The Ret Finger Protein Induces Apoptosis via Its RING Finger-B Box-Coiled-coil Motif
2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês
10.1074/jbc.m304062200
ISSN1083-351X
Autores Tópico(s)Retinoids in leukemia and cellular processes
ResumoThe Ret finger protein (RFP) is a member of the tripartite motif family, which is characterized by a conserved RING finger, a B-box, and a coiled-coil domain (together called RBCC). Although RFP is known to become oncogenic when its RBCC moiety is connected to a tyrosine kinase domain by DNA rearrangement, its biological function is not well defined. Here we show that ectopic expression of RFP in human embryonic kidney 293 cells causes extensive apoptosis, as assessed by multiple criteria. RFP expression activates Jun N-terminal kinase and p38 kinase and also increases caspase-3-like activity. However, RFP failed to release cytochrome c and, therefore, to increase caspase-9-like activity. RFP-induced apoptosis could be blocked by the caspase-8 inhibitor crmA and dominant negative ASK1 but not by Bcl-2. These results reveal a novel RFP death pathway that recruits mitogen-activated protein kinase and caspases independently of mitochondrial events. Domain mapping showed that the intact RBCC moiety is necessary for the pro-apoptotic function of RFP. Moreover, expression of the RBCC moiety further potentiated the pro-apoptotic activity and resulted in a 7-fold increase of caspase activation compared with that induced by full-length RFP. This suggests that a large number of tripartite motif family members sharing the RBCC moiety may participate in the control of cell survival. The Ret finger protein (RFP) is a member of the tripartite motif family, which is characterized by a conserved RING finger, a B-box, and a coiled-coil domain (together called RBCC). Although RFP is known to become oncogenic when its RBCC moiety is connected to a tyrosine kinase domain by DNA rearrangement, its biological function is not well defined. Here we show that ectopic expression of RFP in human embryonic kidney 293 cells causes extensive apoptosis, as assessed by multiple criteria. RFP expression activates Jun N-terminal kinase and p38 kinase and also increases caspase-3-like activity. However, RFP failed to release cytochrome c and, therefore, to increase caspase-9-like activity. RFP-induced apoptosis could be blocked by the caspase-8 inhibitor crmA and dominant negative ASK1 but not by Bcl-2. These results reveal a novel RFP death pathway that recruits mitogen-activated protein kinase and caspases independently of mitochondrial events. Domain mapping showed that the intact RBCC moiety is necessary for the pro-apoptotic function of RFP. Moreover, expression of the RBCC moiety further potentiated the pro-apoptotic activity and resulted in a 7-fold increase of caspase activation compared with that induced by full-length RFP. This suggests that a large number of tripartite motif family members sharing the RBCC moiety may participate in the control of cell survival. The Ret finger protein (RFP) 1The abbreviations used are: RFP, Ret finger protein; TRIM, tripartite motif; RBCC, RING, B-box, coiled-coil; TIF, transcriptional intermediary factor; PML, promyelocytic leukemia protein; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; JNK, Jun N-terminal kinase; TNFR, tumor necrosis factor receptor; FADD, Fas-associated death domain; CMV, cytomegalovirus; HEK, human embryonic kidney; AMC, 7-amino-4-methyl coumarin; MOPS, 4-morpholinepropanesulfonic acid; GST, glutathione S-transferase; EGFP, enhanced green fluorescent protein; ASK1, apoptosis signal-regulating kinase 1. is a member of the tripartite motif (TRIM) protein family, also known as the RING-B box-coiled-coil (RBCC) family (1Takahashi M. Buma Y. Iwamoto T. Inaguma Y. Ikeda H. Hiai H. Oncogene. 