DEDD, a novel death effector domain-containing protein, targeted to the nucleolus
1998; Springer Nature; Volume: 17; Issue: 20 Linguagem: Inglês
10.1093/emboj/17.20.5974
ISSN1460-2075
AutoresAlexander H. Stegh, Olaf Schickling, Andreas Ehret, Carsten Scaffidi, Christoph Peterhänsel, Thomas G. Hofmann, Ingrid Grummt, Peter H. Krammer, Marcus E. Peter,
Tópico(s)NF-κB Signaling Pathways
ResumoArticle15 October 1998free access DEDD, a novel death effector domain-containing protein, targeted to the nucleolus Alexander H. Stegh Alexander H. Stegh Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Olaf Schickling Olaf Schickling Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Andreas Ehret Andreas Ehret Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Carsten Scaffidi Carsten Scaffidi Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Christoph Peterhänsel Christoph Peterhänsel Division of Molecular Biology of the Cell II, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Thomas G. Hofmann Thomas G. Hofmann Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Ingrid Grummt Ingrid Grummt Division of Molecular Biology of the Cell II, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Peter H. Krammer Peter H. Krammer Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Marcus E. Peter Corresponding Author Marcus E. Peter Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Alexander H. Stegh Alexander H. Stegh Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Olaf Schickling Olaf Schickling Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Andreas Ehret Andreas Ehret Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Carsten Scaffidi Carsten Scaffidi Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Christoph Peterhänsel Christoph Peterhänsel Division of Molecular Biology of the Cell II, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Thomas G. Hofmann Thomas G. Hofmann Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Ingrid Grummt Ingrid Grummt Division of Molecular Biology of the Cell II, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Peter H. Krammer Peter H. Krammer Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Marcus E. Peter Corresponding Author Marcus E. Peter Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Author Information Alexander H. Stegh1, Olaf Schickling1, Andreas Ehret1, Carsten Scaffidi1, Christoph Peterhänsel2, Thomas G. Hofmann1, Ingrid Grummt2, Peter H. Krammer1 and Marcus E. Peter 1 1Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany 2Division of Molecular Biology of the Cell II, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:5974-5986https://doi.org/10.1093/emboj/17.20.5974 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The CD95 signaling pathway comprises proteins that contain one or two death effector domains (DED), such as FADD/Mort1 or caspase-8. Here we describe a novel 37 kDa protein, DEDD, that contains an N-terminal DED. DEDD is highly conserved between human and mouse (98.7% identity) and is ubiquitously expressed. Overexpression of DEDD in 293T cells induced weak apoptosis, mainly through its DED by which it interacts with FADD and caspase-8. Endogenous DEDD was found in the cytoplasm and translocated into the nucleus upon stimulation of CD95. Immunocytological studies revealed that overexpressed DEDD directly translocated into the nucleus, where it co-localizes in the nucleolus with UBF, a basal factor required for RNA polymerase I transcription. Consistent with its nuclear localization, DEDD contains two nuclear localization signals and the C-terminal part shares sequence homology with histones. Recombinant DEDD binds to both DNA and reconstituted mononucleosomes and inhibits transcription in a reconstituted in vitro system. The results suggest that DEDD is a final target of a chain of events by which the CD95-induced apoptotic signal is transferred into the nucleolus to shut off cellular biosynthetic activities. Introduction Recently, a new subfamily of the tumor necrosis factor (TNF) receptor superfamily, the death receptors, has been identified (Peter et al., 1998). Death receptors such as TNF-R1, DR3 (APO-3/TRAMP/Wsl-1/LARD), DR4 (TRAIL-R1), DR5 (TRAIL-R2) and CD95 (APO-1/Fas) are characterized by the presence of a death domain (DD) within the cytoplasmic region and have been shown to trigger apoptosis upon binding of their cognate ligands or specific agonistic antibodies. The first event which can be detected during apoptosis mediated by CD95 (APO-1/Fas) is recruitment of the first-level caspase-8 (FLICE/MACH/Mch5) to the death-inducing signaling complex (DISC) (Boldin et al., 1996; Fernandes-Alnemri et al., 1996; Muzio et al., 1996), this being observed within seconds of receptor crosslinking (Kischkel et al., 1995). Binding of caspase-8 to the DISC is mediated by the adaptor molecule FADD/Mort-1 that contains a death effector domain (DED) by which it binds to caspase-8 (Boldin et al., 1995; Chinnaiyan et al., 1995, 1996). Caspase-8 is then activated mainly by association with the DISC (Medema et al., 1997), resulting in the release of active caspase-8 subunits which can then cleave various death substrates such as cytoskeletal proteins (e.g. plectin; A.H.Stegh et al., manuscript submitted) or other caspases (e.g. caspase-3; Scaffidi et al., 1998) that can in turn cleave a large number of intracellular death substrates. Other DED-containing proteins that have been suggested to be involved in signaling of death receptors include caspase-10 (Mch4/FLICE2) (Fernandes-Alnemri et al., 1996; Vincenz and Dixit, 1997) and c-FLIP (FLAME/CASH/I-FLICE/Casper/Mrit/Clarp/Usurpin) (Goltsev et al., 1997; Han et al., 1997; Hu et al., 1997; Inohara et al., 1997; Irmler et al., 1997; Shu et al., 1997; Srinivasula et al., 1997; Rasper et al., 1998). The end point of apoptosis is DNA fragmentation in the nucleus. Between early morphological and nuclear changes a number of events can be detected depending on the type of apoptosis and the cell tested. Essentially all components of a cell are affected. Recently, an endonuclease, CAD, was cloned that was shown to be one of the DNA cleaving activities required for apoptosis (Sakahira et al., 1998). CAD was found in complex with its inhibitor ICAD/DFF45 located in the cytoplasm (Liu et al., 1997; Enari et al., 1998). Upon apoptosis induction, ICAD is cleaved by a caspase and CAD was proposed to translocate to the nucleus where it exerts its activity as an endonuclease. We now describe and functionally characterize a novel protein that contains an N-terminal DED with homology to the DED of FADD, c-FLIP, caspase-8 and caspase-10. This protein binds strongly to DNA with histone-like properties and was therefore called DEDD (for DED containing DNA-binding protein). DEDD weakly induced apoptosis and translocated to the nucleus during CD95-mediated apoptosis. This translocation was in part dependent on activation of caspases. Overexpressed DEDD localized exclusively to nucleoli and recombinant DEDD inhibited transcription of rDNA in a reconstituted in vitro assay. The results suggest that DEDD represents a new link between cytoplasmic and nuclear apoptotic events. Results Cloning of full-length DEDD by 'EST walking' Using the sequence of 22 different DEDs, an algorithm was generated to search the translated non-redundant union of EMBL and GenBank DNA databases for the existence of new DED-containing proteins that might be involved in CD95-mediated apoptosis. This search identified a mouse EST clone (DDBJ\EMBL\GenBank accession number: Aa124451) that contained a DED homologous to the DED of FADD and caspase-8 (Peter et al., 1997). Using the deduced protein sequence of this EST clone in TBlastN searches, other EST sequences containing overlapping identical nucleotide sequences were identified (Figure 1A). These EST sequences were used to search for more overlapping EST sequences. The correct reading frame was identified by comparing human and mouse EST sequences. Reading frames were aligned in a way that most exchanges