Alternatively Spliced Variant of Smad2 Lacking Exon 3

An alternatively spliced variant of Smad2 with a deletion of exon 3 (Smad2Δexon3) is found in various cell types. Here, we studied the function of Smad2Δexon3 and compared it with those of wild-type Smad2 containing exon 3 (Smad2(wt)) and Smad3. When transcriptional activity was measured using the p3TP-lux construct, Smad2Δexon3 was more potent than Smad2(wt), and had activity similar to Smad3. Transcriptional activation of the activin-responsive element (ARE) of Mix.2 gene promoter by Smad2Δexon3 was also similar to that by Smad3, and slightly less potent than that by Smad2(wt). Phosphorylation by the activated transforming growth factor-β type I receptor and heteromer formation with Smad4 occurred to similar extents in Smad2Δexon3, Smad2(wt), and Smad3. However, DNA binding to the activating protein-1 sites of p3TP-lux was observed in Smad2Δexon3 as well as in Smad3, but not in Smad2(wt). In contrast, Smad2(wt), Smad2Δexon3, and Smad3 efficiently formed ARE-binding complexes with Smad4 and FAST1, although Smad2(wt) did not directly bind to ARE. These results suggest that exon 3 of Smad2 interferes with the direct DNA binding of Smad2, and modifies the function of Smad2 in transcription of certain target genes. An alternatively spliced variant of Smad2 with a deletion of exon 3 (Smad2Δexon3) is found in various cell types. Here, we studied the function of Smad2Δexon3 and compared it with those of wild-type Smad2 containing exon 3 (Smad2(wt)) and Smad3. When transcriptional activity was measured using the p3TP-lux construct, Smad2Δexon3 was more potent than Smad2(wt), and had activity similar to Smad3. Transcriptional activation of the activin-responsive element (ARE) of Mix.2 gene promoter by Smad2Δexon3 was also similar to that by Smad3, and slightly less potent than that by Smad2(wt). Phosphorylation by the activated transforming growth factor-β type I receptor and heteromer formation with Smad4 occurred to similar extents in Smad2Δexon3, Smad2(wt), and Smad3. However, DNA binding to the activating protein-1 sites of p3TP-lux was observed in Smad2Δexon3 as well as in Smad3, but not in Smad2(wt). In contrast, Smad2(wt), Smad2Δexon3, and Smad3 efficiently formed ARE-binding complexes with Smad4 and FAST1, although Smad2(wt) did not directly bind to ARE. These results suggest that exon 3 of Smad2 interferes with the direct DNA binding of Smad2, and modifies the function of Smad2 in transcription of certain target genes. Members of the transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF-β, transforming growth factor-β; TβR, TGF-β receptor; Mad, Mothers against decapentaplegic; MH, Mad homology; wt, wild-type; AP, activating protein; ARF, activin-responsive factor; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; PAI, plasminogen activator inhibitor; GST, glutathioneS-transferase; ARE, activin-responsive element; bp, base pair(s). superfamily transduce signals through two different types of serine/threonine kinase receptors, known as type II and type I receptors (1Derynck R. Feng X.-H. Biochim. Biophys. Acta. 1997; 1333: F105-F150Crossref PubMed Scopus (508) Google Scholar). In the TGF-β receptor system, ligand binds to the TGF-β type II receptor (TβR-II), which has a constitutively active kinase. TGF-β type I receptor (TβR-I) is then recruited into the TGF-β·TβR-II complex, and phosphorylated mainly at the glycine/serine-rich domain (GS domain), which results in the activation of TβR-I kinase (2Wrana J.L. Attisano L. Wieser R. Ventura F. Massagué J. Nature. 1994; 370: 341-347Crossref PubMed Scopus (2114) Google Scholar). The TβR-I kinase transduces intracellular signals by activation of various proteins, including Smad proteins. TβR-I thus acts as a downstream component of TβR-II. Mutation in Thr-204 of TβR-I to aspartic acid (TβR-I(TD)) results in the constitutive activation of the TβR-I kinase, which has signaling activity in the absence of TGF-β and TβR-II (3Wieser R. Wrana J.L. Massagué J. EMBO J. 1995; 14: 2199-2208Crossref PubMed Scopus (597) Google Scholar). Smad proteins have recently been shown to comprise a family of proteins that mediate signals for members of the TGF-β superfamily (4Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3348) Google Scholar, 