1988; 3: 571-578PubMed Google Scholar, 2Saurin A.J. Borden K.L. Boddy M.N. Freemont P.S. Trends Biochem. Sci. 1996; 21: 208-214Abstract Full Text PDF PubMed Scopus (613) Google Scholar). RFP is also designated TRIM 27, based on the 37 known members of the TRIM family (3Reymond A. Meroni G. Fantozzi A. Merla G. Cairo S. Luzi L. Riganelli D. Zanaria E. Messali S. Cainarca S. Guffanti A. Minucci S. Pelicci P.G. Ballabio A. EMBO J. 2001; 20: 2140-2151Crossref PubMed Scopus (1053) Google Scholar). The TRIM contains a RING finger (R), one or two B-boxes (B1, B2), followed by a coiled-coil domain (CC); therefore, it is also called the RBCC. In addition to these characteristic features, RFP contains an additional specific carboxyl-terminal region known as the RFP domain. Several TRIM proteins play key roles in regulating gene expression and cell proliferation. For example, transcriptional intermediary factor 1α (TIF1α)/TRIM24, TIF1β/KAP1/TRIM28, and promyelocytic leukemia protein (PML)/TRIM19, modulate transcriptional machinery to control specific gene expression during cell proliferation, differentiation, and development (4Remboutsika E. Lutz Y. Gansmuller A. Vonesch J.L. Losson R. Chambon P. J. Cell Sci. 1999; 112: 1671-1683Crossref PubMed Google Scholar, 5Ryan R.F. Schultz D.C. Ayyanathan K. Singh P.B. Friedman J.R. Fredericks W.J. Rauscher 3rd., F.J. Mol. Cell. Biol. 1999; 19: 4366-4378Crossref PubMed Scopus (315) Google Scholar, 6Jensen K. Shiels C. Freemont P.S. Oncogene. 2001; 20: 7223-7233Crossref PubMed Scopus (382) Google Scholar). TRIMs have been implicated in several human diseases; mutations in Pyrin/TRIM20, MID1/TRIM18, and MUL/TRIM37 have been associated with familial Mediterranean fever, X-linked Opitiz/GBBB syndrome, and mulibrey nanism, respectively (7Consortium T.I.F. 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The RBCC moiety of TIF1α fuses to the kinase domain of B-Raf and Ret tyrosine kinase in mouse hepatocellular carcinoma and human papillary thyroid carcinoma (PTC), respectively (13Le Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat P. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (575) Google Scholar, 14Klugbauer S. Rabes H.M. Oncogene. 1999; 18: 4388-4393Crossref PubMed Scopus (154) Google Scholar). The RBCC moiety of RFP was found to be fused to the Ret tyrosine kinase in transformed NIH3T3 cells (12Takahashi M. Cooper G.M. Mol. Cell. Biol. 1987; 7: 1378-1385Crossref PubMed Scopus (287) Google Scholar). It has been shown that the RBCC moiety is required for the transforming capacities of these TRIM oncogenes (6Jensen K. Shiels C. Freemont P.S. Oncogene. 2001; 20: 7223-7233Crossref PubMed Scopus (382) Google Scholar, 13Le Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat P. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (575) Google Scholar, 14Klugbauer S. Rabes H.M. Oncogene. 1999; 18: 4388-4393Crossref PubMed Scopus (154) Google Scholar, 15Tong Q. Xing S. Jhiang S.M. J. Biol. Chem. 1997; 272: 9043-9047Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 16Hasegawa N. Iwashita T. Asai N. Murakami H. Iwata Y. Isomura T. Goto H. Hayakawa T. Takahashi M. Biochem. Biophys. Res. Commun. 1996; 225: 627-631Crossref PubMed Scopus (34) Google Scholar). RFP often localizes in discrete nuclear structures called PML nuclear bodies (17Cao T. Borden K.L. Freemont P.S. Etkin L.D. J. Cell Sci. 1997; 110: 1563-1571Crossref PubMed Google Scholar, 18Cao T. Duprez E. Borden K.L. Freemont P.S. Etkin L.D. J. Cell Sci. 1998; 111: 1319-1329Crossref PubMed Google Scholar), where it binds directly to PML (another TRIM family member) as well as Int-6, expression of which induces malignancy in transfected cell lines (19Rasmussen S.B. Kordon E. Callahan R. Smith G.H. Oncogene. 2001; 20: 5291-5301Crossref PubMed Scopus (62) Google Scholar). PML acts as a cell growth and tumor suppressor through its ability to control apoptosis (20Borden K.L. CampbellDwyer E.J. Salvato M.S. FEBS Lett. 1997; 418: 30-34Crossref PubMed Scopus (73) Google Scholar, 21Wang Z.G. Ruggero D. Ronchetti S. Zhong S. Gaboli M. Rivi R. Pandolfi P.P. Nat. Genet. 