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Subcellular compartmentalization of FADD as a new levelof regulation in death receptor signalingNiko Fo¨ger1, Silvia Bulfone-Paus1, Andrew C. Chan2and Kyeong-Hee Lee11 Department of Immunology and Cell Biology, Research Center Borstel, Leibniz Center for Medicine and Biosciences, Germany2 Department of Immunology, Genentech, Inc., San Francisco, CA, USAIntroductionCD95 (Fas ⁄ Apo-1 ⁄ TNFRSF6) is a prototypic deathreceptor belonging to the tumor necrosis factor recep-tor superfamily. CD95 is expressed on the surface ofcells as preassociated homotrimers and, upon CD95Lbinding, undergoes a conformational change to revealits cytoplasmic death domain (DD) to favor homotypicinteractions with other DD-containing proteins. Fas-associated protein with DD (FADD) is the most proxi-mal adaptor molecule transmitting the death signalmediated by CD95 [1]. As a DD-containing anddeath effector domain-containing proapoptotic adaptormolecule, FADD is essential to recruit the initiatorcaspases-8 and -10 to instigate formation of the death-inducing signal complex (DISC), which mediatesdeath receptor-induced apoptosis [2,3]. Expression of adominant-negative form of FADD, consisting of theN-terminal DD only, impairs the relay of the apoptoticsignal from death receptors [4]. Moreover, FADD-deficient mice display profound defects in apoptoticpathways, particularly in the immune system [5]. FADDis a multifunctional protein that, in addition to itsprominent role in cell death, has also been implicatedin the regulation of cell survival ⁄ proliferation andcell cycle progression, as well as embryonic develop-ment [5–7].In our previous work, we demonstrated that CD95internalization plays a role in CD95-induced apoptosis[8]. Upon ligand binding, CD95 is internalized anddelivered to endosomal compartments, which thenserve as major sites for CD95-mediated DISC forma-tion and caspase-8 activation. Given that the key roleof FADD in apoptotic signaling is efficient DISCKeywordsapoptosis; CD95; compartmentalization;FADD; nuclear traffickingCorrespondenceK H. Lee, Department of Immunology andCell Biology, Research Center Borstel,Leibniz Center for Medicine andBiosciences, Parkallee 22, 23845 Borstel,GermanyFax: +49 4537 1884904Tel: +49 4537 188585E-mail: klee@fz-borstel.de(Received 30 April 2009, accepted 4 June2009)doi:10.1111/j.1742-4658.2009.07134.xFas-associated protein with death domain (FADD) is an essential adaptorprotein in death receptor-mediated signal transduction. During apoptoticsignaling, FADD functions in the cytoplasm, where it couples activatedreceptors with initiator caspase-8. However, in resting cells, FADD is pre-dominantly stored in the nucleus. In this study, we examined the modalitiesof FADD intracellular trafficking. We demonstrate that, upon CD95 acti-vation, FADD redistributes from the nucleus to the cytoplasm. This induc-ible nuclear–cytoplasmic translocation of FADD is independent of CD95internalization, formation of the death-inducing signaling complex, andcaspase-8 activation. In contrast to nuclear export of FADD, its subse-quent recruitment and accumulation at endosomes containing internalizedCD95 requires a caspase-8-dependent feedback loop. These data indicatethe existence of differential pathways directing FADD nuclear export andcytoplasmic trafficking, and identify subcellular compartmentalization ofFADD as a novel regulatory mechanism in death receptor signaling.AbbreviationsBFA, brefeldin A; DAPI, 4¢,6-diamidino-2-phenylindole; DD, death domain; DISC, death-inducing signaling complex; EEA-1, early endosomeantigen 1; FADD, Fas-associated protein with death domain; GFP, green fluorescent protein; INP54p, Saccharomyces cerevisiae inositolpolyphosphate 5-phosphatase; PtdIns(4,5)P2,phosphatidylinositol 4,5-bisphosphate.4256 FEBS Journal 276 (2009) 4256–4265 ª 2009 The Authors Journal compilation ª 2009 FEBSassembly at endosomal structures, FADD is expectedto function within the cytoplasm. However, FADDcarries strong nuclear localization and nuclear exportsignals, and has been reported to primarily localize tothe nucleus in a variety of different cell types[9–12]. This raises the question of how a predomi-nantly nuclear protein such as FADD is involved inDISC formation occurring at endosomes in thecytoplasm.Here, we demonstrate that CD95 stimulationinduces translocation of nuclear FADD to the cyto-plasm. Employing a combination of biochemical, cellbiological and genetic methods, we investigated therole of ‘classic’ apoptotic signal