Nuclear Retention of the Tumor Suppressor cpml by the Homeodomain Protein TGIF Restricts TGF-b Signaling

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1 Molecular Cell 23, , August 18, 2006 ª2006 Elsevier Inc. DOI /j.molcel Nuclear Retention of the Tumor Suppressor cpml by the Homeodomain Protein TGIF Restricts TGF-b Signaling Su Ryeon Seo, 1,4 Nathalie Ferrand, 1,4 Nourdine Faresse, 1 Céline Prunier, 3 Lucile Abécassis, 2 Marcia Pessah, 1 Marie-Francoise Bourgeade, 2 and Azeddine Atfi 1, * 1 INSERM U673 Hôpital St-Antoine 184 Rue du Faubourg St-Antoine Paris 2 INSERM U542 Hôpital Paul Brousse 14 Avenue Paul Vaillant Couturier Villejuif France 3 Department of Cell Biology Lerner Research Institute Cleveland, Ohio Summary The homeodomain protein TGIF has been implicated in the negative regulation of TGF-b signaling. In this study, we report an unexpected role of TGIF in the inhibition of Smad2 phosphorylation, which occurs by a mechanism independent of its association with Smad2. This inhibitory function of TGIF is executed in concert with c-jun, which facilitates the interaction of TGIF with cpml, resulting in the nuclear sequestration of cpml and the disruption of the cpml-sara complex. Notably, knockdown of TGIF by sirna caused increased association of cpml with SARA and cytoplasmic accumulation of cpml. Furthermore, c-jun 2/2 fibroblasts exhibit enhanced association of cpml with SARA. c-jun 2/2 fibroblasts also lose their sensitivity to TGIF-mediated disruption of the cpml-sara complex and of nuclear sequestration of cpml. We suggest that the interaction of TGIF with cpml through c-jun may negatively regulate TGF-b signaling through controlling the localization of cpml and, consequently, the assembly of the cpml-sara complex. Introduction *Correspondence: atfi@st-antoine.inserm.fr 4 These authors contributed equally to this work. Transforming growth factor (TGF-b) has been implicated in a wide range of cellular functions, such as proliferation, apoptosis, and differentiation (Derynck et al., 2001; Massague and Chen, 2000; Whitman, 1998). TGFb initiates responses by contacting two types of transmembrane serine/threonine kinases called receptors I (TbRI) and II (TbRII), promoting activation of TbRI by the TbRII kinase (Heldin et al., 1997; Wrana, 2000). The activated TbRI then propagates the signal to the nucleus by phosphorylating Smad2 and Smad3 at their extreme C termini on two serine residues in an SSXS motif, and this is facilitated by an adaptor protein called SARA (Smad anchor for receptor activation) (Wrana, 2000). The phosphorylation of Smad2 is also under regulation by the cytoplasmic promyelocytic leukemia protein (cpml), a tumor suppressor that plays a crucial role in TGF-b signaling by mediating the association of SARA with Smad2 (Lin et al., 2004). Once phosphorylated, Smad2 associates with Smad4, and the two accumulate in the nucleus, where they regulate the expression of TGF-b target genes through interactions with transcriptional cofactors (Shi and Massague, 2003; Wrana, 2000). Because of its critical role in cell fate determination, TGF-b signaling is subject to many levels of positive and negative regulation, targeting both the receptors and intracellular mediators. For example, Smad signaling can be attenuated by c-jun, a transcriptional factor regulated by TGF-b and other signal inputs through activation of the c-jun NH2-terminal kinase (JNK) pathway (Dennler et al., 2000; Pessah et al., 2001; Ventura et al., 2004). Once activated, JNK phosphorylates c-jun, and in turn, phosphorylated c-jun regulates the expression ofa numberoftargetgenes(davis, 2000). The importance of JNK cascade in negative regulation of TGF-b signaling is manifested by the recent finding that JNK deficiency causes increased autocrine signaling by TGF-b, leading to sustained phosphorylation of Smad2 and expression of TGF-b responsive genes (Ventura et al., 2004). The mechanism that accounts for upregulation of TGF-b signaling may be initiated by the loss of c-jun function caused by JNK inhibition. However, the JNK pathway can also act in another mechanistic mode to inhibit TGF-b signaling through regulation of the association of c-jun with TG-interacting factor (TGIF) (Pessah et al., 2001). TGIF is a homeodomain protein that functions as a negative modulator in TGF-b signaling (Wotton et al., 1999). The mechanism of TGIF-mediated inhibition of TGF-b signaling has been primarily attributed to the ability of TGIF to recruit to Smad2 a transcriptional corepressor containing histone deacetylases (HDACs) (Wotton et al., 1999). However, the role of the HDAC complex in this process is unclear, since recent works have shown that expression of a holoprosencephaly mutant form of TGIF (TGIF.P63R) that still associates with both Smad2 and the HDAC complex failed to repress TGF-b-induced transcription (Gripp et al., 2000; Melhuish and Wotton, 2000). In this study, we show that TGIF functions in TGF-b signaling by preventing Smad2 phosphorylation, and this event occurs by a mechanism that is independent of its association with Smad2. Furthermore, we found that TGIF acts in partnership with c-jun to sequester cpml into the nucleus, thereby preventing the formation of a functional complex between cpml and SARA. These results provide evidence for a mechanism for the silencing of TGF-b signaling by TGIF. Results TGIF Represses TGF-b-Induced Transcription by a Mechanism Independent of Its Association with Smad2 To gain further insight into the mechanism of action of TGIF, we investigated whether it could function by

2 Molecular Cell 548 Figure 1. TGIF Represses TGF-b-Induced Gene Expression Independently of Its Association with Smad2 (A) 293 cells transfected with the indicated combinations of Myc-Smad2, HA-TGIF, and HA-TGIFDSBD were treated with TGF-b for 1 hr, and cell lysates were immunoprecipitated (IP) with anti-ha and immunoblotted (IB) with anti-myc. In this and all of the following experiments, the expression of proteins was determined by direct immunoblotting. (B) HepG2 cells were transfected with CAGA 9 -Lux or ARE 3 -Lux together with FAST1 and increasing amounts of TGIF or TGIFDSBD. Cells were treated with TGF-b for 16 hr and analyzed for luciferase activity. Luciferase was expressed as mean 6SD of triplicates from a representative experiment performed at least three times. (C) MDCK cells were transfected with the indicated combinations of pegfp, Flag-TGIF, HA-TbRI, constitutively activated TbRI (HA-TbRI.act), and sirna to msin3 or Tiul1. After 48 hr, cells were sorted by FACS based on GFP and the expression of endogenous PAI-1, JunB, Tiul1, and msin3, or transfected proteins were assessed by immunoblotting. Nsp, nonspecific.