1998; 20: 266-272Crossref PubMed Scopus (100) Google Scholar, 22Quignon F. De Bels F. Koken M. Feunteun J. Ameisen J.C. de The H. Nat. Genet. 1998; 20: 259-265Crossref PubMed Scopus (340) Google Scholar). Although the biological function of RFP is largely unknown, it is possible that it influences apoptotic pathways in a manner similar to PML. Apoptosis is a physiological cell suicide process essential in development and homeostasis. Execution of apoptosis involves activation of signaling by mitogen-activated protein (MAP) kinases and/or caspases. Various stresses activate two well-defined MAP kinase signaling modules, JNK and p38. MAP kinase kinase kinases such as MEKK1 and ASK1 activate two different subgroups of MAP kinase kinase, SEK1 (or MKK4) and MKK3/MKK6, which in turn activate the JNK and p38 subgroups of MAP kinase (MAPK), respectively (23Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3646) Google Scholar, 24Schaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1404) Google Scholar). JNK and p38 up-regulate Fas ligands, activate Bid, and/or inactivate Bcl-2 to trigger receptor- or mitochondria-mediated apoptotic pathways. Caspases, a class of cysteine protease, are activated by at least two mechanisms. One involves a direct pathway via stimulation of death receptors such as Fas and tumor necrosis factor receptor 1 (TNFR1), recruitment and activation of caspase-8 through the adaptor protein Fas-associated death domain (FADD), and subsequent activation of caspase-3, -6, and -7. The other passes through mitochondria (25Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 26Loeffler M. Kroemer G. Exp. Cell Res. 2000; 256: 19-26Crossref PubMed Scopus (333) Google Scholar). Pro-apoptotic Bcl-2 family members, once activated by intracellular stresses such as cytokine deprivation and genotoxic damage, permeabilize the outer mitochondrial membrane and release cytochrome c. In the cytosol, cytochrome c controls the assembly of an apoptosome composed of oligomers of Apaf-1 and procaspase-9, thereby triggering activation of caspase-9 and subsequent activation of caspase-3. In addition to MAP kinase, caspase, and Bcl-2 families, a variety of signaling molecules have been suggested to regulate apoptosis, and overexpression of some of them induces apoptosis. For instance, overexpression of PML, p53, Daxx, or HEF1 triggers apoptosis through various mechanisms (22Quignon F. De Bels F. Koken M. Feunteun J. Ameisen J.C. de The H. Nat. Genet. 1998; 20: 259-265Crossref PubMed Scopus (340) Google Scholar, 27Li P.F. Dietz R. von Harsdorf R. EMBO J. 1999; 18: 6027-6036Crossref PubMed Scopus (427) Google Scholar, 28Yang X. Khosravi-Far R. Chang H.Y. Baltimore D. Cell. 1997; 89: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (826) Google Scholar, 29Law S.F. O'Neill G.M. Fashena S.J. Einarson M.B. Golemis E.A. Mol. Cell. Biol. 2000; 20: 5184-5195Crossref PubMed Scopus (79) Google Scholar). Here, we demonstrate for the first time that the RBCC moiety of RFP triggers a rapid apoptosis through activation of stress-activated MAP kinases and caspases. Considering the known pro-apoptotic activity of PML (21Wang Z.G. Ruggero D. Ronchetti S. Zhong S. Gaboli M. Rivi R. Pandolfi P.P. Nat. Genet. 1998; 20: 266-272Crossref PubMed Scopus (100) Google Scholar, 22Quignon F. De Bels F. Koken M. Feunteun J. Ameisen J.C. de The H. Nat. Genet. 1998; 20: 259-265Crossref PubMed Scopus (340) Google Scholar), another TRIM member, our results suggest that a large number of TRIM family members sharing the RBCC moiety may participate in the control of cell survival. Plasmid Construction—The full-length human RFP cDNA (provided by M. Takahashi) was used as a template to generate deletion constructs by PCR. The cDNA fragments encoding the RING finger (amino acids 1–62), RING finger-B box (amino acids 1–132), RING finger-B box-coiled-coil (amino acids 1–317), B box-coiled-coil (amino acids 63–317), coiled-coil (amino acids 133–317), RFP domain (amino acids 