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X-linked Inhibitor of Apoptosis Protein Functions as a Cofactor in Transforming Growth Factor-β Signaling

X-linked Inhibitor of Apoptosis Protein Functions as a Cofactor in Transforming Growth Factor-β... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 28, Issue of July 13, pp. 26542–26549, 2001 Printed in U.S.A. X-linked Inhibitor of Apoptosis Protein Functions as a Cofactor in b Signaling* Transforming Growth Factor- Received for publication, January 12, 2001, and in revised form, April 26, 2001 Published, JBC Papers in Press, May 16, 2001, DOI 10.1074/jbc.M100331200 Stephanie Birkey Reffey‡, Jens U. Wurthner§, W. Tony Parks§, Anita B. Roberts§, and Colin S. Duckett‡¶ From the ‡Metabolism Branch and the §Laboratory of Cell Regulation and Carcinogenesis, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892 X-linked inhibitor of apoptosis protein (XIAP) is a po- The IAPs were first discovered in the genomes of baculovi- ruses, where they were found to maintain viability of virus tent suppressor of apoptotic cell death, which functions by directly inhibiting caspases, the principal effectors of infected cells and thus enhance virus replication (7, 8). Certain apoptosis. Here we report that XIAP can also function as baculovirus IAPs can function in mammalian cells to suppress a cofactor in the regulation of gene expression by trans- apoptosis (9, 10), and IAP-related genes have subsequently b (TGF-b). XIAP, but not the re- forming growth factor- been identified in many metazoan genomes (11), indicating a lated proteins c-IAP1 or c-IAP2, associated with several high degree of evolutionary conservation. IAPs are defined by a b receptor super- members of the type I class of the TGF- domain of ;70 amino acids known as the baculovirus IAP b-induced signaling. Al- family and potentiated TGF- repeat (BIR) (7, 11). Members of the IAP family contain one to though XIAP-mediated activation of c-Jun N-terminal three imperfect BIR repeats arranged in tandem. These BIR kB was found to require the kinase and nuclear factor domains are required for the ability of IAPs to suppress apo- b signaling intermediate Smad4, the ability of XIAP TGF- ptosis, and have been shown to bind caspases directly (6). The to suppress apoptosis was found to be Smad4-independ- mammalian IAP family (6) includes c-IAP1, c-IAP2, X-linked b-me- ent. These data implicate a role for XIAP in TGF- IAP (XIAP), neuronal apoptosis inhibitory protein (NAIP), sur- diated signaling that is distinct from its anti-apoptotic vivin, BRUCE, and ML-IAP (12). Of these, c-IAP1, c-IAP2, and functions. XIAP exhibit the most structural homology, possessing three tandem BIR repeats and a C-terminal RING finger, which is involved in ubiquitin conjugation (13). Apoptosis is an evolutionarily conserved process that plays a XIAP (14), also known as ILP (9), or MIHA (15), is a broad- critical role during development and tissue homeostasis, and ranging suppressor of apoptosis (14, 16). XIAP has been shown also serves to remove damaged or extraneous cells from an to bind to and directly inhibit the activity of specific caspases organism (1–3). Apoptotic cells undergo a regulated autodiges- (17, 18). However, emerging data suggest that the cellular tion, which involves the disruption of cytoskeletal integrity, cell activities of XIAP are not limited to caspase binding and inhi- shrinkage, nuclear condensation, and the activation of endo- bition. A previous report (19) implicated XIAP in the regulation nucleases. The chief effectors of the apoptotic cell death path- of the stress-induced kinase, c-Jun N-terminal kinase (JNK). way are the caspase family of cysteine proteases. Caspases are Similarly, XIAP has recently been identified as an activator of synthesized as inactive precursors that are cleaved at specific nuclear factor kB (NF-kB), a pleiotropic transcription factor aspartate residues to generate the active subunits. Zymogen that regulates expression of a range of acute phase and imme- cleavage can occur by several mechanisms including proximity- diate-early genes (20). Other members of the IAP family have induced autoprocessing or cleavage by other caspases, reveal- also been implicated in signal transduction. For example, c- ing a caspase cascade with upstream initiator caspases such as IAP1 and c-IAP2 associate with the type 2 tumor necrosis caspases -8, -9, and -10 and downstream, effector caspases, factor receptor (TNFR2) signaling machinery (21, 22) through such as caspases-3, -6, and -7 (4, 5). The activity of these physical interactions with members of the TNFR-associated caspases is regulated by several families of both pro- and anti- factors (TRAFs). The TRAFs and c-IAPs are therefore thought apoptotic cellular proteins including the inhibitor of apoptosis 1 to be signal transduction intermediates that are involved in (IAP) proteins (6). JNK and NF-kB activation (23–25). However, XIAP does not interact with any of the known TRAF proteins or with any * The costs of publication of this article were defrayed in part by the other components of the TNFR2 signaling pathway (16, 26), payment of page charges. This article must therefore be hereby marked suggesting that XIAP plays a role in the cell that is separate “advertisement” in accordance with 18 U.S.C. Section 1734 solely to from those of c-IAP1 or c-IAP2. indicate this fact. Several members of the IAP family have been reported to be ¶ To whom correspondence should be addressed: Metabolism Branch, involved in signaling cascades that are unrelated to the tumor Center for Cancer Research, NCI, National Institutes of Health, 10 Center Dr., Room 6B-05, Bethesda, MD 20892-1578. Tel.: 301-594-1127; necrosis factor pathway. For example, the Drosophila IAPs, E-mail: [email protected]. dIAP-1 and dIAP-2/dILP, are known to interact with Thick- The abbreviations used are: IAP, inhibitor of apoptosis protein; veins (Tkv), a type I serine-threonine kinase receptor homolo- XIAP, X-linked inhibitor of apoptosis protein; GST, glutathione S-trans- gous to the bone morphogenetic protein (BMP) type I receptor ferase; TGF-b, transforming growth factor-b;TbRI , transforming growth factor-b type I receptor; HA, hemagglutinin; BIR, baculovirus inhibitor of apoptosis protein repeat; TBS-T, Tris-buffered saline plus Tween 20; FBS, fetal bovine serum; DN, dominant negative; PAI-1, nuclear factor kB; TRAF, tumor necrosis factor receptor-associated plasminogen activator inhibitor-1; TNFR, tumor necrosis factor recep- factor; MOPS, 4-morpholinepropanesulfonic acid; PBS, phosphate-buff- tor; EMEM, Eagle’s medium containing Earle’s salts; DMEM, Dulbec- ered saline; TAB1, transforming growth factor-b-activated kinase-bind- co’s modified Eagle’s medium; JNK, c-Jun N-terminal kinase; NF-kB, ing protein 1; BMP, bone morphogenetic protein. 26542 This paper is available on line at http://www.jbc.org This is an open access article under the CC BY license. XIAP Is a Cofactor in TGF-b Signaling 26543 sequenced in its entirety and found to be identical to human TAK1a (27). XIAP has been implicated in BMP signaling (28) as a (43). The K63W dominant negative mutant (DN TAK1) was constructed bridging molecule between the BMP type I receptor and a by site-directed mutagenesis using a QuikChange mutagenesis kit downstream signaling molecule, TGF-b-activated kinase-bind- (Stratagene, La Jolla, CA), and was confirmed by sequencing. ing protein 1 (TAB1). Cells and Transfections—Human embryonic kidney 293 cells and The TGF-b superfamily encodes a group of cytokines that HeLa (human adenocarcinoma) cells were maintained in Dulbecco’s includes BMP, TGF-b, and the activins/inhibins, and is in- modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS). HepG2 cells (ATCC, Manassas, VA) were maintained in Eagle’s me- volved in diverse cellular responses such as apoptosis, differ- dium containing Earle’s salts (EMEM) with 10% FBS. All media were entiation, and cell cycle arrest (29, 30). Signaling initiated by supplemented with 2 mM glutamine, and all cells were maintained at this family of cytokines involves two classes of receptors. The 37 °C in 5% CO . type II receptors bind their specific ligand and then phospho- Transfection of 293 cells was performed by the calcium phosphate rylate a type I receptor (31, 32), which propagates the signal precipitation procedure as described previously (44). HepG2 cells were through a specific subset of the Smad family of signal trans- transfected using LipofectAMINE reagents (Life Technologies, Inc.) using 10 ml of LipofectAMINE with 200 ml of serum-free DMEM for each duction intermediates and transcription factors (33, 34). One of well of a six-well plate, for 5 h. Following transfection, the medium was these intermediate signaling proteins, Smad4, is common to all changed to EMEM with 10% FBS, and cells were incubated for 16 h. of these signaling pathways in that it serves as the obligatory The medium was then changed to EMEM 1 0.2% FBS with or without partner of the pathway-restricted Smad proteins and, together 5 ng/ml TGF-b and allowed to incubate for another 24 h. Transfection of with them, translocates to the nucleus to regulate gene expres- HeLa cells was performed as described above, except that 6 mlof sion by binding to cognate sites in the promoters of target genes LipofectAMINE was incubated for 3 h, and following transfection, the medium was changed to DMEM with 10% FBS and incubated for 16 h (35, 36). prior to harvest. Interestingly, both TGF-b and XIAP have been reported to Luciferase Assays—For 3TP-Lux reporter assays, cells were trans- specifically activate JNK (19, 37). Furthermore, Smad proteins fected with 200 ng/well reporter plasmid together with 2 mg/well indi- have been shown to cooperate with the AP-1 transcription cated expression vector, in a six-well plate. For 2kB-luc reporter assays, factor, a heterodimer of c-Fos and c-Jun that binds specific 50 ng/well reporter was transfected along with 2 mg/well indicated sequences in its target promoters (38). AP-1 activity is stimu- expression vectors. Cells were transfected for 6 – 8 h, after which time the medium was replaced with fresh DMEM 1 10% FBS and cells were lated by phosphorylation of the c-Jun transactivation domain incubated for 16 h. In all transfections, the amount of DNA was kept by JNK (38, 39). Moreover, recent data have shown a direct, constant by the addition of an empty control vector. TGF-b-inducible interaction between Smad3 and c-Jun (36). Cells were harvested by washing once in phosphate-buffered saline Because both TGF-b and XIAP can activate JNK, and the (PBS) and then lysing in 0.5 ml of 13 reporter lysis buffer (Promega, TGF-b and JNK signaling pathways are closely linked, we Madison, WI) according to the manufacturer’s specifications. Luciferase examined the possibility of a role for XIAP in TGF-b-mediated activity was quantified using the Luciferase Assay System (Promega, Madison, WI) on a Tropix TR717 microplate luminometer (PE Applied signaling. Biosystems, Bedford, MA). All assays were performed in triplicate, and Data presented here reveal the involvement of XIAP in sig- all data shown are representative of at least three independent naling through the TGF-b type I receptor (TbRI). XIAP was experiments. found to co-localize with TbRI and to interact with this recep- GST Coprecipitations—293 cells were transiently transfected with a tor. XIAP also activated transcription of both TGF-b- and kB- total of 10 mg of plasmid DNA in a 10-cm dish. Following transfection, responsive promoters. Furthermore, the signaling properties of cells were washed once in 5 ml of PBS and lysed for 10 min at room temperature in 1.0 ml of 1% Triton X-100 buffer (45). Following lysis, XIAP were found to be distinct from its anti-apoptotic proper- 400 ml of lysate were incubated with 20 ml of a 50% slurry of glutathi- ties in that signaling by XIAP was inhibited by a dominant one-Sepharose beads (Amersham Pharmacia Biotech) in Triton X-100 negative mutant of Smad4, whereas its anti-apoptotic proper- buffer for1hat4 °C. Beads were then washed four times with 1 ml of ties were unaffected by dominant negative Smad4. These data Triton X-100 lysis buffer and analyzed by immunoblotting. suggest that XIAP is involved in both caspase inhibition and Immunoblotting—Proteins were resolved by sodium dodecyl sulfate signaling through the TbRI receptor. (SDS) 4 –12% gradient gel electrophoresis in 13 MOPS buffer (Invitro- gen, Carlsbad, CA) and transferred to nitrocellulose membranes by EXPERIMENTAL PROCEDURES electrophoretic blotting transfer buffer (Invitrogen) containing 20% Plasmids—The pEBB expression vector has been described previ- methanol. The membrane was blocked for1hin Tris-buffered saline ously (40), and the pEBB-Flag, pEBB-HA, and pEBB-T7 expression with 0.2% Tween (TBS-T) and 5% milk. Proteins were visualized by vectors are all derivatives