transduction events inthe nuclear–cytoplasmic relocalization of FADD andits subsequent recruitment to endosomal compart-ments, where FADD promotes efficient DISC forma-tion. The regulation of the subcellular localization ofFADD adds a new level of complexity to the apoptoticsignaling cascade.ResultsNuclear–cytoplasmic redistribution of FADD inresponse to CD95L stimulationTo explore whether FADD shuttles between thenucleus and the cytoplasm in response to an apoptoticstimulus, we analyzed the subcellular distribution ofFADD in resting versus CD95L-treated BJAB cells, ahuman B-cell Burkitt’s lymphoma cell line (Fig. 1A).In agreement with previous reports on other cell lines,FADD colocalizes with the nuclear stain 4¢,6-diamidi-no-2-phenylindole (DAPI) in resting BJAB cells, aswell as in human peripheral blood CD4+T-lympho-cytes, indicating preferential nuclear localization ofFADD (Fig. 1A,B, left panels). In response to CD95receptor triggering, however, FADD redistributedfrom a predominantly nuclear to a nuclear and cyto-plasmic pattern. In BJAB cells, within 5 min ofCD95L treatment, a significant proportion of FADDrelocalized from the nucleus to the cytoplasm andexhibited dispersed fine punctuate patterns in the cyto-plasm (Fig. 1A, middle panel). These structuresbecame more pronounced and enlarged at 15–30 minafter CD95L stimulation (Fig. 1A, right panel). Asimilar redistribution of FADD was also observedin human peripheral blood CD4+T-lymphocytes(Fig. 1B, right panel).These observations indicate that FADD undergoesregulated redistribution from the nucleus to the cyto-plasm in response to CD95 triggering. Notably, we didnot observe recruitment of FADD to the plasma mem-brane, but, instead, FADD relocalized to vesicularstructures in the cytoplasm. This specific vesicularlocalization of FADD is probably due to functionalassociation of FADD with internalized CD95, whichpredominantly occurs at endosomal compartments andconstitutes an essential step in CD95-mediated apop-totic signaling [8].0 minCD95L: 15 minFADDBFADD/DAPI0 minCD95L: 5 min15 minAFADD/DAPIFADDFluorescence intensityLowHigh Fig. 1. Nuclear–cytoplasmic translocation ofFADD in response to CD95 stimulation. (A)BJAB cells were stimulated with CD95L forthe indicated times. Cells were stained forFADD (red), and nuclei were counterstainedwith DAPI (blue). Overlay fluorescence isshown in the upper panel. Quantitativeimage analysis with relative pixel intensitiesrecorded for FADD fluorescence signals isshown in the lower panel. (B) Activatedhuman peripheral blood CD4+T-cells werestimulated with CD95L for 15 min. SingleFADD staining (upper panel, red) and overlayfluorescence (lower panel) of FADD andDAPI are shown. Fluorescence images weregenerated by deconvolution microscopy.The data shown are representative of> 150 cells analyzed.N. Fo¨ger et al. FADD trafficking and CD95 signalingFEBS Journal 276 (2009) 4256–4265 ª 2009 The Authors Journal compilation ª 2009 FEBS 4257Expression of a plasma membrane-localizedphosphatidylinositol 4,5-bisphosphate[PtdIns(4,5)P2]-specific 5¢-phosphatase inhibitsCD95 endocytosis and apoptosis, but not thenuclear–cytoplasmic translocation of FADDAs FADD translocation from the nucleus to the cyto-plasm occurred within 2–5 min following CD95L stim-ulation, prior to significant CD95 internalization, weanalyzed whether FADD translocation required CD95internalization. To this end, we utilized Saccharomy-ces cerevisiae inositol polyphosphate 5-phosphatase(INP54p), an enzyme that hydrolyzes PtdIns(4,5)P2tophosphatidylinositol 4-phosphate [13]. Cellular levels ofPtdIns(4,5)P2are tightly regulated, and it plays impor-tant roles in a multitude of cellular functions, includingclathrin-mediated endocytosis [14–16]. Expression of agreen fluorescent protein (GFP)-tagged plasma mem-brane-targeted INP54p (FynC–GFP–INP54p) in BJABcells specifically reduces PtdIns(4,5)P2levels in theplasma membrane, and results in the inhibition ofCD95L-induced CD95 receptor endocytosis and apop-tosis [8] (Fig. 2A,B). BJAB cells transfected withFynC–GFP–INP54p did, however, still relocalizeFADD from the nucleus to the cytoplasm in responseto CD95L stimulation (Fig. 2C). Whereas the overalldegree of the CD95L-induced FADD nuclear–cytoplas-mic translocation was similar between FynC–GFP–INP54p+cells and control cells, the pattern of FADDstaining was qualitatively distinct in FynC–GFP–INP54p+cells. At 15 min following CD95 activation,FynC–GFP–INP54p+cells (Fig. 2C, middle panel)showed only a diffuse staining pattern of cytoplasmicFADD and did