318–513), and full-length RFP (amino acids 1–513) were amplified by PCR, subcloned into the pFLAG-CMV-2 plasmid, and confirmed by direct sequencing. Cell Culture and Immunoblots—HEK 293 and HEK 293T cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) with 10% fetal calf serum (Invitrogen) in a 5% CO2 atmosphere at 37 °C. Cells were transfected by the calcium phosphate precipitation method. At indicated times after transfection, cells were harvested and lysed in a lysis buffer containing 20 mm HEPES, pH 7.5, 50 mm NaCl, 10% glycerol, and 0.5% Triton X-100. Cell extracts were separated with SDS-PAGE and transferred to a nitrocellulose membrane (Schleicher and Schuell). After blocking with 5% skim milk in 20 mm Tris, pH 7.4, and 150 mm NaCl containing 0.05% Tween 20, the membranes were probed with rabbit anti-mouse RFP antiserum, mouse anti-FLAG antibody (Sigma), goat anti-actin antibody (Santa Cruz Biotechnology), rabbit anti-phospho-SEK1/MKK4 (Thr-261; New England Biolabs), rabbit anti-phospho-MKK3/MKK6 (Ser-189/207; New England Biolabs), or rabbit anti-phospho-p38 MAP kinase (Thr-180/Tyr-182; New England Biolabs). Blots were washed three times with Tris-buffered saline/Tween 20 and incubated with peroxidase-conjugated anti-mouse IgG antibody (Pierce), anti-goat IgG antibody (Sigma), or anti-rabbit IgG antibody (Upstate Biotechnologies), then developed with the use of a chemiluminescence detection system (Pierce). Caspase Assay—Approximately 106 cells were used for measurement of caspase activities. Caspase-3-, -8-, and -9-like activities were measured using the synthetic substrates DEVD-7-amino-4-methyl coumarin (AMC; Peptron), IETD-AMC (Peptron), and LEHD-7-amino-4-trifluoromethyl coumarin (Calbiochem), respectively. Activities were assayed according to the manufacturer's instructions. The fluorescence of the released AMC was measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The excitation and emission wavelengths for 7-amino-4-trifluoromethyl coumarin were 400 nm and 505 nm, respectively. The results are expressed as fold-increase in the caspase activity of sample cells compared with control cells transfected with empty vectors. Immunocomplex Kinase Assays—Cells were washed with phosphate-buffered saline and lysed in a lysis buffer containing 20 mm HEPES, pH 7.5, 50 mm NaCl, 10% glycerol, 0.5% Triton X-100, and 0.1 mm sodium orthovanadate. The lysis buffer was supplemented with a mixture of protease inhibitors (0.1 mm 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 2.0 μg/ml aprotinin, 2.0 μg/ml leupeptin) before use. The cell lysates were immunoprecipitated with anti-JNK1 (BD Pharmingen) or anti-MEKK1 (C-22; Santa Cruz Biotechnology) antibodies, bound to protein G agarose (Sigma) and washed twice with lysis buffer, twice with LiCl buffer (100 mm Tris-HCl, pH 7.6, 500 mm LiCl, and 0.1% Triton X-100) and then twice with kinase reaction buffer (20 mm MOPS, pH 7.2, 2 mm EGTA, 10 mm MgCl2, 1 mm dithiothreitol, and 0.1 mm sodium orthovanadate). To measure the kinase reaction, beads were incubated with 2 μCi of [γ-32P]ATP and 1 μg of GST-c-Jun or GST-SEK1(K129R). The samples were subjected to 12% SDS-PAGE, and the phosphorylation of substrate proteins was analyzed by exposing the gels to x-ray film or a BAS 1500 phosphorescence image analyzer (Fuji). Other protein kinase activities were examined by immunoblot analysis of the cell lysates with antibodies against phospho-JNK, phospho-SEK1, phospho-p38, and phospho-MKK3/6. Annexin V and Propidium Iodide Staining—Apoptosis was monitored by measuring the distribution of plasma membrane phosphatidylserine and hypodiploid DNA content. Cells were trypsinized, collected by centrifugation, and washed with phosphate-buffered saline. Aliquots of cells were incubated with Annexin V-fluorescein isothiocyanate (BD Pharmingen) for 15 min according to the manufacturer's instructions. For propidium iodide staining of DNA, aliquots of cells were fixed in 70% ethanol and