between human and mouse EST sequences were in the wobble position. Seven such searches identified 23 EST sequences covering the full-length clones for both human and murine cDNAs. To confirm the existence and the identity of this open reading frame, the cDNA was amplified from a human (Jurkat) and a murine (EL4) cell line by reverse transcription–polymerase chain reaction (RT–PCR) and sequenced. Open reading frames in both man and mouse code for a 318 amino acid protein (Figure 1B). The protein was designated DEDD, for death effector domain containing DNA binding protein (see below). On the amino acid level, DEDD is 98.7% identical between man and mouse and three of the four amino acid exchanges represent replacements with homologous residues. On the DNA level, DEDD is 94.1% identical between man and mouse. On the RNA level, DEDD is expressed ubiquitously (Figure 2). All tissues tested express a 2.3 kb transcript (Figure 2A). In some tissues an additional transcript of 4.2 kb was found. Moreover, using RT–PCR, DEDD was found in all cell lines tested (Figure 2B), confirming that DEDD mRNA is expressed ubiquitously. Figure 1.Identification of DEDD as a new death effector domain (DED)-containing protein with high homology between mouse and man. (A) A database search for new DED-containing proteins revealed a mouse EST clone (DDBJ/EMBL/GenBank accession number: Aa124451) with a DED homologous to the DED of FADD and caspase-8. Identification of overlapping EST nucleotide sequences resulted in the determination of the full-length DEDD protein sequence. For the human ESTs, the tissue from which each EST clone was derived is given underneath each clone. All mouse EST sequences (except Aa163060) were derived from mouse embryos. For each EST clone, only the DEDD coding areas are shown and numbers indicate amino acids as found in the complete DEDD protein sequence. Stippled lines indicate corresponding sequences located outside the ORF. Steps in lines represent frameshifts caused by missequenced bases found in the EST database. (B) Comparison of murine and human DEDD on DNA as well as on protein level. Differences in nucleotides are shown by open boxes and differences on the protein level by closed boxes. Positions of two putative nuclear localization signals are shown by lines. Download figure Download PowerPoint Figure 2.mRNA distribution of DEDD in several tissues and cell lines. (A) Northern blot analysis with a poly(A)+ RNA hybridized membrane probed with a DEDD oligonucleotide encoding the N-terminal DED–containing domain (N-DEDD) The individual tissues analyzed are indicated above the lanes. β-actin is shown as a loading control. (B) RT–PCR of DEDD from several cell lines of lymphoid and non–lymphoid origin. β-actin primers were used as an internal control. Download figure Download PowerPoint DEDD contains a DED and two nuclear translocation signals (NLS) DEDD is a protein with mostly acidic domains (Figure 3A). Only two putative NLS (Figure 1B) and an area in between predominantly contain basic amino acids. Alignment of DEDD with proteins involved in death receptor signaling pathways revealed that DEDD contains a DED (amino acid positions 23–98) that is homologous to the DEDs of the human proteins FADD, caspase-8, caspase-10 and c-FLIP (Figure 3B). A hydrophilicity plot of 22 DED sequences (Peter et al., 1997) revealed that all DED contained two characteristic hydrophilic stretches. Comparison of the putative DED of DEDD with the DED of all these proteins showed that the locations of the two hydrophilic regions were most similar to the ones found in the DED of FADD and the first DED of caspase-8 (Figure 3C and data not shown). Independent alignments using the Lipman–Pearson method revealed homologies to DNA binding proteins at the C terminus such as SAF-B, a SAR binding protein (Renz and Fackelmayer, 1996), and three histones, H1, H2A and H4 within the center of the histone fold (Luger and Richmond, 1998) (Figure 3D). Figure 3.Structural organization of the DEDD