of pEBB that have been modified to incorpo- incubation with a 1:1000 dilution of primary antibody in TBS-T with 5% rate the appropriate epitope tag and translational termination codes in milk for 1 h, followed by secondary incubation with horseradish perox- all three reading frames. The full-length, DRING, and 3xBIR XIAP idase-conjugated secondary antibody (1:2000) in TBS-T with 5% milk vectors were generated by subcloning into pEBB or one of its epitope- for 1 h. The blot was washed twice for 10 min and once for 30 min in tagged derivatives, as described previously (16). The pEBG mammalian TBS-T and resolved using the enhanced chemiluminescence (ECL) glutathione S-transferase (GST) fusion vector (41) was kindly provided Western blotting detection system (Amersham Pharmacia Biotech). by Dr. B. Mayer. The GST-XIAP/hILP, GST-c-IAP1, and GST-c-IAP2 Primary antibodies used were anti-HA antibody (Mono HA.11; Co- fusion proteins have been previously described (16). Deletions of XIAP vance, Berkeley, CA), anti-GST (Santa Cruz, Santa Cruz, CA), anti-Myc were subcloned into pEBG in-frame with the GST reading frame to (9E10; Covance, Berkeley, CA), anti-T7 tag (horseradish peroxidase- generate pEBG-DRING and pEBG-3xBIR. conjugated; Novagen, Madison, WI), anti-hILP (H59520; Transduction The 2kB-luc reporter construct has been described previously (42). Laboratories, San Diego, CA). The 3TP-Lux reporter was kindly provided by Dr. J. Massague ´ . The Immunofluorescence—HepG2 cells were plated at 1 3 10 cells/well wild type and dominant negative Smad4 (DM4) constructs were gifts of onto sterilized glass coverslips (Corning, Charlotte, NC), incubated for Dr. M. de Caestecker. The HA-tagged expression vectors encoding wild 16 h, and transiently transfected with the indicated plasmids. Cells type, constitutively active (T204D), and kinase-deficient (K232R) TbRI were washed once in PBS, fixed in 3.5% paraformaldehyde for 5 min, receptors, as well as the constitutively active ALK mutants, were gifts permeabilized with 0.5% Triton X-100 in PBS for 10 min, washed once from Dr. J. Wrana and Dr. L. Attisano. The Bax expression vector was in PBS, and blocked in 10% goat serum in PBS for 30 min at room kindly provided by Dr. S. Korsmeyer. The HA-JNK expression vector temperature. Cells were then incubated for 30 min at room temperature was a gift from Dr. M. G. Sanna. with either of the following primary antibodies: anti-hILP/XIAP mouse The full-length open reading frame of TAK1 was amplified by monoclonal antibody (Transduction Laboratories, San Diego, CA) or polymerase chain reaction utilizing the following two primers: 59-ATA- rabbit anti-HA polyclonal antibody (1:200, sc-805, Santa Cruz, Santa GGATCCATGTCTACAGCCTCTGCCGCCTCC-39 and 59-ATTATCGA- Cruz, CA). Cells were washed three times with PBS prior to incubation TATTTCAAAATGTAACGGTCCCAGAGAATC-39 using standard con- with the second primary antibody and then incubated for 30 min with ditions. The polymerase chain reaction product was cloned into pCR2.1 the following secondary antibodies: fluorescein isothiocyanate-conju- (Invitrogen, Carlsbad, CA) and subcloned into pEBB-T7. This clone was gated goat anti-mouse antibody (1:1000, Kirkegaard & Perry, Gaithers- 26544 XIAP Is a Cofactor in TGF-b Signaling FIG.1. XIAP associates with the TGF-b RI receptor. The indi- cated expression vectors were transiently transfected into 293 cells and coprecipitations were performed with glutathione-Sepharose beads, fol- lowed by immunoblotting with anti-HA antibody. The expression of the GST fusion proteins was also confirmed by using an anti-GST antibody (data not shown). A, coprecipitation of XIAP with the constitutively active mutants (denoted by *) of the TGF-b receptor superfamily. B, coprecipitation of XIAP with wild type, constitutively active, or kinase- deficient versions of TbRI. burg, MD) or rhodamine-conjugated goat anti-rabbit antibody (1:1000, Jackson Immunoresearch, West Grove, PA) in PBS with 10% goat serum. Cells were mounted with medium containing 4,6-diamidino-2- phenylindole (Vectashield mounting medium H-1000, Vector Laborato- ries, Burlingame, CA) and then visualized with a Zeiss confocal microscope. Kinase Assays—293 cells were transiently transfected with 0.5 mgof FIG.2. XIAP colocalizes with TbRI. HepG2 cells were transfected HA-tagged JNK and 2 mg of the indicated plasmid in a six-well plate. with both XIAP and HA-TbRI expression vectors. Cells were immuno- Following 18 h of incubation, cells were lysed in 0.3 ml of M2 buffer (19) stained with either anti-hILP/XIAP (mouse) or anti-HA (rabbit) anti- on ice for 30 min. For assays involving p38 inhibitors, cells were treated bodies followed by secondary staining with either fluorescein isothio- with 50 mM SB203580 or 30 mM SB202190 (Calbiochem, La Jolla, CA) cyanate- or rhodamine-conjugated secondary antibodies as described and allowed to incubate for 24 h prior to lysis. Protein concentrations under “Experimental Procedures,” and were visualized by fluorescence were standardized by the method of Bradford (46) using a commercial microscopy for XIAP and/or TbRI localization. The subcellular localiza- kit (Bio-Rad) followed by immunoprecipitation of HA-JNK with protein tion of XIAP is shown in green (top left), TbRI in red (top right), and A-Sepharose and a monoclonal anti-HA antibody (12CA5, Roche Molec- areas of co-localization in yellow (bottom right). A representative field is ular Biochemicals) for1hat4 °C. In vitro kinase assays were per- shown. formed exactly as described (23). Activation of JNK was measured as phosphorylation of GST-c-Jun. Expression of HA-JNK, XIAP, Myc-DN deficient) were compared for their ability to associate with Smad4, and T7-DN TAK1 was confirmed by immunoblotting. Caspase Assays—293 cells were transiently transfected with 2 mgof XIAP in 293 cells. XIAP was found to interact equivalently with the indicated expression vector along with 0.5 mg of a Bax expression all versions of TbRI (Fig. 1B), indicating that the receptor need vector in a six-well plate. Following 16 h of incubation at 37 °C, the cells not be activated to interact with XIAP. were harvested and resuspended in 100 ml of caspase assay cell lysis Since there are currently no antibodies available to allow the buffer (BIOSOURCE, Camarillo, CA). These cells were lysed for 10 min detection of endogenous TbRI, immunofluorescence analysis at room temperature with gentle rocking, and the cell debris was was performed on HepG2 cells transfected with expression removed by centrifugation for 5 min at 14,000 rpm in an Eppendorf microcentrifuge. Protein concentrations were standardized, and vectors encoding both XIAP and HA-tagged TbRI, in order to caspase assays were performed using the ApoTarget protease assay confirm the interaction between TbRI and XIAP. Transfected (BIOSOURCE) according to the manufacturer’s specifications. Samples cells were visualized by confocal microscopy. XIAP and TbRI were read every 5 min for2hona Cytofluor 4000 fluorescence plate were observed to co-localize predominantly in the cytoplasm reader (Perseptive Biosystems, Framingham, MA) with an excitation (Fig. 2), although interestingly a small proportion of XIAP was wavelength of 400 nm and an emission wavelength of 508 nm. detected in the nucleus of transfected cells. These data provide RESULTS further evidence for an interaction between these proteins. XIAP Associates with the TGF-b RI Receptor—To explore the To further explore the possibility of nuclear localization or possibility that XIAP might associate with members of the translocation of XIAP, immunofluorescence analysis was per- TGF-b receptor superfamily, constitutively active mutant formed on several cell lines transfected with epitope-tagged members of the TGF-b type I receptor superfamily were co- XIAP and subsequently treated with either a media control, or transfected with a mammalian GST-XIAP expression vector media containing TGF-b. The subcellular localization of XIAP into human embryonic kidney 293 cells. Cell lysates were pre- was observed by confocal microscopy, and no change in local- cipitated with glutathione-Sepharose beads, and associated re- ization was seen upon treatment with TGF-b (data not shown). ceptors were detected by immunoblot analysis. XIAP was found To determine whether other IAP family members, in addi- to coprecipitate with several members of the receptor super- tion to XIAP, could interact with TbRI, coprecipitations were family, most notably TSR1 (ALK1), activin RIb (ALK4), and performed with several members of the IAP family (XIAP, TGF-b RI (ALK5) receptors (Fig. 1A). Weaker interactions were c-IAP1, and c-IAP2) and TbRI. TbRI was found to coprecipitate also observed between GST-XIAP and activin RI (ALK2) and with GST-XIAP, but not with the other IAP family members or BMP RIa (ALK3) receptors upon longer exposures (data not with the GST control vector (Fig. 3A). These data suggest that shown). The BMP RIb (ALK6) receptor did not coprecipitate TbRI associates specifically with XIAP. with XIAP. XIAP contains three N-terminal BIR domains and a C-ter- To determine whether the activation state of TbRI affected minal RING finger, separated by an amphipathic spacer region its interaction with XIAP, constitutively active (ALK5*), wild (6). The BIR domains are involved in caspase binding and type TbRI (ALK5), and kinase-deficient TbRI (ALK5 kinase- inhibition (17, 18, 47, 48), whereas the C-terminal RING finger XIAP Is a Cofactor in TGF-b Signaling 26545 FIG.3. Coprecipitation of IAPs with TbRI. Expression vectors for the indicated IAPs (A) or the indicated deletion constructs (B) were cotransfected with wild type HA-tagged TbRI into 293 cells. Coprecipi- tations were performed as described in the legend to Fig. 1. Expression of the GST-IAP proteins was confirmed by immunoblot analysis with an antibody specific for GST (data not shown). FIG.4. XIAP activates transcription from a TGF-b-responsive reporter. A, human embryonic kidney 293 cells, HepG2 cells, or HeLa can promote E2-dependent ubiquitination (13, 49 –51). To de- cells were transiently transfected with a control vector or XIAP along with the 3TP-Lux reporter and luciferase activity was determined after termine which domains of XIAP are involved in binding the 16 h. B, the indicated XIAP plasmids were cotransfected with the TbRI receptor, sequential deletions of the C-terminal domains 3TP-Lux reporter plasmid into 293 cells and luciferase activity was of XIAP fused to the mammalian GST protein were utilized in determined after 16 h. C, HepG2 cells were transiently transfected with coprecipitation experiments. A weak interaction was detected XIAP or a vector control, stimulated with TGF-b, and assayed for luciferase activity as described under “Experimental Procedures.” All between TbRI and the XIAP DRING construct, while a strong data are representative of at least three independent experiments, and interaction was observed between TbRI receptor and the 3xBIR all experiments were performed in triplicate. domain of XIAP, which lacks both the RING finger and the spacer region (Fig. 3B). These data suggest that the XIAP-TbRI interaction involves the BIR domain of XIAP. ducers in this pathway (33, 34). These Smad proteins, once XIAP Activates Transcription from TGF-b-responsive Pro- activated, form a complex with the common mediator of TGF-b moters—Since an interaction between XIAP and TbRI was signal transduction, Smad4 (29), which then translocates to the observed, the possibility was tested that XIAP might be in- nucleus and regulates gene expression. To investigate whether volved in regulating transcription from TGF-b-responsive pro- XIAP signaling was Smad4-dependent, 293 cells were trans- moters. Several cell lines were examined to determine whether fected with the 3TP-Lux reporter plasmids and either a control XIAP could activate a TGF-b-responsive reporter, 3TP-Lux, vector or XIAP, with or without a dominant negative mutant of which contains TGF-b-responsive elements of the plasminogen Smad4 (DN Smad4). The DN Smad4 mutation effectively activator inhibitor-1 (PAI-1) and collagenase promoters (31). blocked the activation of 3TP-Lux by XIAP (Fig. 5A), suggest- Transient transfection of XIAP into either 293 cells, HepG2 ing that XIAP transduces signals from TbRI through a Smad4- cells, or HeLa cells resulted in activation of the 3TP-Lux re- dependent pathway, and thus utilizes the known TGF-b porter (Fig. 4A), with the most significant activation seen in the signaling machinery. Similar results were obtained from co- 293 cell line. expression of XIAP and DN Smad4 in HepG2 cells (data not To determine which domains of XIAP are involved in activa- shown). tion of the 3TP-Lux reporter, 293 cells were transiently trans- Since XIAP has previously been identified as a TAB1-asso- fected with the 3TP-Lux reporter and expression vectors con- ciated protein (28), and since TAB1 is