3 Negative Regulation of cpml by TGIF 549 a mechanism that is independent of its association with Smad2. To approach this question, we made use of a deletion mutant, TGIFDSBD, lacking the Smad binding site (Figure 1A). We compared the effect of wild-type TGIF (TGIF.wt) with the effect of TGIFDSBD on the TGF-b/ Smad2 responsive reporter ARE 3 -Lux (Chen et al., 1997). As shown in Figure 1B, expression of TGIFDSBD inhibited the TGF-b-dependent transcription with an efficiency approaching that elicited by TGIF.wt. A similar inhibition by TGIFDSBD was also observed on the TGF-b/Smad3 responsive reporter CAGA 9 -Lux (Dennler et al., 2000). These results suggest the interesting possibility that TGIF may exert its inhibitory role through a mechanism that does not involve its interaction with Smad2. Previous studies have suggested that TGIF inhibits TGF-b signaling either by recruiting to Smad2 a corepressor complex containing msin3 or by targeting Smad2 for degradation through association with the ubiquitin ligase Tiul1 (Seo et al., 2004; Wotton et al., 2001). In an attempt to determine the mechanism underlying the inhibitory function of TGIF, we found that expression of sirna to msin3 had no effect on the ability of TGIF to repress the TGF-b-dependent expression of endogenous PAI-1 and JunB (Figure 1C), which are targets of the Smad signaling pathway (Dennler et al., 2000; Jonk et al., 1998). Furthermore, expression of Tiul1 sirna only modestly attenuates the inhibitory effect of TGIF on PAI-1 and JunB (Figure 1C). We confirmed these results by showing that expression of sirna to Tiul1 or msin3 had little or no effect on the ability of TGIF or TGIFDSBD to inhibit ARE 3 -Lux activity (see Figure S1 in the Supplemental Data available with this article online). In a control experiment, we observed that expression of sirna to msin3 or Tiul1 can relieve repression by SnoN or Smad7 (Figure S1), both of which inhibit TGF-b signaling through association with msin3 and Tiul1, respectively (Seo et al., 2004; Stroschein et al., 1999). When taken together, these data suggest that TGIF may limit TGF-b signaling through a mechanism that is independent of its association with Smad2, msin3, or Tiul1. TGIF Suppresses TGF-b-Mediated Phosphorylation of Smad2 We next examined whether TGIF affects the ability of Smad2 to bind Smad4 or FAST1. We observed that expression of TGIF induced a decrease in the TGF-b-dependent association of Smad2 with Smad4 (Figure 1D) or FAST1 (Figure 1E). Consistent with this, we observed that stable expression of TGIF in MDCK cells (see Figure 7E) inhibited the nuclear accumulation of Smad2 in response to TGF-b. Furthermore, we observed that expression of TGIF inhibited the TGF-b-dependent phosphorylation of Smad2 (Figure 2A). A similar inhibitory effect of TGIF was observed when the phosphorylation of Smad3 was examined (Figure 2B). The effect of TGIF on Smad2 phosphorylation depended on the amount of TGIF expressed (Figure 2C) and was observed in cells treated with TGF-b for various time periods (Figure 2D). The suppression of Smad2 phosphorylation by TGIF was also observed in Mv1Lu, HepG2, and HaCat cells (Figure 2E). To investigate the inhibitory effect of TGIF under physiological conditions, we generated MDCK clones in which the expression of endogenous TGIF was reduced by sirna (Figure 2F). We observed that the reduction of TGIF was associated with an increase in both TGF-b-induced expression of PAI-1 or JunB (Figure 2G) and the phosphorylation of endogenous Smad2 (Figure 2H). A similar increase in the phosphorylation of endogenous Smad2 was also observed in 293 cells transiently transfected with a TGIF sirna (see Figure 5D and Figure S2A). In a control experiment, we observed that the inhibitory effect of a nontargetable form of TGIF (TGIF.NT) on Smad2 phosphorylation was not relieved by TGIF sirna (Figure S2B), demonstrating the specificity of TGIF sirna. Next, we investigated whether TGIF inhibits Smad2 phosphorylation by a mechanism that is independent of its association with Smad2. In fact, expression of TGIFDSBD inhibited Smad2 phosphorylation with an activity similar to that of TGIF.wt (Figure 3A). Consistent with this result, expression of TGIFDSBD decreased the association of Smad2 with FAST1 (Figure 3B). In an alternative approach, we took advantage of the availability of the holoprosencephaly mutant TGIF.P63R, which retains its ability to associate with Smad2 but no longer inhibits TGF-b-induced transcription (Gripp et al., 2000)(Figures S3A and S3B). In contrast to TGIF.wt, the level of phosphorylated Smad2 was not decreased by TGIF.P63R (Figure 3C). Thus, it is likely that the interaction of TGIF with Smad2 is not involved in its inhibitory role on Smad2 phosphorylation. Similar to Smad2, the association of Smad3 with TGIF is not required for the inhibition of Smad3 phosphorylation because TGIFDSBD retains its ability to inhibit Smad3 phosphorylation (Figure 3D). Because the interaction of TGIF with Smad2 is increased by TGF-b (Pessah et al., 2001; Wotton et al., 1999), it is tempting to speculate that the phosphorylation of Smad2 mediates its association with TGIF. Paradoxically, we found that TGIF can inhibit Smad2 phosphorylation. Therefore, we attempted to resolve this discrepancy by using the recently developed mutant TbRImL45.act, which retains a constitutively active kinase domain but is unable to induce Smad2 phosphorylation (Yu et al., 2002) (Figure S3C). Expression of TbRImL45.act induced the association of TGIF with Smad2 as potently as expression of the original TbRI.act (Figure 3E). Furthermore, we never observed phosphorylated Smad2 associated with TGIF (Figure 3F). As a control, we observed that Smad4 was able to coprecipitate phosphorylated Smad2. We conclude from these experiments that the association of TGIF with Smad2 occurs independently of Smad2 phosphorylation. Inhibition of Smad2 Phosphorylation by c-jun Recently, Ventura and colleagues reported that JNK deficiency caused increased phosphorylation of Smad2 and that this effect is initiated by the loss of c-jun (D and E) COS-7 cells were transfected with the indicated expression vectors, and the association of Smad2 with Smad4 (D) or FAST1 (E) was analyzed by blotting the anti-myc immunoprecipitate with anti-ha or anti-flag, respectively. (F) MDCK cells stably expressing vector or TGIF were treated with TGF-b for 1 hr, and the localization of endogenous Smad2 was revealed by immunofluorescence with anti-smad2.