polypeptide. (A) Schematic representation of the subdomains of DEDD. DED, death effector domain; NLS, nuclear localization signal; P-rich, proline-rich region. The isoelectric points (pI) of the several subdomains are indicated below. (B) The DED of DEDD contains an area of significant homology to the DED of FADD (18.4% identity, 48.7% conservation), to the first DED of caspase-8 (CASP-8) (21.0% identity, 40.7% conservation), to the first DED of caspase-10 (CASP-10) (18.4% identity, 42.3% conservation), and to the first DED of c-FLIP (19.7% identity, 41.7% conservation). Identical amino acid residues are depicted in black boxes; conservative exchanges are indicated by white boxes. The asterisk marks the position of the conserved phenylalanine residue important for DED/DED interaction-mediated cytotoxicity (Eberstadt et al., 1998). (C) Hydrophilicity plot of the DED of DEDD, FADD and caspase-8. The location of a recently identified hydrophobic patch containing the conserved phenylalanine residue essential for DED-mediated interactions (Eberstadt et al., 1998) is labeled by a shaded box. (D) The C terminus of DEDD shows homology to histone H1 (36 aa, 19.4% identity, 55.5% conservation), H2A (38 aa, 25.6% identity, 52.6% conservation), H4 (34 aa, 28.6% identity, 55.9% conservation), and to the SAR-binding protein SAF-B (29 aa, 34.5% identity, 44.8% conservation). Download figure Download PowerPoint DEDD induces apoptosis through its DED To determine whether the putative DED in DEDD was functionally active, two DEDD truncation mutants were generated (Figure 4A). N-DEDD spans positions 1–114, including the DED and the adjacent NLS1. C-DEDD spans position 109–318 containing the proline-rich region, NLS2 and the C-terminal half of DEDD. To minimize the risk of interference with the functions of these proteins, N-DEDD was tagged with a FLAG peptide at its C terminus, whereas C-DEDD was constructed with an N–terminal FLAG tag. To test whether DEDD could activate the apoptosis machinery through its DED, 293T cells were transiently transfected with an expression vector encoding DEDD and nuclear fragmentation was quantified. Wild-type DEDD weakly induced apoptosis (Figure 4B), the degree of apoptosis being comparable with that induced by caspase-8, but lower when compared with FADD. Removing 60% of the C terminus of DEDD resulted in a protein which was much more potent in induction of apoptosis than wild-type DEDD (N-DEDD in Figure 4B), demonstrating that the DED of DEDD is functional and that the C-terminal half of DEDD has anti-apoptotic activity. Both wild-type DEDD- and N-DEDD-induced apoptosis could be blocked by co-expression of the serpin caspase inhibitor crmA, indicating that DEDD-induced apoptosis requires caspase activation, as do other DED-containing apoptosis signaling molecules. To test for functional association of DEDD with FADD or caspase-8, suboptimal non-cytotoxic concentrations of FADD or caspase-8 were co-transfected with suboptimal concentrations of the DEDD deletion mutants (Figure 4C). Under these conditions, FADD could enhance DEDD-induced apoptosis only in the absence of the C-terminal part of DEDD, i.e. N-DEDD was much more cytotoxic in the presence of small amounts of FADD when compared with wild-type or C-DEDD. Co-transfection of caspase-8 did not have any effect, even at higher concentrations (data not shown). The observation that C-DEDD-mediated apoptosis could not be enhanced by FADD suggests that the DED in DEDD is required for induction of apoptosis. Figure 4.The DED in DEDD is functionally active. (A) Scheme showing the structure of N-DEDD and C-DEDD. A FLAG tag was added to the 3′ end of wild-type DEDD and N-DEDD or to the 5′ end of C-DEDD. (B) Induction of apoptosis by transient transfection of DEDD mutants into 293T cells. The amount of DNA used for each DEDD construct was 3 μg and for the pFM91-crmA plasmid was 4 μg. All DEDD constructs had a 3′FLAG tag. Only C-DEDD was tagged at the 5′ end to allow potential interaction with the C