October 1, 1997Journal of Biological Chemistry162 citationsOpen Access

Induction of Apoptosis by DPC4, a Transcriptional Factor Regulated by Transforming Growth Factor-β through Stress-activated Protein Kinase/c-Jun N-terminal Kinase (SAPK/JNK) Signaling Pathway

AAAzeddine Atfi MBMaud Buisine AMAnne Mazars

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Abstract

Many of the actions of serine/threonine kinase receptors for the transforming growth factor-β (TGFβ) are mediated by DPC4, a human MAD-related protein identified as a tumor suppressor gene in pancreatic carcinoma. Overexpression of DPC4 is sufficient to induce the activation of gene expression and cell cycle arrest, characteristic of the TGFβ response. The stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) is also one of the downstream targets required for TGFβ-mediated signaling. Here we report that expression of the dominant-interfering mutant of various components of the SAPK/JNK cascade specifically blocked both TGFβ and DPC4-induced gene expression. These dominant-interfering mutants also inhibited TGFβ-stimulated DPC4 transcriptional activity. Moreover, we find that overexpression of DPC4 causes transfected cells to undergo the morphological changes typical of apoptosis. These findings define a mechanism whereby TGFβ signals mediated by DPC4 and SAPK/JNK cascade are integrated in the nucleus to activate gene expression and identify a new cellular function for DPC4. Many of the actions of serine/threonine kinase receptors for the transforming growth factor-β (TGFβ) are mediated by DPC4, a human MAD-related protein identified as a tumor suppressor gene in pancreatic carcinoma. Overexpression of DPC4 is sufficient to induce the activation of gene expression and cell cycle arrest, characteristic of the TGFβ response. The stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) is also one of the downstream targets required for TGFβ-mediated signaling. Here we report that expression of the dominant-interfering mutant of various components of the SAPK/JNK cascade specifically blocked both TGFβ and DPC4-induced gene expression. These dominant-interfering mutants also inhibited TGFβ-stimulated DPC4 transcriptional activity. Moreover, we find that overexpression of DPC4 causes transfected cells to undergo the morphological changes typical of apoptosis. These findings define a mechanism whereby TGFβ signals mediated by DPC4 and SAPK/JNK cascade are integrated in the nucleus to activate gene expression and identify a new cellular function for DPC4. The transforming growth factor-β (TGFβ) 1The abbreviations used are: TGFβ, transforming growth factor-β; Smad, mothers against decapentaplegic; SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal kinases; GFP, green fluorescent protein; GST, glutathione S-transferase; MAPK, mitogen-activated protein kinase; MEKK1, MAPK kinase kinase 1; MKK4, MAPK kinase 4; MDCK, Madin-Darby canine kidney; PBS, phosphate-buffered saline. is a multifunctional factor that regulates a variety of cellular processes including extracellular matrix formation, cell proliferation, differentiation, and apoptosis (1Massagué J. Attisano L. Wrana J.L. Trends Cell Biol. 1994; 4: 172-178Abstract Full Text PDF PubMed Scopus (528) Google Scholar, 2Massagué J. Cell. 1992; 69: 1067-1070Abstract Full Text PDF PubMed Scopus (641) Google Scholar, 3Roberts A.B. Sporn M.B. Growth Factors. 1993; 8: 1-9Crossref PubMed Scopus (751) Google Scholar, 4Massagué J. Cell. 1996; 85: 947-950Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar, 5Kingsley D.M. Trends Genet. 1994; 10: 16-21Abstract Full Text PDF PubMed Scopus (294) Google Scholar). Two types of single transmembrane serine/threonine kinase receptors, the type I and type II, have been found to mediate the cellular effects of the TGFβ family ligands (4Massagué J. Cell. 1996; 85: 947-950Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar, 6Wrana J.L. Attisano L. Càrcamo J. Zentella A. Doody J. Laiho M. Wang X.-F. Massagué J. Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1372) Google Scholar, 7Wrana J.L. Attisano L. Wieser R. Ventura R. Massagué J. Nature. 1994; 370: 341-347Crossref PubMed Scopus (2120) Google Scholar). The signaling pathways downstream of the TGFβ receptor complex that lead to the pleiotropic effects of TGFβ are still poorly understood. However, the recent identification of DPC4 and the related protein Smad3 (mothers against decapentaplegic) have provided initial insight into the mechanism of the TGFβ receptor signal transduction (8Zhang Y. Feng X.