involved in activating taining HA-tagged XIAP or sequential C-terminal deletions of TAK1 (43, 52, 53), it seemed possible that the ability of XIAP to XIAP. Whereas the full-length XIAP activated transcription activate 3TP-Lux might require TAK1, as well as Smad4. To from the 3TP-Lux reporter, the DRING and 3xBIR constructs test this possibility, a dominant negative mutation of TAK1 were unable to do so (Fig. 4B), suggesting that the C-terminal (DN TAK1) that destroys its kinase activity (43, 52, 53) was RING domain of XIAP is required for this activity. utilized in 3TP-Lux reporter assays. XIAP activation of 3TP- The effect of XIAP on TGF-b-mediated activation of 3TP-Lux Lux was not inhibited by the expression of DN TAK1 in 293 was then examined by utilizing the HepG2 cell line since this cells (Fig. 5B). Therefore, XIAP-dependent activation of 3TP- line is known to express TbRI. TGF-b stimulation activated Lux expression requires Smad4, but is independent of TAK1. transcription directed by the 3TP-Lux reporter plasmid ;5-fold Activation of JNK by XIAP Is Both Smad4- and TAK1-de- (Fig. 4C), whereas transient transfection with XIAP in addition pendent—Since XIAP utilizes a Smad-dependent pathway to to TGF-b stimulation activated transcription ;20-fold. These activate TGF-b-regulated gene transcription, the DN Smad4 data suggest that XIAP and TGF-b can cooperatively activate expression vector was tested for its effect on XIAP-mediated transcription of TGF-b-responsive genes. JNK activation. DN Smad4 abrogated JNK activation by XIAP Activation of 3TP-Lux by XIAP Is Smad4-dependent, but (Fig. 6A), indicating that XIAP activates JNK in a Smad-de- TAK1-independent—TGF-b-mediated signal transduction is pendent manner. initiated by phosphorylation of TbRI by TbRII in response to To determine the role of TAK1 on XIAP-induced JNK acti- ligand binding (31). The activated receptor complex can then vation, DN TAK1 was coexpressed with XIAP, and lysates were activate one of several pathway-specific Smad proteins, which evaluated for JNK activity. Interestingly, DN TAK1 blocked have recently been identified as the main cytoplasmic trans- the activation of JNK by XIAP (Fig. 6B), indicating that TAK1 26546 XIAP Is a Cofactor in TGF-b Signaling is involved in JNK activation by XIAP. These data suggest that XIAP are involved in the activation of TGF-b-responsive gene JNK activation can be distinguished from activation of the activity and JNK activation. 3TP-Lux reporter by XIAP, because DN TAK1 did not inhibit XIAP Activates NF-kB-dependent Transcription through a XIAP-induced 3TP-Lux activation (Fig. 5B). Smad-dependent, but TAK1-independent Pathway—A recent report indicated that TGF-b transactivates kB sites in a Smad- Because the activation of JNK by XIAP was inhibited by DN TAK1, and TAK1 is known to activate the p38 pathway (54), dependent manner (55). Therefore, XIAP was examined to de- termine whether its ability to activate NF-kB was also Smad- the effects of the p38 inhibitors, SB203580 and SB202190, on JNK activation by XIAP were tested (Fig. 6C). Neither inhibi- dependent. XIAP was co-expressed in 293 cells with a reporter construct containing tandem kB sites (2kB-luc), with or with- tor blocked the activation of JNK by XIAP. Rather, each p38 out the DN Smad4 expression vector. Activation of NF-kBby inhibitor augmented XIAP’s ability to activate JNK. These data XIAP was blocked by co-expression of DN Smad4 (Fig. 7A), suggest that JNK activation by XIAP is not the result of p38 suggesting that XIAP activates kB-dependent gene expression activation. in a Smad-dependent manner. To further characterize this To determine which domains of XIAP are involved in the pathway, the effects of DN TAK1 on the activation of 2kB-luc activation of JNK, C-terminal deletions of XIAP were tested for by XIAP were evaluated. Interestingly, DN TAK1 did not in- their ability to activate JNK. The DRING and 3xBIR constructs hibit the activation of 2kB-luc by XIAP (Fig. 7B). These data were able to activate JNK to levels equal to that of full-length are similar to results obtained with the 3TP-Lux reporter, in XIAP, indicating that the BIR domains of XIAP are necessary that activation of both reporters by XIAP can be blocked by and sufficient for JNK activation (Fig. 6D). These data also co-expression with DN Smad4 but not DN TAK1 (Fig. 5). These suggest that, although the C-terminal RING finger domain of data also support the observation that the ability of XIAP to XIAP is necessary for activation of the 3TP-lux reporter, it is activate transcription is separate from that of JNK activation, not required for activation of JNK. Thus, distinct domains of because DN TAK1 blocked JNK activation by XIAP (Fig. 6B). To determine the domains of XIAP required for the activa- tion of NF-kB, luciferase activity was evaluated from lysates of 293 cells transfected with the 2kB-luc reporter plasmid and either full-length XIAP or the DRING and 3xBIR mutants (Fig. 7C). Full-length XIAP significantly activated the 2kB-luc re- porter, but neither the DRING nor the 3xBIR deletion mutants were capable of this transactivation. These data are similar to those obtained for the 3TP-Lux reporter and suggest that the C-terminal domains of XIAP are required for transcriptional activation of either promoter by this protein. XIAP Does Not Directly Associate with Smad4 —To test for a possible interaction between XIAP and Smad4, 293 cells were transfected with epitope-tagged Smad4 and GST-XIAP. XIAP is known to associate with itself; therefore, XIAP was used as a control for these experiments. Coprecipitations were per- formed, and Smad4 did not coprecipitate with GST-XIAP or with the GST control vector, whereas XIAP coprecipitated with GST-XIAP (Fig. 8). These data suggest that XIAP does not FIG.5. Activation of TGF-b-directed gene expression by XIAP is blocked by DN Smad4, but not by DN TAK1. The indicated directly associate with Smad4. However, an indirect associa- expression vectors were cotransfected with 3TP-Lux reporter plasmid tion between these proteins cannot be ruled out. into 293 cells, and luciferase activity was determined after 16 h. A, The Anti-apoptotic Properties of XIAP Are Independent of effect of DN Smad4 on activation of 3TP-Lux by XIAP. B, effect of DN Both Smad4 and TAK1—To investigate whether the anti-ap- TAK1 on activation of 3TP-lux by XIAP. All data are representative of at least three independent experiments, and all experiments were per- optotic properties of XIAP are dependent on either Smad4 or formed in triplicate. TAK1, the ability of XIAP to suppress Bax-induced apoptosis FIG.6. XIAP activation of JNK is inhibited by both DN Smad4 and DN TAK1. The indicated expression vectors were cotransfected with HA-JNK into 293 cells, and Jun kinase activity of JNK immunoprecipitates was assayed using a GST-c-Jun-(1–79) substrate (top panel)as described under “Experimental Procedures.” Lysates were immunoblotted with an anti-HA antibody (middle panel) to confirm equal loading. Complexes were evaluated by phosphorimage analysis (lower panel). XIAP was coexpressed with DN Smad4 (A) or DN TAK1 (B), or was treated with 50 mM SB203580 or 30 mM SB202190 for 24 h after transfection to block p38 (C), or alternatively the indicated XIAP deletion mutants were tested (D). All data are representative of at least three independent experiments. XIAP Is a Cofactor in TGF-b Signaling 26547 FIG.7. XIAP activates NF-kB in a Smad4-dependent manner. The indicated expression vectors were cotransfected with a kB lucifer- ase reporter plasmid, and reporter activity was measured after 16 h. XIAP was coexpressed with DN Smad4 (A), DN TAK1 (B), or the indicated XIAP deletion mutants were tested (C). All data are repre- sentative of at least three independent experiments, and each experi- ment was performed in triplicate. FIG.8. XIAP does not co-precipitate with Smad4. The indicated expression vectors were transiently transfected into 293 cells, and co- precipitations were performed with glutathione-Sepharose beads fol- lowed by immunoblotting with a mixture of anti-Myc and anti-HA antibodies. The expression of the GST fusion proteins was also con- firmed by using an anti-GST antibody (data not shown). was examined in the presence of DN Smad4 or DN TAK1. Human embryonic kidney 293 cells were transfected with ex- pression vectors encoding Bax, a pro-apoptotic member of the Bcl-2 family (56), along with XIAP and DN Smad4 or DN TAK1. Cell lysates were prepared, and the ability of XIAP to FIG.9. Inhibition of caspase enzymatic activity by XIAP is inhibit caspase activity was evaluated with a fluorogenic sub- unaffected by DN Smad4 or DN TAK1. The indicated plasmids were strate AFC-DEVD as a measure of caspase activity (Fig. 9). cotransfected into 293 cells and assayed for caspase activity as de- scribed under “Experimental Procedures.” Samples were measured ev- Neither DN Smad4 (Fig. 9A) nor DN TAK1 (Fig. 9B) had any ery 5 min for a total of 2 h. Open symbols represent the control trans- affect on the ability of XIAP to inhibit caspase activity, sug- fection, whereas closed symbols represent cells cotransfected with Bax. gesting that the anti-apoptotic properties of XIAP, at least A, 293 cells cotransfected with XIAP either with or without HA-DN against Bax-induced death, are independent of its roles in both Smad4. E and l, control transfection; M and f, XIAP alone; ‚ and Œ, XIAP with DN Smad4. B, 293 cells cotransfected with XIAP either with, JNK activation and TGF-b-regulated signal transduction. or without T7-DN TAK1. E and l, vector transfection; M and f, XIAP DISCUSSION alone; ƒ and , XIAP with DN TAK1. Although the IAP gene family was initially discovered based way-specific Smad protein (33). This phosphorylated Smad can on the antiapoptotic properties of several of its members (7, 8), then associate with the common mediator, Smad4, which is a number of subsequent findings raised the possibility that involved in signaling through all pathways within the TGF-b they may play multiple roles within the cell. The data pre- superfamily. This complex then translocates to the nucleus, sented here reveal an involvement of XIAP in signal transduc- where it participates in various transcriptional complexes of tion mediated by TGF-b. XIAP was found to co-localize (Fig. 2) specific DNA binding ability (30, 35, 57). and to associate with the TGF-b type I receptor, as well as Several TGF-b-responsive promoters have been identified. other members of the TGF-b receptor superfamily, including These include PAI-1 and fibronectin (58, 59), as well as collagen ALK1 and ALK4 (Fig. 1A). Moreover, the ability to interact type I and type VII (60, 61). Smad-binding elements have been with these receptors was unique to XIAP and not shared by the identified in several of these promoters, including the PAI-1 related apoptotic inhibitors c-IAP1 and c-IAP2 (Fig. 3A). This promoter. However, Smads have a relatively low binding affin- superfamily of receptor serine/threonine kinases is activated by ity and specificity and frequently regulate transcription by TGF-b, activins, and BMPs, as well as other ligands, and uti- functional cooperation with various transcription factors bound lizes a common pathway for signaling that involves ligand- to adjacent sites, or by direct association with DNA-bound specific type II receptors, which recruit a type I receptor to the transcription factors. For example, Smad3 and Smad4 interact complex upon ligand binding. The type II receptor phosphoryl- with the Jun family of transcription factors and synergistically ates the type I receptor, which in turn phosphorylates a path- activate AP-1 promoter sequences (36, 62). Likewise, an NF-kB 26548 XIAP Is a Cofactor in TGF-b Signaling site, located in the 39 enhancer region of junB, has been iden- required the BIR domain, but did not require the RING finger or spacer regions (Fig. 6B). However, these same deletions tified as a TGF-b-responsive site (55). Activation of this site by TGF-b required an intact NF-kB pathway and was mediated by revealed that both the activation of 3TP-Lux, the TGF-b-re- sponsive reporter, and the activation of NF-kB by XIAP re- Smad family members through direct interactions between quired the C-terminal RING and spacer regions, as deletion of Smad proteins and the NF-kB subunit. these regions completely abrogated these functions. These data Data presented here suggest that XIAP is involved not only suggest that XIAP is multifunctional, with the N-terminal BIR in the activation of a TGF-b-responsive promoter, 3TP-Lux domains participating in receptor association and JNK activa- (Fig. 4), which contains both PAI-1 and collagenase promoter tion, and the C-terminal RING finger and spacer being in- elements (31), but also in the activation of both JNK and NF-kB volved in the activation of NF-kB and TGF-b-mediated (Fig. 6), transcriptional mediators of TGF-b signaling. Activa- transcription. tion of all three pathways was efficiently blocked by co-expres- Further evidence for the multifunctionality of XIAP comes sion of DN Smad4 along with XIAP, indicating that XIAP from the fact that, although its signaling properties were all utilizes a Smad-dependent pathway for activation of not only a Smad4-dependent, its ability to inhibit the enzymatic activity TGF-b-responsive promoter (Fig. 5A), but also of JNK (Fig. 6A) of caspases was completely independent of Smad4 (Fig. 9A). and NF-kB (Fig. 7A). These data suggest a role for XIAP at a These data were obtained by using Bax to induce apoptosis in point upstream of Smad4 signaling. 