terminus of C-DEDD. All experiments were performed with 3′FLAG-tagged DEDD and N-DEDD. 5′FLAG-tagged DEDD or N-DEDD were also tested and found to be much less potent in induction of apoptosis (data not shown). Untagged DEDD behaved exactly like 3′FLAG-DEDD, excluding the possibility that the 3′FLAG tag interfered with apoptosis-inducing activity of DEDD. DNA fragmentation was determined as described in Materials and methods. Means (± SD) of three independent experiments are shown. (C) Enhancement of N-DEDD-induced apoptosis by FADD. Suboptimal, non-cytotoxic amounts (0.5 μg) of pcDNA3-3′FLAG-DEDD, pcDNA3-3′FLAG-N-DEDD or pcDNA3-5′FLAG-C-DEDD were co-transfected with suboptimal DNA concentrations (0.3 μg) of either pcDNA3-AU1-FADD or pcDNA3-caspase-8. DNA fragmentation was determined as described in Materials and methods. Means (± SD) of three independent experiments are shown. (D) DEDD interacts with DED containing GST fusion proteins of caspase-8 and FADD. Several GST fusion proteins as indicated were incubated with either in vitro-translated [35S]caspase-8 and [35S]FADD or [35S]DEDD. The precipitates were subjected to 12% PAGE and subsequently analyzed by autoradiography. The migration positions of caspase-8 (CASP-8), FADD and DEDD are indicated. Download figure Download PowerPoint DEDD binds to FADD and caspase-8 through DED/DED interaction The cooperative effect of FADD to enhance N-DEDD-mediated apoptosis suggested that DEDD was involved in death receptor signaling through DED/DED interactions. To test whether DEDD interacts with FADD and/or caspase-8, precipitation experiments were performed using glutathione S-transferase (GST)–FADD and GST–caspase-8 fusion constructs incubated with 35S-labeled in vitro-translated FADD, caspase-8 or DEDD. [35S]DEDD bound only to the DED containing GST fusion proteins, i.e. GST–FADD, GST–N-FADD, GST–caspase-8 and GST–N-caspase-8 (Figure 4D). None of the in vitro-translated proteins bound to GST alone, to GST–C-FADD or to GST–C-caspase-8, demonstrating that DEDD binds to the DED of FADD and caspase-8 in vitro. In summary, the data suggest that DEDD is functional, binds to other DED-containing proteins like caspase-8 or FADD, induces apoptosis when expressed in 293T cells, and that this apoptosis can be enhanced by FADD. FADD may therefore be a physiological interaction partner for DEDD. DEDD is located in nucleoli The presence of two putative NLS in DEDD suggests that it could be localized within the nucleus. To test this, pcDNA3-DEDD was transiently transfected into 293T cells and the subcellular localization of DEDD determined by Western blotting using an affinity-purified DEDD-specific anti-peptide rabbit antiserum after fractionation into nuclear, cytoplasmic and microsomal fractions. As shown in Figure 5A, DEDD was found only in the nuclear fraction of cells transfected with the DEDD expression vector. Detection of this band was abolished by preincubation of the anti-DEDD antibody with the peptide used for antibody generation (Figure 5A, lane 7). Consistent with their cytosolic location, both FADD and caspase-8 were found predominantly in the cytoplasm. Figure 5.Subcellular distribution of DEDD. (A) DEDD is localized in the nuclear compartment. 293T cells transfected either with 5 μg pcDNA3 or pcDNA3-DEDD were fractionated into nuclei (N), cytoplasm (C) and membrane fractions (M) and the distribution of DEDD was analyzed by Western blotting using an affinity-purified polyclonal anti-DEDD antibody (top panel), anti-FADD mAb (center panel) and anti-caspase-8 mAb (bottom panel). Lane 7, nuclear extracts from pcDNA3-DEDD-transfected 293T cells developed with the polyclonal anti-DEDD antibody preincubated with the DEDD peptide used for immunization. The nuclear localization of DEDD was additionally confirmed by transfecting a GFP–DEDD fusion protein into 293T cells. GFP–DEDD was again found only in the nuclei (data not shown). (B) 293T cells transfected with 3 μg pcDNA3-5′FLAG-DEDD were subjected to immunofluorescence microscopy