not exhibit the intense coalescence ofFADD with larger endocytic structures that is observedin FynC–GFP–INP54p)cells (Fig. 2C, right panel).This may reflect a lack of internalized CD95 to concen-trate FADD within endocytic vesicles.0CD95L: 0 min 15 min 15 min102030405060708090100GFP – GFP + GFP – GFP +FynC–GFP–INP54 FynC–GFPA100101102103104100101102103104FynC–GFP+FynC–GFP–INP54+Annexin VCell countBCD95 internalization (%)CGFPFADDDAPIFADDLowHighFluorescence intensity654312Fig. 2. FADD translocation into the cytoplasm is independent of CD95 internalization. (A, B) BJAB cells transiently expressing FynC–GFP–INP54p, a PtdIns(4,5)P2-specific 5¢-phosphatase–GFP fusion construct, or the control construct FynC–GFP were analyzed for CD95 internali-zation (A) and apoptosis (B) following CD95L stimulation for 30 min (A) and 6 h (B), respectively. (A) The remaining surface CD95 wasdetected by FACS analysis, and the percentage of CD95 downregulation was calculated for the GFP+and GFP)populations. (B) Apoptosisin GFP+(red) cells was assessed by annexin V staining and FACS analysis. Nonstimulated cells are shown in gray. The data shown arerepresentative of three experiments. (C) BJAB cells transiently transfected with FynC–GFP–INP54p were stimulated with CD95L for 0 min(1, 4) and 15 min (2, 3, 5, 6). Panels 1, 2, 4, 5 show FynC–GFP–INP54p-expressing cells (GFP+), and FynC–GFP–INP54p-non-expressingcells are shown in the right panel (3, 6). Cells were stained for DAPI (blue) and FADD (red). Overlay fluorescence is shown in the upperpanel (1–3), and quantitative image analysis of CD95 fluorescence signals is shown in the lower panel (4–6). The data shown are representa-tive of > 50 cells analyzed.FADD trafficking and CD95 signaling N. Fo¨ger et al.4258 FEBS Journal 276 (2009) 4256–4265 ª 2009 The Authors Journal compilation ª 2009 FEBSCD95 internalization promotes endosomaltargeting of FADDTo investigate whether internalized CD95 provides adocking signal to recruit FADD to endosomes, weanalyzed the subcellular localization of FADD andCD95 in CD95L-activated FynC–GFP–INP54p+andFynC–GFP–INP54p)BJAB cells. Following stimula-tion for 30 min with CD95L, colocalization of cyto-plasmic FADD with internalized CD95 was readilydetected at intracellular compartments in FynC–GFP–INP54p)cells (Fig. 3A, panels 5–8). In con-trast, in CD95-activated but endocytosis-defectiveFynC–GFP–INP54p+BJAB cells, CD95 had formedmicroaggregates in the plasma membrane, and nosignificant colocalization between FADD and CD95was observed, although FADD could be readilydetected in the cytoplasm (Fig. 3A, panels 1–4).There was minimal overlap of staining for FADDwith the early endosome marker early endosomeantigen 1 (EEA-1) in resting cells (Fig. 3B, panels1–3). Overlap of staining for FADD and EEA-1was, however, readily detected in CD95L-stimulatedcontrol FynC–GFP–INP54p)cells (Fig. 3B, panels9–11), whereas in FynC–GFP–INP54p+BJAB cells,FADD largely failed to accumulate at EEA-1+en-dosomes (Fig. 3B, panels 5–8).An internalization-defective CD95 mutantdisrupts apoptotic signaling but still inducesFADD nuclear–cytoplasmic translocationTo further analyze the interrelationship between CD95internalization and FADD nuclear–cytoplasmic relo-calization, we specifically interfered with CD95receptor endocytosis by employing the internalization-defective CD95(Y291F) mutant [8]. The ability of thismutant form of CD95, in which Tyr291 within theconsensus AP-2-binding motif of CD95 has beenmutated to Phe, to internalize in murine A20 B-lym-phoma cells following stimulation with a mAb againsthuman CD95 (CH-11) was significantly reduced ascompared to wild-type CD95 (Fig. 4A). Concomi-tantly, the ability of CD95(Y291F)-expressing cells toactivate caspase-8 in response to CD95 stimulationwas similarly compromised (Fig. 4C). However, despitethe relative inability of CD95(Y291F) to internalizeand to induce classic proximal apoptotic signalingBFADDEEA-1FADD / EEA-1 FADD / DAPI123847659101112CD95L0 min(GFP +)30 min(GFP +)30 min(GFP–)AGFPCD95FADDCD95 / FADD4321CD95L30 min30 min8765Fig. 3. CD95 internalization promotesendosomal targeting of FADD. (A) BJABcells were transfected with FynC–GFP–INP54p and stimulated with CD95L for30 min. Cells were stained for CD95 (red)and FADD (blue). Panels 1–4 represent aFynC–GFP–INP54p-expressing (GFP+) cell,and panels 5–8 show a FynC–GFP–INP54p-non-expressing (GFP)) cell. The data shownare representative of > 50 cells analyzed.