5Massagué J. Hata A. Liu F. Trends Cell Biol. 1997; 7: 187-192Abstract Full Text PDF PubMed Scopus (271) Google Scholar, 6Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 1998; 10: 188-194Crossref PubMed Scopus (178) Google Scholar). Thus far, eight mammalian Smad proteins have been identified, termed Smad1 through Smad8. Smads are classified into three subgroups based on their structure and function, i.e. pathway-restricted Smads, common mediator Smads, and inhibitory Smads. Pathway-restricted Smads can be further subdivided into those involved in the TGF-β and activin signaling pathways, and those activated by the bone morphogenetic protein pathway. Smad2 and Smad3 serve as pathway-restricted Smads for the TGF-β/activin signaling pathways. Smad1, Smad5, and possibly Smad8/MADH6 are activated by bone morphogenetic protein receptors. Smad4 is a common mediator Smad; thus far only one common mediator Smad has been identified in mammals. Smad6 and Smad7 are inhibitory Smads. Pathway-restricted Smads are phosphorylated by serine/threonine kinase receptors, and form hetero-oligomers with Smad4. Phosphorylation of pathway-restricted Smads occurs at the C-terminal Ser-Ser-X-Ser motif (7Macı́as-Silva M. Abdollah S. Hoodless P.A. Pirone R. Attisano L. Wrana J.L. Cell. 1996; 87: 1215-1224Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 8Souchelnytskyi S. Tamaki K. Engström U. Wernstedt C. ten Dijke P. Heldin C.-H. J. Biol. Chem. 1997; 272: 28107-28115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 9Abdollah S. Macı́as-Silva M. Tsukazaki T. Hayashi H. Attisano L. Wrana J.L. J. Biol. Chem. 1997; 272: 27678-27685Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). The heteromers then translocate into the nucleus and activate the transcription of various target genes. Smad proteins have been shown to interact with DNA-binding proteins, such as winged-helix transcription factors,Xenopus, and human FAST1 and mouse FAST2 (10Chen X. Rubock M.J. Whitman M. Nature. 1996; 383: 691-696Crossref PubMed Scopus (632) Google Scholar, 11Zhou S. Zawel L. Lengauer C. Kinzler K.W. Vogelstein B. Mol. Cell. 1998; 2: 121-127Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 12Labbé E. Silvestri C. Hoodless P.A. Wrana J.L. Attisano L. Mol. Cell. 1998; 2: 109-120Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar), and also to directly bind to specific DNA sequences (13Kim J. Johnson K. Chen H.J. Carroll S. Laughon A. Nature. 1997; 388: 304-308Crossref PubMed Scopus (449) Google Scholar, 14Yingling J.M. Datto M.B. Wong C. Frederick J.P. Liberati N.T. Wang X.-F. Mol. Cell. Biol. 1997; 17: 7019-7028Crossref PubMed Google Scholar, 15Zawel L. Dai J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar, 16Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.-M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1581) Google Scholar, 17Jonk L.J.C. Itoh S. Heldin C.-H. ten Dijke P. Kruijer W. J. Biol. Chem. 1998; 273: 21145-21152Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar). Smads have conserved N- and C-terminal regions known as Mothers against decapentaplegic (Mad) homology domain-1 (MH1) and -2 (MH2), respectively, which are linked by a linker region of variable length and amino acid sequence. The MH2 domain is a functional domain, which has transactivation activity when fused to the Gal4-DNA binding domain (18Liu F. Hata A. Baker J.C. Doody J. Cárcamo J. Harland R.M. Massagué J. Nature. 1996; 381: 620-623Crossref PubMed Scopus (592) Google Scholar, 19Meersseman G. Verschueren K. Nelles L. Blumenstock C. Kraft H. Wuytens G. Remacle J. Kozak C.A. Tylzanowski P. Niehrs C. Huylebroeck D. Mech. 1997; PubMed Scopus Google Scholar). The MH2 domain also in with type I receptors Chen Massagué J. EMBO J. 1998; 17: PubMed Scopus Google Scholar), and by Smad proteins Feng X.-H. R. Mol. Cell. Biol. 1997; 17: PubMed Scopus Google Scholar, T. R. Curr. Biol. 1997; 7: Full Text Full Text PDF PubMed Scopus Google Scholar), with a transcription FAST1 X. E. M. G. Whitman M. Nature. 1997; PubMed Scopus Google F. C. Massagué J. 1997; PubMed Scopus Google Scholar), and with transcriptional X.-H. R. 1998; PubMed Scopus Google Scholar, R. T. 1998; PubMed Scopus Google Scholar, D. T. U. S. A. 1998; PubMed Scopus Google Scholar, A. J. J. S. Miyazono K. M. 1998; PubMed Scopus Google Scholar). The domain has been shown to interact with the MH2 domain, and the activity of the A. D. G. Massagué J. Nature. 