293 cells. Bax is known to promote the release of cytochrome c Since XIAP activated these signaling pathways in a Smad4- from the mitochondria, which, along with Apaf-1 and ATP/ dependent manner, it is possible that overexpression of wild dATP, catalyzes the processing of caspase-9 (65, 66). Recently, type Smad4 would enhance this activation. However, data sug- TGF-b was found to activate the caspase cascade in a similar gest that it is the amount of activated Smad4 present in the manner, through the release of cytochrome c and subsequent cell, and not the absolute levels of protein that affect Smad-de- processing of caspase-9 (67). XIAP has been shown to bind to, pendent pathways (36, 63, 64). Also, the cell lines utilized in and inhibit the enzymatic activity of caspases-3, -7, and -9, all this study express very high levels of endogenous Smad4, and of which are activated by Bax as well as by TGF-b (67). There- it is therefore unlikely that Smad4 is limiting in any of these fore, these functions are separable in that the signaling prop- responses. Therefore, simple overexpression of Smad4 did not erties are Smad4-dependent, whereas the anti-apoptotic prop- enhance signaling (data not shown). erties are Smad4-independent. Recent studies have revealed the existence of both Smad-de- Recent reports have described the identification of a negative pendent and Smad-independent pathways of JNK activation, regulator of XIAP function, Smac/DIABLO (68, 69), which has in response to TGF-b (37). In the primary, Smad-independent been shown to bind to XIAP and prevent its caspase inhibitory pathway, JNK activity peaked within 10 min following stimu- function (70). It will be of great interest to determine whether lation with TGF-b, whereas in the secondary, Smad-dependent Smac/DIABLO is capable of regulating the function of XIAP in pathway, JNK activity exhibited a slow, sustained peak over the TGF-b signaling pathway. 12–16 h (37). Because the results presented in Fig. 6A were Taken together, these data place XIAP at a central location obtained by cotransfection of XIAP and DN Smad4 and cells for coordinating signaling from the TGF-b type I receptor for were harvested ;18 h after transfection, the possibility that the activation of transcription, as well as the activation of both XIAP may also activate JNK through the Smad-independent JNK and NF-kB, co-factors involved in transcribing subsets of pathway cannot be eliminated. TGF-b-responsive genes, since each of these activities is de- Interestingly, the activation of JNK by XIAP was inhibited pendent on Smad4. Since no association was observed between by DN TAK1 as well as by DN Smad4 (Fig. 6, A and B), whereas XIAP and Smad4 (Fig. 8), XIAP could be a signaling co-factor both activation of NF-kB (Fig. 7B) and the TGF-b-responsive that is involved indirectly in the activation of Smad4, perhaps promoter (Fig. 5B) were TAK1-independent. TAK1 is an up- through association with another Smad protein, or other sig- stream MAP3K that activates the p38 pathway (54) and has naling intermediates. Further studies will be required to de- also been shown to be a mediator of TGF-b-dependent signaling termine the mechanisms of these functional interactions. (43, 52). Data presented here show that the ability of XIAP to Acknowledgments—We thank Drs. B. Mayer, J. Massague ´ , M. de activate JNK does not require the p38 pathway, despite the fact Caestecker, J. Wrana, L. Attisano, S. Korsmeyer, and M. G. Sanna for that DN TAK1 abrogates this activity (Fig. 6C). TAK1 has plasmids. We also thank L. Eiben, B. Richter, and J. Lewis for critical previously been reported to indirectly associate with XIAP reading of the manuscript. through mutual interaction with TAB1, the TAK1-binding pro- REFERENCES tein (28). Subsequently, XIAP was found to activate NF-kBina 1. Kerr, J. F., Wyllie, A. H., and Currie, A. R. (1972) Br. J. Cancer 26, 239 –257 TAK1-dependent manner (20) in endothelial cells. In 293 cells, 2. Vaux, D. L., Haecker, G., and Strasser, A. (1994) Cell 76, 777–779 however, XIAP was found to activate NF-kB independent of 3. Nagata, S. (1996) Curr. Biol. 6, 1241–1243 4. Salvesen, G. S., and Dixit, V. M. (1997) Cell 91, 443– 446 TAK1 (Fig. 7B), and a recent report described the activation of 5. Thornberry, N. A., and Lazebnik, Y. (1998) Science 281, 1312–1316 NF-kB-directed gene expression by TGF-b in a TAK1-inde- 6. Deveraux, Q. L., and Reed, J. C. (1999) Genes Dev. 13, 239 –252 pendent fashion (55), suggesting the existence of cell type var- 7. Crook, N. E., Clem, R. J., and Miller, L. K. (1993) J. Virol. 67, 2168 –2174 8. Miller, L. K. (1999) Trends Cell Biol. 9, 323–328 iation of this pathway. 9. Duckett, C. S., Nava, V. E., Gedrich, R. W., Clem, R. J., Van Dongen, J. L., XIAP contains two distinct structural motifs found in several Gilfillan, M. C., Shiels, H., Hardwick, J. M., and Thompson, C. B. (1996) EMBO J. 15, 2685–2694 cellular IAPs. Three imperfectly repeated BIR domains are 10. Hawkins, C. J., Uren, A. G., Ha ¨ cker, G., Medcalf, R. L., and Vaux, D. L. (1996) contained in the N terminus of XIAP. The BIR is the defining Proc. Natl. Acad. Sci. U. S. A. 93, 13786 –13790 11. Uren, A. G., Coulson, E. J., and Vaux, D. L. (1998) Trends Biochem. Sci. 23, motif of the IAP family and is involved in caspase binding and 159 –162 inhibition (6). The second motif is the C-terminal RING finger 12. Vucic, D., Stennicke, H. R., Pisabarro, M. T., Salvesen, G. S., and Dixit, V. M. domain, which has been implicated in E2-dependent ubiquitin (2000) Curr. Biol. 10, 1359 –1366 13. Joazeiro, C. A., and Weissman, A. M. (2000) Cell 102, 549 –552 conjugation (13). Deletion analysis of XIAP revealed that the 14. Liston, P., Roy, N., Tamai, K., Lefebvre, C., Baird, S., Cherton-Horvat, G., BIR domain is involved in the association between XIAP and Farahani, R., McLean, M., Ikeda, J.-E., MacKenzie, A., and Korneluk, R. G. (1996) Nature 379, 349 –353 the TbRI receptor, since deletions lacking the RING finger or 15. Uren, A., Pakusch, M., Hawkins, C., Puls, K. L., and Vaux, D. L. (1996) Proc. both the RING and spacer regions were still able to associate Natl. Acad. Sci. U. S. A. 93, 4974 – 4978 with this receptor (Fig. 3B). Likewise, JNK activation by XIAP 16. Duckett, C. S., Li, F., Wang, Y., Tomaselli, K. J., Thompson, C. B., and XIAP Is a Cofactor in TGF-b Signaling 26549 Armstrong, R. C. (1998) Mol. Cell. Biol. 18, 608 – 615 44. Perkins, N. D., Agranoff, A. B., Duckett, C. S., and Nabel, G. J. (1994) J. Virol. 17. Deveraux, Q. L., Takahashi, R., Salvesen, G. S., and Reed, J. C. (1997) Nature 68, 6820 – 6823 388, 300 –304 45. Duckett, C. S., and Thompson, C. B. (1997) Genes Dev. 11, 2810 –2821 18. Roy, N., Deveraux, Q. L., Takahashi, R., Salvesen, G. S., and Reed, J. C. (1997) 46. Bradford, M. M. (1976) Anal. Biochem. 72, 248 –254 EMBO J. 16, 6914 – 6925 47. Deveraux, Q. L., Leo, E., Stennicke, H. R., Welsh, K., Salvesen, G. S., and 19. Sanna, M. G., Duckett, C. S., Richter, B. W. M., Thompson, C. B., and Ulevitch, Reed, J. C. (1999) EMBO J. 18, 5242–5251 R. J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 6015– 6020 48. Takahashi, R., Deveraux, Q., Tamm, I., Welsh, K., Assa-Munt, N., Salvesen, 20. Hofer-Warbinek, R., Schmid, J. A., Stehlik, C., Binder, B. R., Lipp, J., and de G. S., and Reed, J. C. (1998) J. Biol. Chem. 273, 7787–7790 Martin, R. (2000) J. Biol. Chem. 275, 22064 –22068 49. Huang, H.-K., Joazeiro, C. A. P., Bonfoco, E., Kamada, S., Leverson, J. D., and 21. Rothe, M., Wong, S. C., Henzel, W. J., and Goeddel, D. V. (1994) Cell 78, Hunter, T. (2000) J. Biol. Chem. 275, 26661–26664 681– 692 50. Yang, Y., Fang, S., Jensen, J. P., Weissman, A. M., and Ashwell, J. D. (2000) 22. Rothe, M., Pan, M.-G., Henzel, W. J., Ayres, T. M., and Goeddel, D. V. (1995) Science 288, 874 – 877 Cell 83, 1243–1252 51. Lorick, K. L., Jensen, J. P., Fang, S., Ong, A. M., Hatakeyama, S., and 23. Liu, Z. G., Hsu, H., Goeddel, D. V., and Karin, M. (1996) Cell 87, 565–576 Weissman, A. M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11364 –11369 24. Rothe, M., Sarma, V., Dixit, V. M., and Goeddel, D. V. (1995) Science 269, 52. Shibuya, H., Yamaguchi, K., Shirakabe, K., Tonegawa, A., Gotch, Y., Ueno, N., 1424 –1427 Irie, K., Nishida, E., and Matsumoto, K. (1996) Science 272, 1179 –1182 25. Natoli, G., Costanzo, A., Ianni, A., Templeton, D. J., Woodgett, J. R., Balsano, 53. Shibuya, H., Iwata, H., Masuyama, N., Gotoh, Y., Yamaguchi, K., Irie, K., C., and Levrero, M. (1997) Science 275, 200 –203 Matsumoto, K., Nishida, E., and Ueno, N. (1998) EMBO J. 17, 1019 –1028 26. Deveraux, Q. L., Roy, N., Stennicke, H. R., Van Arsdale, T., Zhou, Q., 54. Hanafusa, H., Ninomiya-Tsuji, J., Masuyama, N., Nishita, M., Fujisawa, J., Srinivasula, S. M., Alnemri, E. S., Salvesen, G. S., and Reed, J. C. (1998) Shibuya, H., Matsumoto, K., and Nishida, E. (1999) J. Biol. Chem. 274, EMBO J. 17, 2215–2223 27161–27167 27. Oeda, E., Oka, Y., Miyazono, K., and Kawabata, M. (1998) J. Biol. Chem. 273, 55. Lo ´ pez-Rovira, T., Chalaux, E., Rosa, J. L., Bartrons, R., and Ventura, F. (2000) 9353–9356 J. Biol. Chem. 275, 28937–28946 28. Yamaguchi, K., Nagai, S., Ninomiya-Tsuji, J., Nishita, M., Tamai, K., Irie, K., 56. Oltvai, Z. N., Milliman, C. L., and Korsmeyer, S. J. (1993) Cell 74, 609 – 619 Ueno, N., Nishida, E., Shibuya, H., and Matsumoto, K. (1999) EMBO J. 18, 57. Zawel, L., Dai, J. L., Buckhaults, P., Zhou, S., Kinzler, K. W., Vogelstein, B., 179 –187 and Kern, S. E. (1998) Mol. Cell. 1, 611– 617 29. Wrana, J. L. (2000) Cell 100, 189 –192 58. Dennler, S., Itoh, S., Vivien, D., ten Dijke, P., and Gauthier, J.-M. (1998) 30. Massague ´ , J., and Chen, Y. G. (2000) Genes Dev. 14, 627– 644 EMBO J. 17, 3091–3100 31. Wrana, J. L., Attisano, L., Carcamo, J., Zentella, A., Doody, J., Laiho, M., 59. Hocevar, B. A., Brown, T. L., and Howe, P. H. (1999) EMBO J. 18, 1345–1356 Wang, X. F., and Massague ´ , J. (1992) Cell 71, 1003–1014 60. Chung, K.-Y., Agarwal, A., Uitto, J., and Mauviel, A. (1996) J. Biol. Chem. 271, 32. Carcamo, J., Zentella, A., and Massague ´ , J. (1995) Mol. Cell. Biol. 15, 3272–3278 1573–1581 61. Vindevoghel, L., Kon, A., Lechleider, R. J., Uitto, J., Roberts, A. B., and 33. Heldin, C. H., Miyazono, K., and ten Dijke, P. (1997) Nature 390, 465– 471 Mauviel, A. (1998) J. Biol. Chem. 273, 13053–13057 34. Derynck, R., Zhang, Y., and Feng, X. H. (1998) Cell 95, 737–740 62. Liberati, N. T., Datto, M. B., Frederick, J. P., Shen, X., Wong, C., Rougier- 35. Jonk, L. J., Itoh, S., Heldin, C. H., ten Dijke, P., and Kruijer, W. (1998) J. Biol. Chapman, E. M., and Wang, X. F. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, Chem. 273, 21145–21152 4844 – 4849 36. Zhang, Y., Feng, X. H., and Derynck, R. (1998) Nature 394, 909 –913 63. Feng, X. H., Lin, X., and Derynck, R. (2000) EMBO J. 19, 5178 –5193 37. Engel, M. E., McDonnell, M. A., Law, B. K., and Moses, H. L. (1999) J. Biol. 64. Zhang, Y., and Derynck, R. (2000) J. Biol. Chem. 275, 16979 –16985 Chem. 274, 37413–37420 65. Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 38. Karin, M. (1995) J. Biol. Chem. 270, 16483–16486 405– 413 39. Yang, D., Tournier, C., Wysk, M., Lu, H.-T., Xu, J., Davis, R. J., and Flavell, 66. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, R. A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3004 –3009 E. S., and Wang, X. (1997) Cell 91, 479 – 489 40. Cheng, G., and Baltimore, D. (1996) Genes Dev. 10, 963–973 67. Freathy, C., Brown, D. G., Roberts, R. A., and Cain, K. (2000) Hepatology 32, 41. Mayer, B. J., Hirai, H., and Sakai, R. (1995) Curr. Biol. 5, 296 –305 750 –760 42. Duckett, C. S., Gedrich, R. W., Gilfillan, M. C., and Thompson, C. B. (1997) 68. Du, C., Fang, M., Li, Y., Li, L., and Wang, X. (2000) Cell 102, 33– 42 Mol. Cell. Biol. 17, 1535–1542 69. Verhagen, A. M., Ekert, P. G., Pakusch, M., Silke, J., Connolly, L. M., Reid, 43. Yamaguchi, K., Shirakabe, K., Shibuya, H., Irie, K., Oishi, I., Ueno, N., G. E., Moritz, R. L., Simpson, R. J., and Vaux, D. L. (2000) Cell 102, 43–53 Taniguchi, T., Nishida, E., and Matsumoto, K. (1995) Science 270, 70. Srinivasula, S. M., Datta, P., Fan, X. J., Fernandes-Alnemri, T., Huang, Z., and 2008 –2011 Alnemri, E. S. (2000) J. Biol. Chem. 275, 36152–36157 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