4 Molecular Cell 550 Figure 2. TGIF Suppresses Smad2 Phosphorylation (A) 293 cells were transfected with Myc-Smad2, HA-TGIF, HA-TbRI, and HA-TbRI.act as indicated, and Smad2 phosphorylation was assessed by blotting the anti-myc immunoprecipitate with anti-phospho-smad2 (anti-psmad2). (B) 293 cells were transfected with Myc-Smad3 in the presence or absence of HA-TGIF and treated with TGF-b for 1 hr. The phosphorylation of Smad3 was assessed by blotting the anti-myc immunoprecipitate with anti-psmad3. (C) 293 cells were transfected with Myc-Smad2 together with HA-TbRI or HA-TbRI.act and increasing amounts of HA-TGIF. (D) 293 cells were transfected with Myc-Smad2 either in the presence or absence of Myc-TGIF and treated with TGF-b for the indicated times. (E) Mv1Lu, HepG2, or HaCat cells were transfected with Myc-Smad2 either in the presence or absence of HA-TGIF and treated with TGF-b for 1 hr. In (C) (E), the phosphorylation of Smad2 was assessed by blotting the anti-myc immunoprecipitate with anti-psmad2. (F) MDCK cells were stably transfected with psuper-tgif (TGIF sirna) or psuper, and clones with reduced expression of TGIF were identified by immunoblotting with anti-tgif. As a control, the membrane was reprobed with anti-jnk1. (G and H) MDCK cells stably expressing psuper-tgif or psuper were treated with TGF-b for 16 hr (G) or 1 hr (H). The expression of PAI-1 or JunB (G) and the phosphorylation of Smad2 (H) were analyzed by immunoblotting with anti-pai-1, anti-c-jun, or anti-psmad2. function (Ventura et al., 2004). Since c-jun binds to TGIF and is required for TGIF-mediated suppression of TGFb signaling (Pessah et al., 2001), we set out to analyze the importance of c-jun in the inhibition of Smad2 phosphorylation by TGIF. We reasoned that if TGIF associates with c-jun to inhibit Smad2 phosphorylation, TGIFDSBD should retain its ability to associate with c- Jun. In fact, we found that TGIF or TGIFDSBD coprecipitated equal amounts of c-jun (Figure 4A). Next, we examined whether c-jun expression, like TGIF expression, affects Smad2 phosphorylation, by employing mouse embryonic fibroblasts (MEFs) derived from c-jun 2/2 and wt mice. In unstimulated cells, substantially more phosphorylated Smad2 accumulated in c-jun 2/2 MEFs, compared with wt MEFs (Figure 4B). In a kinetic experiment, addition of TGF-b further enhanced this phosphorylation at any time examined (Figure S4A). This increased Smad2 phosphorylation was associated with increased association of Smad2 with Smad4 (Figure 4B). Add-back of c-jun into c-jun 2/2 MEFs reduced the phosphorylation of Smad2 to the levels detected in wt MEFs (Figure 4C). To confirm that c-jun can act as an inhibitor of Smad2 phosphorylation, we investigated whether increased expression of endogenous c-jun by tumor necrosis factor a (TNF-a), which is known to antagonize TGF-b effects on gene expression via c-jun (Verrecchia et al., 2000), interferes with Smad2 phosphorylation. In wt MEFs, TGF-b-induced phosphorylation of Smad2 was suppressed by TNF-a (Figure 4D). In contrast, exposure of c-jun 2/2 MEFs to TNF-a failed to inhibit Smad2 phosphorylation. Similar results were obtained with 293 cells in which the expression of c- Jun was reduced by sirna (Figure S4B). In another approach, we used wt and JNK1 2/2 JNK2 2/2 MEFs because TNF-a is known to induce the expression of c-jun through activation of JNK (Ventura et al., 2003)

5 Negative Regulation of cpml by TGIF 551 Figure 3. TGIF Inhibits Smad2 Phosphorylation Independently of Its Association with Smad2 (A C) COS-7 cells were transfected with the indicated expression vectors. The phosphorylation of Smad2 (A and C) was assessed by blotting the anti-myc immunoprecipitate with anti-psmad2. The association of Smad2 with FAST1 (B) was analyzed by blotting the anti-myc immunoprecipitate with anti-flag. (D) 293 cells transfected with Myc-Smad3 and either HA-TGIF or HA-TGIFDSBD were treated with TGF-b for 1 hr, and Smad3 phosphorylation was assessed by blotting the anti-myc immunoprecipitate with anti-psmad3. The association of Smad3 with HA-TGIF or HA-TGIFDSBD was analyzed by blotting the anti-ha immunoprecipitate with anti-myc. (E) COS-7 cells were transfected with the indicated expression vectors, and the association of Smad2 with TGIF was analyzed by blotting the anti-ha immunoprecipitate with anti-myc. (F) 293 cells were transfected with the indicated combinations of Myc-Smad2, Flag-TGIF, and Flag-Smad4. Cells were treated with TGF-b for 1 hr, and the phosphorylation of Smad2 associated with TGIF or Smad4 was analyzed by blotting the anti-flag immunoprecipitate with anti-psmad2. (see also Figure S4C). In contrast to wt MEFs, exposure of JNK1 2/2 JNK2 2/2 MEFs to TNF-a failed to inhibit Smad2 phosphorylation (Figure S4C). During the course of these analyses, we also investigated the effect of interferon-g (IFN-g) on Smad2 phosphorylation. Like TNF-a, IFN-g was shown to induce the expression of c-jun and to inhibit TGF-b-mediated transcriptional activation (Massague and Chen, 2000; Ramana et al., 2001) (see also Figure 4E and Figures S4D and S4E). As shown in Figure 4E, exposure of wt MEFs to IFN-g completely blocked TGF-b-induced phosphorylation of Smad2. This inhibitory effect was greatly relieved, but not abolished, in c-jun 2/2 MEFs, suggesting that IFN-g may interfere with Smad2 phosphorylation, at least in part, by inducing the expression of c-jun. A similar conclusion could be drawn when the expression of c-jun was reduced by transfection of c-jun sirna into the IFN-g-responsive cell line U4A (Figure S4E). Consistent

6 Molecular Cell 552 Figure 4. c-jun Mediates the Inhibitory Effect of TGIF on Smad2 Phosphorylation (A) 293 cells were transfected with the indicated combinations of c-jun, HA-TGIF, and HA-TGIFDSBD. Cells were treated with TGF-b for 1 hr, and the association of c-jun with TGIF mutants was analyzed by blotting the anti-ha immunoprecipitate with anti-c-jun. (B) Wt or c-jun 2/2 MEFs were treated with TGF-b for 1 hr. The phosphorylation of Smad2 and its association with Smad4 were examined by blotting the anti-smad2 immunoprecipitate with anti-psmad2 and anti-smad4. (C) Wt or c-jun 2/2 MEFs transfected with the indicated expression vectors were treated with TGF-b for 1 hr. The phosphorylation of Smad2 was assessed by blotting the anti-myc immunoprecipitate with anti-psmad2. (D and E) Wt or c-jun 2/2 MEFs were treated with TNF-a (D) or IFN-g (E) for 8 hr prior to treatment with TGF-b for 1 hr. The expression of c-jun and the phosphorylation of Smad2 were analyzed by immunoblotting with anti-c-jun and anti-psmad2, respectively. (F) Wt or c-jun 2/2 MEFs transfected with the indicated expression vectors were treated with TGF-b for 1 hr. The phosphorylation of Smad2 was