1997; 388: PubMed Scopus Google Scholar). However, it has been shown that the domain has function in i.e. direct binding to specific DNA Mad in was shown to bind to the of gene (13Kim J. Johnson K. Chen H.J. Carroll S. Laughon A. Nature. 1997; 388: 304-308Crossref PubMed Scopus (449) Google Scholar). Smad3 and Smad4 have also been shown to bind to specific DNA sequences through their (13Kim J. Johnson K. Chen H.J. Carroll S. Laughon A. Nature. 1997; 388: 304-308Crossref PubMed Scopus (449) Google Scholar, 14Yingling J.M. Datto M.B. Wong C. Frederick J.P. Liberati N.T. Wang X.-F. Mol. Cell. Biol. 1997; 17: 7019-7028Crossref PubMed Google Scholar, 15Zawel L. Dai J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar, 16Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.-M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1581) Google Scholar, 17Jonk L.J.C. Itoh S. Heldin C.-H. ten Dijke P. Kruijer W. J. Biol. Chem. 1998; 273: 21145-21152Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar). Smad2 and Smad3 are in amino acid have certain in In to Smad3 and Smad2 not directly bind to DNA E. Silvestri C. Hoodless P.A. Wrana J.L. Attisano L. Mol. Cell. 1998; 2: 109-120Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, J. Johnson K. Chen H.J. Carroll S. Laughon A. Nature. 1997; 388: 304-308Crossref PubMed Scopus (449) Google Scholar, 14Yingling J.M. Datto M.B. Wong C. Frederick J.P. Liberati N.T. Wang X.-F. Mol. Cell. Biol. 1997; 17: 7019-7028Crossref PubMed Google Scholar, 15Zawel L. Dai J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar, 16Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.-M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1581) Google Scholar, 17Jonk L.J.C. Itoh S. Heldin C.-H. ten Dijke P. Kruijer W. J. Biol. Chem. 1998; 273: 21145-21152Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar). to a transcriptional is observed for Smad3 but not for Smad2 M. K. K. T. T. S. K. H. Nature. 1998; PubMed Scopus Google Scholar). functional Smad2 and Smad3 has also been in the of TGF-β and activin on the A. M. A. T. ten Dijke P. Heldin C.-H. M. S. Miyazono K. 1998; PubMed Scopus Google Scholar). TGF-β and activin the growth of but TGF-β is more potent than TGF-β the of Smad2 and Smad3, activin Smad3 A. M. A. T. ten Dijke P. Heldin C.-H. M. S. Miyazono K. 1998; PubMed Scopus Google Scholar). Smad2 has a region with amino acid which is not found in Smad3 Smads in mammals. The Smad2 gene is of and acid region is by exon we found that a Smad2 which exon 3 is in certain and although the of the is of that containing exon 3 S. A. M. K. K. A. Miyazono K. K. 1998; PubMed Scopus Google Scholar). we studied the function of Smad2 exon 3 (Smad2Δexon3) and compared it with those of wild-type Smad2 (Smad2(wt)) and Smad3. In the transcriptional activation using the p3TP-lux construct, Smad2Δexon3 was more potent than Smad2(wt), and had activity similar to Smad3. Phosphorylation by TβR-I and heteromer formation with Smad4 did not Smad2Δexon3 and Smad2(wt). However, Smad2Δexon3, but not Smad2(wt), was to bind to the activating protein sites In contrast, Smad2(wt) and Smad2Δexon3 form the activin-responsive with FAST1 and and the Mix.2 gene The of Smad2(wt), and Smad3 have been A. T. S. M. A. E. Tamaki K. J. Heldin C.-H. Miyazono K. ten Dijke P. EMBO J. 1997; PubMed Scopus Google Scholar, T. M. A. E. J. M. Miyazono K. Nature. 1997; PubMed Scopus Google Scholar). Smad2Δexon3 was by the polymerase chain of Smad2Δexon3 into Smad2(wt). In to of proteins, into 1996; PubMed Scopus Google Scholar). for and the MH2 by the into FAST1 by M. was into of the various cell and The by using and as S. A. M. K. K. A. Miyazono K. K. 1998; PubMed Scopus Google Scholar). The to the using and The was on a and a was using a by J. The in with and in cell using the with p3TP-lux by J.L. H. Abdollah S. J. Wrana J.L. D. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google in the of various of Smad and of gene in the was in for and activity in the cell was with a using a to the The of p3TP-lux and measured as of was measured by the and have been A. T. S. M. A. E. Tamaki K. J. Heldin C.