3 ligases; however, the number of HECT-type E3 ligases is less than that of RING-type E3 ligases.Smurf1 was originally identified as an E3 ligase in which ligand independently induces the ubiquitination and degradation of BMP-specific Smads1 and 5 (14Zhu H. Kavsak P. Abdollah S. Wrana J.L. Thomsen G.H. Nature. 1999; 400: 687-693Crossref PubMed Scopus (674) Google Scholar). Smurf2, a Smurf1-related E3 ubiquitin ligase, also interacts with Smad1/5 as well as with activated Smad2 (15Lin X. Liang M. Feng X.-H. J. Biol. Chem. 2000; 275: 36818-36822Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 16Zhang Y. Chang C. Gehling D.J. Hemmati-Brivanlou A. Derynck R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 974-979Crossref PubMed Scopus (421) Google Scholar). In addition, Smurf2 interacts with a transcriptional co-repressor SnoN and thereby targets SnoN for ubiquitin-mediated degradation by proteasomes (17Bonni S. Wang H.R. Causing C.G. Kavsak P. Stroschein S.L. Luo K. Wrana J.L. Nat. Cell Biol. 2001; 3: 587-595Crossref PubMed Scopus (271) Google Scholar). In addition, Smurf1 and Smurf2 interact with Smad7 in the nucleus and induce translocation of Smad7 to the cytoplasm. The Smurfs·Smad7 complexes then associate with TGF-β-type I receptor (TβR-I) and enhance its turnover (18Kavsak P. Rasmussen R.K. Causing C.G. Bonni S. Zhu H. Thomsen G.H. Wrana J.L. Mol. Cell. 2000; 6: 1365-1375Abstract Full Text Full Text PDF PubMed Scopus (1089) Google Scholar, 19Ebisawa T. Fukuchi M. Murakami G. Chiba T. Tanaka K. Imamura T. Miyazono K. J. Biol. Chem. 2001; 276: 12477-12480Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). However, the mechanism of the nuclear export and the plasma membrane localization of the Smurf1·Smad7 complex have not been elucidated.Smurf1 is composed of a protein kinase C conserved 2 (C2) domain at the N terminus with WW domains in the middle and HECT domain at the C terminus. The WW domains bind to a PY motif in the linker regions of Smads, and the HECT domain is responsible for its ubiquitin protein ligase activity. However, the role of the C2 domain of Smurf1 has not been elucidated. The C2 domain was first identified as the Ca2+ binding site in conventional protein kinase C and has been found in various proteins including those involved in signal transduction and membrane trafficking (20Coussens L. Parker P.J. Rhee L. Yang-Feng L. Chen E. Waterfield M.D. Francke U. Ullrich A. Science. 1986; 233: 859-866Crossref PubMed Scopus (752) Google Scholar, 21Cho W. J. Biol. Chem. 2001; 276: 32407-32410Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The C2 domain of Nedd4, which is structurally similar to Smurf1, was found to bind phospholipids and to be responsible for the localization of Nedd4 at the plasma membrane (22Plant P.J. Yeger H. Staub O. Howard P. Rotin D. J. Biol. Chem. 1997; 272: 32329-32336Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). However, Snyder et al. (23Snyder P.M. Olson D.R. McDonald F.J. Bucher D.B. J. Biol. Chem. 2001; 276: 28321-28326Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) reported that the C2 domain of human Nedd4 is not required for inhibition of the epithelial Na+ channel. Thus, the functional role of the C2 domain of Nedd4 is still unclear. In this study, we examined the role of the C2 domain of Smurf1 in its subcellular localization and biological function in the TGF-β signaling pathway.DISCUSSIONSmurf1 was originally identified as an E3 ubiquitin ligase that specifically degrades the BMP-specific R-Smads, Smad1 and Smad5 (14Zhu H. Kavsak P. Abdollah S. Wrana J.L. Thomsen G.H. Nature. 1999; 400: 687-693Crossref PubMed Scopus (674) Google Scholar). Smurf2 is highly related structurally to Smurf1 and induces the degradation of Smad1 as well as that of a TGF-β-specific R-Smad, Smad2 (15Lin X. Liang M. Feng X.