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Uncoupling of the Signaling and Caspase-inhibitory Properties of X-linked Inhibitor of Apoptosis

2004; Elsevier BV; Volume: 279; Issue: 10 Linguagem: Inglês

10.1074/jbc.m312891200

1083-351X

Jennifer A. Lewis, Ezra Burstein, Stephanie Birkey Reffey, Shawn B. Bratton, Anita B. Roberts, Colin S. Duckett,

interferon and immune responses

In addition to its well described function as an enzymatic inhibitor of specific caspases, X-linked inhibitor of apoptosis (X-linked IAP or XIAP) can function as a cofactor in Smad, NF-κB, and JNK signaling pathways. However, caspases themselves have been shown to regulate the activity of a number of signaling cascades, raising the possibility that the effect of XIAP in these pathways is indirect. Here we examine this question by introducing point mutations in XIAP predicted to disrupt the ability of the molecule to bind to and inhibit caspases. We show that whereas these mutant variants of XIAP lost caspase-inhibitory activity, they maintained their ability to activate Smad, NF-κB, and JNK signaling pathways. Indeed, the signaling properties of the molecule were mapped to domains not directly involved in caspase binding and inhibition. The activation of NF-κB by XIAP was dependent on the E3 ubiquitin ligase activity of the RING domain. On the other hand, the ability of XIAP to activate Smad-dependent signaling was mapped to the third baculoviral IAP repeat (BIR) and loop regions of the molecule. Thus, the anti-apoptotic and signaling properties of XIAP can be uncoupled. In addition to its well described function as an enzymatic inhibitor of specific caspases, X-linked inhibitor of apoptosis (X-linked IAP or XIAP) can function as a cofactor in Smad, NF-κB, and JNK signaling pathways. However, caspases themselves have been shown to regulate the activity of a number of signaling cascades, raising the possibility that the effect of XIAP in these pathways is indirect. Here we examine this question by introducing point mutations in XIAP predicted to disrupt the ability of the molecule to bind to and inhibit caspases. We show that whereas these mutant variants of XIAP lost caspase-inhibitory activity, they maintained their ability to activate Smad, NF-κB, and JNK signaling pathways. Indeed, the signaling properties of the molecule were mapped to domains not directly involved in caspase binding and inhibition. The activation of NF-κB by XIAP was dependent on the E3 ubiquitin ligase activity of the RING domain. On the other hand, the ability of XIAP to activate Smad-dependent signaling was mapped to the third baculoviral IAP repeat (BIR) and loop regions of the molecule. Thus, the anti-apoptotic and signaling properties of XIAP can be uncoupled. The iap (inhibitor of apoptosis) genes were originally described in baculoviruses (1Crook N.E. Clem R.J. Miller L.K. J. Virol. 1993; 67: 2168-2174Crossref PubMed Google Scholar, 2Birnbaum M.J. Clem R.J. Miller L.K. J. Virol. 1994; 68: 2521-2528Crossref PubMed Google Scholar) and have subsequently been identified in a wide range of cellular genomes (3Verhagen A.M. Coulson E.J. Vaux D.L. Genome Biol. 2001; 2: 3009.1-3009.10Crossref Google Scholar). The anti-apoptotic activity of IAPs 1The abbreviations used are: IAP, inhibitor of apoptosis; XIAP, X-linked IAP; BIR, baculoviral IAP repeat; E3, ubiquitin-protein isopeptide ligase; TGF, transforming growth factor; BMP, bone morphogenetic protein; JNK, c-Jun N-terminal kinase; GST, glutathione S-transferase; HA, hemagglutinin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PIPES, piperazine-N,N′-bis-2-ethanosulfonic acid; AFC, 7-amino-4-trifluoromethylcoumarin; BisTris, bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane. was first recognized. This property has been largely attributed to their ability to directly inhibit members of the caspase family of cysteinyl proteases, the central effectors of the apoptotic cascade (4Deveraux Q.L. Roy N. Stennicke H.R. Van Arsdale T. Zhou Q. Srinivasula S.M. Alnemri E.S. Salvesen G.S. Reed J.C. EMBO J. 1998; 17: 2215-2223Crossref PubMed Scopus (1246) Google Scholar, 5Devereaux Q.L. Takahashi R. Salvesen G.S. Reed J.C. 