(B) BJAB cells were transfected withFynC–GFP–INP54p and stimulated withCD95L for 0 min (1–4) and 30 min (5–12).Cells were stained for FADD (green), EEA-1(red), and DAPI (blue). Individual andmerged fluorescence images were obtainedby deconvolution microscopy. FynC–GFP–INP54p-expressing cells (GFP+) are shownin panels 1–8, and a FynC–GFP–INP54p-non-expressing cell (GFP)) is shown in pan-els 9–12. The data shown are representativeof > 100 cells analyzed.N. Fo¨ger et al. FADD trafficking and CD95 signalingFEBS Journal 276 (2009) 4256–4265 ª 2009 The Authors Journal compilation ª 2009 FEBS 4259events, stimulation of CD95(Y291F) still inducednuclear–cytoplasmic relocalization of FADD (Fig. 4A,B).FADD was preferentially localized within the nucleusof resting cells expressing CD95(Y291F). In responseto CD95 stimulation, FADD exhibited a nuclear andcytoplasmic distribution in cells expressing either wild-type CD95 or CD95(Y291F). However, whereas inwild-type human CD95-expressing cells FADDconcentrated and colocalized with internalized CD95at EEA-1-positive endosomal compartments, in cellsexpressing the internalization mutant CD95(Y291F)FADD remained in a diffuse cytoplasmic pattern andshowed no significant colocalization with EEA-1. Thedata on the nuclear–cytoplasmic relocalization ofFADD, as observed by deconvolution microscopy,were further confirmed by biochemical subcellular frac-tionation experiments. Little to no FADD protein wasdetected in the cytoplasmic fraction of nonstimulatedcells transfected with either wild-type human CD95 orCD95(Y291F) (Fig. 4D, lanes 3 and 6). Triggering ofhuman CD95 for 15–30 min induced a significantincrease in the amount of FADD in the cytoplasmicfraction of cells expressing wild-type CD95 (Fig. 4D,lanes 4 and 5). A similar increase in cytoplasmicFADD was also observed in CD95(Y291F)-expressingcells stimulated with antibody against human CD95(Fig. 4D, lanes 7 and 8).Together, these data indicate that CD95L-inducedFADD translocation to the cytoplasm occurs indepen-dently of CD95 internalization. However, internalizedCD95 then probably serves as a scaffold to amplifyand ⁄ or stabilize FADD assembly at endosomal com-partments.Inhibition of caspase-8 activation allows fortransient nuclear–cytoplasmic shuttling of FADDand results in the recycling of CD95To further investigate whether inhibition of apoptoticsignaling affects the subcellular localization of CD95and ⁄ or FADD, BJAB cells were treated with thecaspase-8 inhibitor z-IETD, and FADD localizationB Y291F Y291F WT FADD EEA-1 FADD/EEA-1 1 2 3 6 5 4 7 8 9 C WT Y291F WB: Cas-8 hCD95 CD95 : 0’ 15’ 30’ 60’ 0’ 15’ 30’ 60’CD95: 0’ 0’ 0’15’30’30’0’15’1 2 3 4 5 6 7 8 WB: FADDLamininGDI-RhoWT Y291F WT Y291F Nuclear Cytoplasmic D 1 2 3 4 5 6 7 8 A Y291F WT FADD CD95 CD95/FADD 1 2 3 6 5 4 Fig. 4. Cytoplasmic translocation of FADD in cells expressing theinternalization mutant of human CD95 (hCD95). (A) A20 cellsexpressing the internalization mutant hCD95(Y291F) (1–3) or wild-type (WT) hCD95 (4–6) were activated for 30 min with mAb againsthCD95 (CH-11). Cells were subsequently stained for FADD (green),CD95 (red), and DAPI (blue). (B) A20 cells expressinghCD95(Y291F) (1–6) or wild-type hCD95 (7–9) were activated withCH-11 for 0 min (1–3) or 30 min (4–9). Cells were stained for FADD(green), EEA-1 (red), and DAPI (blue). Images were obtained bydeconvolution microscopy. The data shown are representative of> 60 cells analyzed. (C) A20 cells were transfected with wild-typehCD95 or hCD95(Y291F) and stimulated with biotinylated CH-11 forthe indicated times. Human CD95-associated signaling complexeswere isolated using streptavidin-conjugated beads. Association ofcaspase-8 with hCD95 was analyzed by immunoblotting for cas-pase-8 and hCD95. (D) A20 cells were transfected with wild-typehCD95 or hCD95(Y291F) and stimulated with CH-11 for the indi-cated times. Nuclear (lanes 1 and 2) and cytoplasmic (lanes 3–8)fractions were prepared from total cellular lysates and were immu-noblotted using antibody against FADD. Effective separation ofnuclear and cytoplasmic fractions was controlled for by immuno-blotting for laminin (nuclear marker) and GDI-Rho (cytosolicmarker).FADD trafficking and CD95 signaling N. Fo¨ger et al.4260 FEBS Journal 276 (2009) 4256–4265 ª 2009 The Authors Journal compilation ª 2009 FEBSwas investigated. In unstimulated BJAB cells eithertreated or not treated with the caspase-8 inhibitor z-IETD, FADD was predominantly detected in thenucleus (Fig. 5A, panels 1–3 and 13–15). Within 2 minof stimulation with CD95L, FADD could readily bedetected in the cytoplasm of z-IETD-treated cells(Fig. 5A, panels 16–18), as