-H. J. Biol. Chem. 2000; 275: 36818-36822Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 16Zhang Y. Chang C. Gehling D.J. Hemmati-Brivanlou A. Derynck R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 974-979Crossref PubMed Scopus (421) Google Scholar). In addition, Smurf1 and Smurf2 have been shown to physically interact with I-Smads, Smad6 and Smad7. Smurfs induce nuclear export of Smad7, interact with the TGF-β receptor complex, and facilitate the degradation of Smad7 as well as that of the TGF-β receptor complex (18Kavsak P. Rasmussen R.K. Causing C.G. Bonni S. Zhu H. Thomsen G.H. Wrana J.L. Mol. Cell. 2000; 6: 1365-1375Abstract Full Text Full Text PDF PubMed Scopus (1089) Google Scholar, 19Ebisawa T. Fukuchi M. Murakami G. Chiba T. Tanaka K. Imamura T. Miyazono K. J. Biol. Chem. 2001; 276: 12477-12480Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). Thus, Smurfs negatively regulate the Smad signaling pathways through interaction with both R-Smads and I-Smads.Smad signaling pathways are conserved in vertebrates and inDrosophila (30Das P. Maduzia L.L. Padgett R.W. Cytokine Growth Factor Rev. 1999; 10: 179-186Crossref PubMed Scopus (10) Google Scholar). MAD serves as an R-Smad that specifically transmits signals for decapentaplegia. MEDEA acts as a Co-Smad, and DAD is an I-Smad in Drosophila. DSmurf was recently identified as a Drosophila ortholog of vertebrate Smurf1 and 2 (31Podos S.D. Hanson K.K. Wang Y.C. Ferguson E.L. Dev. Cell. 2001; 1: 567-578Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The function of DSmurf is restricted to the decapentaplegic pathway during development, suggesting critical roles for Smurf ubiquitin ligases in TGF-β superfamily signaling. Similar to Smurf1 and Smurf2, DSmurf appears to control the intracellular pool of MAD and to down-regulate the accumulation of phosphorylated MAD by receptor turnover, possibly through interaction with DAD.In this study, we have shown that the C2 domain of Smurf1 is responsible for the association with the plasma membrane. Although Smurf1 may exhibit activity by degrading R-Smads in the cytoplasm or in the nucleus, membrane localization of Smurf1 appears to be essential for inhibition of TGF-β signaling by the Smurf1·Smad7 complex. Intriguingly, Smurf1(ΔC2) ubiquitinylates Smad7 more efficiently than Smurf1(WT), suggesting that Smurf1 degrades Smad7 and possibly other Smads if it is not targeted to the membrane. Several HECT-type E3 ligases have been identified in mammals including Nedd4 and related Nedd4-like proteins (32Harvey K.F. Kumar S. Trends Cell Biol. 1999; 9: 166-169Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Nedd4 interacts with epithelial Na+ channel and some other proteins (33Murillas R. Simms K.S. Hatakeyama S. Weissman A.M. Kuehn M.R. J. Biol. Chem. 2002; 277: 2897-2907Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Nedd4 is observed throughout the cytoplasm in cells, and its interaction with the plasma membrane is mediated by cytosolic Ca2+ (22Plant P.J. Yeger H. Staub O. Howard P. Rotin D. J. Biol. Chem. 1997; 272: 32329-32336Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). In contrast, membrane localization of Smurf1 was not regulated by cytosolic Ca2+, suggesting that subcellular localization of Smurf1 is controlled by certain other mechanisms. There are many non-Ca2+-binding C2 domains, and some of them including those of PTEN and protein kinase Cε still have abilities to bind to the membrane (34Lee J.O. Yang H. Georgescu M.M., Di Cristofano A. Maehama T. Shi Y. Dixon J.E. Pandolfi P. Pavletich N.P. Cell. 1999; 99: 323-334Abstract Full Text Full Text PDF PubMed Scopus (863) Google Scholar, 35Ochoa W.F. Garcia-Garcia J. Fita I. Corbalan-Garcia S. Verdaguer N. Gomez-Fernandez J.C. J. Mol. Biol. 2001; 311: 837-849Crossref PubMed Scopus (91) Google Scholar). Thus, it will be important to determine how the association of the C2 domain of Smurf1 with the plasma membrane is mediated under physiological conditions.Levels of expression of I-Smads are regulated by various stimuli including TGF-β, BMPs, growth factors, interferon-γ, NFκB signaling, and shear stress (36Miyazono K. ten Dijke P. Heldin C.-H. Adv. Immunol. 