X-linked Inhibitor of Apoptosis Protein Functions as a Cofactor in Transforming Growth Factor-β Signaling

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 28, Issue of July 13, pp. 26542–26549, 2001 Printed in U.S.A. X-linked Inhibitor of Apoptosis Protein Functions as a Cofactor in b Signaling* Transforming Growth Factor- Received for publication, January 12, 2001, and in revised form, April 26, 2001 Published, JBC Papers in Press, May 16, 2001, DOI 10.1074/jbc.M100331200 Stephanie Birkey Reffey‡, Jens U. Wurthner§, W. Tony Parks§, Anita B. Roberts§, and Colin S. Duckett‡¶ From the ‡Metabolism Branch and the §Laboratory of Cell Regulation and Carcinogenesis, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892 X-linked inhibitor of apoptosis protein (XIAP) is a po- The IAPs were first discovered in the genomes of baculovi- ruses, where they were found to maintain viability of virus tent suppressor of apoptotic cell death, which functions by directly inhibiting caspases, the principal effectors of infected cells and thus enhance virus replication (7, 8). Certain apoptosis. Here we report that XIAP can also function as baculovirus IAPs can function in mammalian cells to suppress a cofactor in the regulation of gene expression by trans- apoptosis (9, 10), and IAP-related genes have subsequently b (TGF-b). XIAP, but not the re- forming growth factor- been identified in many metazoan genomes (11), indicating a lated proteins c-IAP1 or c-IAP2, associated with several high degree of evolutionary conservation. IAPs are defined by a b receptor super- members of the type I class of the TGF- domain of ;70 amino acids known as the baculovirus IAP b-induced signaling. Al- family and potentiated TGF- repeat (BIR) (7, 11). Members of the IAP family contain one to though XIAP-mediated activation of c-Jun N-terminal three imperfect BIR repeats arranged in tandem. These BIR kB was found to require the kinase and nuclear factor domains are required for the ability of IAPs to suppress apo- b signaling intermediate Smad4, the ability of XIAP TGF- ptosis, and have been shown to bind caspases directly (6). The to suppress apoptosis was found to be Smad4-independ- mammalian IAP family (6) includes c-IAP1, c-IAP2, X-linked b-me- ent. These data implicate a role for XIAP in TGF- IAP (XIAP), neuronal apoptosis inhibitory protein (NAIP), sur- diated signaling that is distinct from its anti-apoptotic vivin, BRUCE, and ML-IAP (12). Of these, c-IAP1, c-IAP2, and functions. XIAP exhibit the most structural homology, possessing three tandem BIR repeats and a C-terminal RING finger, which is involved in ubiquitin conjugation (13). Apoptosis is an evolutionarily conserved process that plays a XIAP (14), also known as ILP (9), or MIHA (15), is a broad- critical role during development and tissue homeostasis, and ranging suppressor of apoptosis (14, 16). XIAP has been shown also serves to remove damaged or extraneous cells from an to bind to and directly inhibit the activity of specific caspases organism (1–3). Apoptotic cells undergo a regulated autodiges- (17, 18). However, emerging data suggest that the cellular tion, which involves the disruption of cytoskeletal integrity, cell activities of XIAP are not limited to caspase binding and inhi- shrinkage, nuclear condensation, and the activation of endo- bition. A previous report (19) implicated XIAP in the regulation nucleases. The chief effectors of the apoptotic cell death path- of the stress-induced kinase, c-Jun N-terminal kinase (JNK). way are the caspase family of cysteine proteases. Caspases are Similarly, XIAP has recently been identified as an activator of synthesized as inactive precursors that are cleaved at specific nuclear factor kB (NF-kB), a pleiotropic transcription factor aspartate residues to generate the active subunits. Zymogen that regulates expression of a range of acute phase and imme- cleavage can occur by several mechanisms including proximity- diate-early genes (20). Other members of the IAP family have induced autoprocessing or cleavage by other caspases, reveal- also been implicated in signal transduction. For example, c- ing a caspase cascade with upstream initiator caspases such as IAP1 and c-IAP2 associate with the type 2 tumor necrosis caspases -8, -9, and -10 and downstream, effector caspases, factor receptor (TNFR2) signaling machinery (21, 22) through such as caspases-3, -6, and -7 (4, 5). The activity of these physical interactions with members of the TNFR-associated caspases is regulated by several families of both pro- and anti- factors (TRAFs). The TRAFs and c-IAPs are therefore thought apoptotic cellular proteins including the inhibitor of apoptosis 1 to be signal transduction intermediates that are involved in (IAP) proteins (6). JNK and NF-kB activation (23–25). However, XIAP does not interact with any of the known TRAF proteins or with any * The costs of publication of this article were defrayed in part by the other components of the TNFR2 signaling pathway (16, 26), payment of page charges. This article must therefore be hereby marked suggesting that XIAP plays a role in the cell that is separate “advertisement” in accordance with 18 U.S.C. Section 1734 solely to from those of c-IAP1 or c-IAP2. indicate this fact. Several members of the IAP family have been reported to be ¶ To whom correspondence should be addressed: Metabolism Branch, involved in signaling cascades that are unrelated to the tumor Center for Cancer Research, NCI, National Institutes of Health, 10 Center Dr., Room 6B-05, Bethesda, MD 20892-1578. Tel.: 301-594-1127; necrosis factor pathway. For example, the Drosophila IAPs, E-mail: [email protected]. dIAP-1 and dIAP-2/dILP, are known to interact with Thick- The abbreviations used are: IAP, inhibitor of apoptosis protein; veins (Tkv), a type I serine-threonine kinase receptor homolo- XIAP, X-linked inhibitor of apoptosis protein; GST, glutathione S-trans- gous to the bone morphogenetic protein (BMP) type I receptor ferase; TGF-b, transforming growth factor-b;TbRI , transforming growth factor-b type I receptor; HA, hemagglutinin; BIR, baculovirus inhibitor of apoptosis protein repeat; TBS-T, Tris-buffered saline plus Tween 20; FBS, fetal bovine serum; DN, dominant negative; PAI-1, nuclear factor kB; TRAF, tumor necrosis factor receptor-associated plasminogen activator inhibitor-1; TNFR, tumor necrosis factor recep- factor; MOPS, 4-morpholinepropanesulfonic acid; PBS, phosphate-buff- tor; EMEM, Eagle’s medium containing Earle’s salts; DMEM, Dulbec- ered saline; TAB1, transforming growth factor-b-activated kinase-bind- co’s modified Eagle’s medium; JNK, c-Jun N-terminal kinase; NF-kB, ing protein 1; BMP, bone morphogenetic protein. 26542 This paper is available on line at http://www.jbc.org This is an open access article under the CC BY license. XIAP Is a Cofactor in TGF-b Signaling 26543 sequenced in its entirety and found to be identical to human TAK1a (27). XIAP has been implicated in BMP signaling (28) as a (43). The K63W dominant negative mutant (DN TAK1) was constructed bridging molecule between the BMP type I receptor and a by site-directed mutagenesis using a QuikChange mutagenesis kit downstream signaling molecule, TGF-b-activated kinase-bind- (Stratagene, La Jolla, CA), and was confirmed by sequencing. ing protein 1 (TAB1). Cells and Transfections—Human embryonic kidney 293 cells and The TGF-b superfamily encodes a group of cytokines that HeLa (human adenocarcinoma) cells were maintained in Dulbecco’s includes BMP, TGF-b, and the activins/inhibins, and is in- modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS). HepG2 cells (ATCC, Manassas, VA) were maintained in Eagle’s me- volved in diverse cellular responses such as apoptosis, differ- dium containing Earle’s salts (EMEM) with 10% FBS. All media were entiation, and cell cycle arrest (29, 30). Signaling initiated by supplemented with 2 mM glutamine, and all cells were maintained at this family of cytokines involves two classes of receptors. The 37 °C in 5% CO . type II receptors bind their specific ligand and then phospho- Transfection of 293 cells was performed by the calcium phosphate rylate a type I receptor (31, 32), which propagates the signal precipitation procedure as described previously (44). HepG2 cells were through a specific subset of the Smad family of signal trans- transfected using LipofectAMINE reagents (Life Technologies, Inc.) using 10 ml of LipofectAMINE with 200 ml of serum-free DMEM for each duction intermediates and transcription factors (33, 34). One of well of a six-well plate, for 5 h. Following transfection, the medium was these intermediate signaling proteins, Smad4, is common to all changed to EMEM with 10% FBS, and cells were incubated for 16 h. of these signaling pathways in that it serves as the obligatory The medium was then changed to EMEM 1 0.2% FBS with or without partner of the pathway-restricted Smad proteins and, together 5 ng/ml TGF-b and allowed to incubate for another 24 h. Transfection of with them, translocates to the nucleus to regulate gene expres- HeLa cells was performed as described above, except that 6 mlof sion by binding to cognate sites in the promoters of target genes LipofectAMINE was incubated for 3 h, and following transfection, the medium was changed to DMEM with 10% FBS and incubated for 16 h (35, 36). prior to harvest. Interestingly, both TGF-b and XIAP have been reported to Luciferase Assays—For 3TP-Lux reporter assays, cells were trans- specifically activate JNK (19, 37). Furthermore, Smad proteins fected with 200 ng/well reporter plasmid together with 2 mg/well indi- have been shown to cooperate with the AP-1 transcription cated expression vector, in a six-well plate. For 2kB-luc reporter assays, factor, a heterodimer of c-Fos and c-Jun that binds specific 50 ng/well reporter was transfected along with 2 mg/well indicated sequences in its target promoters (38). AP-1 activity is stimu- expression vectors. Cells were transfected for 6 – 8 h, after which time the medium was replaced with fresh DMEM 1 10% FBS and cells were lated by phosphorylation of the c-Jun transactivation domain incubated for 16 h. In all transfections, the amount of DNA was kept by JNK (38, 39). Moreover, recent data have shown a direct, constant by the addition of an empty control vector. TGF-b-inducible interaction between Smad3 and c-Jun (36). Cells were harvested by washing once in phosphate-buffered saline Because both TGF-b and XIAP can activate JNK, and the (PBS) and then lysing in 0.5 ml of 13 reporter lysis buffer (Promega, TGF-b and JNK signaling pathways are closely linked, we Madison, WI) according to the manufacturer’s specifications. Luciferase examined the possibility of a role for XIAP in TGF-b-mediated activity was quantified using the Luciferase Assay System (Promega, Madison, WI) on a Tropix TR717 microplate luminometer (PE Applied signaling. Biosystems, Bedford, MA). All assays were performed in triplicate, and Data presented here reveal the involvement of XIAP in sig- all data shown are representative of at least three independent naling through the TGF-b type I receptor (TbRI). XIAP was experiments. found to co-localize with TbRI and to interact with this recep- GST Coprecipitations—293 cells were transiently transfected with a tor. XIAP also activated transcription of both TGF-b- and kB- total of 10 mg of plasmid DNA in a 10-cm dish. Following transfection, responsive promoters. Furthermore, the signaling properties of cells were washed once in 5 ml of PBS and lysed for 10 min at room temperature in 1.0 ml of 1% Triton X-100 buffer (45). Following lysis, XIAP were found to be distinct from its anti-apoptotic proper- 400 ml of lysate were incubated with 20 ml of a 50% slurry of glutathi- ties in that signaling by XIAP was inhibited by a dominant one-Sepharose beads (Amersham Pharmacia Biotech) in Triton X-100 negative mutant of Smad4, whereas its anti-apoptotic proper- buffer for1hat4 °C. Beads were then washed four times with 1 ml of ties were unaffected by dominant negative Smad4. These data Triton X-100 lysis buffer and analyzed by immunoblotting. suggest that XIAP is involved in both caspase inhibition and Immunoblotting—Proteins were resolved by sodium dodecyl sulfate signaling through the TbRI receptor. (SDS) 4 –12% gradient gel electrophoresis in 13 MOPS buffer (Invitro- gen, Carlsbad, CA) and transferred to nitrocellulose membranes by EXPERIMENTAL PROCEDURES electrophoretic blotting transfer buffer (Invitrogen) containing 20% Plasmids—The pEBB expression vector has been described previ- methanol. The membrane was blocked for1hin Tris-buffered saline ously (40), and the pEBB-Flag, pEBB-HA, and pEBB-T7 expression with 0.2% Tween (TBS-T) and 5% milk. Proteins were visualized by vectors are all derivatives of pEBB that have been modified to incorpo- incubation with a 1:1000 dilution of primary antibody in TBS-T with 