treated with 50 μg/ml propidium iodide in phosphate-buffered saline. The fluorescence emitted by the cells was analyzed using a FACSCalibur flow cytometer (BD Biosciences). Detection of Cytochrome c Release—Approximately 106 cells were trypsinized and collected by centrifugation, and cell pellets were washed with phosphate-buffered saline and resuspended in 500 μl of isotonic solution (10 mm HEPES, pH 8.0, 250 mm sucrose, 1 mm EDTA, 1 mm EGTA, and 1 mm dithiothreitol) supplemented with a mixture of protease inhibitors. The cells were then homogenized with 40 strokes of a loose pestle in a Dounce homogenizer (Wheaton). After removal of unbroken cells, large plasma membrane pieces, and nuclei by centrifugation at 600 × g for 10 min, the mitochondria-enriched fraction was pelleted by centrifugation at 15,000 × g for 20 min. The supernatant was then centrifuged at 50,000 × g for 30 min to generate a cytosolic fraction. The cytosolic and mitochondria-enriched fractions were subjected to immunoblotting with a monoclonal antibody to cytochrome c (BD Pharmingen). RFP Expression Induces Apoptosis—Given that many TRIM proteins play roles in cell proliferation, we examined the effect of RFP overexpression on cell proliferation. Transient transfection of RFP into HEK 293 cells resulted in morphological changes suggestive of apoptosis. On gross examination, we found that cells became round, displayed membrane blebbing, and initiated detachment from the dish within 36 h after transfection. pEGFP (Clontech) was co-transfected with pCMV-RFP to distinguish the transfected cells from nontransfected cells. All EGFP-positive cells showed membrane blebbing, whereas EGFP-negative cells were normal in shape (Fig. 1A). Apoptosis induced by RFP expression was further confirmed by Annexin V assay. pCMV-RFP transfection resulted in increased Annexin V-positive cells compared with empty vector transfection (Fig. 1B), indicating a typical apoptotic event. RFP Activates both JNK and p38 Signaling Pathways— Because JNK and p38 kinase signaling play important roles in apoptotic cell death (23Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3646) Google Scholar, 24Schaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1404) Google Scholar), we investigated JNK and p38 kinase cascades after RFP overexpression in cells. First, we examined the activity of JNK1, the major and ubiquitously expressed JNK, by an in vitro-coupled kinase assay using a recombinant c-Jun protein. An ASK1-expressing plasmid was transiently transfected as a positive control, because ASK1 has been shown to induce JNK activation (30Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2027) Google Scholar). RFP expression in HEK 293 cells activated JNK1 to an extent similar to that of ASK1 when protein levels were normalized (Fig. 2A). Similarly, we found RFP-mediated activation of both exogenous and endogenous forms of the other stress-activated MAPK, p38. This activation was lower than that of JNK1 under the same experimental conditions (Fig. 2C). Then we examined the effects of RFP expression on kinases upstream of JNK1 and p38. Among the tested MAP kinase kinases, SEK1 and MKK6 were highly activated, whereas MKK3 was weakly activated (Fig. 2, D and E). The activations of MAP kinase kinase kinases such as ASK1 and MEKK1 were also examined to determine which MAP kinase kinase kinase might be involved in phosphorylation of SEK1 and MKK3/MKK6 during RFP-induced apoptosis. Expression of RPF did not activate MEKK1, in comparison with clear activation by the positive control, UV irradiation (Fig. 2B). Because of technical difficulties, we were unable to observe ASK1 activation in either the experimental or positive control measurements of autophosphorylation and in vitro kinase reaction using a recombinant GST-SEK1(K129R) kinase-inactive protein substrate (data not shown). Therefore, we used a dominant-negative mutant of ASK1 to determine its involvement in RFP