2000; 75: 115-157Crossref PubMed Google Scholar). Smad3 and Smad4 and BMP-specific R-Smads bind to the promoter regions of Smad7 and Smad6, respectively (37Nagarajan R.P. Zhang J., Li, W. Chen Y. J. Biol. Chem. 1999; 274: 33412-33418Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 38Ishida W. Hamamoto T. Kusanagi K. Yagi K. Kawabata M. Takehara K. Sampath T.K. Kato M. Miyazono K. J. Biol. Chem. 2000; 275: 6075-6079Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). In contrast, Smurf1 and Smurf2 appear to be ubiquitously expressed in various tissues, and the regulation of their levels of expression has not been reported. Smurfs may be constitutively expressed in cells once I-Smads are induced by certain stimuli and Smurfs capture I-Smads, induce their nuclear export, and associate with the TGF-β receptor complex.For determination of the ubiquitination of TβR-I by Smurfs, a constitutively active form of TβR-I was used in this study as well as by others (18Kavsak P. Rasmussen R.K. Causing C.G. Bonni S. Zhu H. Thomsen G.H. Wrana J.L. Mol. Cell. 2000; 6: 1365-1375Abstract Full Text Full Text PDF PubMed Scopus (1089) Google Scholar, 19Ebisawa T. Fukuchi M. Murakami G. Chiba T. Tanaka K. Imamura T. Miyazono K. J. Biol. Chem. 2001; 276: 12477-12480Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). Because TβR-I and TβR-II spontaneously form a complex without ligand stimulation in transfected cells (39Souchelnytskyi S. ten Dijke P. Miyazono K. Heldin C.-H. EMBO J. 1996; 15: 6231-6240Crossref PubMed Scopus (108) Google Scholar), it has not been determined whether ubiquitination of TβR-I by the Smurf1·Smad7 complex is induced only upon ligand stimulation. However, the ubiquitination of TβR-I by Smurf1·Smad7 may be dependent on the activation status of TβR-I by the TβR-II kinase, because Smad7 fails to efficiently associate with TβR-I in the presence of kinase-inactive form of TβR-II (data not shown) (7Hayashi H. Abdollah S. Qiu Y. Cai J., Xu, Y.Y. Grinnell B.W. Richardson M.A. Topper J.N. Gimbrone Jr., M.A. Wrana J.L. Falb D. Cell. 1997; 89: 1165-1173Abstract Full Text Full Text PDF PubMed Scopus (1149) Google Scholar).It is important to note that Smurf1 enhanced the interaction of Smad7 with the TGF-β receptor complex through targeting of Smad7 to the plasma membrane (see Fig. 2 B). This finding suggests that Smurf1 brings Smad7 to the TGF-β receptor complex and allows Smad7 to compete with R-Smads for receptor activation before it begins to degrade the receptor complex (Fig.7 A). After several hours, Smurf1 begins to induce the degradation of Smad7 and the TGF-β receptor complex, leading to further inhibition of TGF-β signaling (Fig. 7 B). Thus, the C2 domain of Smurf1 plays important roles in both the early and late phases of inhibition of TGF-β signaling by the Smurf1·Smad7 complex. Members of the transforming growth factor-β (TGF-β)1 superfamily are multifunctional proteins that regulate a wide spectrum of cellular responses including growth, differentiation, apoptosis, and morphogenesis (1Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Peptide Growth Factors and Their Receptors, Part I. Springer-Verlag New York Inc., New York1990: 419-472Google Scholar). TGF-β and related proteins initiate cellular responses 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 (2Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar). Members of the TGF-β 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) (3Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3316) Google Scholar, 4Derynck R. Zhang Y. Feng X.-H. Cell. 1998; 95: 737-740Abstract Full Text Full Text PDF PubMed Scopus (945) Google Scholar, 5Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (475) Google Scholar). R-Smads and Co-Smad, Smad4, positively regulate signaling by the TGF-β superfamily (3Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3316) Google Scholar). Among R-Smads, Smad2 and Smad3 act in the TGF-β/activin pathway, whereas Smad1, Smad5, and Smad8 function in bone morphogenetic protein (BMP) and anti-Müllerian hormone pathways. In contrast to R-Smads and Co-Smad, I-Smads including Smad6 and Smad7 bind to type I receptors and compete with R-Smads for activation by the type I receptors, resulting in the inhibition of TGF-β superfamily signaling (6Imamura T. Takase M. Nishihara A. Oeda E. Hanai J. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar, 7Hayashi H. Abdollah S. Qiu Y. Cai J., Xu, Y.Y. Grinnell B.W. Richardson M.A. Topper J.N. Gimbrone Jr., M.A. Wrana J.L. Falb D. Cell. 1997; 89: 1165-1173Abstract Full Text Full Text PDF PubMed Scopus (1149) Google Scholar, 8Nakao A. Afrakhte M. Moren A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.-E. Heldin C.-H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1546) Google Scholar). Smad6 also inhibits BMP signaling by forming a complex with Smad1 and by interfering with complex formation between Smad1 and Smad4 (9Hata A. Lagna G. Massagué J. Hemmati-Brivanlou A. Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (577) Google Scholar). Ubiquitin-dependent protein degradation plays key roles in various biological processes including signal transduction, cell cycle progression, and transcriptional regulation (10Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6793) Google Scholar, 11Jesenberger V. Jentsch S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 112-121Crossref PubMed Scopus (308) Google Scholar). Ubiquitination of proteins is induced by an E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases. In the ubiquitin-proteasome pathway, E3 ligases play a crucial role in the recognition of target proteins and subsequent protein degradation by 26 S proteasomes (12Laney J.D. Hochstrasser M. Cell. 1999; 97: 427-430Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). Of the E3 ubiquitin ligases, the RING-type and HECT-type ligases have been well characterized in mammals. Many proteins containing RING finger domains have been found to function as E3 ligases, and some of those are involved in signaling pathways (12Laney J.D. Hochstrasser M. Cell. 1999; 97: 427-430Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). For example, ROC1·Skp1·Cullin1·F-box protein complex containing Fbw1a (also termed βTrCP1) is an E3 ligase for IκB and β-catenin, which participate in the NFκB- and Wnt-signaling pathways, respectively. We have recently shown that the ROC1·Skp1·Cullin1·F-box protein complex containing Fbw1a induced ubiquitination and degradation of activated Smad3 (13Fukuchi M. Imamura T. Chiba T. Ebisawa T. Kawabata M. Tanaka K. Miyazono K. Mol. Biol. Cell. 2001; 12: 1431-1443Crossref PubMed Scopus (176) Google Scholar). Neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) and Smad ubiquitin regulatory factor (Smurf) family proteins represent the HECT-type subclass of E3 ligases; however, the number of HECT-type E3 ligases is less than that of RING-type E3 ligases. Smurf1 was originally identified as an E3 ligase in which ligand independently induces the ubiquitination and degradation of BMP-specific Smads1 and 5 (14Zhu H. Kavsak P. Abdollah S. Wrana J.L. Thomsen G.H. Nature. 1999; 400: 687-693Crossref PubMed Scopus (674) Google Scholar). Smurf2, a Smurf1-related E3 ubiquitin ligase, also interacts with Smad1/5 as well as with activated Smad2 (15Lin X. Liang M. Feng X.-H. J. Biol. Chem. 2000; 275: 36818-36822Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 16Zhang Y. Chang C. Gehling D.J. Hemmati-Brivanlou A. Derynck R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 974-979Crossref PubMed Scopus (421) Google Scholar). In addition, Smurf2 interacts with a transcriptional co-repressor SnoN and thereby targets SnoN for ubiquitin-mediated degradation by proteasomes (17Bonni S. Wang H.R. Causing C.G. Kavsak P. Stroschein S.L. Luo K. Wrana J.L. Nat. Cell Biol. 2001; 3: 587-595Crossref PubMed Scopus (271) Google Scholar). In addition, Smurf1 and Smurf2 interact with Smad7 in the nucleus and induce translocation of Smad7 to the cytoplasm. The Smurfs·Smad7 complexes then associate with TGF-β-type I receptor (TβR-I) and enhance its turnover (18Kavsak P. Rasmussen R.K. Causing C.G. Bonni S. Zhu H. Thomsen G.H. Wrana J.L. Mol. Cell. 2000; 6: 1365-1375Abstract Full Text Full Text PDF PubMed Scopus (1089) Google Scholar, 19Ebisawa T. Fukuchi M. Murakami G. Chiba T. Tanaka K. Imamura T. Miyazono K. J. Biol. Chem. 2001; 276: 12477-12480Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). However, the mechanism of the nuclear export and the plasma membrane localization of the Smurf1·Smad7 complex have not been elucidated. Smurf1 is composed of a protein kinase C conserved 2 (C2) domain at the N terminus with WW domains in the middle and HECT domain at the C terminus. The WW domains bind to a PY motif in the linker regions of Smads, and the HECT domain is responsible for its ubiquitin protein ligase activity. However, the role of the C2 domain of Smurf1 has not been elucidated. The C2 domain was first identified as the Ca2+ binding site in conventional protein kinase C and has been found in various proteins including those involved in signal transduction and membrane trafficking (20Coussens L. Parker P.J. Rhee L. Yang-Feng L. Chen E. Waterfield M.D. Francke U. Ullrich A. Science. 