A Bax/Bak-independent Mitochondrial Death Pathway Triggered by Drosophila Grim GH3 Domain in Mammalian Cells
2004; Elsevier BV; Volume: 279; Issue: 2 Linguagem: Inglês
10.1074/jbc.m309819200
ISSN1083-351X
AutoresCristina Claverı́a, Carlos Martínez‐A, Miguel Torres,
Tópico(s)Heat shock proteins research
ResumoGrim encodes a protein required for programmed cell death in Drosophila, whose proapoptotic activity is conserved in mammalian cells. Two proapoptotic domains are relevant for Grim killing function; the N-terminal region, which induces apoptosis by disrupting inhibitor of apoptosis protein (IAP) blockage of caspase activity, and the internal GH3 domain, which triggers a mitochondrial pathway. We explored the role of these two domains in heterologous killing of mammalian cells by Grim. The GH3 domain is essential for Grim proapoptotic activity in mouse cells, whereas the N-terminal domain is dispensable. The GH3 domain is required and sufficient for Grim targeting to mitochondria and for cytochrome c release in a caspase- and N-terminal-independent, IAP-insensitive manner. These Grim GH3 activities do not require Bax or Bak function, revealing GH3 activity as the first proapoptotic stimulus able to trigger the mitochondrial death pathway in mammalian cells in the absence of multidomain proapoptotic Bcl-2 proteins. Grim encodes a protein required for programmed cell death in Drosophila, whose proapoptotic activity is conserved in mammalian cells. Two proapoptotic domains are relevant for Grim killing function; the N-terminal region, which induces apoptosis by disrupting inhibitor of apoptosis protein (IAP) blockage of caspase activity, and the internal GH3 domain, which triggers a mitochondrial pathway. We explored the role of these two domains in heterologous killing of mammalian cells by Grim. The GH3 domain is essential for Grim proapoptotic activity in mouse cells, whereas the N-terminal domain is dispensable. The GH3 domain is required and sufficient for Grim targeting to mitochondria and for cytochrome c release in a caspase- and N-terminal-independent, IAP-insensitive manner. These Grim GH3 activities do not require Bax or Bak function, revealing GH3 activity as the first proapoptotic stimulus able to trigger the mitochondrial death pathway in mammalian cells in the absence of multidomain proapoptotic Bcl-2 proteins. Multicellular organisms eliminate unwanted or damaged cells by cell death, a process essential in shaping embryonic structures, maintaining adult tissue homeostasis, and eliminating damaged cells. Most cell death processes are executed through a defined pattern of cellular and biochemical events known as apoptosis (1.Green D.R. Cell. 2000; 102: 1-4Abstract Full Text Full Text PDF PubMed Scopus (886) Google Scholar, 2.Kerr J.F. Wyllie A.H. Currie A.R. Br. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12858) Google Scholar, 3.Wyllie A.H. Kerr J.F. Currie A.R. Int. Rev. 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In vertebrates, members of the Bcl-2 homology group comprising both pro- and antiapoptotic molecules play a central role in regulating mitochondrial-mediated cell death (29.Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3251) Google Scholar). Of the four homology blocks found among the Bcl-2 family members, the BH3 domain appears essential for the function of the proapoptotic members. For some of these, BH3 is the only domain shared with the rest of the family (30.Kelekar A. Thompson C.B. Trends Cell Biol. 1998; 8: 324-330Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar), and its amphipathic α-helical structure is an essential feature, required for its biological activity. Recent studies in cells from mice deficient in both Bax and Bak (DKO) demonstrated that BH3-only proteins are unable to kill cells in the absence of Bax and Bak multidomain proapoptotic proteins (31.Cheng E.H. Wei M.C. Weiler S. Flavell R.A. Mak T.W. 