-H. Miyazono K. ten Dijke P. EMBO J. 1997; PubMed Scopus Google Scholar). with for Smads, and the and the cell with the by with protein The then by for 3 in the containing and to gel of the cell directly to to and with the and using the the was as J.M. Datto M.B. Wong C. Frederick J.P. Liberati N.T. Wang X.-F. Mol. Cell. Biol. 1997; 17: 7019-7028Crossref PubMed Google Scholar). cell the with and FAST1 in a containing and proteins as S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.-M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1581) Google Scholar). against Smad2 Smad3 by P. ten A. T. S. M. A. E. Tamaki K. J. Heldin C.-H. Miyazono K. ten Dijke P. EMBO J. 1997; PubMed Scopus Google to the cell Smad proteins. The containing sites was by of the p3TP-lux and and that containing was by of the with I The then to cell containing 3 of proteins with a containing of and of the to activity of M.B. Wang X.-F. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). The of in the was to by and then on a polyacrylamide gel and by Smad2 has a acid region in the of the domain acid which is not found in Smads in various human cell i.e. the cell human cell human cell and human cell cell was using and to the sequences in and and the to using and to results with and S. A. M. K. K. A. Miyazono K. K. 1998; PubMed Scopus Google Scholar), we observed two in and i.e. of and of The only in the two cell DNA we that the to Smad2 exon the into Smad2(wt) we a for The Smad into and the of proteins was by Smad2Δexon3 was efficiently in with protein similar to those with Smad2(wt) and Smad3 not The of Smad2Δexon3 is than that of Smad2(wt), and than that of Smad3, with their we studied the transcriptional activity of Smad2Δexon3 and compared it with those of Smad2(wt) and Smad3 using the p3TP-lux promoter construct, which the promoter region of plasminogen activator and three linked sites J. A. Massagué J. Mol. Cell. Biol. 1995; PubMed Google The which functional used to the transcriptional activity of Smads. In the of of a in the transcriptional activity on p3TP-lux was Smad3 efficiently transcription in the absence of Smad2(wt) was less in transcriptional activation than Smad3. of Smad4 to transcriptional activity of Smad2(wt), although it was less than that of Smad3. Smad2Δexon3 had transcriptional activity similar to that of Smad3 in the absence of which was more potent than that of Smad2(wt). The transcriptional activity of Smad2Δexon3 was also in the gene containing the of Mix.2 promoter in H. Abdollah S. J. Wrana J.L. D. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google in the absence of the specific DNA-binding FAST1 (10Chen X. Rubock M.J. Whitman M. Nature. 1996; 383: 691-696Crossref PubMed Scopus (632) Google Scholar). In to the results using Smad2 was slightly more potent than Smad3 in activity of The by Smad2Δexon3 was similar to that by Smad3, and slightly less than that by In to of the transcriptional activity in p3TP-lux of Smad2Δexon3 than of Smad2(wt), we the of Smad2 and Smad3 by Smad into with various of and with by using Smad2Δexon3 and Smad3 more potent than Smad2(wt) in transcriptional activity on was the of in Smad2Δexon3, Smad2(wt), and Smad3. formation with Smad4 was also studied using Smad2Δexon3, Smad2(wt), and Smad3 with Smad4 was observed in Smad2Δexon3 and Smad2(wt) and was with the of the Smad2 proteins. heteromer formation was in Smad2 and Smad3 by and we found in Smad2Δexon3, Smad2(wt), and Smad3. then studied the formation of DNA-binding complexes by Smad2Δexon3 at the sites of was shown that Smad3 and but not Smad2, bind to p3TP-lux DNA (13Kim J. Johnson K. Chen H.J. Carroll S. Laughon A. Nature. 1997; 388: 304-308Crossref PubMed Scopus (449) Google Scholar). cell with the Smad and and to of the Smad proteins formed DNA-binding complexes in the absence of not In the of Smad3 and Smad2Δexon3, but not Smad2(wt), formed DNA-binding The complexes in the of the Smad4 did not form a DNA-binding in the of but it did in DNA-binding complexes in the of Smad2Δexon3 Smad3, but not of Smad2(wt). The in the of although compared with those with the of and to with a that the DNA-binding complexes Smad4. These results that by deletion of exon Smad2 the to in DNA-binding can directly bind to the sites of Smad3 the MH2 domain was shown to directly bind to DNA E. Silvestri C. Hoodless P.A. Wrana J.L. Attisano L. Mol. Cell. 1998; 2: 109-120Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, 14Yingling J.M. Datto M.B. Wong C. Frederick J.P. Liberati N.T. Wang X.