1986; 233: 859-866Crossref PubMed Scopus (752) Google Scholar, 21Cho W. J. Biol. Chem. 2001; 276: 32407-32410Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The C2 domain of Nedd4, which is structurally similar to Smurf1, was found to bind phospholipids and to be responsible for the localization of Nedd4 at the plasma membrane (22Plant P.J. Yeger H. Staub O. Howard P. Rotin D. J. Biol. Chem. 1997; 272: 32329-32336Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). However, Snyder et al. (23Snyder P.M. Olson D.R. McDonald F.J. Bucher D.B. J. Biol. Chem. 2001; 276: 28321-28326Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) reported that the C2 domain of human Nedd4 is not required for inhibition of the epithelial Na+ channel. Thus, the functional role of the C2 domain of Nedd4 is still unclear. In this study, we examined the role of the C2 domain of Smurf1 in its subcellular localization and biological function in the TGF-β signaling pathway. DISCUSSIONSmurf1 was originally identified as an E3 ubiquitin ligase that specifically degrades the BMP-specific R-Smads, Smad1 and Smad5 (14Zhu H. Kavsak P. Abdollah S. Wrana J.L. Thomsen G.H. Nature. 1999; 400: 687-693Crossref PubMed Scopus (674) Google Scholar). Smurf2 is highly related structurally to Smurf1 and induces the degradation of Smad1 as well as that of a TGF-β-specific R-Smad, Smad2 (15Lin X. Liang M. Feng X.-H. J. Biol. Chem. 2000; 275: 36818-36822Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 16Zhang Y. Chang C. Gehling D.J. Hemmati-Brivanlou A. Derynck R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 974-979Crossref PubMed Scopus (421) Google Scholar). In addition, Smurf1 and Smurf2 have been shown to physically interact with I-Smads, Smad6 and Smad7. Smurfs induce nuclear export of Smad7, interact with the TGF-β receptor complex, and facilitate the degradation of Smad7 as well as that of the TGF-β receptor complex (18Kavsak P. Rasmussen R.K. Causing C.G. Bonni S. Zhu H. Thomsen G.H. Wrana J.L. Mol. Cell. 2000; 6: 1365-1375Abstract Full Text Full Text PDF PubMed Scopus (1089) Google Scholar, 19Ebisawa T. Fukuchi M. Murakami G. Chiba T. Tanaka K. Imamura T. Miyazono K. J. Biol. Chem. 2001; 276: 12477-12480Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). Thus, Smurfs negatively regulate the Smad signaling pathways through interaction with both R-Smads and I-Smads.Smad signaling pathways are conserved in vertebrates and inDrosophila (30Das P. Maduzia L.L. Padgett R.W. Cytokine Growth Factor Rev. 1999; 10: 179-186Crossref PubMed Scopus (10) Google Scholar). MAD serves as an R-Smad that specifically transmits signals for decapentaplegia. MEDEA acts as a Co-Smad, and DAD is an I-Smad in Drosophila. DSmurf was recently identified as a Drosophila ortholog of vertebrate Smurf1 and 2 (31Podos S.D. Hanson K.K. Wang Y.C. Ferguson E.L. Dev. Cell. 2001; 1: 567-578Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The function of DSmurf is restricted to the decapentaplegic pathway during development, suggesting critical roles for Smurf ubiquitin ligases in TGF-β superfamily signaling. Similar to Smurf1 and Smurf2, DSmurf appears to control the intracellular pool of MAD and to down-regulate the accumulation of phosphorylated MAD by receptor turnover, possibly through interaction with DAD.In this study, we have shown that the C2 domain of Smurf1 is responsible for the association with the plasma membrane. Although Smurf1 may exhibit activity by degrading R-Smads in the cytoplasm or in the nucleus, membrane localization of Smurf1 appears to be