assessed by blotting the anti-myc immunoprecipitate with anti-psmad2. (G) Wt or c-jun 2/2 MEFs were transfected with ARE 3 -Lux together with FAST1 in either the absence or presence of TGIF or Smad7. Cells were treated with TGF-b for 16 hr and analyzed for luciferase activity. Luciferase was expressed as mean 6SD of triplicates from a representative experiment. (H) MDCK cells stably expressing psuper or psuper-tgif were treated with TNF-a for 8 hr prior to treatment with TGF-b for 1 hr. The expression of c-jun and the phosphorylation of Smad2 were determined by immunoblotting with anti-c-jun and anti-psmad2, respectively. with these results, knockout of c-jun can partially relieve IFN-g-mediated repression of TGF-b-mediated transcriptional responses (Figure S4D). Thus, it is likely that c-jun plays an important role in the negative regulation of TGF-b signaling in response to other signal inputs. c-jun Is Required for the Inhibition of Smad2 Phosphorylation by TGIF Next, the contribution of c-jun to TGIF-suppressed Smad2 phosphorylation was examined. In wt MEFs coexpressing TGIF, we observed almost a complete block in Smad2 phosphorylation (Figure 4F). In contrast, coexpression of TGIF in c-jun 2/2 MEFs failed to inhibit Smad2 phosphorylation. This loss of function of TGIF does not occur as a consequence of the disruption of the TGIF-Smad2 complex in c-jun 2/2 MEFs (Figure S4F). In another control experiment, we observed that expression of Smad7 in c-jun 2/2 MEFs completely blocked Smad2 phosphorylation (Figure 4F), providing support for the theory that the lack of TGIF effects was not attributable to a nonspecific altered cytokine

7 Negative Regulation of cpml by TGIF 553 responsiveness in these cells. Confirmation of these results was obtained by an experiment showing that TGIF failed to inhibit TGF-b-mediated ARE 3 -Lux activity in c- Jun 2/2 MEFs (Figure 4G). The inability of TGIF to inhibit TGF-b-induced transcription in c-jun 2/2 MEFs is specific, because the ARE 3 -Lux activity was reduced to the background level by Smad7. Taken together, these results suggest that c-jun may have an essential role in the negative regulation of TGF-b signaling by TGIF. We also investigated whether TGIF could play a role in the negative regulation of TGF-b signaling by c-jun. For this, we used our MDCK cell line stably expressing the TGIF sirna (Figure 2F). We observed that TNF-a treatment induced an increase in the expression of c-jun in MDCK stably expressing TGIF sirna with an efficiency similar to that observed in control cells (Figure 4H). In contrast to control cells, TNF-a failed to block Smad2 phosphorylation in MDCK with reduced expression of TGIF (Figure 4H). We concluded from these experiments that TGIF and c-jun act together to inhibit Smad2 phosphorylation in response to another physiological input. TGIF Interacts with cpml We have previously reported that the transcriptional activity of c-jun is dispensable for its inhibitory effect on TGF-b signaling (Dennler et al., 2000). Since c-jun interacts with PML, a component of the TGF-b pathway that facilitates Smad2 phosphorylation (Lin et al., 2004; Salomoni et al., 2005), we speculated that TGIF may act in partnership with c-jun to interfere with cpml. At first, we looked for possible interaction between TGIF and cpml through transient transfection. In the absence of c-jun, we detected a slight interaction between TGIF and cpml only in cells that had been exposed to TGFb (Figure 5A). However, coexpression of c-jun resulted in a strong association of TGIF with cpml, suggesting that c-jun may mediate this association. In support of this notion, in vitro-translated cpml failed to bind GST-TGIF in the absence of purified c-jun (Figure 5B). We also investigated the requirement of endogenous c-jun in the association of endogenous TGIF with endogenous cpml. As expected, a TGF-b-inducible interaction between endogenous TGIF and endogenous cpml could be detected in wt MEFs (Figure 5C). Interestingly, TGIF and cpml no longer interacted in the absence of c-jun. These data are consistent with a model in which c-jun may function as a bridging factor between TGIF and cpml. In the course of these experiments, we confirmed that c-jun and cpml can form physical complexes (Salomoni et al., 2005), the levels of which can be enhanced by TGF-b (Figure 5C). In addition, we observed that cotransfection of TGIF stabilized the cpml-c-jun complex (Figure 5A). Consistent with this, we observed that knockdown of TGIF by sirna was accompanied by a decrease in the association of endogenous cpml with endogenous c-jun (Figure 5D). Here again, we confirmed the specificity of TGIF sirna by demonstrating that it is defective in interfering with the stabilization of c-jun-cpml complex mediated by the nontargetable form of TGIF, TGIF.NT (Figure 5E). To determine whether the interaction of TGIF with cpml through c-jun might play a role in the inhibition of TGF-b signaling, we used PML 2/2 MEFs in gene reporter assays. We observed that PML deficiency caused a significant decrease in TGF-b-mediated transcription (Figure 5F), consistent with previous findings (Lin et al., 2004). In wt MEFs, expression of TGIF and/or c-jun caused a significant inhibition of ARE 3 -Lux. By contrast, PML 2/2 MEFs did not exhibit a further decrease in ARE 3 - Lux activity by expression of either TGIF or c-jun alone, or of both. The inability of TGIF and c-jun to inhibit TGFb-induced transcription in PML-deficient cells is specific, because the ARE 3 -Lux activity was reduced to the background level by expression of Smad7 (Figure 5F). Together, these results suggest that TGIF may act in concert with c-jun to limit TGF-b signaling by interfering with cpml/pml. Next, we sought to analyze the physiological significance of the association of TGIF with cpml through c- Jun. We reasoned that if the complex TGIF-c-JuncPML acts to set a threshold level for TGF-b signaling, the accumulation of this complex would be expected to differ among different cell types that exhibit different sensitivities to TGF-b. To test this hypothesis, we used four cell lines based on their ability to respond to TGFb to a variable extent (Figure 5G and Figure S5B). We observed an inverse correlation between the expression of TGIF and the extent of TGF-b-induced transcription (Figures 5G and 5H) or growth arrest in these cell lines (Figure 5H and Figure S5B). Next, we examined the accumulation of the TGIF-c-Jun-cPML complex by investigating the association between TGIF and cpml, which is mediated by c-jun (Figures 5A 5C). Interestingly, we observed an inverse correlation between the accumulation of the TGIF-c-Jun-cPML complex and the sensitivity of cell lines to TGF-b (Figures 5G and 5H and Figure