in control cells. Inuntreated control cells, FADD remained in the cyto-plasm after 30 and 60 min of CD95 stimulation, andcells started to exhibit signs of apoptosis (Fig. 5A,panels 7–12). In contrast, in z-IETD-treated cells,which do not undergo apoptosis, significant amountsof cytoplasmic FADD could only be detected within30 min of CD95L stimulation (Fig. 5A, panels 19–20).At 60 min, only minimal amounts of FADD hadremained in the cytoplasm of z-IETD-treated cells(Fig. 5A, panels 22–24). Thus, inhibition of caspase-8activation does not affect the initial nuclear–cytoplas-mic translocation of FADD; however, FADD relocal-ization to the cytoplasm is not persistent under theseconditions. Whether, in the absence of caspase-8 acti-vation, FADD shuttles back to the nucleus or isdegraded in the cytoplasm remains to be investigated.As treatment of cells with caspase inhibitors hasbeen reported to be required for CD95 internalizationfollowing receptor activation [17], we next analyzed thekinetics with which caspase inhibition may affectreceptor internalization. Treatment of BJAB cells withthe inhibitors z-IETD (caspase-8 selective), z-VAD (ageneral caspase inhibitor) or z-DEVD (caspase-3 selec-tive) did not affect ligand-mediated CD95 internaliza-tion at 15 min and had moderate effects at 30 min ascompared to untreated cells (Fig. 5B,C). Between30 min and 60 min, control cells further downregulatedCD95, whereas in cells treated with caspase inhibitorsan increase in CD95 surface expression was observed.These kinetics were further supported by microscopystudies, in which CD95 was detected within the cyto-plasm within 30 min following CD95L stimulation,even in the presence of z-IETD (Fig. 5A, panel 20). At60 min following CD95L stimulation, when CD95 hadmaximally internalized and cells already demonstratedmorphological changes associated with apoptosis(Fig. 5A, panel 11), CD95 was detected almost exclu-sively at the cell surface in cells treated with caspaseinhibitors (Fig. 5A, panel 23; Fig. 5B,C), as previouslyreported [17].To analyze the potential contributions of CD95recycling to the plasma membrane, cells were treatedwith brefeldin A (BFA), a fungal metabolite thatblocks protein transport from the endoplasmic reticu-lum to the Golgi and protein recycling, in the presenceor absence of z-VAD. Whereas cells incubated withz-VAD alone again demonstrated significant down-regulation of surface CD95 expression at 30 minfollowed by an increase at 60 min, cells treated withz-VAD and BFA continued to downregulate CD95without any subsequent increase in surface CD95expression (Fig. 5D,E). Thus, CD95 internalizationfollowing receptor engagement is not dependent oncaspase activation, and a significant proportion of thesurface expression of CD95 observed at 30 min and60 min following receptor engagement in the presenceof caspase inhibitors appears to be a consequence ofCD95 receptor recycling when cells are unable toundergo apoptosis. Microscopic analysis of CD95-stimulated cells treated with both BFA and the cas-pase-8 inhibitor z-IETD showed that CD95 largelyaccumulated in the cytoplasm and significant amountsof FADD localized to the cytoplasm, but CD95 andFADD failed to interact with each other under theseconditions (Fig. 5F). These data indicate that nuclear–cytoplasmic shuttling of FADD is independent of cas-pase-8 activity. Further recruitment of FADD toCD95-containing endosomal compartments, however,seems to require an activation loop involving activecaspase-8.DiscussionFADD is an essential adaptor protein in the CD95-mediated apoptotic signaling cascade that couplesactivated receptors with the activation of initiatorcaspase-8 [1,18,19]. Here, we demonstrate that, inresponse to CD95 receptor activation, a significantamount of FADD relocalizes from the nucleus to thecytoplasm.Our data indicate that CD95 receptor triggeringinduces membrane proximal signals to induce nuclearexport of FADD that are independent of CD95 inter-nalization and ‘classic’ apoptotic signaling events, suchas DISC formation and caspase-8 activation. Weemployed two different experimental systems to inhibitCD95 internalization: modulation of PtdIns(4,5)P2levels by INP54p, and the internalization mutantCD95(Y291F). In these systems, CD95-induced DISCformation, caspase-8 activation and apoptosis areseverely compromised [8], whereas CD95 triggering stillinduces translocation of FADD from the nucleus to thecytoplasm. Subsequent recruitment and concentrationof FADD to endosomal compartments, where DISC isstabilized and