essential for inhibition of TGF-β signaling by the Smurf1·Smad7 complex. Intriguingly, Smurf1(ΔC2) ubiquitinylates Smad7 more efficiently than Smurf1(WT), suggesting that Smurf1 degrades Smad7 and possibly other Smads if it is not targeted to the membrane. Several HECT-type E3 ligases have been identified in mammals including Nedd4 and related Nedd4-like proteins (32Harvey K.F. Kumar S. Trends Cell Biol. 1999; 9: 166-169Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Nedd4 interacts with epithelial Na+ channel and some other proteins (33Murillas R. Simms K.S. Hatakeyama S. Weissman A.M. Kuehn M.R. J. Biol. Chem. 2002; 277: 2897-2907Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Nedd4 is observed throughout the cytoplasm in cells, and its interaction with the plasma membrane is mediated by cytosolic Ca2+ (22Plant P.J. Yeger H. Staub O. Howard P. Rotin D. J. Biol. Chem. 1997; 272: 32329-32336Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). In contrast, membrane localization of Smurf1 was not regulated by cytosolic Ca2+, suggesting that subcellular localization of Smurf1 is controlled by certain other mechanisms. There are many non-Ca2+-binding C2 domains, and some of them including those of PTEN and protein kinase Cε still have abilities to bind to the membrane (34Lee J.O. Yang H. Georgescu M.M., Di Cristofano A. Maehama T. Shi Y. Dixon J.E. Pandolfi P. Pavletich N.P. Cell. 1999; 99: 323-334Abstract Full Text Full Text PDF PubMed Scopus (863) Google Scholar, 35Ochoa W.F. Garcia-Garcia J. Fita I. Corbalan-Garcia S. Verdaguer N. Gomez-Fernandez J.C. J. Mol. Biol. 2001; 311: 837-849Crossref PubMed Scopus (91) Google Scholar). Thus, it will be important to determine how the association of the C2 domain of Smurf1 with the plasma membrane is mediated under physiological conditions.Levels of expression of I-Smads are regulated by various stimuli including TGF-β, BMPs, growth factors, interferon-γ, NFκB signaling, and shear stress (36Miyazono K. ten Dijke P. Heldin C.-H. Adv. Immunol. 2000; 75: 115-157Crossref PubMed Google Scholar). Smad3 and Smad4 and BMP-specific R-Smads bind to the promoter regions of Smad7 and Smad6, respectively (37Nagarajan R.P. Zhang J., Li, W. Chen Y. J. Biol. Chem. 1999; 274: 33412-33418Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 38Ishida W. Hamamoto T. Kusanagi K. Yagi K. Kawabata M. Takehara K. Sampath T.K. Kato M. Miyazono K. J. Biol. Chem. 2000; 275: 6075-6079Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). In contrast, Smurf1 and Smurf2 appear to be ubiquitously expressed in various tissues, and the regulation of their levels of expression has not been reported. Smurfs may be constitutively expressed in cells once I-Smads are induced by certain stimuli and Smurfs capture I-Smads, induce their nuclear export, and associate with the TGF-β receptor complex.For determination of the ubiquitination of TβR-I by Smurfs, a constitutively active form of TβR-I was used in this study as well as by others (18Kavsak P. Rasmussen R.K. Causing C.G. Bonni S. Zhu H. Thomsen G.H. Wrana J.L. Mol. Cell. 2000; 6: 1365-1375Abstract Full Text Full Text PDF PubMed Scopus (1089) Google Scholar, 19Ebisawa T. Fukuchi M. Murakami G. Chiba T. Tanaka K. Imamura T. Miyazono K. J. Biol. Chem. 2001; 276: 12477-12480Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). Because TβR-I and TβR-II spontaneously form a complex without ligand stimulation in transfected cells (39Souchelnytskyi S. ten Dijke P. Miyazono K. Heldin C.-H. EMBO J. 1996; 15: 6231-6240Crossref PubMed Scopus (108) Google Scholar), it has not been determined whether ubiquitination of TβR-I by the Smurf1·Smad7 complex is induced only upon ligand stimulation. However, the ubiquitination of TβR-I by Smurf1·Smad7 may be dependent on the activation status of TβR-I by the TβR-II kinase, because Smad7 fails to efficiently associate with TβR-I in the presence of kinase-inactive form of TβR-II (data not shown) (7Hayashi H. Abdollah S. Qiu Y. Cai J., Xu, Y.Y. Grinnell B.W. Richardson M.A. Topper J.N. Gimbrone Jr., M.A. Wrana J.L. Falb D. Cell. 1997; 89: 1165-1173Abstract Full Text Full Text PDF PubMed Scopus (1149) Google Scholar).It