S5B), suggesting that the formation of the TGIF-c-Jun-cPML complex might act to set a threshold level for TGF-b signaling. To test this possibility directly, we chose to interfere with the accumulation of the TGIFc-Jun-cPML complex by depleting c-jun with sirna (Figure S5A). We found that knockdown of c-jun enhanced TGF-b-induced ARE 3 -Lux activity and growth arrest in all cell lines, although the extent was more pronounced in the less-responsive cell lines that show the accumulation of the highest levels of the TGIF-c-JuncPML complex (Figure 5G and Figure S5B). In several experiments, we consistently observed that depletion of the TGIF-c-Jun-cPML complex enabled all cell lines to reach the same extent of responsiveness to TGF-b, irrespective of their original sensitivity to TGF-b (Figure 5G and Figure S5B). Taken together, these results suggest that the formation of the TGIF-c-Jun-cPML complex may act to set a threshold level for TGF-b-mediated transcriptional responses and growth inhibitory signals. TGIF Disrupts the cpml-sara Complex In light of the observation that the association of cpml with SARA is required for Smad2 phosphorylation (Lin et al., 2004), we focused next on determining whether TGIF modulates the formation of the cpml-sara complex in a c-jun-dependent manner. Experiments using transfected 293 cells demonstrated a constitutive association between cpml and SARA that was decreased in response to TGF-b (Figure 6A), similar to previous observations (Lin et al., 2004). The constitutive association of cpml with SARA was significantly inhibited by expression of either TGIF or c-jun alone, and expression

8 Molecular Cell 554 Figure 5. Association of TGIF with cpml (A) 293 cells transfected with the indicated combinations of Flag-TGIF, Myc-cPML, and HA-c-Jun were treated with TGF-b for 1 hr. The association of TGIF or c-jun with cpml was analyzed by blotting the anti-flag or anti-ha immunoprecipitate with anti-myc. (B) In vitro interaction of cpml with TGIF in the presence or absence of His 6 -c-jun was examined by incubating [ 35 S]methionine-labeled cpml with Sepharose bound GST-TGIF. Bound material was visualized by SDS-PAGE and PhosphorImager. (C) Wt or c-jun 2/2 MEFs were treated with TGF-b for 1 hr. TGIF bound to cpml was detected by blotting the anti-cpml immunoprecipitate with anti-tgif. cpml bound to c-jun was detected by blotting the anti-c-jun immunoprecipitate with anti-cpml. (D) 293 cells were transfected with either TGIF sirna or scrambled sirna, and, 24 hr after transfection, they were treated with TGF-b for 1 hr. The expression of TGIF and the phosphorylation of Smad2 were assessed by immunoblotting with anti-tgif and anti-psmad2. The association of cpml with c-jun was analyzed by blotting the anti-cpml immunoprecipitate with anti-c-jun. (E) 293 cells were transfected with the indicated combinations of Myc-cPML, c-jun, HA-TGIF, HA-TGIF.NT, and TGIF sirna. The association of cpml with c-jun was analyzed by blotting the anti-myc immunoprecipitate with anti-c-jun. (F) Wt or PML 2/2 MEFs were transfected with the indicated combinations of ARE 3 -Lux, FAST1, TGIF, c-jun, and Smad7. (G) Mv1Lu, CaCO2, HepG2, and HaCat cells were transfected with ARE 3 -Lux together with FAST2 in either the absence or presence of c-jun sirna or scrambled sirna. In (F) and (G), cells were treated with TGF-b for 16 hr and analyzed for luciferase activity. Luciferase was expressed as mean 6SD of triplicates from a representative experiment. (H) Mv1Lu, CaCO2, HepG2, and HaCat cells were treated with TGF-b for 1 hr. cpml bound to TGIF and c-jun bound to TGIF were detected by blotting the anti-tgif immunoprecipitate with anti-pml and anti-c-jun. of both proteins completely blocked this association (Figure 6A). We also examined the effects of TGIF or c-jun on the association of cpml with SARA under physiological conditions. Comparative studies using wt and c-jun 2/2 MEFs indicated that c-jun deficiency caused increased association of endogenous cpml with endogenous SARA (Figure 6B). Similarly, knockdown of TGIF by stable expression of sirna was

9 Negative Regulation of cpml by TGIF 555 Figure 6. TGIF Interferes with the Association of cpml with SARA (A) 293 cells were transfected with the indicated combinations of Myc-cPML, Flag-SARA, HA-c-Jun, and GFP-TGIF. Cells were treated with TGFb for 1 hr, and the association of cpml with SARA was analyzed by blotting the anti-flag immunoprecipitate with anti-myc. (B) Wt or c-jun 2/2 MEFs were treated with TGF-b for 1 hr, and cell lysates were immunoprecipitated with anti-sara. cpml bound to SARA was detected by blotting with anti-pml. (C) MDCK cells stably expressing psuper or psuper-tgif were treated with TGF-b for 1 hr, and cpml bound to SARA was detected by blotting the anti-sara immunoprecipitate with anti-cpml. (D) Wt or c-jun 2/2 MEFs transfected with the indicated combinations of Myc-cPML, Flag-SARA, c-jun, and HA-TGIF were treated with TGF-b for 1 hr. cpml bound to SARA was detected by blotting the anti-flag immunoprecipitate with anti-myc. (E) 293 cells were transfected with Myc-Smad2 and Flag-SARA in the absence or presence of HA-TGIF. Cells were treated with TGF-b for 1 hr, and the association of Smad2 with SARA was analyzed by blotting the anti-flag immunoprecipitate with anti-myc. (F) Cytoplasmic and nuclear fractions from MDCK cells stably expressing psuper or psuper-tgif were analyzed by immunoblotting with anti- PML. As controls, the fractions were reprobed for a-tubulin (cytoplasmic) and lamin-b (nuclear). (G) MDCK cells stably expressing TGIF, psuper-tgif, or vector (control) were stained with anti-pml, anti-tgif, or DAPI. The localization of PML (red), TGIF (green), or the nuclei (blue) was visualized with a fluorescence microscope. (H) Wt or c-jun 2/2 MEFs were transfected with the indicated combinations of Myc-cPML and Flag-TGIF. Cytoplasmic and nuclear fractions were immunoblotted with the indicated antibodies. associated with an increase in the association of endogenous cpml with endogenous SARA (Figure 6C). The ability of TGIF to restrict the formation of the cpml- SARA complex is likely to be mediated by c-jun since TGIF failed to inhibit the association of cpml with SARA in c-jun 2/2 MEFs (Figure 6D). Add-back of c-jun in