5% rate the appropriate epitope tag and translational termination codes in milk for 1 h, followed by secondary incubation with horseradish perox- all three reading frames. The full-length, DRING, and 3xBIR XIAP idase-conjugated secondary antibody (1:2000) in TBS-T with 5% milk vectors were generated by subcloning into pEBB or one of its epitope- for 1 h. The blot was washed twice for 10 min and once for 30 min in tagged derivatives, as described previously (16). The pEBG mammalian TBS-T and resolved using the enhanced chemiluminescence (ECL) glutathione S-transferase (GST) fusion vector (41) was kindly provided Western blotting detection system (Amersham Pharmacia Biotech). by Dr. B. Mayer. The GST-XIAP/hILP, GST-c-IAP1, and GST-c-IAP2 Primary antibodies used were anti-HA antibody (Mono HA.11; Co- fusion proteins have been previously described (16). Deletions of XIAP vance, Berkeley, CA), anti-GST (Santa Cruz, Santa Cruz, CA), anti-Myc were subcloned into pEBG in-frame with the GST reading frame to (9E10; Covance, Berkeley, CA), anti-T7 tag (horseradish peroxidase- generate pEBG-DRING and pEBG-3xBIR. conjugated; Novagen, Madison, WI), anti-hILP (H59520; Transduction The 2kB-luc reporter construct has been described previously (42). Laboratories, San Diego, CA). The 3TP-Lux reporter was kindly provided by Dr. J. Massague ´ . The Immunofluorescence—HepG2 cells were plated at 1 3 10 cells/well wild type and dominant negative Smad4 (DM4) constructs were gifts of onto sterilized glass coverslips (Corning, Charlotte, NC), incubated for Dr. M. de Caestecker. The HA-tagged expression vectors encoding wild 16 h, and transiently transfected with the indicated plasmids. Cells type, constitutively active (T204D), and kinase-deficient (K232R) TbRI were washed once in PBS, fixed in 3.5% paraformaldehyde for 5 min, receptors, as well as the constitutively active ALK mutants, were gifts permeabilized with 0.5% Triton X-100 in PBS for 10 min, washed once from Dr. J. Wrana and Dr. L. Attisano. The Bax expression vector was in PBS, and blocked in 10% goat serum in PBS for 30 min at room kindly provided by Dr. S. Korsmeyer. The HA-JNK expression vector temperature. Cells were then incubated for 30 min at room temperature was a gift from Dr. M. G. Sanna. with either of the following primary antibodies: anti-hILP/XIAP mouse The full-length open reading frame of TAK1 was amplified by monoclonal antibody (Transduction Laboratories, San Diego, CA) or polymerase chain reaction utilizing the following two primers: 59-ATA- rabbit anti-HA polyclonal antibody (1:200, sc-805, Santa Cruz, Santa GGATCCATGTCTACAGCCTCTGCCGCCTCC-39 and 59-ATTATCGA- Cruz, CA). Cells were washed three times with PBS prior to incubation TATTTCAAAATGTAACGGTCCCAGAGAATC-39 using standard con- with the second primary antibody and then incubated for 30 min with ditions. The polymerase chain reaction product was cloned into pCR2.1 the following secondary antibodies: fluorescein isothiocyanate-conju- (Invitrogen, Carlsbad, CA) and subcloned into pEBB-T7. This clone was gated goat anti-mouse antibody (1:1000, Kirkegaard & Perry, Gaithers- 26544 XIAP Is a Cofactor in TGF-b Signaling FIG.1. XIAP associates with the TGF-b RI receptor. The indi- cated expression vectors were transiently transfected into 293 cells and coprecipitations were performed with glutathione-Sepharose beads, fol- lowed by immunoblotting with anti-HA antibody. The expression of the GST fusion proteins was also confirmed by using an anti-GST antibody (data not shown). A, coprecipitation of XIAP with the constitutively active mutants (denoted by *) of the TGF-b receptor superfamily. B, coprecipitation of XIAP with wild type, constitutively active, or kinase- deficient versions of TbRI. burg, MD) or rhodamine-conjugated goat anti-rabbit antibody (1:1000, Jackson Immunoresearch, West Grove, PA) in PBS with 10% goat serum. Cells were mounted with medium containing 4,6-diamidino-2- phenylindole (Vectashield mounting medium H-1000, Vector Laborato- ries, Burlingame, CA) and then visualized with a Zeiss confocal microscope. Kinase Assays—293 cells were transiently transfected with 0.5 mgof FIG.2. XIAP colocalizes with TbRI. HepG2 cells were transfected HA-tagged JNK and 2 mg of the indicated plasmid in a six-well plate. with both XIAP and HA-TbRI expression vectors. Cells were immuno- Following 18 h of incubation, cells were lysed in 0.3 ml of M2 buffer (19) stained with either anti-hILP/XIAP (mouse) or anti-HA (rabbit) anti- on ice for 30 min. For assays involving p38 inhibitors, cells were treated bodies followed by secondary staining with either fluorescein isothio- with 50 mM SB203580 or 30 mM SB202190 (Calbiochem, La Jolla, CA) cyanate- or rhodamine-conjugated secondary antibodies as described and allowed to incubate for 24 h prior to lysis. Protein concentrations under “Experimental Procedures,” and were visualized by fluorescence were standardized by the method of Bradford (46) using a commercial microscopy for XIAP and/or TbRI localization. The subcellular localiza- kit (Bio-Rad) followed by immunoprecipitation of HA-JNK with protein tion of XIAP is shown in green (top left), TbRI in red (top right), and A-Sepharose and a monoclonal anti-HA antibody (12CA5, Roche Molec- areas of co-localization in yellow (bottom right). A representative field is ular Biochemicals) for1hat4 °C. In vitro kinase assays were per- shown. formed exactly as described (23). Activation of JNK was measured as phosphorylation of GST-c-Jun. Expression of HA-JNK, XIAP, Myc-DN deficient) were compared for their ability to associate with Smad4, and T7-DN TAK1 was confirmed by immunoblotting. Caspase Assays—293 cells were transiently transfected with 2 mgof XIAP in 293 cells. XIAP was found to interact equivalently with the indicated expression vector along with 0.5 mg of a Bax expression all versions of TbRI (Fig. 1B), indicating that the receptor need vector in a six-well plate. Following 16 h of incubation at 37 °C, the cells not be activated to interact with XIAP. were harvested and resuspended in 100 ml of caspase assay cell lysis Since there are currently no antibodies available to allow the buffer (BIOSOURCE, Camarillo, CA). These cells were lysed for 10 min detection of endogenous TbRI, immunofluorescence analysis at room temperature with gentle rocking, and the cell debris was was performed on HepG2 cells transfected with expression removed by centrifugation for 5 min at 14,000 rpm in an Eppendorf microcentrifuge. Protein concentrations were standardized, and vectors encoding both XIAP and HA-tagged TbRI, in order to caspase assays were performed using the ApoTarget protease assay confirm the interaction between TbRI and XIAP. Transfected (BIOSOURCE) according to the manufacturer’s specifications. Samples cells were visualized by confocal microscopy. XIAP and TbRI were read every 5 min for2hona Cytofluor 4000 fluorescence plate were observed to co-localize predominantly in the cytoplasm reader (Perseptive Biosystems, Framingham, MA) with an excitation (Fig. 2), although interestingly a small proportion of XIAP was wavelength of 400 nm and an emission wavelength of 508 nm. detected in the nucleus of transfected cells. These data provide RESULTS further evidence for an interaction between these proteins. XIAP Associates with the TGF-b RI Receptor—To explore the To further explore the possibility of nuclear localization or possibility that XIAP might associate with members of the translocation of XIAP, immunofluorescence analysis was per- TGF-b receptor superfamily, constitutively active mutant formed on several cell lines transfected with epitope-tagged members of the TGF-b type I receptor superfamily were co- XIAP and subsequently treated with either a media control, or transfected with a mammalian GST-XIAP expression vector media containing TGF-b. The subcellular localization of XIAP into human embryonic kidney 293 cells. Cell lysates were pre- was observed by confocal microscopy, and no change in local- cipitated with glutathione-Sepharose beads, and associated re- ization was seen upon treatment with TGF-b (data not shown). ceptors were detected by immunoblot analysis. XIAP was found To determine whether other IAP family members, in addi- to coprecipitate with several members of the receptor super- tion to XIAP, could interact with TbRI, coprecipitations were family, most notably TSR1 (ALK1), activin RIb (ALK4), and performed with several members of the IAP family (XIAP, TGF-b RI (ALK5) receptors (Fig. 1A). Weaker interactions were c-IAP1, and c-IAP2) and TbRI. TbRI was found to coprecipitate also observed between GST-XIAP and activin RI (ALK2) and with GST-XIAP, but not with the other IAP family members or BMP RIa (ALK3) receptors upon longer exposures (data not with the GST control vector (Fig. 3A). These data suggest that shown). The BMP RIb (ALK6) receptor did not coprecipitate TbRI associates specifically with XIAP. with XIAP. XIAP contains three N-terminal BIR domains and a C-ter- To determine whether the activation state of TbRI affected minal RING finger, separated by an amphipathic spacer region its interaction with XIAP, constitutively active (ALK5*), wild (6). The BIR domains are involved in caspase binding and type TbRI (ALK5), and kinase-deficient TbRI (ALK5 kinase- inhibition (17, 18, 47, 48), whereas the C-terminal RING finger XIAP Is a Cofactor in TGF-b Signaling 26545 FIG.3. Coprecipitation of IAPs with TbRI. Expression vectors for the indicated IAPs (A) or the indicated deletion constructs (B) were cotransfected with wild type HA-tagged TbRI into 293 cells. Coprecipi- tations were performed as described in the legend to Fig. 1. Expression of the GST-IAP proteins was confirmed by immunoblot analysis with an antibody specific for GST (data not shown). FIG.4. XIAP activates transcription from a TGF-b-responsive reporter. A, human embryonic kidney 293 cells, HepG2 cells, or HeLa can promote E2-dependent ubiquitination (13, 49 –51). To de- cells were transiently transfected with a control vector or XIAP along with the 3TP-Lux reporter and luciferase activity was determined after termine which domains of XIAP are involved in binding the 16 h. B, the indicated XIAP plasmids were cotransfected with the TbRI receptor, sequential deletions of the C-terminal domains 3TP-Lux reporter plasmid into 293 cells and luciferase activity was of XIAP fused to the mammalian GST protein were utilized in determined after 16 h. C, HepG2 cells were transiently transfected with coprecipitation experiments. A weak interaction was detected XIAP or a vector control, stimulated with TGF-b, and assayed for luciferase activity as described under “Experimental Procedures.” All between TbRI and the XIAP DRING construct, while a strong data are representative of at least three independent experiments, and interaction was observed between TbRI receptor and the 3xBIR all experiments were performed in triplicate. domain of XIAP, which lacks both the RING finger and the spacer region (Fig. 3B). These data suggest that the XIAP-TbRI interaction involves the BIR domain of XIAP. ducers in this pathway (33, 34). These Smad proteins, once XIAP Activates Transcription from TGF-b-responsive Pro- activated, form a complex with the common mediator of TGF-b moters—Since an interaction between XIAP and TbRI was signal transduction, Smad4 (29), which then translocates to the observed, the possibility was tested that XIAP might be in- nucleus and regulates gene expression. To investigate whether volved in regulating transcription from TGF-b-responsive pro- XIAP signaling was Smad4-dependent, 293 cells were trans- moters. Several cell lines were examined to determine whether fected with the 3TP-Lux reporter plasmids and either a control XIAP could activate a TGF-b-responsive reporter, 3TP-Lux, vector or XIAP, with or without a dominant negative mutant of which contains TGF-b-responsive elements of the plasminogen Smad4 (DN Smad4). The DN Smad4 mutation effectively activator inhibitor-1 (PAI-1) and collagenase promoters (31). blocked the activation of 3TP-Lux by XIAP (Fig. 5A), suggest- Transient transfection of XIAP into either 293 cells, HepG2 ing that XIAP transduces signals from TbRI through a Smad4- cells, or HeLa cells resulted in activation of the 3TP-Lux re- dependent pathway, and thus utilizes the known TGF-b porter (Fig. 4A), with the most significant activation seen in the signaling machinery. Similar results were obtained from co- 293 cell line. expression of XIAP and DN Smad4 in HepG2 cells (data not To determine which domains of XIAP are involved in activa- shown). tion of the 3TP-Lux reporter, 293 cells were transiently trans- Since XIAP has previously been identified as a TAB1-asso- fected with the 3TP-Lux reporter and expression vectors con- ciated protein (28), and since TAB1 is involved in activating taining HA-tagged XIAP or sequential C-terminal deletions of TAK1 (43, 52, 53), it seemed