is important to note that Smurf1 enhanced the interaction of Smad7 with the TGF-β receptor complex through targeting of Smad7 to the plasma membrane (see Fig. 2 B). This finding suggests that Smurf1 brings Smad7 to the TGF-β receptor complex and allows Smad7 to compete with R-Smads for receptor activation before it begins to degrade the receptor complex (Fig.7 A). After several hours, Smurf1 begins to induce the degradation of Smad7 and the TGF-β receptor complex, leading to further inhibition of TGF-β signaling (Fig. 7 B). Thus, the C2 domain of Smurf1 plays important roles in both the early and late phases of inhibition of TGF-β signaling by the Smurf1·Smad7 complex. Smurf1 was originally identified as an E3 ubiquitin ligase that specifically degrades the BMP-specific R-Smads, Smad1 and Smad5 (14Zhu H. Kavsak P. Abdollah S. Wrana J.L. Thomsen G.H. Nature. 1999; 400: 687-693Crossref PubMed Scopus (674) Google Scholar). Smurf2 is highly related structurally to Smurf1 and induces the degradation of Smad1 as well as that of a TGF-β-specific R-Smad, Smad2 (15Lin X. Liang M. Feng X.-H. J. Biol. Chem. 2000; 275: 36818-36822Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 16Zhang Y. Chang C. Gehling D.J. Hemmati-Brivanlou A. Derynck R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 974-979Crossref PubMed Scopus (421) Google Scholar). In addition, Smurf1 and Smurf2 have been shown to physically interact with I-Smads, Smad6 and Smad7. Smurfs induce nuclear export of Smad7, interact with the TGF-β receptor complex, and facilitate the degradation of Smad7 as well as that of the TGF-β receptor complex (18Kavsak P. Rasmussen R.K. Causing C.G. Bonni S. Zhu H. Thomsen G.H. Wrana J.L. Mol. Cell. 2000; 6: 1365-1375Abstract Full Text Full Text PDF PubMed Scopus (1089) Google Scholar, 19Ebisawa T. Fukuchi M. Murakami G. Chiba T. Tanaka K. Imamura T. Miyazono K. J. Biol. Chem. 2001; 276: 12477-12480Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). Thus, Smurfs negatively regulate the Smad signaling pathways through interaction with both R-Smads and I-Smads. Smad signaling pathways are conserved in vertebrates and inDrosophila (30Das P. Maduzia L.L. Padgett R.W. Cytokine Growth Factor Rev. 1999; 10: 179-186Crossref PubMed Scopus (10) Google Scholar). MAD serves as an R-Smad that specifically transmits signals for decapentaplegia. MEDEA acts as a Co-Smad, and DAD is an I-Smad in Drosophila. DSmurf was recently identified as a Drosophila ortholog of vertebrate Smurf1 and 2 (31Podos S.D. Hanson K.K. Wang Y.C. Ferguson E.L. Dev. Cell. 2001; 1: 567-578Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The function of DSmurf is restricted to the decapentaplegic pathway during development, suggesting critical roles for Smurf ubiquitin ligases in TGF-β superfamily signaling. Similar to Smurf1 and Smurf2, DSmurf appears to control the intracellular pool of MAD and to down-regulate the accumulation of phosphorylated MAD by receptor turnover, possibly through interaction with DAD. In this study, we have shown that the C2 domain of Smurf1 is responsible for the association with the plasma membrane. Although Smurf1 may exhibit activity by degrading R-Smads in the cytoplasm or in the nucleus, membrane localization of Smurf1 appears to be essential for inhibition of TGF-β signaling by the Smurf1·Smad7 complex. Intriguingly, Smurf1(ΔC2) ubiquitinylates Smad7 more efficiently than Smurf1(WT), suggesting that Smurf1 degrades Smad7 and possibly other Smads if it is not targeted to the membrane. Several HECT-type E3 ligases have been identified in mammals including Nedd4 and related Nedd4-like proteins (32Harvey K.F. Kumar S. Trends Cell Biol. 1999; 9: 166-169Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Nedd4 interacts with epithelial Na+ channel and some other proteins (33Murillas R. Simms K.S. Hatakeyama S. Weissman A.M. Kuehn M.R. J. Biol. Chem. 2002; 277: 2897-2907Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Nedd4 is observed throughout the cytoplasm in cells, and its interaction with the plasma membrane is mediated by cytosolic Ca2+ (22Plant P.J. Yeger H. Staub O. Howard P. Rotin D. J. Biol. Chem. 1997; 272: 