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Powers S.E. McCleland M.L. Kuang J. Stukenberg P.T. Mol. Biol. Cell. 2002; 13: 3064-3077Crossref PubMed Scopus (276) Google Scholar). IAPs contain between one and three imperfect repeats of an ∼65-residue motif termed the baculoviral IAP repeat (BIR), which exhibits structural similarity to zinc fingers (17Sun C. Cai M. Gunasekera A.H. Meadows R.P. Wang H. Chen J. Zhang H. Wu W. Xu N. Ng S.C. Fesik S.W. Nature. 1999; 401: 818-822Crossref PubMed Scopus (298) Google Scholar, 18Hinds M.G. Norton R.S. Vaux D.L. Day C.L. Nat. Struct. Biol. 1999; 6: 648-651Crossref PubMed Scopus (154) Google Scholar). Many IAPs also contain a carboxyl-terminal RING finger domain that possesses E3 ubiquitin ligase activity (10Li X. Yang Y. Ashwell J.D. Nature. 2002; 416: 345-347Crossref PubMed Scopus (395) Google Scholar, 19Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (873) Google Scholar). One mammalian member of this family is X-linked IAP (XIAP), a 56-kDa protein composed of three amino-terminal BIRs and a RING domain at the carboxyl terminus (20Holcik M. Korneluk R.G. Nat. Rev. Mol. Cell Biol. 2001; 2: 550-556Crossref PubMed Scopus (234) Google Scholar). XIAP has potent antiapoptotic properties and has been shown to directly suppress the enzymatic activity of caspase-3, -7, and -9 in vitro and in intact cells (4Deveraux Q.L. Roy N. Stennicke H.R. Van Arsdale T. Zhou Q. Srinivasula S.M. Alnemri E.S. Salvesen G.S. Reed J.C. EMBO J. 1998; 17: 2215-2223Crossref PubMed Scopus (1246) Google Scholar, 5Devereaux Q.L. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1724) Google Scholar, 21Takahashi R. Deveraux Q. Tamm I. Welsh K. Assa-Munt N. Salvesen G.S. Reed J.C. J. Biol. Chem. 1998; 273: 7787-7790Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). Recent studies have determined the crystal structure of XIAP interacting with caspase-3, -7, and -9 and revealed great detail of the molecular determinants necessary for these interactions. The linker region between BIR 1 and BIR 2 is important for binding to caspase-3 and -7 (6Chai J. Shiozaki E. Srinivasula S.M. Wu Q. Dataa P. Alnemri E.S. Shi Y. Cell. 2001; 104: 769-780Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar, 7Riedl S.J. Renatus M. Schwarzenbacher R. Zhou Q. Sun C. Fesik S.W. Liddington R.C. Salvesen G.S. Cell. 2001; 104: 791-800Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar, 8Huang Y. Park Y.C. Rich R.L. Segal D. Myszka D.G. Wu H. Cell. 2001; 104: 781-790Abstract Full Text Full Text PDF PubMed Google Scholar). The amino acids in the linker region upstream of BIR 2 bind to the active site of caspase-3 and -7 in an antiparallel orientation, inhibiting the enzymatic activity of these caspases. The residue Asp148 in XIAP is particularly important for the ability of XIAP to bind to these caspases (7Riedl S.J. Renatus M. Schwarzenbacher R. Zhou Q. Sun C. Fesik S.W. Liddington R.C. Salvesen G.S. Cell. 2001; 104: 791-800Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar, 17Sun C. Cai M. Gunasekera A.H. Meadows R.P. Wang H. Chen J. Zhang H. Wu W. Xu N. Ng S.C. Fesik S.W. Nature. 1999; 401: 818-822Crossref PubMed Scopus (298) Google Scholar). The interactions between XIAP and caspase-9 depend on a different region of the molecule (22Sun C. Cai M. Meadows R.P. Xu N. Gunasekera A.H. Herrmann J. Wu J.C. Fesik S.W. J. Biol. Chem. 2000; 275: 33777-33781Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 23Srinivasula S.M. Hegde R. Saleh A. Datta P. Shiozaki E. Chai J. Lee R.A. Robbins P.D. Fernandes-Alnemri T. Shi Y. Alnemri E.S. Nature. 