-F. Mol. Cell. Biol. 1997; 17: 7019-7028Crossref PubMed Google Scholar, 15Zawel L. Dai J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar, 16Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.-M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1581) Google Scholar, 17Jonk L.J.C. Itoh S. Heldin C.-H. ten Dijke P. Kruijer W. J. Biol. Chem. 1998; 273: 21145-21152Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar). and MH2 used for gel to the results with cell with Smads and Smad2Δexon3 and Smad3 to bind the but Smad2(wt) did The in the of Smad although the and compared with that with the of the of the Smad Smad2 with Smad4 and FAST1 to form ARF, which binds to of gene promoter X. E. M. G. Whitman M. Nature. 1997; PubMed Scopus Google Scholar, F. C. Massagué J. 1997; PubMed Scopus Google Scholar). Smad2(wt) was slightly more potent in transcriptional activity on than Smad3 formation with FAST1 was observed in Smad2(wt), Smad2Δexon3, and Smad3 in the and absence of are in the DNA-binding of the complexes containing different Smads. containing Smad2(wt), and FAST1 efficiently in to TGF-β receptor similar to those containing Smad3 Smad2Δexon3 of to of the that the DNA-binding complexes Smad2, Smad3, Smad2Δexon3, with Smad4 and FAST1 was shown to interact with Smad2 through the MH2 domain X. E. M. G. Whitman M. Nature. 1997; PubMed Scopus Google Scholar, F. C. Massagué J. 1997; PubMed Scopus Google Scholar). These that the of exon 3 of Smad2 not with the formation of DNA-binding containing further the of different Smad proteins to directly bind to DNA containing the in the absence of to binding to the sites and the but did not The in the of Smad of the was more with the of the of the and of These that in to binding to DNA as Smad3 and Smad2Δexon3, but not Smad2(wt), have to directly bind to Smad proteins have and MH2 which are linked by a linker Smads have MH2 but have The MH2 domain in various of Smads, i.e. with type I receptors, and with DNA-binding proteins, and with transcriptional (4Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3348) Google Scholar, 5Massagué J. Hata A. Liu F. Trends Cell Biol. 1997; 7: 187-192Abstract Full Text PDF PubMed Scopus (271) Google Scholar, 6Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 1998; 10: 188-194Crossref PubMed Scopus (178) Google Scholar, X.-H. R. 1998; PubMed Scopus Google Scholar, R. T. 1998; PubMed Scopus Google Scholar, D. T. U. S. A. 1998; PubMed Scopus Google Scholar, A. J. J. S. Miyazono K. M. 1998; PubMed Scopus Google Scholar). Smad2, which the domain, is constitutively in the nucleus and target J.C. Harland R.M. 1996; 10: PubMed Scopus Google Scholar). The MH2 domain with type I receptors, and the a acid region in the MH2 domain, a in Chen Massagué J. EMBO J. 1998; 17: PubMed Scopus Google Scholar). In of the MH2 domain has recently been shown to the binding to DNA-binding proteins such as FAST1 Hata A. D. Massagué J. 1998; PubMed Scopus Google Scholar). The domain as a of the function of the MH2 In the domain has binding to specific DNA Mad, of mammalian Smad1 and directly binds to the of gene (13Kim J. Johnson K. Chen H.J. Carroll S. Laughon A. Nature. 1997; 388: 304-308Crossref PubMed Scopus (449) Google Scholar). Smad3 and but not Smad2, have been shown to bind to the sites of p3TP-lux J.M. Datto M.B. Wong C. Frederick J.P. Liberati N.T. Wang X.-F. Mol. Cell. Biol. 1997; 17: 7019-7028Crossref PubMed Google Scholar, M. H. A. T. Miyazono K. EMBO J. 1998; 17: PubMed Scopus Google Scholar). Smad3 and Smad4 have been shown to bind to specific DNA and containing of specific DNA sequences have been shown to be activated by Smad3 and Smad4 L. Dai J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar, 16Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.-M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1581) Google Scholar, 17Jonk L.J.C. Itoh S. Heldin C.