32329-32336Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). In contrast, membrane localization of Smurf1 was not regulated by cytosolic Ca2+, suggesting that subcellular localization of Smurf1 is controlled by certain other mechanisms. There are many non-Ca2+-binding C2 domains, and some of them including those of PTEN and protein kinase Cε still have abilities to bind to the membrane (34Lee J.O. Yang H. Georgescu M.M., Di Cristofano A. Maehama T. Shi Y. Dixon J.E. Pandolfi P. Pavletich N.P. Cell. 1999; 99: 323-334Abstract Full Text Full Text PDF PubMed Scopus (863) Google Scholar, 35Ochoa W.F. Garcia-Garcia J. Fita I. Corbalan-Garcia S. Verdaguer N. Gomez-Fernandez J.C. J. Mol. Biol. 2001; 311: 837-849Crossref PubMed Scopus (91) Google Scholar). Thus, it will be important to determine how the association of the C2 domain of Smurf1 with the plasma membrane is mediated under physiological conditions. Levels of expression of I-Smads are regulated by various stimuli including TGF-β, BMPs, growth factors, interferon-γ, NFκB signaling, and shear stress (36Miyazono K. ten Dijke P. Heldin C.-H. Adv. Immunol. 2000; 75: 115-157Crossref PubMed Google Scholar). Smad3 and Smad4 and BMP-specific R-Smads bind to the promoter regions of Smad7 and Smad6, respectively (37Nagarajan R.P. Zhang J., Li, W. Chen Y. J. Biol. Chem. 1999; 274: 33412-33418Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 38Ishida W. Hamamoto T. Kusanagi K. Yagi K. Kawabata M. Takehara K. Sampath T.K. Kato M. Miyazono K. J. Biol. Chem. 2000; 275: 6075-6079Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). In contrast, Smurf1 and Smurf2 appear to be ubiquitously expressed in various tissues, and the regulation of their levels of expression has not been reported. Smurfs may be constitutively expressed in cells once I-Smads are induced by certain stimuli and Smurfs capture I-Smads, induce their nuclear export, and associate with the TGF-β receptor complex. For determination of the ubiquitination of TβR-I by Smurfs, a constitutively active form of TβR-I was used in this study as well as by others (18Kavsak P. Rasmussen R.K. Causing C.G. Bonni S. Zhu H. Thomsen G.H. Wrana J.L. Mol. Cell. 2000; 6: 1365-1375Abstract Full Text Full Text PDF PubMed Scopus (1089) Google Scholar, 19Ebisawa T. Fukuchi M. Murakami G. Chiba T. Tanaka K. Imamura T. Miyazono K. J. Biol. Chem. 2001; 276: 12477-12480Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). Because TβR-I and TβR-II spontaneously form a complex without ligand stimulation in transfected cells (39Souchelnytskyi S. ten Dijke P. Miyazono K. Heldin C.-H. EMBO J. 1996; 15: 6231-6240Crossref PubMed Scopus (108) Google Scholar), it has not been determined whether ubiquitination of TβR-I by the Smurf1·Smad7 complex is induced only upon ligand stimulation. However, the ubiquitination of TβR-I by Smurf1·Smad7 may be dependent on the activation status of TβR-I by the TβR-II kinase, because Smad7 fails to efficiently associate with TβR-I in the presence of kinase-inactive form of TβR-II (data not shown) (7Hayashi H. Abdollah S. Qiu Y. Cai J., Xu, Y.Y. Grinnell B.W. Richardson M.A. Topper J.N. Gimbrone Jr., M.A. Wrana J.L. Falb D. Cell. 1997; 89: 1165-1173Abstract Full Text Full Text PDF PubMed Scopus (1149) Google Scholar). It is important to note that Smurf1 enhanced the interaction of Smad7 with the TGF-β receptor complex through targeting of Smad7 to the plasma membrane (see Fig. 2 B). This finding suggests that Smurf1 brings Smad7 to the TGF-β receptor complex and allows Smad7 to compete with R-Smads for receptor activation before it begins to degrade the receptor complex (Fig.7 A). After several hours, Smurf1 begins to induce the degradation of Smad7 and the TGF-β receptor complex, leading to further inhibition of TGF-β signaling (Fig. 7 B). Thus, the C2 domain of Smurf1 plays important roles in both the early and late phases of inhibition of TGF-β signaling by the Smurf1·Smad7 complex. We thank Y. Inada and M. Yanagiwatari for technical assistance. Supplementary Material Download .pdf (.02 MB) Help with pdf files Download .pdf (.02 MB) Help with pdf files
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