2001; 410: 112-116Crossref PubMed Scopus (863) Google Scholar). A hydrophobic pocket in the surface groove of BIR 3 of XIAP mediates this interaction. This pocket is occupied by the first four amino acids of processed caspase-9 (p12 subunit). The residue Trp310 in BIR 3 forms part of this pocket and is critical for binding to caspase-9 (22Sun C. Cai M. Meadows R.P. Xu N. Gunasekera A.H. Herrmann J. Wu J.C. Fesik S.W. J. Biol. Chem. 2000; 275: 33777-33781Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 24Bratton S.B. Lewis J. Butterworth M. Duckett C.S. Cohen G.M. Cell Death Differ. 2002; 9: 881-892Crossref PubMed Scopus (118) Google Scholar). The antiapoptotic activity of XIAP can be suppressed by two nuclear encoded, mitochondrially localized proteins, Smac/DIABLO and Omi/HtrA2 (25Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2941) Google Scholar, 26Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1985) Google Scholar, 27Hegde R. Srinivasula S.M. Zhang Z. Wassell R. Mukattash R. Cilenti L. DuBois G. Lazebnik Y. Zervos A.S. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2001; 277: 432-438Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar, 28Martins L.M. Iaccarino I. Tenev T. Gschmeissner S. Totty N.F. Lemoine N.R. Savopoulos J. Gray C.W. Creasy C.L. Dingwall C. Downward J. J. Biol. Chem. 2001; 277: 439-444Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 29Suzuki Y. Imai Y. Nakayama H. Takahashi K. Takio K. Takahashi R. Mol. Cell. 2001; 8: 613-621Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar, 30van Loo G. van Gurp M. Depuydt B. Srinivasula S.M. Rodriguez I. Alnemri E.S. Gevaert K. Vandekerckhove J. Declercq W. Vandenabeele P. Cell Death Differ. 2002; 9: 20-26Crossref PubMed Scopus (277) Google Scholar). These molecules are released from mitochondria into the cytoplasm during apoptosis, where they bind to the same hydrophobic pocket in BIR 3 that is responsible for XIAP interactions with caspase-9 (23Srinivasula S.M. Hegde R. Saleh A. Datta P. Shiozaki E. Chai J. Lee R.A. Robbins P.D. Fernandes-Alnemri T. Shi Y. Alnemri E.S. Nature. 2001; 410: 112-116Crossref PubMed Scopus (863) Google Scholar, 31Wu G. Chai J. Suber T.L. Wu J.W. Du C. Wang X. Shi Y. Nature. 2000; 408: 1008-1012Crossref PubMed Scopus (715) Google Scholar, 32Liu Z. Sun C. Olejniczak E.T. Meadows R.P. Betz S.F. Oost T. Herrmann J. Wu J.C. Fesik S.W. Nature. 2000; 408: 1004-1008Crossref PubMed Scopus (548) Google Scholar). This binding is mediated by tetrapeptide sequences present in the amino-terminal portion of the mature form of these proteins that have significant similarity to amino-terminal sequences present in mature caspase-9 (p12 subunit). The binding of these molecules or caspase-9 to XIAP is mutually exclusive (17Sun C. Cai M. Gunasekera A.H. Meadows R.P. Wang H. Chen J. Zhang H. Wu W. Xu N. Ng S.C. Fesik S.W. Nature. 1999; 401: 818-822Crossref PubMed Scopus (298) Google Scholar, 23Srinivasula S.M. Hegde R. Saleh A. Datta P. Shiozaki E. Chai J. Lee R.A. Robbins P.D. Fernandes-Alnemri T. Shi Y. Alnemri E.S. Nature. 2001; 410: 112-116Crossref PubMed Scopus (863) Google Scholar, 32Liu Z. Sun C. Olejniczak E.T. Meadows R.P. Betz S.F. Oost T. Herrmann J. Wu J.C. Fesik S.W. Nature. 2000; 408: 1004-1008Crossref PubMed Scopus (548) Google Scholar). Thus, Smac/DIABLO and Omi/HtrA2 are negative regulators of XIAP that function to release XIAP from caspases; in the case of Omi/HtrA2, proteolytic cleavage of XIAP also participates in this negative regulation (33Srinivasula S.M. Gupta S. Datta P. Zhang Z. Hegde R. Cheong N. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2003; 278: 31469-31472Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 34Yang Q.H. Church-Hajduk R. Ren J. Newton M.L. Du C. Genes Dev. 2003; 17: 1487-1496Crossref PubMed Scopus (272) Google Scholar). In addition to the ability of XIAP to inhibit caspases, other roles for this molecule have been demonstrated, including its participation in a number of signaling pathways. XIAP has been found to be a cofactor in transforming growth factor-β and bone morphogenetic protein (TGF-β/BMP) signaling pathways. The TGF-β superfamily is a group of cytokines that function as growth regulators with diverse effects depending on the target tissue (35de Caestecker M.P. Piek E. Roberts A.B. J. Natl. Cancer Inst. 2000; 92: 1388-1402Crossref PubMed Google Scholar, 36Letterio J.J. Roberts A.B. Annu. Rev. Immunol. 