amplified, however, requires CD95 inter-nalization. Consequently, in endocytosis-defective cells,FADD did not accumulate at endosomal structures inresponse to CD95 stimulation, but exhibited more dif-fuse localization in the cytoplasm. Thus, internalizedN. Fo¨ger et al. FADD trafficking and CD95 signalingFEBS Journal 276 (2009) 4256–4265 ª 2009 The Authors Journal compilation ª 2009 FEBS 426101020304050607080900 204060No inhibitorz-IETD (Cas-8)z-VAD (general)z-DEVD (Cas-3 & Cas-7)(min)MFINo inhibitorz-IETDz-VADz-DEVDCell countCD950 min5 min30 minCD95Cell countz-VADz-VAD+ BFA0 min5 min30 minBFANo inhibitor0204060801001200 204060No inhibitorBFAz-VADz-VAD + BFA(min)MFIFADD CD95 CD95/FADDCD95L (30 min)z-IETDBFACD95L (30 min)BFAz-IETD (caspase-8 inhibitor)No inhibitorCD95LABCDEF0 min2 min30 min60 minFADDCD95 CD95/FADDFADDCD95 CD95/FADD12345678910 11 1213 14 1516 17 1819 20 2122 23 24Fig. 5. FADD translocation is independent of caspase-8 activation. (A) BJAB cells were stimulated with CD95L for the indicated times in theabsence (left, 1–12) or presence (right, 13–24) of 50 lM caspase-8 inhibitor z-IETD. Cells were stained for FADD (green), CD95 (red), andDAPI (blue). Images were obtained by deconvolution microscopy. The data shown are representative of > 30 cells analyzed. (B, C) BJABcells were pretreated with the caspase inhibitor zIETD-fmk, zVAD-fmk or zDEVD-fmk for 1 h. Cells were then stimulated with CD95L for theindicated times, and surface CD95 expression was assessed by FACS. Changes in mean fluorescence intensity (MFI) are quantified in (C).(D, E) BJAB cells were pretreated with either BFA (10 lgÆmL)1), 50 lM z-VAD-fmk or both for 30 min. Cells were then stimulated withCD95L for the indicated times, and surface CD95 expression was assessed by FACS (D). Changes in MFI are quantified in (E). The datashown are representative of three independent experiments. (F) BJAB cells were stimulated with CD95L for 30 min in the presence of BFA(10 lgÆmL)1) (upper panel) or with the combination of BFA (10 lgÆmL)1) and 50 lM caspase-8 inhibitor z-IETD (lower panel). Cells werestained for FADD (green), CD95 (red), and DAPI (blue). Individual and merged fluorescence images were obtained by deconvolution micros-copy. The data shown are representative of > 50 cells analyzed.FADD trafficking and CD95 signaling N. Fo¨ger et al.4262 FEBS Journal 276 (2009) 4256–4265 ª 2009 The Authors Journal compilation ª 2009 FEBSCD95 within the endosome appears to provide a local-izing signal for further recruitment of FADD. Thisspecific recruitment of FADD to internalized CD95is, however, severely compromised in the presence ofa caspase-8 inhibitor, even when accumulation ofinternalized CD95 is forced by treatment of cells withBFA. Hence, CD95 internalization is required, but isnot sufficient, for endosomal accumulation of FADD.Noteworthy, in BJAB cells treated with caspase-8inhibitors, internalized CD95 appears to recycle to thecell surface, and CD95-induced FADD shuttling to thecytoplasm is only of a transient nature.Our data suggest a sequential model of signaling inwhich CD95 receptor activation generates early signalsat the plasma membrane that lead to the translocationof nuclear FADD to the cytoplasm. In a process thatdepends on a positive feedback loop involving caspase-8 activation, cytoplasmic FADD is then furtherrecruited to internalized CD95 at endosomal struc-tures, leading to efficient DISC assembly and amplifi-cation and eventually to apoptotic cell death.Nuclear localization of FADD can be regulated byphosphorylation at Ser194, which is required for theinteraction of FADD with the nuclear–cytoplasmictransport receptor exportin-5 [10]. The phosphoryla-tion of FADD does not, however, appear to play asignificant role in the induction of apoptosis by CD95[20], but is, rather, involved in the nonapoptoticfunctions of FADD, such as regulation of cell cycleprogression [21,22]. Another signaling event potentiallyinvolved in the translocation of FADD from thenucleus to the cytoplasm is CD95-induced generationof ceramide. A recent report has implicated ceramidein the regulation of nucleocytoplasmic trafficking insmooth muscle cells [23]. It is currently unclearwhether CD95-induced ceramide exhibits a similarregulatory function during apoptosis. Also, whether ornot CD95-mediated ceramide generation, like CD95-mediated FADD translocation, is independent ofcaspase-8 activation is still controversial [24–26]. Thus,the molecular mechanisms involved in the regulationof FADD subcellular localization during apoptoticsignaling await