-H. Wu R.-Y. Derynck R. Nature. 1996; 383: 168-172Crossref PubMed Scopus (759) Google Scholar, 9Lagna G. Hata A. Hemmati-Brivanlou A. Massagué J. Nature. 1996; 383: 832-836Crossref PubMed Scopus (809) Google Scholar, 10Hahn S.A. Schutte M. Hogque A. Moskaluk C. daCosta L. Rosenblum E. Weinstein C. Fischer A. Yeo C. Hruban R. Kem S. Science. 1996; 271: 350-353Crossref PubMed Scopus (2176) Google Scholar). Smad3 but not DPC4 associates with the TGFβ receptors and is directly phosphorylated by the ligand-bound receptor complex (8Zhang Y. Feng X.-H. Wu R.-Y. Derynck R. Nature. 1996; 383: 168-172Crossref PubMed Scopus (759) Google Scholar). Coexpression of Smad3 along with DPC4 resulted in the synergetic activation of TGFβ-like responses suggesting that Smad3 act in partnership with DPC4 to initiate TGFβ signaling (8Zhang Y. Feng X.-H. Wu R.-Y. Derynck R. Nature. 1996; 383: 168-172Crossref PubMed Scopus (759) Google Scholar). Since DPC4 does not associate with nor become phosphorylated by the TGFβ receptors, these data raise the possibility that DPC4 may fulfill some function that is not regulated by receptor-dependent phosphorylation. Because TGFβ receptor can also trigger stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) activation (11Atfi A. Djelloul S. Chastre E. Davis R. Gespach C. J. Biol. Chem. 1997; 272: 1429-1432Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 12Frey R.S. Mulder K.M. Cancer Res. 1997; 57: 628-633PubMed Google Scholar), we investigated whether the TGFβ-regulated function of DPC4 involved the activation of SAPK/JNK signaling pathway. The p3TP-Lux reporter construct (a gift from Dr. Joan Massagué) contains three consecutive 12-O-tetradecanoylphorbol-13-acetate response elements, the plasminogen activator inhibitor promoter, and a luciferase reporter gene. pRK5-Flag-DPC4 and pRK5-Flag-Smad3 were kindly provided by Dr. Rick Derynck. Expression plasmids for the dominant-negative mutant of c-Jun (pCMVTAM67), glutathione S-transferase-Jun (GST-Jun), the kinase-inactive MEKK1 mutant (pCMV5 MEKK1(K432A)), and the dominant-interfering pcDNA3-Flag-MKK4(Ala) mutant have been described previously (11Atfi A. Djelloul S. Chastre E. Davis R. Gespach C. J. Biol. Chem. 1997; 272: 1429-1432Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Gal4-DPC4 was a gift from Dr. Joan Massagué. G15E1b-luc and Gal4-VP16 were kindly provided by Dr. Roger Davis and Michael R. Green, respectively. The pEGFP vector encoding for the green fluorescence protein (GFP) was purchased fromCLONTECH. GST-Jun-(1–79) was expressed inEscherichia coli as described (13Minden A. Lin A. Claret F.G. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1447) Google Scholar). MDCK cells were transfected with expression vectors by the LipofectAMINETM method (Life Technologies, Inc.). Cells were subsequently incubated in the presence or absence of human TGFβ1 (2 ng/ml) for 12 h. The luciferase activities were normalized on the basis of β-galactosidase expression from pCMV5.LacZ-Control vector and protein content. Assays for SAPK/JNK activity were carried out as described (11Atfi A. Djelloul S. Chastre E. Davis R. Gespach C. J. Biol. Chem. 1997; 272: 1429-1432Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). We analyzed MDCK cells for apoptosis by phase-contrast microscopy 48 h after transfection. Transfected MDCK cells were fixed in PBS containing 0.5% glutaraldehyde for 15 min followed by washing in PBS with 5 mm MgCl2. Fixed cells were stained overnight with PBS containing 1 mg/ml 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside, 5 mm potassium ferricyanide, 5 mm potassium ferrocyanide, 2 mmMgCl2, 0.02% Nonidet P-40. The number of blue-staining cells was determined microscopically. For the GFP, cells were fixed in PBS containing 4% paraformaldehyde, stained with Hoechst (2 μg/ml), and mounted. DNA fragmentation (TUNEL staining) was determined using the in situ cell death detection kit (Boehringer Mannheim). The TUNEL-stained cells were visualized by microscopy. To assess the apoptotic DNA damage, cells were collected, rinsed with Ca2+- and Mg2+-free PBS, lysed in lysis buffer (10 mm EDTA, 50 mm Tris (pH 8.0), 0.5% sodium lauryl sarcosine, 0.5 mg of proteinase K/ml), and incubated at 50 °C for 1 h. RNase A (0.5 mg/ml) was added, and lysates were incubated for an additional hour. DNA was electrophoresed in a 1% agarose gel in 0.5 × TBE running buffer (4.5 mm Tris, 4.5 mm boric acid, 62.5 μm EDTA). In comparison with the immediate and transient SAPK/JNK activation induced by other stimuli such as tumor necrosis factor-α (5 min) and anisomycin (13Minden A. Lin A. Claret F.G. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1447) Google Scholar, 14Coso O.A. Chiariello M. Yu J.-C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.C. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1570) Google Scholar), treatment of MDCK cells with human TGFβ1 induces a delayed and persistent increase in SAPK/JNK activity that peaks at 8 h and reaches a maximal value of about 3–5-fold over the basal activity (11Atfi A. Djelloul S. Chastre E. Davis R. Gespach C. J. Biol. Chem. 1997; 272: 1429-1432Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Therefore, we used conditioned medium from cells treated with TGFβ under conditions in which SAPK/JNK activation was readily observed (8 h) to address the question of whether TGFβ-induced SAPK/JNK activation occurs via a paracrine mechanism. This possibility seems more unlikely, since conditioned medium stimulation did not significantly induce the activation of SAPK/JNK at any time up to 30 min, although the addition of anisomycin to these cells led to the increase in SAPK/JNK activity under these experimental conditions (data not shown). In contrast with SAPK/JNK, TGFβ failed to activate p38 kinase under the same experimental conditions (data not shown). Thus, the increase in SAPK/JNK activity most likely resulted from phosphorylation and activation by an upstream kinase such as mitogen-activated protein kinase (MAPK) kinase 4 (MKK4, also termed Sek1 or JNKK), a dual specificity protein kinase, which is structurally related to MKK3, the kinase that phosphorylates and promotes activation of p38 MAP kinase (15Dérijard B. Raingeaud J. Barrett T. Wu I.-H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1415) Google Scholar, 16Sanchez I. Hughes R.T. Mayer B.J. Yee K. Woodgett J.R. Kyriakis J.M. Zon L.I. Nature. 1994; 372: 794-798Crossref PubMed Scopus (917) Google Scholar, 17Minden A. Lin A. McMahon M. Lange-Carter C. Dérijard B. Davis R.J. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1012) Google Scholar, 18Yan M. Dai T. Deak J.C. Kyriakis J.M. Zon L.I. Woodgett J.R. Templeton D.J. Nature. 1994; 372: 798-800Crossref PubMed Scopus (660) Google Scholar). The activation of SAPK/JNK indicated by our observations agrees with recent studies showing that TGFβ activates a novel MAPK kinase kinase (MAPKKK), known as TAK1, that may be involved in signal transduction by members of the TGFβ superfamily (15Dérijard B. Raingeaud J. Barrett T. Wu I.-H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1415) Google Scholar, 19Yamagushi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1178) Google Scholar). Activated TAK1 phosphorylates and promotes activation of MKK4, the kinase that controls activation of SAPK/JNK (19Yamagushi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1178) Google Scholar). Whether TAK1 kinase is downstream of the TGFβ receptor in the biochemical route to SAPK/JNK warrants further investigation. To investigate whether the SAPK/JNK signaling pathway participates in TGFβ signaling, we examined the effect of dominant-negative mutants of MEKK1, MKK4, and c-Jun expression on transcriptional activation by TGFβ. We made use of p3TP-Lux, a reporter construct that directs luciferase expression in response to TGFβ (6Wrana J.L. Attisano L. Càrcamo J. Zentella A. Doody J. Laiho M. Wang X.-F. Massagué J. Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1372) Google Scholar). Expression of dominant-negative mutants MEKK1(K432A), MKK4(Ala), and c-Jun(TAM67) suppressed the TGFβ-induced reporter gene activity; similar results were obtained with all of the SAPK/JNK signaling pathway components in HepG2 and CHO cells ((11) and data not shown). Further evidence for the specificity of JNK signaling pathways in mediating these processes is provided by the inability of the dominant-interfering mutant of MKK3(Ala) to inhibit reporter gene activation by TGFβ (data not shown). DPC4 and the related protein Smad3 are also regulated by the TGFβ signal transduction pathway and may function to transmit TGFβ signals from the cytoplasm to the nucleus (4Massagué J. Cell. 1996; 85: 947-950Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar, 8Zhang Y. Feng X.-H. Wu R.-Y. Derynck R. Nature. 