1998; 16: 137-161Crossref PubMed Scopus (1686) Google Scholar). XIAP can bind to the cytoplasmic domain of the type I BMP receptor (ALK-3) and can affect BMP-regulated dorsal-ventral polarity in a Xenopus developmental model (11Yamaguchi K. Nagai S. Ninomiya-Tsuji J. Nishita M. Tamai K. Irie K. Ueno N. Nishida E. Shibuya H. Matsumoto K. EMBO J. 1999; 18: 179-187Crossref PubMed Scopus (326) Google Scholar). Similarly, an association between XIAP and the type I TGF-β receptor (ALK-5) results in synergistic activation of TGF-β-dependent transcription by XIAP (12Birkey Reffey S. Wurthner J.U. Parks W.T. Roberts A.B. Duckett C.S. J. Biol. Chem. 2001; 276: 26542-26549Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). In addition, ectopic expression of XIAP has been shown to activate stress-responsive signaling pathways, such as the N-terminal c-Jun kinase (JNK) pathway (37Sanna M.G. da Silva Correia J. Ducrey O. Lee J. Nomoto K. Schrantz N. Deveraux Q.L. Ulevitch R.J. Mol. Cell Biol. 2002; 22: 1754-1766Crossref PubMed Scopus (211) Google Scholar, 38Sanna M.G. Duckett C.S. Richter B.W.M. Thompson C.B. Ulevitch R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6015-6020Crossref PubMed Scopus (117) Google Scholar) and the transcription factor NF-κB (12Birkey Reffey S. Wurthner J.U. Parks W.T. Roberts A.B. Duckett C.S. J. Biol. Chem. 2001; 276: 26542-26549Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 39Levkau B. Garton K.J. Ferri N. Kloke K. Nofer J.R. Baba H.A. Raines E.W. Breithardt G. Circ. Res. 2001; 88: 282-290Crossref PubMed Scopus (123) Google Scholar, 40Hofer-Warbinek R. Schmid J.A. Stehlik C. Binder B.R. Lipp J. de Martin R. J. Biol. Chem. 2000; 275: 22064-22068Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). The activation of JNK1 was reported to contribute to the protective effect conferred by XIAP (41Sanna M.G. Da Silva Correia J. Luo Y. Chuang B. Paulson L.M. Nguyen B. Deveraux Q.L. Ulevitch R.J. J. Biol. Chem. 2002; Google Scholar) and is thought to result from interactions between XIAP and factors such as TAB1 (11Yamaguchi K. Nagai S. Ninomiya-Tsuji J. Nishita M. Tamai K. Irie K. Ueno N. Nishida E. Shibuya H. Matsumoto K. EMBO J. 1999; 18: 179-187Crossref PubMed Scopus (326) Google Scholar) and ILPIP (41Sanna M.G. Da Silva Correia J. Luo Y. Chuang B. Paulson L.M. Nguyen B. Deveraux Q.L. Ulevitch R.J. J. Biol. Chem. 2002; Google Scholar) that activate the MAP3 kinase TAK1. The activation of NF-κB in endothelial cells can result from activation of IκB kinase by TAK1 (40Hofer-Warbinek R. Schmid J.A. Stehlik C. Binder B.R. Lipp J. de Martin R. J. Biol. Chem. 2000; 275: 22064-22068Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), but in other cell models it is not mediated by this pathway (12Birkey Reffey S. Wurthner J.U. Parks W.T. Roberts A.B. Duckett C.S. J. Biol. Chem. 2001; 276: 26542-26549Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Whether the caspase-inhibitory properties of XIAP play any role in its signaling properties is not known. Caspases not only play a central role in apoptosis but can regulate other signaling cascades. Caspase-1, -4, and -5 participate in IL-1β and IL-18 processing and maturation; similarly, a role for caspase-8 in signaling events involved in T-cell proliferation has been demonstrated (42Newton K. Strasser A. Genes Dev. 2003; 17: 819-825Crossref PubMed Scopus (69) Google Scholar). Caspases can participate in NF-κB signaling through the ability of caspase-3 to cleave the amino-terminal portion of IκB-α, an inhibitory factor that prevents nuclear translocation of NF-κB, leading to a stabilization of IκB-α and blockade of κB-mediated transcription (43Barkett