further investigation.What is the biological function of nuclear FADDand its nuclear–cytoplasmic translocation? FunctionalDISC assembly and activation of caspase-8 is generallyconsidered to be a ‘point of no return’ in the apoptoticsignaling cascade. Thus, trapping FADD in the nucleusand away from the cytoplasm, where the other compo-nents of DISC can be found, may serve as a safetymechanism to protect cells from unwanted spontaneousDISC formation and apoptosis. Mutation of thenuclear export signal within FADD, such that FADDis retained within the nucleus, reduces the death-inducing efficacy of FADD. Only upon specificCD95-induced signals does FADD relocalize to thecytoplasm, promoting CD95–FADD association, whichin turn leads to DISC assembly, caspase-8 activation,and apoptotic cell death. In addition, nuclear FADDmay be involved in other, nonapoptotic functions ofFADD, such as the control of cell cycling and prolifer-ation of lymphoid cells or embryonic development[5,7,21,27]. Nuclear FADD has also been implicated ingenome surveillance through its association with theDNA repair molecule MBD4 [10]. Like FADD, thetumor necrosis factor receptor 1-associated DD-containing adaptor protein TRADD also rapidly shut-tles between the nucleus and the cytoplasm. Whereascytoplasmic TRADD mediates apoptosis throughFADD and caspase-8 activation, nuclear TRADD actsthrough a mitochondrial apoptosis pathway [28].Our study provides, for the first time, experimentalevidence for the regulation of nuclear cytoplasmic shut-tling of FADD by CD95-mediated signals, suggesting anew level of regulation in death receptor signaling. Asthe specific relocalization of FADD from the nucleusto the cytoplasm is independent of CD95 receptorinternalization, DISC assembly at endosomes and cas-pase activation, our data indicate that CD95 triggeringinduces additional, plasma membrane proximal signals.The elucidation of the molecular pathways involved inconnecting CD95 signaling to the compartmentaliza-tion of FADD will help us to better understand theregulatory mechanisms in death receptor signaling andmay lead to new avenues in apoptosis research.Experimental proceduresCellsHuman Burkitt lymphoma BJAB cells and murine A20B-lymphoma cells were cultured in RPMI-1640 supple-mented with 10% fetal bovine serum, penicillin ⁄ streptomycin(50 lgÆmL)1each) and 2 mml-glutamine (RPMI standardmedium). Cells were maintained in 5% CO2 at 37 °C. CD4+human peripheral blood T-lymphocytes were isolated fromheparinized blood of healthy donors with the Rosette SepKit (Stem Cell Technologies, Vancouver, Canada) andsubsequent Ficoll-Hypaque density centrifugation. Freshlyisolated CD4+ human peripheral blood T-lymphocyteswere activated with mAbs against CD3 (1 lgÆmL)1, UCHT1;BD Pharmingen, Franklin Lakes, NJ, USA) and CD28(5 lgÆmL)1, CD28.2; BD Pharmingen), and maintained inRPMI-1640 standard medium containing recombinanthuman interleukin-2 (R&D Systems, Minneapolis, MN,USA; 25 UÆmL)1).N. Fo¨ger et al. FADD trafficking and CD95 signalingFEBS Journal 276 (2009) 4256–4265 ª 2009 The Authors Journal compilation ª 2009 FEBS 4263DNA constructs and transfectionThe DNA constructs have been described previously [8].The catalytic domain of INP54p was cloned into the modi-fied pEGFP-C1 (Clontech, Mountain View, CA, USA)vector following the C-terminus of GFP. The first 10 aminoacids of Fyn were engineered in frame N-terminal to GFP(FynC–GFP–INP54p). Human CD95 was inserted intopcDNA4 ⁄ TO (Invitrogen, Carlsbad, CA, USA), and thespecific amino acid mutation (Y291F) was generated usingthe QuickChange site-directed mutagenesis kit (Stratagene,La Jolla, CA, USA). Plasmids were transfected using theNucleofector (Lonza, Ko¨ln, Germany) transfection systemaccording to the manufacturer’s instructions.Cell stimulation and apoptosis assayFor induction of apoptosis, cells were cultured with50 ngÆmL)1recombinant human CD95L (AXXORA,Lo¨rrach, Germany) or 200 ngÆmL)1antibody against humanCD95 (CH-11) for the time periods described in the figurelegends. Apoptosis was determined by annexin V ⁄ 7-AADstaining according to the manufacturer’s instructions (BDPharmingen). Apoptotic cells were quantified on a FACSCalibur flow cytometer and analyzed using cellquestsoftware (Becton Dickinson, Franklin Lakes, NJ, USA).CD95 receptor downregulationCells were incubated with CD95L on ice for 30 min in thepresence or absence of caspase inhibitors (Biozol, Eching,Germany) and ⁄ or BFA (Epicenter Technologies, Madison,WI, USA). Cells were then stimulated by subjecting them toa