signaling. Dominant-negative mutants of ASK1 and SEK1 significantly inhibited RFP-induced apoptosis (Fig 3A), suggesting that ASK1 activation is required for this apoptosis. Taken together, these results suggest that RFP expression induces stress-activated MAPK signaling cascades likely to originate with ASK1. Caspases Are Activated during RFP-triggered Apoptosis— Caspase induction is a specific indicator of apoptotic cell death. In response to apoptotic stimuli, initiator caspases such as caspase-2, -8, -9, and -10 are activated by self-cleavage and in turn cleave effector caspases such as caspase-3, -6, and -7, leading to degradation of specific cellular components and ultimately to cell death (31Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5154) Google Scholar, 32Salvesen G.S. Dixit V.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10964-10967Crossref PubMed Scopus (774) Google Scholar). To determine whether caspases are activated by RFP expression, we examined the activities of caspase-3-, -8-, and -9-like proteases by detecting cleavage of the fluorogenic substrates DEVD-AMC, IETD-AMC and LEHD-7-amino-4-trifluoromethyl coumarin, respectively (33Talanian R.V. Quinlan C. Trautz S. Hackett M.C. Mankovich J.A. Banach D. Ghayur T. Brady K.D. Wong W.W. J. Biol. Chem. 1997; 272: 9677-9682Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar). Expression of RFP in HEK 293 cells strongly induced caspase-3-like activity (∼4-fold that of control cells; Fig. 3B), and weakly induced caspase-8-like activity (less than 2-fold; data not shown), whereas there was little induction of caspase-9-like activity at 36 h after transfection. We next assessed whether caspase-8 and/or caspase-3 are required for RFP-induced apoptosis. To test the requirement of caspase-8, we co-expressed RFP with the viral protein crmA, which is known to inhibit caspase-8. To test the requirement of caspase-3, we treated the RFP-expressing cells with the peptidyl caspase-3 inhibitor benzyloxycarbonyl-DEVD-fluoromethyl ketone. Both inhibitors markedly suppressed RFP-induced apoptosis (Fig. 3A), implying that activation of caspase-8 and the subsequent activation of caspase-3 contribute a great deal to this apoptosis. Taken together, our results suggest that the MAPK and caspase pathways are both required for RFP-induced apoptosis and that neither of them is sufficient to initiate apoptosis alone. Two Distinct RFP-mediated Signaling Pathways—We next considered the relationship between the two pathways in RFP-induced apoptosis. For example, MAPKs activated by ASK1 expression can induce caspase activation via cytochrome c release from mitochondria, as shown by the triggering of apoptosis in Mv1Lu and MEF cells by a constitutively active form of ASK1 (34Hatai T. Matsuzawa A. Inoshita S. Mochida Y. Kuroda T. Sakamaki K. Kuida K. Yonehara S. Ichijo H. Takeda K. J. Biol. Chem. 2000; 275: 26576-26581Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). Conversely, caspase-3 can cleave MEKK1, which in turn activates downstream MAPKs in the Fas-mediated apoptosis of vascular smooth muscle cells (35Suhara T. Kim H.S. Kirshenbaum L.A. Walsh K. Mol. Cell. Biol. 2002; 22: 680-691Crossref PubMed Scopus (135) Google Scholar). Therefore, we questioned whether caspases might be activated by MAPKs or vice versa in RFP-induced apoptosis. However, the observation that caspases were active even when MAPKs were blocked by expression of ASK1- or SEK1 dominant-negative mutants suggests that the RFP-induced caspase cascade does not occur through the activation of MAPKs (Fig. 3B). In addition, the observation that JNK1/2 was not inhibited by caspase inhibitor crmA expression or DEVD treatment suggests that RFP does not initiate MAPK cascades via caspase activation, either (Fig. 3C). Thus, the MAPK and caspase cascades may mediate apoptotic signaling downstream of RFP by two distinct pathways. Several reports have indicated that JNK activity and caspase activity are both required for apoptosis. For example, the amyloid β-peptide (Aβ 17–42) leads to apoptosis in human neuroblastoma cells through activation of caspase-8/-3 as well as JNK (36Wei W. Norton D.D. Wang X. Kusiak J.W. Brain. 