M. Xue D. Horvitz H.R. Gilmore T.D. J. Biol. Chem. 1997; 272: 29419-29422Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 44Reuther J.Y. Baldwin Jr., A.S. J. Biol. Chem. 1999; 274: 20664-20670Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Additionally, Relish, a Drosophila homolog of NF-κB, has been found to be cleaved by the Drosophila caspase Dredd, leading to Relish activation (45Stoven S. Silverman N. Junell A. Hedengren-Olcott M. Erturk D. Engstrom Y. Maniatis T. Hultmark D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5991-5996Crossref PubMed Scopus (260) Google Scholar). Similarly, the mammalian NF-κB subunit c-Rel can also be cleaved by caspase-3, although the functional significance of this event is unclear (46Barkett M. Dooher J.E. Lemonnier L. Simmons L. Scarpati J.N. Wang Y. Gilmore T.D. Biochim. Biophys. Acta. 2001; 1526: 25-36Crossref PubMed Scopus (19) Google Scholar). Therefore, we set out to determine whether the caspase-inhibitory activity of XIAP was required for its signaling properties. Here we report that point mutations in XIAP that abrogate its antiapoptotic activity do not affect its signaling activities, demonstrating that these properties are separate and independent of each other. In addition, the data presented demonstrate that the various signaling properties of XIAP can be mapped to different regions of the molecule. Cell Culture and Transfections—Human embryonic kidney 293 cells and the Smad4-deficient human breast cancer cell line, MDA-MB-468, were obtained from the American Tissue Culture Collection. All cells were cultured at 37 °C with 5% CO2 in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 2 mm glutamine. Transfection of 293 cells was performed using the calcium phosphate precipitation procedure as described previously (47Duckett C.S. Gedrich R.W. Gilfillan M.C. Thompson C.B. Mol. Cell Biol. 1997; 17: 1535-1542Crossref PubMed Google Scholar). MDA-MB-468 cells were transfected using Fugene 6 reagent following the manufacturer's instructions (Roche Applied Science) with a 3:1 ratio of Fugene reagent to DNA. Plasmids—The pEBB expression vector and the 2κB-luc reporter have been described previously (47Duckett C.S. Gedrich R.W. Gilfillan M.C. Thompson C.B. Mol. Cell Biol. 1997; 17: 1535-1542Crossref PubMed Google Scholar). Construction of the pEBB XIAP and HA-JNK1 plasmids have been previously reported (12Birkey Reffey S. Wurthner J.U. Parks W.T. Roberts A.B. Duckett C.S. J. Biol. Chem. 2001; 276: 26542-26549Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 38Sanna M.G. Duckett C.S. Richter B.W.M. Thompson C.B. Ulevitch R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6015-6020Crossref PubMed Scopus (117) Google Scholar). The SBE-JONK reporter expression vector was kindly provided by B. Vogelstein. The Bax expression vector was kindly provided by S. Korsmeyer. The Fas expression vector was kindly provided by R. Siegel. The Myc-tagged Smad4 expression vector was kindly provided by M. de Caestecker. The XIAP caspase-binding and RING mutants were generated using the PCR-based QuikChange™ site-directed mutagenesis kit (Stratagene) following the manufacturer’s instructions as described previously (24Bratton S.B. Lewis J. Butterworth M. Duckett C.S. Cohen G.M. Cell Death Differ. 2002; 9: 881-892Crossref PubMed Scopus (118) Google Scholar). The bacterial GST expression vectors for the XIAP constructs were prepared by subcloning the inserts into the BamHI and NotI sites of pGEX 4T-1 (Amersham Biosciences). The HA-tagged XIAP plasmids were constructed by subcloning the inserts from the respective pEBG constructs previously described (48Duckett C.S. Li F. Wang Y. Tomaselli K.J. Thompson C.B. Armstrong R.C. Mol. Cell Biol. 1998; 18: 608-615Crossref PubMed Scopus (193) Google Scholar, 49Richter B.W.M. Mir S.S. Eiben L.J. Lewis J. Reffey S.B. Frattini A. Tian L. Frank S. Youle R.J. Nelson D.L. Notarangelo L.D. Vezzoni P. Fearnhead H.O. Duckett C.S. Mol. Cell