using an anti-FLAG antibody (red fluorescence, right panel). The transfected cells were additionally stained with the DNA-intercalating dye Hoechst 33258 (blue fluorescence) marking size and form of the nuclei. Globular structures within nuclei intensively stained by the anti-FLAG mAb also visible in phase contrast are labeled by arrow heads. (C) Co-localization of DEDD with UBF. 293T cells transfected with 3 μg pcDNA3-3′FLAG-DEDD were stained for DEDD using an anti-FLAG antibody with a FITC-labeled secondary antibody (green fluorescence, left panel) and for the nucleolar transcription factor UBF using a human anti-UBF serum and a Texas red-coupled secondary antibody (red fluorescence, center panel). The overlay of both images is shown in the right panel. The localizations of DEDD and UBF in the single fluorescence and in the overlay are indicated by arrowheads. Download figure Download PowerPoint To demonstrate the nuclear localization of DEDD more directly, a FLAG-tagged DEDD was transfected into 293T cells and visualized by immunofluorescence microscopy (Figure 5B). These experiments revealed that DEDD was not uniformly distributed within the nucleoplasm, but rather accumulated in distinct globular structures which appeared to be nucleoli. To prove this, the cells were co-stained with both the anti-FLAG mAb and an antibody against the upstream binding factor (UBF), a basal factor for RNA polymerase I transcription, which is exclusively located in nucleoli (Chan et al., 1991) (Figure 5C, center panel). Superimposition of both stainings revealed that DEDD co-localized precisely with UBF (Figure 5C, right panel). Thus, DEDD overexpressed in 293T cells is found exclusively in nucleoli. In vivo, DEDD is localized in the cytoplasm and translocates to the nucleus upon stimulation of CD95 The experiments on the intracellular localization of DEDD described so far have been performed in cells overexpressing DEDD. To investigate the intracellular localization of endogenous DEDD, we determined its distribution in a number of lymphoid cells using the affinity-purified anti-DEDD rabbit antibody. In all cells tested, a specific protein of 37 kDa was detected with the same electrophoretic mobility as DEDD overexpressed in 293T cells (Figure 6A). In contrast to the latter, endogenous DEDD was found exclusively in the cytoplasm. Since overexpression of DEDD and in vitro association with FADD and caspase-8 indicated that DEDD was involved in death receptor signaling, we treated Jurkat and CEM T cells with anti-APO-1 to induce apoptosis and analyzed the localization of DEDD at different times after stimulation (Figure 6B and C). In both cell lines DEDD appeared in the nuclear fraction first detectable after 10 min. Specificity of the detection of DEDD in Western blot was again established by competition with the DEDD peptide (Figure 6B, bottom panel). Consistent with the overexpression experiment in the 293T cells, in vivo translocation of DEDD to the nucleus required activation of caspases as it could be partially blocked by pretreating CEM cells with the broad-spectrum caspase inhibitor zVAD-fmk (Figure 6C, lanes 7 and 14). The data show that during CD95-mediated apoptosis, up to 80% of cytoplasmic DEDD (in the Jurkat cells) translocates to the nucleus. Quantitative Western blots revealed that DEDD is an abundant protein, being present in ∼200 000 copies in the cytoplasm of Jurkat cells. Hence, up to 160 000 molecules of DEDD may accumulate in the nucleus of these cells during CD95-mediated apoptosis. Figure 6.In vivo localization of DEDD. (A) Expression of endogenous DEDD in the cytoplasm of lymphoid cell lines. Anti-DEDD Western blot of nuclear (N) and cytoplasmic (C) fractions from 5×106 cells. The nuclear compartment of 105 293T cells transfected with pcDNA3-DEDD is shown as a migration control. (B) Translocation of DEDD from the cytosol to nucleus during CD95-mediated apoptosis. Jurkat cells (5×106) were treated with anti-APO-1 for the indicated time periods, subjected