2002; 125: 2036-2043Crossref PubMed Google Scholar). Fas- and UV-induced apoptosis in HEK 293 cells is mediated by independent actions of the JNK and FADD/caspase pathways (28Yang X. Khosravi-Far R. Chang H.Y. Baltimore D. Cell. 1997; 89: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (826) Google Scholar, 37Wu S. Loke H.N. Rehemtulla A. Neoplasia. 2002; 4: 486-492Crossref PubMed Scopus (38) Google Scholar), and overexpression of the docking protein HEF1 causes apoptosis by simultaneously activating the JNK and caspase pathways (29Law S.F. O'Neill G.M. Fashena S.J. Einarson M.B. Golemis E.A. Mol. Cell. Biol. 2000; 20: 5184-5195Crossref PubMed Scopus (79) Google Scholar). RFP Expression Leads to Apoptosis Independent of a Mitochondrial Event—Apoptotic signals often lead to mitochondrial dysfunction (25Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 26Loeffler M. Kroemer G. Exp. Cell Res. 2000; 256: 19-26Crossref PubMed Scopus (333) Google Scholar), which includes loss of membrane potential, production of reactive oxygen species, opening of the permeability transition pores, and the release of the intermembrane space protein, cytochrome c. To investigate whether RFP causes the release of cytochrome c from the mitochondria, we examined the distribution of cytochrome c after RFP transfection. Subcellular fractions including either cytosol or mitochondria-enriched heavy membranes (HM) were prepared, and cytochrome c protein levels were measured by immunoblotting. Cytosolic cytochrome c increased significantly in a positive control experiment using staurosporine as an apoptotic reagent (Fig. 4A). In contrast, there was no significant change of cytosolic cytochrome c in RFP-expressing cells (Fig. 4A). This is consistent with our earlier observation that caspase-9-like activity, inducible by cytochrome c release, was little changed during RFP-induced apoptosis. We therefore suggest that cytochrome c release does not occur during RFP-mediated apoptosis. Death pathways mediated by endogenous gene products such as p53 (27Li P.F. Dietz R. von Harsdorf R. EMBO J. 1999; 18: 6027-6036Crossref PubMed Scopus (427) Google Scholar) and MEKK1 (38Gibson E.M. Henson E.S. Villanueva J. Gibson S.B. J. Biol. Chem. 2002; 277: 10573-10580Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) are similar examples of apoptotic inductions that are not accompanied by cytochrome c release. To further investigate the involvement of mitochondria in RFP-induced apoptosis, we next determined whether the expression of Bcl-2 or Bcl-xL, which blocks the mitochondrial permeability transition and subsequent release of cytochrome c (25Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar), inhibited RFP-mediated apoptosis. We found that neither Bcl-2 nor Bcl-xL blocked apoptosis induced by RFP (Fig. 4B), whereas Bcl-2 and Bcl-xL suppressed up to 52 and 58% of staurosporine-induced apoptosis, respectively. In addition, we determined intracellular production of reactive oxygen species, a known marker for mitochondria-dependent apoptosis (25Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 26Loeffler M. Kroemer G. Exp. Cell Res. 2000; 256: 19-26Crossref PubMed Scopus (333) Google Scholar), in RFP-expressing cells by staining with the reactive oxygen species-sensitive fluorescent dye 2′,7′-dichlorofluorescein diacetate. Reactive oxygen species levels were not changed significantly even after the RFP-transfected cells showed apoptotic morphology (data not shown). Taken together, our results indicate that RFP induces apoptosis in a mitochondria-independent fashion. RBCC Is Sufficient to Induce Cell Death—To further dissect RFP signaling, we investigated which region of RFP is required fo
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