Biol. 2001; 21: 4292-4301Crossref PubMed Scopus (87) Google Scholar) using the BamHI and NotI sites in the pEBB-HA mammalian expression vector. In addition, pEBB-HA-BIR 1, pEBB-HA-BIR 2, pEBB-HA-BIR 3, pEBB-HA-BIR 1-2, pEBB-HA-BIR 2-3, and pEBB-HA-BIR 1-2-3 were generated by PCR using pEBB-XIAP as template with the boundaries for each construct as indicated in Fig. 5A. Luciferase Assays—For all reporter assays, cells were seeded into 6-well plates, and all treatment groups were performed in triplicate. For the NF-κB reporter assays in 293 cells, 50 ng of 2κB-luc reporter along with 2 μg of the indicated expression vectors were transfected into each well. Total DNA amount was equalized with control vector. At 24 h post-transfection, the cells were washed once with PBS and lysed in 0.5 ml of reporter lysis buffer (Promega). Luciferase activity was measured as described previously (12Birkey Reffey S. Wurthner J.U. Parks W.T. Roberts A.B. Duckett C.S. J. Biol. Chem. 2001; 276: 26542-26549Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) using the luciferase assay system (Promega) and a TR717 Applied Biosystems microplate luminometer. For the TGF-β-mediated reporter experiments in MDA-MB-468 cells, 200 ng of SBE-JONK reporter, 20 ng of Myc-Smad4, and 1 μg of the indicated expression vector were transfected into each well. At 24 h post-transfection, the medium was changed to Dulbecco’s modified Eagle’s medium plus 0.2% FCS with or without 5 ng/ml human TGF-β1 (Roche Applied Science). The luciferase assay was performed 24 h later as described above. Western Blot—Human embryonic kidney 293 cells were plated into 6-well dishes and were transfected with 2 μg of the indicated expression vector and lysed 24–48 h later using Triton X-100 lysis buffer (49Richter B.W.M. Mir S.S. Eiben L.J. Lewis J. Reffey S.B. Frattini A. Tian L. Frank S. Youle R.J. Nelson D.L. Notarangelo L.D. Vezzoni P. Fearnhead H.O. Duckett C.S. Mol. Cell Biol. 2001; 21: 4292-4301Crossref PubMed Scopus (87) Google Scholar). Protein samples were resolved using 4–12% gradient Novex BisTris gels (Invitrogen), transferred to nitrocellulose membranes (Invitrogen), and blocked with 5% milk solution in TBS plus 0.2% Tween 20. For the XIAP Western blots, the membranes were incubated with a monoclonal antibody to XIAP (H59520; Transduction Laboratories) followed by incubation with horseradish peroxidase-conjugated sheep anti-mouse secondary antibody (Amersham Biosciences). Detection of the HA-tagged XIAP mutants was performed using a monoclonal antibody directed against the HA tag (HA11; Covance). Antibody detection was performed by the ECL Western blot detection system (Amersham Biosciences). Caspase Assays and Recombinant Proteins—The recombinant GST-XIAP fusion proteins were prepared by transforming the pGEX-4T1 vectors into BL21 (DE3)-competent cells (Stratagene). Cultures were allowed to grow until A600 = 0.6 and were induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside. The cultures were kept shaking at 30 °C for an additional 3 h. Bacterial pellets were lysed using GST lysis buffer (50 mm Tris, pH 8.0, 1 mm EDTA, 100 mm NaCl, and 1% Triton X-100) followed by sonication. The clarified lysates were run over a glutathione-Sepharose column (Amersham Biosciences), washed with GST lysis buffer, and eluted in a buffer containing free glutathione (100 mm Tris, pH 8.0, 0.615 g/dl of glutathione). The eluates were then dialyzed overnight at 4 °C (25 mm HEPES, pH 7.9, 50 mm NaCl, 5 mm EGTA, 1 mm MgCl2, 10% glycerol, 0.1% CHAPS) and stored at -80 °C until use. The S-100 extracts were prepared from 293 cells as described previously (49Richter B.W.M. Mir S.S. Eiben L.J. Lewis J. Reffey S.B. Frattini A. Tian L. Frank S. Youle R.J. Nelson D.L. Notarangelo L.D. Vezzoni P. Fearnhead H.O. Duckett C.S. Mol. Cell Biol. 2001; 21: 4292-4301Crossref PubMed Scopus (87) Google Scholar). The caspase assays were set up by mixing 125 ng of the indicated GST fusion protein with 