-H. ten Dijke P. Kruijer W. J. Biol. Chem. 1998; 273: 21145-21152Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar). Smad2 and Smad3 are and serve as pathway-restricted Smads in the TGF-β and activin signaling pathways. However, functional Smad2 and Smad3 have been In to the in DNA-binding Smad3, but not Smad2, binds to a transcriptional which the activity of Smad3 M. K. K. T. T. S. K. H. Nature. 1998; PubMed Scopus Google Scholar). In the cell TGF-β of Smad2 and Smad3, activin Smad3 A. M. A. T. ten Dijke P. Heldin C.-H. M. S. Miyazono K. 1998; PubMed Scopus Google Scholar). TGF-β and activin have different in the growth and of cell and M. the in Smad2 and Smad3 at in for their Smad2 Smad3 in a acid region in the of the have shown in the S. A. M. K. K. A. Miyazono K. K. 1998; PubMed Scopus Google and in the that exon 3 is in various and of the Smad2 exon 3 to cell types. be of to the Smad2Δexon3 is certain and In the we have shown that Smad2 exon 3 has transcriptional activity on p3TP-lux than Smad2(wt). The domain the function of the MH2 Mutation of in Smad2 in of the and MH2 and was less active than the wild-type Smad2 A. D. G. Massagué J. Nature. 1997; 388: PubMed Scopus Google Scholar). results for Smad4 with at A. D. G. Massagué J. Nature. 1997; 388: PubMed Scopus Google Scholar). In the deletion of exon 3 of Smad2 did not to in by heteromer formation with that exon 3 is not directly involved in the activity of the Smad2Δexon3 is to bind to DNA containing the a not observed for Smad2(wt). These suggest that the direct binding of to DNA is a in the transcriptional activation of the p3TP-lux In cell DNA-binding formation was observed in the and absence of Smad4. DNA binding of Smad3 and Smad2Δexon3 was when proteins MH2 These with those of E. Silvestri C. Hoodless P.A. Wrana J.L. Attisano L. Mol. Cell. 1998; 2: 109-120Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, J. Johnson K. Chen H.J. Carroll S. Laughon A. Nature. 1997; 388: 304-308Crossref PubMed Scopus (449) Google Scholar, 14Yingling J.M. Datto M.B. Wong C. Frederick J.P. Liberati N.T. Wang X.-F. Mol. Cell. Biol. 1997; 17: 7019-7028Crossref PubMed Google Scholar, 15Zawel L. Dai J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar, 16Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.-M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1581) Google Scholar, 17Jonk L.J.C. Itoh S. Heldin C.-H. ten Dijke P. Kruijer W. J. Biol. Chem. 1998; 273: 21145-21152Abstract Full Text Full Text PDF PubMed Scopus (511) Google suggest that the through the domain, when by the MH2 domain is Smad2Δexon3 has transcriptional activity than Smad2(wt), direct DNA binding of Smads their transcriptional activity on p3TP-lux However, Smad2(wt) to transcriptional activation of Smad proteins can activate target through a which not the direct DNA binding of Smads. In to the of DNA binding of in the transcriptional activation of transcription of gene to be less by direct DNA binding of The Mix.2 promoter a similar to the element L. Dai J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar, 17Jonk L.J.C. Itoh S. Heldin C.-H. ten Dijke P. Kruijer W. J. Biol. Chem. 1998; 273: 21145-21152Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar), to which Smad3, and possibly Smad2Δexon3, Smad2(wt), which to directly bind the Mix.2 was in transcriptional activation of the binding of Smad2 to FAST1 occurs through the MH2 domain, in the structure of the domain have less on the transcription of the Mix.2 Smad2 activated promoter with Smad4 and mouse a winged-helix transcription to FAST1, Smad3 transactivation of promoter E. Silvestri C. Hoodless P.A. Wrana J.L. Attisano L. Mol. Cell. 1998; 2: 109-120Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). Smad3 and but not Smad2, directly to the sequences of the These suggest that direct DNA binding of Smads the domain as well as binding DNA-binding FAST1 and the transcription of target genes. DNA binding of Smads in transcription of certain target such as it is less acts for i.e. Mix.2 and of Smad3 to be compared with that of Smad2 S. A. M. K. K. A. Miyazono K. K. 1998; PubMed Scopus Google Scholar). In certain cell types that the of Smad3, Smad2Δexon3 function as a The region exon 3 is observed only in Smad2 but not in Smads in mammals. 3 of Smad2 a in the function of Smad2 by with the direct DNA binding to target genes. are to H. H. M. C.-H. and P. ten Dijke for and and A. and for also J. A. for J. L. Wrana for and M. Whitman for