1996; 383: 168-172Crossref PubMed Scopus (759) Google Scholar, 9Lagna G. Hata A. Hemmati-Brivanlou A. Massagué J. Nature. 1996; 383: 832-836Crossref PubMed Scopus (809) Google Scholar). Consistent with previous studies, overexpression of wild-type DPC4, but not Smad3, into MDCK cells strongly increased the basal level of p3TP-Lux expression and potentiated the ability of TGFβ to induce reporter gene activity (8Zhang Y. Feng X.-H. Wu R.-Y. Derynck R. Nature. 1996; 383: 168-172Crossref PubMed Scopus (759) Google Scholar,9Lagna G. Hata A. Hemmati-Brivanlou A. Massagué J. Nature. 1996; 383: 832-836Crossref PubMed Scopus (809) Google Scholar). Coexpression of Smad3 along with DPC4 resulted in the synergetic activation of gene reporter expression, and this effect was enhanced by TGFβ addition (Fig. 1 A). The ability of the dominant-interfering mutant of c-Jun (TAM67) to inhibit TGFβ-induced transcriptional activation allows us to address the functional role of SAPK/JNK pathways in Smad3 and DPC4-mediated signaling events. TAM67 acts as a dominant-interfering mutant because of a deletion in the N-terminal transactivation domain of c-Jun that includes the binding site for SAPK/JNK (20Dérijard B. Hibi M. Wu I.-H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2957) Google Scholar, 21Rapp U.L. Troppmair J. Beck T. Birrer M.J. Oncogene. 1994; 9: 3493-3498PubMed Google Scholar). Expression of TAM67 is sufficient to block the superinduction of the gene reporter activity by TGFβ in cells coexpressing wild-type Smad3 and DPC4. Immunoblotting with anti-Flag antibody confirms that expression of TAM67 did not alter expression of Flag-Smad3 and Flag-DPC4 proteins (data not shown). Similar results were obtained with the dominant-negative mutant MEKK1(K432A) (Fig. 1 A), which is consistent with the hypothesis that SAPK/JNK function is required for transcriptional activation by Smad3 and DPC4 proteins. To determine the potential mechanism underlying the dominant-negative activity of TAM67, we investigated whether this mutant may alter the regulation of DPC4 transcriptional activity by TGFβ. When fused to the DNA binding domain of the yeast transactivator Gal4-(1–147), the C-terminal domain of DPC4 activated transcription from a promoter containing Gal4-binding sites (22Liu F. Hata A. Baker J. Doody J. Càrcamo J. Harland R. Massagué J. Nature. 1996; 381: 620-623Crossref PubMed Scopus (591) Google Scholar), thus raising the interesting possibility that DPC4 may act as a transcriptional activator to initiate TGFβ responses. In support of this hypothesis, we show that a Gal4 fusion protein containing the full-length DPC4 failed to stimulate transcription in this assay, but a strong increase of Gal4-DPC4 transcriptional activity was detected in MDCK cells expressing both Gal4-DPC4 and full-length Smad3 in the presence of TGFβ (Fig. 1 B). Similar to its effect on p3TP-Lux promoter activity, expression of TAM67 inhibits both basal and TGFβ-induced Gal4-DPC4 transcriptional activity (Fig. 1 B). As a control (Fig. 1 B), transfection of TAM67 produces little or no effect on a Gal4 fusion containing the potent acidic activating region of the herpes simplex virus VP16 protein (23Liu F. Green M.R. Nature. 1994; 368: 520-524Crossref PubMed Scopus (225) Google Scholar). From these results, it is becoming evident that TAM67 acts specifically as a dominant-negative inhibitor in TGFβ signaling by blocking the function of endogenous DPC4 and Smad3 proteins and that inhibition of DPC4 transcriptional activity occurs at a level downstream of the SAPK/JNK signaling pathway. Our analysis of the regulation of DPC4 transcriptional activity by c-Jun was consistent with the possibility that c-Jun and DPC4 could interact and produce trans-activation of the 3TP-Lux reporter. One attractive possibility is that the dominant-interfering mutant of c-Jun (TAM67) binds and sequesters DPC4, thus removing it from its natural target. However, we were unable to detect any interactions between DPC4 and TAM67 using different methods, including immunoprecipitation followed by Western blotting and in vitro binding assays with GST-DPC4 produced in bacteria. Because association could be transient or unstable, these negative results do not completely rule out direct interactions of DPC4 and c-Jun. Another important biological activity mediated by the SAPK/JNK signaling pathway is the induction of programmed cell death (24Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5045) Google Scholar, 25Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R. Nature. 