to subcellular fractionation, and the Western blot developed with the polyclonal anti-DEDD antibody (upper panel) or preincubated with the DEDD peptide (bottom panel). The migration position of DEDD is indicated. (C) CEM cells (5×106) treated with anti-APO-1 for different time points were analyzed as in (B). Samples in lanes 7 and 14 were preincubated with zVAD-fmk (+ zVAD). The decrease of the total amount of DEDD in Jurkat and CEM cells after 240 min of anti-APO-1 treatment is due to a general loss of proteins at this advanced stage of apoptosis. Download figure Download PowerPoint Translocation of DEDD to the nucleus requires caspase activation The results presented so far indicate that DEDD could translocate to the nucleus both in vivo and in an overexpression system. To test whether the quantitative translocation of overexpressed DEDD was linked to its apoptotic activity, the effect of blocking apoptosis by co-transfection of crmA (Figure 4B) on the translocation of DEDD was tested. Upon co-transfection of DEDD and crmA, in addition to cells exhibiting the nuclear staining (Figure 7A, arrowhead in top row), ∼50% of the transfected cells showed a pronounced cytoplasmic distribution of DEDD (Figure 7A, arrows in top row). This effect of crmA suggested again that apoptosis and caspase activation were needed for translocation of DEDD to the nucleus. Since overexpression of DEDD resulted in weak apoptosis, DEDD itself may have provided the necessary apoptosis signal facilitating its translocation to the nucleus. When 293T cells were transfected with DEDD and crmA, or treated with zVAD-fmk, a less efficient translocation of DEDD into the nucleus was observed (Figure 7B, top row). Both N-DEDD and C-DEDD, each of which contain one NLS (NLS1 or NLS2, respectively), were still found in the nucleus as revealed by anti-FLAG immunofluorescence (Figure 7A, middle and bottom rows) and subcellular fractionation (Figure 7B, middle and bottom rows). However, blocking of apoptosis with crmA or zVAD-fmk only affected the localization of C-DEDD, resulting in a pronounced cytoplasmic distribution in immunofluorescence microscopy (Figure 7A, bottom row) and subcellular fractionation (Figure 7B, lower panel). The nuclear localization of N-DEDD did not change under apoptosis-inhibiting conditions (Figure 7A and B, middle rows). These data indicate that NLS2 is inducible and requires activation of caspases. Interestingly, the subnuclear localization of N-DEDD and C-DEDD appeared different. Similar to wild-type DEDD, N-DEDD containing NLS1 was localized in nucleoli, whereas C-DEDD having NLS2 showed a more diffuse nuclear localization (Figure 7A, left column, center and bottom panel). The data suggest that both NLS can individually direct DEDD to the nucleus. However, in this overexpression system NLS1 is constitutively active and mainly responsible for the nucleolar localization of DEDD, whereas NLS2 requires activation of caspases to be functional. The contribution of both NLS in DEDD to nuclear and nucleolar localization were confirmed by using NLS1, NLS2 or NLS1/2 deletion mutants in immunofluorescence microscopy (data not shown). Figure 7.Caspase inhibition prevents translocation of DEDD into the nucleus. (A) Analysis of the localization of DEDD and DEDD deletion mutants by immunofluorescence microscopy. 293T cells were transfected with 3 μg pcDNA3-3′FLAG-DEDD, pcDNA3-3′FLAG-N-DEDD and pcDNA3-5′FLAG-C-DEDD either alone or together with 4 μg pFM91-crmA, respectively, and DEDD was analyzed by immunostaining with an anti-FLAG antibody and a FITC-labeled secondary antibody. Nucleolar localization is indicated by small arrowheads, nucleoplasmic staining by large arrowheads, and cytoplasmic distribution by arrows. (B) Localization of wild-type DEDD, N-DEDD and C-DEDD. Nuclear (N) and cytoplasmic (C) compartments of the transfected 293T cells co-transfected with DEDD and crmA [see (A)] or transfected with DEDD in the presence of
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