possible that the ability of XIAP to XIAP. Whereas the full-length XIAP activated transcription activate 3TP-Lux might require TAK1, as well as Smad4. To from the 3TP-Lux reporter, the DRING and 3xBIR constructs test this possibility, a dominant negative mutation of TAK1 were unable to do so (Fig. 4B), suggesting that the C-terminal (DN TAK1) that destroys its kinase activity (43, 52, 53) was RING domain of XIAP is required for this activity. utilized in 3TP-Lux reporter assays. XIAP activation of 3TP- The effect of XIAP on TGF-b-mediated activation of 3TP-Lux Lux was not inhibited by the expression of DN TAK1 in 293 was then examined by utilizing the HepG2 cell line since this cells (Fig. 5B). Therefore, XIAP-dependent activation of 3TP- line is known to express TbRI. TGF-b stimulation activated Lux expression requires Smad4, but is independent of TAK1. transcription directed by the 3TP-Lux reporter plasmid ;5-fold Activation of JNK by XIAP Is Both Smad4- and TAK1-de- (Fig. 4C), whereas transient transfection with XIAP in addition pendent—Since XIAP utilizes a Smad-dependent pathway to to TGF-b stimulation activated transcription ;20-fold. These activate TGF-b-regulated gene transcription, the DN Smad4 data suggest that XIAP and TGF-b can cooperatively activate expression vector was tested for its effect on XIAP-mediated transcription of TGF-b-responsive genes. JNK activation. DN Smad4 abrogated JNK activation by XIAP Activation of 3TP-Lux by XIAP Is Smad4-dependent, but (Fig. 6A), indicating that XIAP activates JNK in a Smad-de- TAK1-independent—TGF-b-mediated signal transduction is pendent manner. initiated by phosphorylation of TbRI by TbRII in response to To determine the role of TAK1 on XIAP-induced JNK acti- ligand binding (31). The activated receptor complex can then vation, DN TAK1 was coexpressed with XIAP, and lysates were activate one of several pathway-specific Smad proteins, which evaluated for JNK activity. Interestingly, DN TAK1 blocked have recently been identified as the main cytoplasmic trans- the activation of JNK by XIAP (Fig. 6B), indicating that TAK1 26546 XIAP Is a Cofactor in TGF-b Signaling is involved in JNK activation by XIAP. These data suggest that XIAP are involved in the activation of TGF-b-responsive gene JNK activation can be distinguished from activation of the activity and JNK activation. 3TP-Lux reporter by XIAP, because DN TAK1 did not inhibit XIAP Activates NF-kB-dependent Transcription through a XIAP-induced 3TP-Lux activation (Fig. 5B). Smad-dependent, but TAK1-independent Pathway—A recent report indicated that TGF-b transactivates kB sites in a Smad- Because the activation of JNK by XIAP was inhibited by DN TAK1, and TAK1 is known to activate the p38 pathway (54), dependent manner (55). Therefore, XIAP was examined to de- termine whether its ability to activate NF-kB was also Smad- the effects of the p38 inhibitors, SB203580 and SB202190, on JNK activation by XIAP were tested (Fig. 6C). Neither inhibi- dependent. XIAP was co-expressed in 293 cells with a reporter construct containing tandem kB sites (2kB-luc), with or with- tor blocked the activation of JNK by XIAP. Rather, each p38 out the DN Smad4 expression vector. Activation of NF-kBby inhibitor augmented XIAP’s ability to activate JNK. These data XIAP was blocked by co-expression of DN Smad4 (Fig. 7A), suggest that JNK activation by XIAP is not the result of p38 suggesting that XIAP activates kB-dependent gene expression activation. in a Smad-dependent manner. To further characterize this To determine which domains of XIAP are involved in the pathway, the effects of DN TAK1 on the activation of 2kB-luc activation of JNK, C-terminal deletions of XIAP were tested for by XIAP were evaluated. Interestingly, DN TAK1 did not in- their ability to activate JNK. The DRING and 3xBIR constructs hibit the activation of 2kB-luc by XIAP (Fig. 7B). These data were able to activate JNK to levels equal to that of full-length are similar to results obtained with the 3TP-Lux reporter, in XIAP, indicating that the BIR domains of XIAP are necessary that activation of both reporters by XIAP can be blocked by and sufficient for JNK activation (Fig. 6D). These data also co-expression with DN Smad4 but not DN TAK1 (Fig. 5). These suggest that, although the C-terminal RING finger domain of data also support the observation that the ability of XIAP to XIAP is necessary for activation of the 3TP-lux reporter, it is activate transcription is separate from that of JNK activation, not required for activation of JNK. Thus, distinct domains of because DN TAK1 blocked JNK activation by XIAP (Fig. 6B). To determine the domains of XIAP required for the activa- tion of NF-kB, luciferase activity was evaluated from lysates of 293 cells transfected with the 2kB-luc reporter plasmid and either full-length XIAP or the DRING and 3xBIR mutants (Fig. 7C). Full-length XIAP significantly activated the 2kB-luc re- porter, but neither the DRING nor the 3xBIR deletion mutants were capable of this transactivation. These data are similar to those obtained for the 3TP-Lux reporter and suggest that the C-terminal domains of XIAP are required for transcriptional activation of either promoter by this protein. XIAP Does Not Directly Associate with Smad4 —To test for a possible interaction between XIAP and Smad4, 293 cells were transfected with epitope-tagged Smad4 and GST-XIAP. XIAP is known to associate with itself; therefore, XIAP was used as a control for these experiments. Coprecipitations were per- formed, and Smad4 did not coprecipitate with GST-XIAP or with the GST control vector, whereas XIAP coprecipitated with GST-XIAP (Fig. 8). These data suggest that XIAP does not FIG.5. Activation of TGF-b-directed gene expression by XIAP is blocked by DN Smad4, but not by DN TAK1. The indicated directly associate with Smad4. However, an indirect associa- expression vectors were cotransfected with 3TP-Lux reporter plasmid tion between these proteins cannot be ruled out. into 293 cells, and luciferase activity was determined after 16 h. A, The Anti-apoptotic Properties of XIAP Are Independent of effect of DN Smad4 on activation of 3TP-Lux by XIAP. B, effect of DN Both Smad4 and TAK1—To investigate whether the anti-ap- TAK1 on activation of 3TP-lux by XIAP. All data are representative of at least three independent experiments, and all experiments were per- optotic properties of XIAP are dependent on either Smad4 or formed in triplicate. TAK1, the ability of XIAP to suppress Bax-induced apoptosis FIG.6. XIAP activation of JNK is inhibited by both DN Smad4 and DN TAK1. The indicated expression vectors were cotransfected with HA-JNK into 293 cells, and Jun kinase activity of JNK immunoprecipitates was assayed using a GST-c-Jun-(1–79) substrate (top panel)as described under “Experimental Procedures.” Lysates were immunoblotted with an anti-HA antibody (middle panel) to confirm equal loading. Complexes were evaluated by phosphorimage analysis (lower panel). XIAP was coexpressed with DN Smad4 (A) or DN TAK1 (B), or was treated with 50 mM SB203580 or 30 mM SB202190 for 24 h after transfection to block p38 (C), or alternatively the indicated XIAP deletion mutants were tested (D). All data are representative of at least three independent experiments. XIAP Is a Cofactor in TGF-b Signaling 26547 FIG.7. XIAP activates NF-kB in a Smad4-dependent manner. The indicated expression vectors were cotransfected with a kB lucifer- ase reporter plasmid, and reporter activity was measured after 16 h. XIAP was coexpressed with DN Smad4 (A), DN TAK1 (B), or the indicated XIAP deletion mutants were tested (C). All data are repre- sentative of at least three independent experiments, and each experi- ment was performed in triplicate. FIG.8. XIAP does not co-precipitate with Smad4. The indicated expression vectors were transiently transfected into 293 cells, and co- precipitations were performed with glutathione-Sepharose beads fol- lowed by immunoblotting with a mixture of anti-Myc and anti-HA antibodies. The expression of the GST fusion proteins was also con- firmed by using an anti-GST antibody (data not shown). was examined in the presence of DN Smad4 or DN TAK1. Human embryonic kidney 293 cells were transfected with ex- pression vectors encoding Bax, a pro-apoptotic member of the Bcl-2 family (56), along with XIAP and DN Smad4 or DN TAK1. Cell lysates were prepared, and the ability of XIAP to FIG.9. Inhibition of caspase enzymatic activity by XIAP is inhibit caspase activity was evaluated with a fluorogenic sub- unaffected by DN Smad4 or DN TAK1. The indicated plasmids were strate AFC-DEVD as a measure of caspase activity (Fig. 9). cotransfected into 293 cells and assayed for caspase activity as de- scribed under “Experimental Procedures.” Samples were measured ev- Neither DN Smad4 (Fig. 9A) nor DN TAK1 (Fig. 9B) had any ery 5 min for a total of 2 h. Open symbols represent the control trans- affect on the ability of XIAP to inhibit caspase activity, sug- fection, whereas closed symbols represent cells cotransfected with Bax. gesting that the anti-apoptotic properties of XIAP, at least A, 293 cells cotransfected with XIAP either with or without HA-DN against Bax-induced death, are independent of its roles in both Smad4. E and l, control transfection; M and f, XIAP alone; ‚ and Œ, XIAP with DN Smad4. B, 293 cells cotransfected with XIAP either with, JNK activation and TGF-b-regulated signal transduction. or without T7-DN TAK1. E and l, vector transfection; M and f, XIAP DISCUSSION alone; ƒ and , XIAP with DN TAK1. Although the IAP gene family was initially discovered based way-specific Smad protein (33). This phosphorylated Smad can on the antiapoptotic properties of several of its members (7, 8), then associate with the common mediator, Smad4, which is a number of subsequent findings raised the possibility that involved in signaling through all pathways within the TGF-b they may play multiple roles within the cell. The data pre- superfamily. This complex then translocates to the nucleus, sented here reveal an involvement of XIAP in signal transduc- where it participates in various transcriptional complexes of tion mediated by TGF-b. XIAP was found to co-localize (Fig. 2) specific DNA binding ability (30, 35, 57). and to associate with the TGF-b type I receptor, as well as Several TGF-b-responsive promoters have been identified. other members of the TGF-b receptor superfamily, including These include PAI-1 and fibronectin (58, 59), as well as collagen ALK1 and ALK4 (Fig. 1A). Moreover, the ability to interact type I and type VII (60, 61). Smad-binding elements have been with these receptors was unique to XIAP and not shared by the identified in several of these promoters, including the PAI-1 related apoptotic inhibitors c-IAP1 and c-IAP2 (Fig. 3A). This promoter. However, Smads have a relatively low binding affin- superfamily of receptor serine/threonine kinases is activated by ity and specificity and frequently regulate transcription by TGF-b, activins, and BMPs, as well as other ligands, and uti- functional cooperation with various transcription factors bound lizes a common pathway for signaling that involves ligand- to adjacent sites, or by direct association with DNA-bound specific type II receptors, which recruit a type I receptor to the transcription factors. For example, Smad3 and Smad4 interact complex upon ligand binding. The type II receptor phosphoryl- with the Jun family of transcription factors and synergistically ates the type I receptor, which in turn phosphorylates a path- activate AP-1 promoter sequences (36, 62). Likewise, an NF-kB 26548 XIAP Is a Cofactor in TGF-b Signaling site, located in the 39 enhancer region of junB, has been iden- required the BIR domain, but did not require the RING finger or spacer regions (Fig. 6B). However, these same deletions tified as a TGF-b-responsive site (55). Activation of this site by TGF-b required an intact NF-kB pathway and was mediated by revealed that both the activation of 3TP-Lux, the TGF-b-re- sponsive reporter, and the activation of NF-kB by XIAP re- Smad family members through direct interactions between quired the C-terminal RING and spacer regions, as deletion of Smad proteins and the NF-kB subunit. these regions completely abrogated these functions. These data Data presented here suggest that XIAP is involved not only suggest that XIAP is multifunctional, with the N-terminal BIR in the activation of a TGF-b-responsive promoter, 3TP-Lux domains participating in receptor association and JNK activa- (Fig. 4), which contains both PAI-1 and collagenase promoter tion, and the C-terminal RING finger and spacer being in- elements (31), but also in the activation of both JNK and NF-kB volved in the activation of NF-kB and TGF-b-mediated (Fig. 6), transcriptional mediators of TGF-b signaling. Activa- transcription. tion of all three pathways was efficiently blocked by co-expres- Further evidence for the multifunctionality of XIAP comes sion of DN Smad4 along with XIAP, indicating that XIAP from the fact that, although its signaling properties were all utilizes a Smad-dependent pathway for activation of not only a Smad4-dependent, its ability to inhibit the enzymatic activity TGF-b-responsive promoter (Fig. 5A), but also of JNK (Fig. 6A) of caspases was completely independent of Smad4 (Fig. 9A). and NF-kB (Fig. 7A). These data suggest a role for XIAP at a These data were obtained by using Bax to induce apoptosis in point upstream of Smad4 signaling. 