10 Molecular Cell 556 c-jun 2/2 MEFs restored the inhibitory function of TGIF on the association of cpml with SARA (Figure 6D). To provide further evidence that TGIF inhibits the association of cpml with SARA, we investigated the effect of TGIF on the association of Smad2 with SARA, a process that is facilitated by cpml. In the absence of TGIF, we observed a constitutive association between Smad2 and SARA that was decreased by TGF-b (Figure 6E), as previously described (Tsukazaki et al., 1998). Interestingly, this constitutive association was decreased by expression of TGIF. Because SARA facilitates the association of Smad2 with TGF-b receptor, we examined whether TGIF could also interfere with the association of Smad2 with TbRI. For this, we used an unphosphorylable form of Smad2, Smad2.3SA, to trap the receptor, because the interaction between wt Smad2 and the receptor is transient and thus difficult to detect (Macías-Silva et al., 1996). We observed that the association of Smad2.3SA and TbRI was decreased by expression of TGIF (Figure S6A). All together, these results suggest that TGIF may inhibit the cpml-sara association and, consequently, the assembly of Smad2-SARA and Smad2-receptor complexes. Nuclear Retention of cpml by TGIF Because both c-jun and TGIF localize in the nucleus (Bohmann et al., 1987; Wotton et al., 1999), we speculated that TGIF and c-jun might act together to sequester cpml into the nucleus. Analysis of cytoplasmic and nuclear lysates from control MDCK cells indicated that cpml was localized in the cytoplasm and the nucleus (Figure 6F). Notably, knockdown of endogenous TGIF by stable expression of sirna caused the redistribution of endogenous cpml into the cytoplasm. Under these conditions, two nuclear isoforms of PML could be detected in MDCK cells, and their localization was not changed by stable expression of TGIF sirna (Figure 6F). We also performed immunofluorescence experiments to confirm that expression of TGIF induces nuclear retention of endogenous cpml. In agreement with previous findings (Lin et al., 2004), we detected distinctive PML-nuclear bodies as well as punctate cytoplasmic staining of PML in control cells (Figure 6G, see also Figure S6B, in which the pictures were enlarged to provide more details on the distribution of endogenous PML). In cells stably expressing TGIF, we observed a marked decrease in the cytoplasmic staining of endogenous PML. Conversely, knockdown of endogenous TGIF by stable expression of TGIF sirna was associated with an increase in the cytoplasmic staining of PML (Figure 6G), confirming that endogenous TGIF can function under physiological conditions to sequester endogenous cpml into the nucleus. Next, we used wt and c-jun 2/2 MEFs to investigate the requirement of c-jun in the TGIF-dependent nuclear retention of cpml. Like in MDCK cells, cpml was localized in the cytoplasm and the nucleus in wt MEFs (Figure 6H). However, in c-jun 2/2 MEFs, cpml was found predominantly in the cytoplasm, suggesting that c-jun may retain cpml in the nucleus. As expected, expression of TGIF in wt MEFs caused redistribution of cpml into the nucleus (Figure 6H). Unlike wt MEFs, c-jun 2/2 MEFs lost their sensitivity to the TGIF-dependent redistribution of cpml into the nucleus. These data indicate that c-jun functions in partnership with TGIF to sequester cpml into the nucleus. Our model predicts that the relative amount of cpml in the cytoplasm may regulate the sensitivity of cells to TGIF-induced suppression of TGF-b signaling. Indeed, we found that overexpression of cpml can relieve the inhibitory effects of TGIF on (1) Smad2 and Smad3 phosphorylation (Figure 7A and Figure S6C), (2) the association of Smad2 with Smad4 (Figure 7B), and (3) the TGFb-dependent expression of PAI-1 and JunB (Figure 7C). Consistent with these results, expression of cpml can also relieve TGIF repression of TGF-b-induced ARE 3 - Lux activity (Figure 7D). Similar results were obtained in this gene reporter assay when TGIFDSBD was used. Taken together, these data indicate that the retention of cpml in the nucleus is essential in the negative regulation of TGF-b signaling by TGIF. Expression of TGIF Causes Anchorage-Independent Growth Our findings indicate that TGIF might restrict TGF-b signaling by functioning as a negative regulator of the tumor suppressors cpml and Smad2. The ability of TGIF to interfere with a complex tumor suppressor network that integrates cytostatic signals suggests that it might play a role in cellular transformation. To test this hypothesis, assays of anchorage-independent growth in soft agar were conducted. We found that MDCK cells expressing TGIF formed colonies, whereas control cells or cells stably expressing sirna to TGIF did not (Figures 7E and 7F). To provide further evidence for a role of TGIF in cellular transformation, we examined whether it could cooperate with a constitutively active form of Ras, Ras.V12, to stimulate anchorage-independent growth. We observed that dual expression of Ras.V12 and TGIF caused increased colony formation as compared with expression of each protein alone (Figures 7E and 7F). We also investigated the anchorage-independent growth of MDCK cells stably expressing Ras.V12 and TGIF sirna (Figure 7E). In three independent side-byside experiments, the Ras.V12-TGIF sirna yielded fewer colonies in soft agar than the Ras.V12 cells expressing the control vector (Figure 7F). Taken together, these data indicate that TGIF cooperates with Ras to increase anchorage-independent growth, one hallmark of cellular transformation. Discussion In the present study, we have shown that TGIF exerts its inhibitory function in TGF-b signaling by a mechanism that is independent of its association with Smad2. We have provided evidence that TGIF acts by preventing the ligand-dependent phosphorylation of Smad2 and Smad3. The molecular mechanism that accounts for the inhibition of Smad2 phosphorylation may be mediated by the association of TGIF with c-jun, which functions as a bridging factor between TGIF and cpml. We proposed a model in which TGIF acts in concert with c-jun to sequester cpml into the nucleus, thereby preventing the formation of a functional complex between cpml and SARA, which is required for the phosphorylation of Smad2 by the activated TGF-b receptor (Figure 7G). Thus, by functioning to restrict TGF-b-mediated