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Here we explore the relevance of the GH3 domain in the conservation of Grim proapoptotic function in vertebrate cells. Our results show that GH3 activity is conserved in evolution, triggering a mitochondrial-cytochrome c death pathway in mammalian cells, whereas the N-terminal domain appears irrelevant for Grim killing activity in this context. Furthermore, Grim-induced mitochondrial death pathway is independent of Bax and Bak function, identifying Grim as the first molecule able to trigger this pathway in mammalian cells in the absence of both multidomain proapoptotic Bcl-2 family members. Expression Vectors and Site-directed Mutagenesis—WT mouse bad was PCR-amplified from pEBG-mbad plasmid (New England Biolabs) with 5′-CTCTCTCTGCGGCCGCCATGGGAACCCCAAAGCAGCC-3′ and 5′-GCTCGGATCCTCACTGGGAGGGGGTGGAGC-3′ primers, which introduced NotI and BamHI sites. BadΔBH3 was amplified from pEBG-mbad with 5′-CTCTCTCTGCGGCCGCCATGGGAACCCCAAAGCAGCC-3′ and 5′-CCTTTAAAGGAGTAGCGCTGCGCTG-3′ primers for the 5′ region and 5′-TCCTTTAAAGGACTTCCTCGCCCAAAG-3′ and 5′-GCTCGGATCCTCACTGGGAGGGGGTGGAGC-3′ primers for the 3′ region; a DraI site was introduced without changing the amino acids coded, and both fragments were fused using this site, resulting in deletion of Bad amino acids 148-160. BadGH3 was amplified from pEBG-mbad with 5′-CTCTCTCTGCGGCCGCCATGGGAACCCCAAAGCAGCC-3′ and 5′-CTCAGATCTGGGCCAAAAGATCCCAGTAGCGCTGCGCTGCCCAG-3′ primers for the 5′ region and 5′-CTCAGATCTTCTGCTACGCTCTGCGATCCTTCAAGGGACTTCCTCGC-3′ and 5′-GCTCGGATCCTCACTGGGAGGGGGTGGAGC-3′ primers for the 3′ region; a BglII site present in the GH3 domain was used to fuse both fragments, resulting in replacement of WT Bad amino acids 148-160 with Grim 86-98 amino acids. The BadGH3LE 5′-end was amplified from pEBG-mbad with 5′-CTCTCTCTGCGGCCGCCATGGGAACCCCAAAGCAGCC-3′ and 5′-CTCAGATCTGGGCCTCAAGATCCCAGTAGCGCTGCGCTGCCCAG-3′ primers and fused to the BadGH3 3′-end using the BglII site. WT bad and the mutant forms were cloned in the pcDNA3.1 mammalian expression vector (Invitrogen) using NotI and BamHI sites, and the sequence was confirmed. The truncated mouse Bid form (tBid) was PCR-amplified from mouse embryo cDNA using 5′-CTCTCTCTGCGGCCGCCATGGGCAGCCAGGCCAGCCG-3′ and 5′-GCTCGGATCCTCAGTCCATCTCGTTTCTAACC-3′ primers, which introduced NotI and BamHI sites; it was cloned in the pcDNA3.1 mammalian expression vector using these sites, and the sequence was confirmed. Cell Culture and Death Assays—NIH 3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium (Bio-Whittaker) supplemented with 10% newborn calf serum and transfected using LipofectAMINE Plus (Invitrogen) as recommended by the manufacturer. For Grim mutants death assays, cells were cotransfected with pcDNA3.1 empty vector (control) or pcDNA3.1-grim (WT or mutants) expression vector (19.Clavería C. Caminero E. Martínez A.C. Campuzano S. Torres M. EMBO J. 2002; 21: 3327-3336Crossref PubMed Scopus (67) Google Scholar), together with pcDNA3.1-lacZ expression vector at a 2:1 molar ratio. After 30 h, cells were fixed in 0.2% glutaraldehyde, washed in PBS, and X-gal-stained following standard protocols. For Bad mutants death assays, cells were cotransfected with pcDNA3.1 empty vector (control) or pcDNA3.1-bad (WT or mutants) expression vector together with pcDNA3.1-lacZ expression vector at a 2:1 molar ratio. After 48 h, cells were fixed in 0.2% glutaraldehyde, washed in PBS, and X-gal-stained as above. WT and bax-/-bak-/- (DKO) simian virus 40 (SV40)-transformed mouse embryonic fibroblasts (MEF) were cultured as described previously (32.Wei M.C. Zong W.X. Cheng E.H. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Crossref PubMed Scopus (3352) Google Scholar) and transfected using LipofectAMINE Plus. For death assays, cells were cotransfected with pcDNA3.1 empty vector (control), pcDNA3.1-bad or pcDNA3.1-grim (WT or mutants) expression vector, together with pcDNA3.1-lacZ expression vector at a 2:1 molar ratio. After 30 h, cells were fixed in 0.2% glutaraldehyde, washed in PBS, and X-gal-stained as above. Western Blotting and Immunoprecipitation—To test the stability of the Grim protein and its mutant forms, NIH 3T3 fibroblasts were transfected with pcDNA3.1 empty vector or pcDNA3.1-grim (WT or mutants) expression vector. Cells were treated with 50 μm zVAD-fmk (Bachem) and, after 20 h, they were lysed in 0.2% Nonidet P-40 isotonic lysis buffer with freshly added protease inhibitors, then proteins were eluted and analyzed in SDS-PAGE. Western blotting analyses were performed using rabbit anti-grim IgG (18.Clavería C. Albar J.P. Serrano A. Buesa J.M. Barbero J.L. Martínez A.C. Torres M. EMBO J. 1998; 17: 7199-7208Crossref PubMed Scopus (63) Google Scholar) and HPR-conjugated goat anti-rabbit IgG (Dako) and developed by enhanced chemiluminescence (ECL; Amersham Biosciences). For immunoprecipitations, NIH 3T3 fibroblasts were transfected with pcDNA3.1 empty vector or pcDNA3.1-grim (WT or mutants) expression vector together with pcDNA3.1 empty vector or pcDNA-6myc-hNAIP expression vector at a 1:1 molar ratio. Cells were treated with 50 μm zVAD-fmk, and, after 24 h, they were lysed in 0.2% Nonidet P-40 isotonic lysis buffer with freshly added protease inhibitors. Equal amounts of cell lysates were incubated with an affinity-purified fraction of rabbit anti-Grim IgG or with a mouse anti-c-Myc monoclonal antibody (overnight, 4 °C), mixed with 30 μl of a 1:1 slurry of Gammabind G-Sepharose (Amersham Biosciences) and incubated (45 min, 4 °C). The Sepharose beads were washed twice in 0.2% Nonidet P-40 lysis buffer, once in 0.25 m LiCl washing buffer, and twice in 50 mm Tris-HCl, pH 7.5, washing buffer before proteins were eluted and analyzed in SDS-PAGE. Western blotting analyses were performed using rabbit anti-grim IgG and protein G-HPR conjugate (Bio-Rad) or mouse anti-c-Myc monoclonal antibody and HPR-conjugated goat anti-mouse IgG (Dako), then developed by ECL. Immunofluorescence—NIH 3T3 fibroblasts were cultured in microscope slide chambers (Cultek) and transfected with pcDNA3.1-grim (WT or mutants) or pcDNA3.1-bad (WT or mutants) expression vector. When indicated, cells were treated with 100 μm zVAD-fmk. After 24 h, cells were washed in PBS, fixed in 4% paraformaldehyde/PBS (15 min, room temperature), and washed three times in PBS. Cells were permeabilized in 0.05% Triton X-100/PBS for 15 min, washed three times in PBS, preincubated in 10% goat serum/0.1% Tween 20/PBS for 1 h, and incubated successively with the primary and secondary antibodies for 1 h each, with three 5-min washes in 0.1% Tween 20/PBS between incubations. Grim was detected with rabbit anti-grim IgG, mitochondria were detected with human anti-mitochondrial serum (18.Clavería C. Albar J.P. Serrano A. Buesa J.M. Barbero J.L. Martínez A.C. Torres M. EMBO J. 1998; 17: 7199-7208Crossref PubMed Scopus (63) Google Scholar), cytochrome c with mouse anti-cytochrome c monoclonal antibody (monoclonal antibody) (clone 6H2.B4, BD Pharmingen), and Bad with rabbit anti-bad Ab (New England Biolabs). Secondary antibodies used were goat anti-rabbit IgG-Alexa 488, goat anti-mouse IgG-Cy3, goat anti-human IgG-Cy3, biotinylated goat anti-human IgG, biotinylated goat anti-mouse IgG and streptavidin-Cy5 (all from Jackson ImmunoResearch). MitoTracker staining was performed by incubating cells with MitoTracker Red CMXRos (Molecular Probes) before fixing, as recommended by the manufacturer. Optical sections were obtained using an Ar-Kr laser and TCS-NT Leica confocal imaging systems. For the Bax conformational change assay, NIH 3T3 fibroblasts were transfected with pEGFP-C1 (Clontech), pcDNA3.1-grim, or pcDNA3.1-tBid expression vector, this last together with pEGFP-C1 expression vector at a 2:1 molar ratio. Cells were treated with 100 μm zVAD-fmk (22 h), then washed in PBS, fixed in 4% paraformaldehyde/PBS (15 min, room temperature), and washed three times in PBS. Cells were permeabilized in 0.2% Triton X-100/PBS (15 min) and immunofluorescence was performed as described. Anti-Grim antibody was biotinylated