293 cells. Bax is known to promote the release of cytochrome c Since XIAP activated these signaling pathways in a Smad4- from the mitochondria, which, along with Apaf-1 and ATP/ dependent manner, it is possible that overexpression of wild dATP, catalyzes the processing of caspase-9 (65, 66). Recently, type Smad4 would enhance this activation. However, data sug- TGF-b was found to activate the caspase cascade in a similar gest that it is the amount of activated Smad4 present in the manner, through the release of cytochrome c and subsequent cell, and not the absolute levels of protein that affect Smad-de- processing of caspase-9 (67). XIAP has been shown to bind to, pendent pathways (36, 63, 64). Also, the cell lines utilized in and inhibit the enzymatic activity of caspases-3, -7, and -9, all this study express very high levels of endogenous Smad4, and of which are activated by Bax as well as by TGF-b (67). There- it is therefore unlikely that Smad4 is limiting in any of these fore, these functions are separable in that the signaling prop- responses. Therefore, simple overexpression of Smad4 did not erties are Smad4-dependent, whereas the anti-apoptotic prop- enhance signaling (data not shown). erties are Smad4-independent. Recent studies have revealed the existence of both Smad-de- Recent reports have described the identification of a negative pendent and Smad-independent pathways of JNK activation, regulator of XIAP function, Smac/DIABLO (68, 69), which has in response to TGF-b (37). In the primary, Smad-independent been shown to bind to XIAP and prevent its caspase inhibitory pathway, JNK activity peaked within 10 min following stimu- function (70). It will be of great interest to determine whether lation with TGF-b, whereas in the secondary, Smad-dependent Smac/DIABLO is capable of regulating the function of XIAP in pathway, JNK activity exhibited a slow, sustained peak over the TGF-b signaling pathway. 12–16 h (37). Because the results presented in Fig. 6A were Taken together, these data place XIAP at a central location obtained by cotransfection of XIAP and DN Smad4 and cells for coordinating signaling from the TGF-b type I receptor for were harvested ;18 h after transfection, the possibility that the activation of transcription, as well as the activation of both XIAP may also activate JNK through the Smad-independent JNK and NF-kB, co-factors involved in transcribing subsets of pathway cannot be eliminated. TGF-b-responsive genes, since each of these activities is de- Interestingly, the activation of JNK by XIAP was inhibited pendent on Smad4. Since no association was observed between by DN TAK1 as well as by DN Smad4 (Fig. 6, A and B), whereas XIAP and Smad4 (Fig. 8), XIAP could be a signaling co-factor both activation of NF-kB (Fig. 7B) and the TGF-b-responsive that is involved indirectly in the activation of Smad4, perhaps promoter (Fig. 5B) were TAK1-independent. TAK1 is an up- through association with another Smad protein, or other sig- stream MAP3K that activates the p38 pathway (54) and has naling intermediates. Further studies will be required to de- also been shown to be a mediator of TGF-b-dependent signaling termine the mechanisms of these functional interactions. (43, 52). Data presented here show that the ability of XIAP to Acknowledgments—We thank Drs. B. Mayer, J. Massague ´ , M. de activate JNK does not require the p38 pathway, despite the fact Caestecker, J. Wrana, L. Attisano, S. Korsmeyer, and M. G. Sanna for that DN TAK1 abrogates this activity (Fig. 6C). TAK1 has plasmids. We also thank L. Eiben, B. Richter, and J. Lewis for critical previously been reported to indirectly associate with XIAP reading of the manuscript. through mutual interaction with TAB1, the TAK1-binding pro- REFERENCES tein (28). Subsequently, XIAP was found to activate NF-kBina 1. Kerr, J. F., Wyllie, A. H., and Currie, A. R. (1972) Br. J. Cancer 26, 239 –257 TAK1-dependent manner (20) in endothelial cells. In 293 cells, 2. Vaux, D. L., Haecker, G., and Strasser, A. (1994) Cell 76, 777–779 however, XIAP was found to activate NF-kB independent of 3. Nagata, S. (1996) Curr. Biol. 6, 1241–1243 4. Salvesen, G. S., and Dixit, V. M. (1997) Cell 91, 443– 446 TAK1 (Fig. 7B), and a recent report described the activation of 5. Thornberry, N. A., and Lazebnik, Y. (1998) Science 281, 1312–1316 NF-kB-directed gene expression by TGF-b in a TAK1-inde- 6. Deveraux, Q. L., and Reed, J. C. (1999) Genes Dev. 13, 239 –252 pendent fashion (55), suggesting the existence of cell type var- 7. Crook, N. E., Clem, R. J., and Miller, L. K. (1993) J. Virol. 67, 2168 –2174 8. Miller, L. K. (1999) Trends Cell Biol. 9, 323–328 iation of this pathway. 9. Duckett, C. S., Nava, V. E., Gedrich, R. W., Clem, R. J., Van Dongen, J. L., XIAP contains two distinct structural motifs found in several Gilfillan, M. C., Shiels, H., Hardwick, J. M., and Thompson, C. B. (1996) EMBO J. 15, 2685–2694 cellular IAPs. Three imperfectly repeated BIR domains are 10. Hawkins, C. J., Uren, A. G., Ha ¨ cker, G., Medcalf, R. L., and Vaux, D. L. (1996) contained in the N terminus of XIAP. The BIR is the defining Proc. Natl. Acad. Sci. U. S. A. 93, 13786 –13790 11. Uren, A. G., Coulson, E. J., and Vaux, D. L. (1998) Trends Biochem. Sci. 23, motif of the IAP family and is involved in caspase binding and 159 –162 inhibition (6). The second motif is the C-terminal RING finger 12. Vucic, D., Stennicke, H. R., Pisabarro, M. T., Salvesen, G. S., and Dixit, V. M. domain, which has been implicated in E2-dependent ubiquitin (2000) Curr. Biol. 10, 1359 –1366 13. Joazeiro, C. A., and Weissman, A. M. (2000) Cell 102, 549 –552 conjugation (13). Deletion analysis of XIAP revealed that the 14. Liston, P., Roy, N., Tamai, K., Lefebvre, C., Baird, S., Cherton-Horvat, G., BIR domain is involved in the association between XIAP and Farahani, R., McLean, M., Ikeda, J.-E., MacKenzie, A., and Korneluk, R. G. (1996) Nature 379, 349 –353 the TbRI receptor, since deletions lacking the RING finger or 15. Uren, A., Pakusch, M., Hawkins, C., Puls, K. L., and Vaux, D. L. (1996) Proc. both the RING and spacer regions were still able to associate Natl. Acad. Sci. U. S. A. 93, 4974 – 4978 with this receptor (Fig. 3B). Likewise, JNK activation by XIAP 16. Duckett, C. S., Li, F., Wang, Y., Tomaselli, K. J., Thompson, C. B., and XIAP Is a Cofactor in TGF-b Signaling 26549 Armstrong, R. C. (1998) Mol. Cell. Biol. 18, 608 – 615 44. Perkins, N. D., Agranoff, A. B., Duckett, C. S., and Nabel, G. J. (1994) J. Virol. 17. Deveraux, Q. L., Takahashi, R., Salvesen, G. S., and Reed, J. C. (1997) Nature 68, 6820 – 6823 388, 300 –304 45. Duckett, C. S., and Thompson, C. B. (1997) Genes Dev. 11, 2810 –2821 18. Roy, N., Deveraux, Q. L., Takahashi, R., Salvesen, G. S., and Reed, J. C. (1997) 46. Bradford, M. M. (1976) Anal. Biochem. 72, 248 –254 EMBO J. 16, 6914 – 6925 47. Deveraux, Q. L., Leo, E., Stennicke, H. R., Welsh, K., Salvesen, G. S., and 19. Sanna, M. G., Duckett, C. S., Richter, B. W. M., Thompson, C. B., and Ulevitch, Reed, J. C. (1999) EMBO J. 18, 5242–5251 R. J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 6015– 6020 48. Takahashi, R., Deveraux, Q., Tamm, I., Welsh, K., Assa-Munt, N., Salvesen, 20. Hofer-Warbinek, R., Schmid, J. A., Stehlik, C., Binder, B. R., Lipp, J., and de G. S., and Reed, J. C. (1998) J. Biol. Chem. 273, 7787–7790 Martin, R. (2000) J. Biol. Chem. 275, 22064 –22068 49. Huang, H.-K., Joazeiro, C. A. P., Bonfoco, E., Kamada, S., Leverson, J. D., and 21. Rothe, M., Wong, S. C., Henzel, W. J., and Goeddel, D. V. (1994) Cell 78, Hunter, T. (2000) J. Biol. Chem. 275, 26661–26664 681– 692 50. Yang, Y., Fang, S., Jensen, J. P., Weissman, A. M., and Ashwell, J. D. (2000) 22. Rothe, M., Pan, M.-G., Henzel, W. J., Ayres, T. M., and Goeddel, D. V. (1995) Science 288, 874 – 877 Cell 83, 1243–1252 51. Lorick, K. L., Jensen, J. P., Fang, S., Ong, A. M., Hatakeyama, S., and 23. Liu, Z. G., Hsu, H., Goeddel, D. V., and Karin, M. (1996) Cell 87, 565–576 Weissman, A. M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11364 –11369 24. Rothe, M., Sarma, V., Dixit, V. M., and Goeddel, D. V. (1995) Science 269, 52. Shibuya, H., Yamaguchi, K., Shirakabe, K., Tonegawa, A., Gotch, Y., Ueno, N., 1424 –1427 Irie, K., Nishida, E., and Matsumoto, K. (1996) Science 272, 1179 –1182 25. Natoli, G., Costanzo, A., Ianni, A., Templeton, D. J., Woodgett, J. R., Balsano, 53. Shibuya, H., Iwata, H., Masuyama, N., Gotoh, Y., Yamaguchi, K., Irie, K., C., and Levrero, M. (1997) Science 275, 200 –203 Matsumoto, K., Nishida, E., and Ueno, N. (1998) EMBO J. 17, 1019 –1028 26. Deveraux, Q. L., Roy, N., Stennicke, H. R., Van Arsdale, T., Zhou, Q., 54. Hanafusa, H., Ninomiya-Tsuji, J., Masuyama, N., Nishita, M., Fujisawa, J., Srinivasula, S. M., Alnemri, E. S., Salvesen, G. S., and Reed, J. C. (1998) Shibuya, H., Matsumoto, K., and Nishida, E. (1999) J. Biol. Chem. 274, EMBO J. 17, 2215–2223 27161–27167 27. Oeda, E., Oka, Y., Miyazono, K., and Kawabata, M. (1998) J. Biol. Chem. 273, 55. Lo ´ pez-Rovira, T., Chalaux, E., Rosa, J. L., Bartrons, R., and Ventura, F. (2000) 9353–9356 J. Biol. Chem. 275, 28937–28946 28. Yamaguchi, K., Nagai, S., Ninomiya-Tsuji, J., Nishita, M., Tamai, K., Irie, K., 56. Oltvai, Z. N., Milliman, C. L., and Korsmeyer, S. J. (1993) Cell 74, 609 – 619 Ueno, N., Nishida, E., Shibuya, H., and Matsumoto, K. (1999) EMBO J. 18, 57. Zawel, L., Dai, J. L., Buckhaults, P., Zhou, S., Kinzler, K. W., Vogelstein, B., 179 –187 and Kern, S. E. (1998) Mol. Cell. 1, 611– 617 29. Wrana, J. L. (2000) Cell 100, 189 –192 58. Dennler, S., Itoh, S., Vivien, D., ten Dijke, P., and Gauthier, J.-M. (1998) 30. Massague ´ , J., and Chen, Y. G. (2000) Genes Dev. 14, 627– 644 EMBO J. 17, 3091–3100 31. Wrana, J. L., Attisano, L., Carcamo, J., Zentella, A., Doody, J., Laiho, M., 59. Hocevar, B. A., Brown, T. L., and Howe, P. H. (1999) EMBO J. 18, 1345–1356 Wang, X. F., and Massague ´ , J. (1992) Cell 71, 1003–1014 60. Chung, K.-Y., Agarwal, A., Uitto, J., and Mauviel, A. (1996) J. Biol. Chem. 271, 32. Carcamo, J., Zentella, A., and Massague ´ , J. (1995) Mol. Cell. Biol. 15, 3272–3278 1573–1581 61. Vindevoghel, L., Kon, A., Lechleider, R. J., Uitto, J., Roberts, A. B., and 33. Heldin, C. H., Miyazono, K., and ten Dijke, P. (1997) Nature 390, 465– 471 Mauviel, A. (1998) J. Biol. Chem. 273, 13053–13057 34. Derynck, R., Zhang, Y., and Feng, X. H. (1998) Cell 95, 737–740 62. Liberati, N. T., Datto, M. B., Frederick, J. P., Shen, X., Wong, C., Rougier- 35. Jonk, L. J., Itoh, S., Heldin, C. H., ten Dijke, P., and Kruijer, W. (1998) J. Biol. Chapman, E. M., and Wang, X. F. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, Chem. 273, 21145–21152 4844 – 4849 36. Zhang, Y., Feng, X. H., and Derynck, R. (1998) Nature 394, 909 –913 63. Feng, X. H., Lin, X., and Derynck, R. (2000) EMBO J. 19, 5178 –5193 37. Engel, M. E., McDonnell, M. A., Law, B. K., and Moses, H. L. (1999) J. Biol. 64. Zhang, Y., and Derynck, R. (2000) J. Biol. Chem. 275, 16979 –16985 Chem. 274, 37413–37420 65. Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 38. Karin, M. (1995) J. Biol. Chem. 270, 16483–16486 405– 413 39. Yang, D., Tournier, C., Wysk, M., Lu, H.-T., Xu, J., Davis, R. J., and Flavell, 66. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, R. A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3004 –3009 E. S., and Wang, X. (1997) Cell 91, 479 – 489 40. Cheng, G., and Baltimore, D. (1996) Genes Dev. 10, 963–973 67. Freathy, C., Brown, D. G., Roberts, R. A., and Cain, K. (2000) Hepatology 32, 41. Mayer, B. J., Hirai, H., and Sakai, R. (1995) Curr. Biol. 5, 296 –305 750 –760 42. Duckett, C. S., Gedrich, R. W., Gilfillan, M. C., and Thompson, C. B. (1997) 68. Du, C., Fang, M., Li, Y., Li, L., and Wang, X. (2000) Cell 102, 33– 42 Mol. Cell. Biol. 17, 1535–1542 69. Verhagen, A. M., Ekert, P. G., Pakusch, M., Silke, J., Connolly, L. M., Reid, 43. Yamaguchi, K., Shirakabe, K., Shibuya, H., Irie, K., Oishi, I., Ueno, N., G. E., Moritz, R. L., Simpson, R. J., and Vaux, D. L. (2000) Cell 102, 43–53 Taniguchi, T., Nishida, E., and Matsumoto, K. (1995) Science 270, 70. Srinivasula, S. M., Datta, P., Fan, X. J., Fernandes-Alnemri, T., Huang, Z., and 2008 –2011 Alnemri, E. S. (2000) J. Biol. Chem. 275, 36152–36157

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Published: Jul 1, 2001

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