11 Negative Regulation of cpml by TGIF 557 Figure 7. Restoration of cpml Suppressed TGIF-Mediated Inhibition of TGF-b Signaling (A and B) 293 cells were transfected with the indicated expression vectors and treated with TGF-b for 1 hr. The phosphorylation of Smad2 (A) and its association with Smad4 (B) were analyzed by blotting the anti-flag immunoprecipitate with anti-psmad2 and anti-ha, respectively. (C) MDCK cells were transfected with pegfp and the indicated expression vectors. After 48 hr, cells were sorted by FACS based on GFP and the expression of endogenous JunB or PAI-1, or transfected proteins were assessed by immunoblotting. (D) HepG2 cells were transfected with ARE 3 -Lux together with FAST1 and the indicated expression vectors. Cells were treated with TGF-b for 16 hr and analyzed for luciferase activity. Luciferase was expressed as mean 6SD of triplicates from a representative experiment. (E and F) MDCK cells were stably transfected with the indicated expression vectors. Several clones stably expressing the appropriate cdna were pooled, and the expression of TGIF or Ras (E) was examined by immunoblotting with anti-tgif or anti-ras. The anchorage-independent growth of the different cell lines (F) was determined in soft agar. (G) A model for inhibition of TGF-b signaling by TGIF. phosphorylation of Smad2, TGIF acts in an important regulatory position in the pathway. This function of TGIF might act to set a threshold level for TGF-b-mediated responses, depending on cell types or the level of TGIF expressed. The present work also demonstrates that TGIF inhibits Smad2 and Smad3 phosphorylation by a mechanism that is independent of its association with these receptor-regulated Smads (R-Smads). Our data differ significantly from data reported by Wotton and colleagues (Wotton et al., 1999), which suggest that the association of TGIF with Smad2 is required for suppression of TGFb signaling in L17 cells. They also demonstrated that TGIF can not only stabilize the interaction of Smad2 with Smad4 or FAST1 but can also interfere with TGFb-mediated association of Smad2 with the coactivator p300 (Wotton et al., 1999). These results, however, provide no clue to the mechanisms by which TGIF interferes with the assembly of Smad2-p300 complex. It is also not clear how the ability of TGIF to stabilize the

12 Molecular Cell 558 Smad2-Smad4 and Smad2-FAST complexes relates to its inhibitory effect on TGF-b signaling, since the formation of these complexes is required for TGF-b signaling. At present, the apparent discrepancy between the previous findings and our findings might be due to different experimental strategies. In any event, our data clearly indicate that TGIF functions as an antagonist of TGFb signaling by suppressing Smad2 phosphorylation. As the association of Smad2 with p300 requires phosphorylation of Smad2 (Massague and Chen, 2000), our data provide an explanation for the ability of TGIF to repress the TGF-b-dependent association of Smad2 with p300. How does TGIF inhibit the TGF-b-dependent phosphorylation of Smad2? Despite the importance of TGIF in the negative regulation of TGF-b signaling, its biochemical activities are poorly understood. Most, but not all, studies correlate TGIF inhibitory function with its ability to bind the HDAC complex (Gripp et al., 2000; Wotton et al., 1999). However, it is unlikely that TGIF-mediated inhibition of TGF-b signaling is primarily mediated through transcriptional repression. In fact, we observed that TGIF suppressed the ability of TGF-b to induce transcription by interfering with the phosphorylation of Smad2 and Smad3 through a mechanism that is independent of its association with these R-Smads. As TGIF localizes in the nucleus (Wotton et al., 1999), it is reasonable to suggest that TGIF might function to sequester in the nucleus components of the TGF-b signaling pathway that facilitate Smad2 phosphorylation by TbRI. cpml represents a strong candidate for such a component, since disruption of the gene encoding PML can prevent Smad2 phosphorylation and enforced expression of cpml can increase the sensitivity to TGFb-mediated phosphorylation of Smad2 (Lin et al., 2004). However, cpml cannot be the only TGIF target, since cpml functions in TGF-b signaling by mediating the association of Smad2 with SARA, the latter being involved in the recruitment of the former to TGF-b receptors (Lin et al., 2004; Tsukazaki et al., 1998). The findings outlined in the present study indicate that TGIF interacts with cpml and that expression of TGIF leads to the sequestration of cpml into the nucleus, resulting in the disruption of the SARA-cPML complex and the subsequent formation of Smad2-SARA and Smad2-receptor complexes. Thus, our results provide a mechanistic explanation for the inhibitory effect of TGIF on Smad2 phosphorylation and establish TGIF as a negative modulator of the SARA-cPML complex. Although TGIF and cpml can interact under physiological conditions, we found that c-jun was required for this interaction in vivo and in vitro. Comparative studies using wt and c-jun 2/2 MEFs indicated that c-jun deficiency abolished the association of TGIF with cpml, leading to increased association of cpml with SARA. In addition, c-jun 2/2 MEFs exhibit enhanced phosphorylation of Smad2 and loss their sensitivity to TGIF-mediated disruption of the cpml-sara complex and inhibition of Smad2 phosphorylation as well. Like c-jun 2/2 fibroblasts, JNK-deficient fibroblasts also exhibit enhanced phosphorylation of Smad2 (Ventura et al. [2004] and this study). Since activation of JNK can stabilize the TGIF-c-Jun complex (Pessah et al., 2001), our model also provides additional insights into the negative regulation of TGF-b signaling by the JNK pathway. The pml gene plays an important role in cancer, although its functions and regulation are not well understood. PML initially was identified in patients with acute promyelocytic leukemia (APL), where it is fused to the retinoic acid receptor a (RARa) as a result of the reciprocal t(15;17) chromosomal translocation (Piazza et al., 2001). Expression of the resulting fusion proteins in mice is sufficient to produce leukemia with features of APL (Piazza et al., 2001). PML loss also contributes to cellular transformation, and PML inactivation has been noted in cancer patients (Salomoni and Pandolfi, 2002). Our demonstration that TGIF acts in concert with oncogenic Ras to induce anchorage-independent growth and functions as a negative modulator of cpml provides additional insights into the regulation and roles of the tumor suppressor PML and the TGF-b pathway in malignant transformation. Experimental Procedures Cell Lines and Expression Vectors For details of cell lines, vectors, and antibodies, see the Supplemental Data. Gene Expression Analysis Cells were transfected with expression vectors by using FuGENE (Roche). After 36 hr, cells were treated with or without TGF-b1 (2 ng/ml) for 16 hr. Luciferase activity was measured (Promega) and was normalized for transfection efficiency using a b-galactosidase-expressing vector and the Galacto-Star system (PerkinElmer). Immunoprecipitation and Immunoblotting Cells were resuspended in TNMG buffer (Prunier et al., 2001). Cell lysates were subjected to immunoprecipitation with the appropriate antibody for 2 hr, followed by adsorption to Sepharose-coupled protein G for 1 hr. Immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. Subcellular Fractionation Cells were removed from the plate and pelleted at g. The cell pellet was resuspended in lysis buffer containing 25 mm HEPES (ph 7.4), 1 mm PMSF, 1 mm EDTA, 10 mm benzamidine, 