using EZ-Link Sulfo-NHS-Biotinylation kit (Pierce), and was used to detect Grim in this assay. Bax was detected with anti-Bax NT rabbit polyclonal antibody (Upstate Biotechnology), and cytochrome c as described above. Secondary antibodies used were NeutrAvidin-Alexa 488, goat anti-rabbit IgG-Cy3, and goat anti-mouse IgG-Cy5 (all from Jackson ImmunoResearch). WT and DKO SV40-transformed MEF were cultured in microscope slide chambers and transfected with rtTA-grim expression vector (18.Clavería C. Albar J.P. Serrano A. Buesa J.M. Barbero J.L. Martínez A.C. Torres M. EMBO J. 1998; 17: 7199-7208Crossref PubMed Scopus (63) Google Scholar). After lipofection, cells were treated with 2 μg/ml of doxycycline and 100 μm zVAD-fmk, and after 24 h, cells were processed for immunofluorescence as described. The GH3 Domain Is Essential for Grim Proapoptotic Function in Mammalian Cells—We recently described the GH3 domain as essential for Grim proapoptotic activity in flies (19.Clavería C. Caminero E. Martínez A.C. Campuzano S. Torres M. EMBO J. 2002; 21: 3327-3336Crossref PubMed Scopus (67) Google Scholar); therefore, we tested whether GH3 integrity was a requisite for its conserved activity in mouse 3T3 cells. As previously described (18.Clavería C. Albar J.P. Serrano A. Buesa J.M. Barbero J.L. Martínez A.C. Torres M. EMBO J. 1998; 17: 7199-7208Crossref PubMed Scopus (63) Google Scholar), wild type (WT) Grim induced cell death when overexpressed in mouse 3T3 fibroblasts (Fig. 1B). In contrast, a Grim mutant form with a 13-amino acid deletion that removes the GH3 domain (Δ86-98, Fig. 1A) only marginally induced apoptosis in this assay (Fig. 1B). A 5′-shifted 11-amino acid deletion, such that the 3′ part of the domain is preserved (Δ83-93, Fig. 1A), was less effective in eliminating the proapoptotic activity than complete GH3 deletion (Fig. 1B). The elimination of four internal amino acids common to the two larger deletions (Δ89-92, Fig. 1A) also resulted in effective impairment of Grim killing ability (Fig. 1B). The consequences of removing these regions are similar to those observed in assays in cultured Drosophila SL2 cells and transgenic flies (19.Clavería C. Caminero E. Martínez A.C. Campuzano S. Torres M. EMBO J. 2002; 21: 3327-3336Crossref PubMed Scopus (67) Google Scholar). Furthermore, point mutations in the GH3 domain that alter Grim activity in flies similarly affect Grim activity in mouse cells. A nonconservative replacement of Leu-89 by glutamic acid (L89E, Fig. 1A) impaired GH3 killing ability nearly to the level of the Δ89-92 mutant (Fig. 1B), whereas a semi-conservative replacement of Leu-89 by alanine (L89A, Fig. 1A) had only a mild effect on Grim proapoptotic function (Fig. 1B). These results show that, as in the fly, the GH3 domain is required for Grim proapoptotic function in mouse cells and that the residues relevant for GH3-mediated killing in flies are equally important for its conserved activity in mammalian cells. Because residual proapoptotic function was observed even in the most extreme case of Grim function impairment, we tested whether the conserved N-terminal domain was responsible for the residual killing activity. As reported (18.Clavería C. Albar J.P. Serrano A. Buesa J.M. Barbero J.L. Martínez A.C. Torres M. EMBO J. 1998; 17: 7199-7208Crossref PubMed Scopus (63) Google Scholar), the N-terminal domain was unnecessary for Grim killing in mouse 3T3 fibroblasts (Δ2-14, Fig. 1B). Combinations of the N-terminal deletion with either the complete GH3 deletion (Δ2-14, Δ86-98) or the internal four-amino acid deletion (Δ2-14, Δ89-92) did not further reduce Grim function (Fig. 1B). We therefore could not demonstrate a proapoptotic function for the N-terminal domain in this context. Deletion of the GH3 domain, alone or in combination with the N-terminal domain deletion, did not compromise Grim ability to bind mammalian, viral, or insect IAPs (Fig. 2 and not shown). Furthermore, IAPs only show a residual rescue capacity of Grim killing activity