293 S-100 extracts and incubating this mixture for 30 min at 4 or 37 °C. Following the 30-min incubation, dATP was added to a final concentration of 1 mm, and the reaction mixture was incubated for an additional 20 min. An aliquot of the reaction was analyzed for caspase-3 activity, an indirect measurement of apoptosome and caspase-9 activation. The assay was performed in a caspase reaction buffer (50 mm PIPES, pH 7.0, 0.1 mm EDTA, 1 mm dithiothreitol, 10% glycerol) with the addition of 20 μm DEVD-AFC; cleavage was measured using a fluorescence plate reader (Cytofluor 4000; Perseptive Biosystems). To measure direct inhibition of caspase-3 activity, increasing amounts of recombinant GST fusion proteins were added to 10 ng of purified recombinant caspase-3 (BD Biosciences) in caspase reaction buffer with the addition of DEVD-AFC, and cleavage was monitored as described above. Kinase Assays—Human embryonic kidney 293 cells were transfected with 0.5 μg of HA-JNK1 and 2 μg of the indicated plasmids. HA-JNK1 was precipitated with a monoclonal anti-HA antibody (12CA5; Roche Applied Science), and kinase assays were performed as previously described (12Birkey Reffey S. Wurthner J.U. Parks W.T. Roberts A.B. Duckett C.S. J. Biol. Chem. 2001; 276: 26542-26549Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Activation of JNK1 was determined by in vitro phosphorylation of recombinant GST-c-Jun-(1–79) and was quantified using a PhosphorImager (Amersham Biosciences). Expression of HA-JNK1 and XIAP was confirmed by immunoblotting of lysates with HA- and XIAP-specific antibodies as described above. The -fold activation was normalized to the total amount of HA-JNK1 detected in each case. Generation of XIAP Mutants—XIAP is capable of binding to and inhibiting the enzymatic activity of key caspases involved in the activation of cell death pathways, conferring protection from intrinsic and extrinsic apoptotic stimuli (Fig. 1A). In order to evaluate the potential contribution of caspase inhibition to the signaling properties of XIAP, mutations intended to disrupt the ability of XIAP to bind to and inhibit caspases were introduced in the molecule as previously described (24Bratton S.B. Lewis J. Butterworth M. Duckett C.S. Cohen G.M. Cell Death Differ. 2002; 9: 881-892Crossref PubMed Scopus (118) Google Scholar). Using site-directed mutagenesis, the mutations D148A and W310A were introduced to disrupt the binding to caspase-3 and -7 or caspase-9, respectively (Fig. 1B). These mutations were introduced independently (XIAP D148A or XIAP W310A) or simultaneously (XIAP D148A/W310A) to generate three XIAP caspase-binding mutants. In order to assess the contribution of the RING domain of XIAP and its E3 ubiquitination activity to its ability to activate signal transduction, two additional mutants of XIAP were generated. A mutation creating a stop codon just prior to the RING domain at amino acid 448 (XIAP ΔRING) was introduced. In addition, a point mutation at histidine 467 (H467A), which results in loss of E3 ubiquitin ligase activity (19Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (873) Google Scholar), was also generated. Functional Characterization of XIAP Caspase-binding Mutants—The caspase-inhibitory activity of XIAP proteins, including the wild type form and the point mutations expected to affect caspase binding, was tested in vitro. The ability of recombinant XIAP proteins to directly inhibit purified recombinant caspase-3 was tested first. To determine whether there was a difference in inhibitory activity between the various recombinant XIAP proteins, increasing amounts of recombinant XIAP were incubated with 10 ng of purified recombinant caspase-3. Both the wild type and W310A mutant showed caspase-3-inhibitory activity starting at 10 ng of recombinant XIAP. The mutants that contained the D148A mutation targeting the caspase-3 binding motif in XIAP were unable to inhibit recombinant caspase-3 activity even at the highest con