10 mg/ml aprotinin, 2 mg/ml pepstatin, and 0.08% b-mercaptoethanol. The cells were homogenized in a tight-fitting Dounce homogenizer, and nuclei were pelleted by a 30 min centrifugation at g. After washing in lysis buffer, nuclei were resuspended in SDS lysis buffer. Purity of the fractions was monitored by immunoblot analysis with anti-lamin B, anti-a-tubulin, anti-tgif, or anti-c-jun antibodies. Immunofluorescence Analysis Cells were fixed in 4% paraformaldehyde for 30 min and permeabilized in 0.1% Triton X-100. They were incubated with antibodies to TGIF or PML, washed, and incubated with the secondary antibody conjugated to Texas Red or FITC. The slides were mounted and examined on a fluorescence microscope. In Vitro Protein Interaction Assay The in vitro transcription and translational reactions were performed using the TNT-coupled reticulocyte lysate system (Promega). Translation of cpml was carried out in the presence of [ 35 S]methionine, and the labeled protein was incubated in the presence or absence of His 6 -c-jun (purified from bacteria) for 2 hr at 4 C in LSLD buffer (Wotton et al., 1999). Then, samples were incubated with immobilized GST-TGIF, GST-c-Jun, or GST and washed. Samples were resolved by SDS-PAGE, and bound cpml was visualized by autoradiography. Supplemental Data Supplemental Data include Supplemental Experimental Procedures and six figures and can be found with this article online at

13 Negative Regulation of cpml by TGIF 559 Acknowledgments We thank Dr. E. Wagner for providing wt and c-jun 2/2 MEFs, Dr. P. Pandolfi for providing wt and PML 2/2 MEFs, Dr. A. Mauviel for providing wt and JNK1 2/2 JNK2 2/2 MEFs, and Drs. M. Muenke, J. Wrana, and Y.E. Zhang for providing expression vectors. This work was supported by Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS), la Ligue contre le Cancer Comité de Paris, and la Fondation pour la Recherche Médicale. Received: July 22, 2005 Revised: May 2, 2006 Accepted: June 15, 2006 Published: August 17, 2006 References Bohmann, D., Bos, T.J., Admon, A., Nishimura, T., Vogt, P.K., and Tjian, R. (1987). Human proto-oncogene c-jun encodes a DNA binding protein with structural and functional properties of transcription factor AP-1. Science 238, Chen, X., Weisberg, E., Fridmacher, V., Watanabe, M., Naco, G., and Whitman, M. (1997). Smad4 and FAST-1 in the assembly of activinresponsive factor. Nature 389, Davis, R.J. (2000). Signal transduction by the JNK group of MAP kinases. Cell 103, Dennler, S., Prunier, C., Ferrand, N., Gauthier, J.M., and Atfi, A. (2000). c-jun inhibits TGF-b-mediated transcription by repressing Smad3 transcriptional activity. J. Biol. Chem. 275, Derynck, R., Akhurst, R.J., and Balmain, A. (2001). TGF-b signaling in tumor suppression and cancer progression. Nat. Genet. 29, Gripp, K.W., Wotton, D., Edwards, M.C., Roessler, E., Ades, L., Meinecke, P., Richieri-Costa, A., Zackai, E.H., Massague, J., Muenke, M., and Elledge, S.J. (2000). Mutations in TGIF cause holoprosencephaly and link NODAL signalling to human neural axis determination. Nat. Genet. 25, Heldin, C.H., Miyazono, K., and ten Dijke, P. (1997). TGF-b signalling from cell membrane to nucleus through SMAD proteins. Nature 390, Jonk, L.J., Itoh, S., Heldin, C.H., ten Dijke, P., and Kruijer, W. (1998). Identification and functional characterization of a Smad binding element (SBE) in the JunB promoter that acts as a TGF-b, activin, and bone morphogenetic protein-inducible enhancer. J. Biol. Chem. 273, Lin, H.K., Bergmann, S., and Pandolfi, P.P. (2004). Cytoplasmic PML function in TGF-b signalling. Nature 431, Macías-Silva, M., Abdollah, S., Hoodless, P.A., Pirone, R., Attisano, L., and Wrana, J.L. (1996). MADR2 is a substrate of the TGFb receptor and its phosphorylation is required for nuclear accumulation and signaling. Cell 87, Massague, J., and Chen, Y.G. (2000). Controlling TGF-b signaling. Genes Dev. 14, Melhuish, T.A., and Wotton, D. (2000). The interaction of the carboxyl terminus-binding protein with the Smad corepressor TGIF is disrupted by a holoprosencephaly mutation in TGIF. J. Biol. Chem. 275, Pessah, M., Prunier, C., Marais, J., Ferrand, N., Mazars, A., Lallemand, F., Gauthier, J.M., and Atfi, A. (2001). c-jun interacts with the corepressor TG-interacting factor (TGIF) to suppress Smad2 transcriptional activity. Proc. Natl. Acad. Sci. USA 98, Piazza, F., Gurrieri, C., and Pandolfi, P.P. (2001). The theory of APL. Oncogene 20, Prunier, C., Ferrand, N., Frottier, B., Pessah, M., and Atfi, A. (2001). Mechanism for mutational inactivation of the tumor suppressor Smad2. Mol. Cell. Biol. 21, Ramana, C.V., Gil, M.P., Han, Y., Ransohoff, R.M., Schreiber, R.D., and Stark, G.R. (2001). Stat1-independent regulation of gene expression in response to IFN-g. Proc. Natl. Acad. Sci. USA 98, Salomoni, P., and Pandolfi, P.P. (2002). The role of PML in tumor suppression. Cell 108, Salomoni, P., Bernardi, R., Bergmann, S., Changou, A., Tuttle, S., and Pandolfi, P.P. (2005). The promyelocytic leukemia protein PML regulates c-jun function in response to DNA damage. Blood 105, Seo, S.R., Lallemand, F., Ferrand, N., Pessah, M., L Hoste, S., Camonis, J., and Atfi, A. (2004). The novel E3 ubiquitin ligase Tiul1 associates with TGIF to target Smad2 for degradation. EMBO J. 23, Shi, Y., and Massague, J. (2003). Mechanisms of TGF-b signaling from cell membrane to the nucleus. Cell 113, Stroschein, S.L., Wang, W., Zhou, S., Zhou, Q., and Luo, K. (1999). Negative feedback regulation of TGF-b signaling by the SnoN oncoprotein. Science 286, Tsukazaki, T., Chiang, T.A., Davison, A.F., Attisano, L., and Wrana, J.L. (1998). SARA, a FYVE domain protein that recruits Smad2 to the TGFb receptor. Cell 95, Ventura, J.J., Kennedy, N.J., Lamb, J.A., Flavell, R.A., and Davis, R.J. (2003). c-jun NH(2)-terminal kinase is essential for the regulation of AP-1 by tumor necrosis factor. Mol. Cell. Biol. 23, Ventura, J.J., Kennedy, N.J., Flavell, R.A., and Davis, R.J. (2004). JNK regulates autocrine expression of TGF-b1. Mol. Cell 15, Verrecchia, F., Pessah, M., Atfi, A., and Mauviel, A. (2000). TNF-a inhibits TGF-b/Smad signaling in human dermal fibroblasts via AP-1 activation. J. Biol. Chem. 275, Whitman, M. (1998). Smads and early developmental signaling by the TGFb superfamily. Genes Dev. 12, Wotton, D., Lo, R.S., Lee, S., and Massague, J. (1999). A Smad transcriptional corepressor. Cell 97, Wotton, D., Knoepfler, P.S., Laherty, C.D., Eisenman, R.N., and Massague, J. (2001). The Smad transcriptional corepressor TGIF recruits msin3. Cell Growth Differ. 12, Wrana, J.L. (2000). Regulation of Smad activity. Cell 100, Yu, L., Hebert, M.C., and Zhang, Y.E. (2002). TGF-b receptor-activated p38 MAP kinase mediates Smad-independent TGF-b responses. EMBO J. 21,