1996; 380: 75-79Crossref PubMed Scopus (1718) Google Scholar, 26Chen Y.-R. Wang X. Templeton D. Davis R.J. Tan T-H. J. Biol. Chem. 1996; 271 (31929): 31929Abstract Full Text Full Text PDF PubMed Scopus (856) Google Scholar). One mechanism by which JNK activation may contribute to cell death is by phosphorylating and enhancing the activity of c-Jun, which in turn regulates the activity of cell-killing genes. Consistent with this observation, death of cells does not new gene although of c-Jun activity by of or by expression of dominant-negative TAM67 inhibits apoptotic death (24Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5045) Google Scholar). Since the activity of DPC4 been to be under the control of c-Jun, we were in whether DPC4 and Smad3 were to trigger cell To this hypothesis, we used a to determine the effects of DPC4 and Smad3 overexpression on cell The MDCK cells expressing DPC4, but not Smad3, morphological typical of cells becoming and from the (Fig. DNA with Hoechst that of the cells apoptotic a not control vector or Smad3 expression plasmids were used A). DNA fragmentation analysis with the method confirms that the death of MDCK cells induced by overexpression of DPC4 by an apoptotic mechanism (Fig. 2 B). To further the effects of expression of DPC4 and Smad3 on cell in a were with vector expressing β-galactosidase of In this assay, MDCK cells which by apoptosis up and from the the number of cells a of DPC4 and Smad3 function P. E. B. Cell. 1995; 81: Full Text PDF PubMed Scopus Google Scholar, H. B. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). 48 h after over of cells from of DPC4 and the β-galactosidase morphological changes consistent with of the cells that been transfected with β-galactosidase or in with Smad3 such a (Fig. A). However, of Smad3 along with DPC4 resulted in a in cell suggesting that Smad3 may act in partnership with DPC4 to trigger cell death (Fig. A). A similar could be the were using the detection method (Fig. B). In addition to morphological the effects of DPC4 and Smad3 on apoptosis were with a increase in DNA because apoptosis is by the of DNA at which results in a analyzed by agarose gel (Fig. B). these data the that DPC4 and Smad3 may function in a to induce cell by TGFβ family members is at the level of receptor and the biological responses are becoming in of gene targets that are regulated by these signaling pathways (1Massagué J. Attisano L. Wrana J.L. Trends Cell Biol. 1994; 4: 172-178Abstract Full Text PDF PubMed Scopus (528) Google Scholar, 2Massagué J. Cell. 1992; 69: 1067-1070Abstract Full Text PDF PubMed Scopus (641) Google Scholar, 3Roberts A.B. Sporn M.B. Growth Factors. 1993; 8: 1-9Crossref PubMed Scopus (751) Google Scholar, 4Massagué J. Cell. 1996; 85: 947-950Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar, 5Kingsley D.M. Trends Genet. 1994; 10: 16-21Abstract Full Text PDF PubMed Scopus (294) Google Scholar, A. Djelloul S. Chastre E. Davis R. Gespach C. J. Biol. Chem. 1997; 272: 1429-1432Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, A. K. P. A. S. S. A. 1995; PubMed Scopus Google Scholar). The recent of the protein family and SAPK/JNK signaling pathways provided an important as to members of the TGFβ receptor superfamily signal downstream responses (4Massagué J. Cell. 1996; 85: 947-950Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar, 8Zhang Y. Feng X.-H. Wu R.-Y. Derynck R. Nature. 1996; 383: 168-172Crossref PubMed Scopus (759) Google Scholar, 9Lagna G. Hata A. Hemmati-Brivanlou A. Massagué J. Nature. 1996; 383: 832-836Crossref PubMed Scopus (809) Google Scholar, A. Djelloul S. Chastre E. Davis R. Gespach C. J. Biol. Chem. 1997; 272: 1429-1432Abstract Full Text Full Text PDF PubMed Scopus (273) Google R.S. Mulder K.M. Cancer Res. 1997; 57: 628-633PubMed Google Scholar, K. H. R. P. H. B. S. Wrana J. Attisano L. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. P. R. Attisano L. Wrana Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The findings in the evidence that the activation of SAPK/JNK signaling pathways is required for the function of proteins. The that we to a number of for including the identification of downstream targets of DPC4 and Smad3 and to determine these targets are regulated to initiate TGFβ responses and apoptotic cell We M. R. R. M. M. R. Green, J. and D. Templeton for

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