in mouse cells (Ref. 18.Clavería C. Albar J.P. Serrano A. Buesa J.M. Barbero J.L. Martínez A.C. Torres M. EMBO J. 1998; 17: 7199-7208Crossref PubMed Scopus (63) Google Scholar and not shown), suggesting an IAP-independent mechanism of action for the GH3 domain. This conclusion is also validated by the fact that GH3-induced proapoptotic events are independent of caspase activity (see below), which would not be the case for an IAP-dependent pathway. We conclude that the proapoptotic activity of the GH3 domain is required for Grim function in mammalian cells and is not related to an IAP-inhibitory pathway, whereas N-terminal domain function does not appear to be essential in this context. Grim Targets Mitochondria and Induces Cytochrome c Release in Mammalian Cells—We previously showed that Grim localizes diffusely in the cytoplasm of preapoptotic mouse 3T3 fibroblasts and progressively colonizes mitochondria as apoptosis progresses (18.Clavería C. Albar J.P. Serrano A. Buesa J.M. Barbero J.L. Martínez A.C. Torres M. EMBO J. 1998; 17: 7199-7208Crossref PubMed Scopus (63) Google Scholar). To identify the mechanism involved in Grim cell killing in mammalian cells, we tested for cytochrome c release and mitochondrial membrane potential (MMP) state in 3T3 fibroblasts overexpressing Grim. We found that cytochrome c was undetectable in mitochondria of the large majority of cells expressing Grim (Fig. 3, A-D). This Grim-induced cytochrome c release is unaffected by the presence of the broad-spectrum caspase inhibitor zVAD-fmk (Fig. 3, E-H) or p35 coexpression (not shown). Cytochrome c release was detected in Grim-expressing cells in which no other symptom of apoptosis was observed. In contrast, most Grim-transfected cells retain MMP, as measured by MitoTracker incorporation (Fig. 3, I-L). Conservation of MMP extends into apoptosis progression, when changes in cell shape and organelle distribution have already taken place (Fig. 3, I-L). MMP loss was only observed in cells in which the apoptotic process was very advanced (not shown). To confirm that cytochrome c release preceded MMP loss, we stained simultaneously with the cytochrome c antibody and MitoTracker and observed that most Grim-expressing cells retained MMP but had released cytochrome c, whereas the reverse was never found (Fig. 3, M-P). These results show that in mammalian cells cytochrome c release is the first Grim-induced proapoptotic event and that it does not require the previous activation of cytosolic caspases; MMP loss occurs later as a consequence of mitochondrial dysfunction or advanced apoptosis. The GH3 Domain Is Required for Grim Targeting to Mitochondria and Cytochrome c Release in Mammalian Cells—Because the GH3 domain triggers a mitochondrial death pathway in flies, we tested the involvement of the GH3 domain in the mitochondrial changes induced by Grim in mammalian cells. Complete or partial elimination of the GH3 domain, or even the L89E substitution, abolished mitochondrial localization (Fig. 4, C and E and not shown) and cytochrome c release (Fig. 4, D and F, and not shown). In all cases, however, the subcellular distribution of the mutant proteins was not diffuse, but concentrated in prominent cytoplasmic dots (Fig. 4, C-F). In contrast, the Δ2-14 mutant, which did not appreciably impair Grim function in cultured cells, retained mitochondrial localization and provoked cytochrome c release (Fig. 4, G and H). Furthermore, NAIP or OpIAP coexpression does not impair the ability of Grim or any of its mutant proteins to induce apoptosis or to release cytochrome c in 3T3 cells (not shown). The L89A Grim mutant, which showed slightly reduced proapoptotic activity, displayed mitochondrial staining and induced cytochrome c release, although in a lower proportion of transfected cells than that observed for WT Grim, suggesting that greater concentrations of the mutant protein were required to achieve functional levels (not shown). When
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