temperature of 37 °C for the time periods described in thefigure legends. Stimulation-induced internalization was ter-minated by adding ice-cold 0.5% azide containing RPMImedium and placing the cells on ice. Nonspecific interactionswere blocked by preincubation with isotype-matched IgG1,and cell surface CD95 was stained with a mAb againsthuman CD95 (DX2; BD Pharmingen) on ice. Cells werethen fixed with 2% paraformaldehyde for analysis by flowcytometry. Alternatively, cells were stimulated with Alexa647-labeled CH-11 at 37 °C and analyzed by fluorescencemicroscopy.Immunofluorescence microscopyCells were fixed with 4% PFA and permeabilized with either0.2% Triton X-100 for detection of FADD and CD95 or0.2% Triton X-100 and 0.2% sodium citrate for EEA-1detection. Immunofluorescence labeling was performedaccording to standard procedures, using specific mAbsagainst FADD [clone 1 (Becton Dickinson) or clone A66-2(BD Pharmingen)], CD95 (CH-11; MBL, Woburn, MA,USA), and EEA-1 (clone 14; Becton Dickinson). All primaryantibodies were directly labeled with Alexa 488, Alexa 546,or Alexa 647, or biotinylated according to the manufac-turer’s recommendations (Invitrogen). To block nonspecificstaining, cells were preincubated with isotype-matched mouseIgG1or IgG2aprior to staining with specific antibodies.Alexa 546-conjugated or Alexa 647-conjugated streptavidinand DAPI were purchased from Invitrogen.Images were obtained using a deconvolution microscope(Applied Precision, Issaquah, WA, USA) equipped withinverted fluorescence optics and a CCD camera. Deconvo-luted images from 60 z-serial sections were subsequentlygenerated by softworx software (Applied Precision).Quantitative analysis of images to determine relative pixelvalues of fluorescence intensity was performed using iVisionsoftware (Biovision Technologies, Exton, PA, USA).Immunoprecipitation and western blottingCells were stimulated for the indicated times with500 ngÆmL)1CH-11 (MBL) at 37 °C, and lysed with buffercontaining 50 mm Tris ⁄ HCl (pH 7.4), 150 mm NaCl, 1%NP-40, 1 mm Na3Vo4,10mm NaF, and complete proteaseinhibitor cocktail (Boehringer, Mannheim, Germany). Toisolate the CD95-associated signaling complex, cell lysateswere immunoprecipitated using specific antibody against theDD of human CD95 (G254-274; BD Pharmingen) and pro-tein A ⁄ G plus agarose (Thermo Fisher Scientific, Rockford,IL, USA). Immunoprecipitates were subjected to westernblot analysis using antibodies against human CD95 (C20;Santa Cruz Biotechnology, Heidelberg, Germany), FADD[clone 1F7 (Millipore, Schwalbach, Germany) or H-181(Santa Cruz Biotechnology)], caspase-8 (C15; Alexis Bio-chemicals, Farmingdale, NY, USA), Laminin A ⁄ C (clone 14;Millipore), and GDI-Rho (clone 16; BD Pharmingen).Membrane fractionationA20 cells expressing either full-length human CD95 ormutant human CD95(Y291F) were incubated with500 ngÆmL)1antibody against human CD95 (CH11; MBL)for the indicated times at 37 °C. Stimulation was termi-nated by adding ice-cold homogenization buffer (BioVision,Mountain View, CA, USA) containing 0.5% azide. Nuclearand cytoplasmic membrane fractions were subsequentlyseparated using a nuclear ⁄ cytosol protein extraction kit(BioVision), according to the manufacturer’s instructions.References1 Chinnaiyan AM, O’Rourke K, Tewari M & Dixit VM(1995) FADD, a novel death domain-containingprotein, interacts with the death domain of Fas andinitiates apoptosis. Cell 81, 505–512.FADD trafficking and CD95 signaling N. Fo¨ger et al.4264 FEBS Journal 276 (2009) 4256–4265 ª 2009 The Authors Journal compilation ª 2009 FEBS2 Ashkenazi A & Dixit VM (1998) Death receptors:signaling and modulation. Science 281, 1305–1308.3 Tibbetts MD, Zheng L & Lenardo MJ (2003) The deatheffector domain protein family: regulators of cellularhomeostasis. Nat Immun 4, 404–409.4 Chinnaiyan AM, Tepper CG, Seldin MF, O’Rourke K,Kischkel FC, Hellbardt S, Krammer PH, Peter ME &Dixit VM (1996) FADD ⁄ MORT1 is a common media-tor of CD95 (Fas ⁄ APO-1) and tumor necrosis factorreceptor-induced apoptosis. 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CurrBiol 8, 1399–1402.17 Algeciras-Schimnich A & Peter ME (2003) Actin depen-dent CD95 internalization is specific for type I cells.FEBS Lett 546, 185–188.18 Boldin MP, Goncharov TM, Goltsev YV & Wallach D(1996) Involvement of MACH, a novel MORT1 ⁄FADD-interacting protease, in Fas ⁄ APO-1- and TNFreceptor-induced cell death. Cell 85, 803–815.19 Kischkel FC, Hellbardt S, Behrmann I, Germer M,Pawlita M, Krammer PH & Peter ME (1995) Cytotox-icity-dependent APO-1 (Fas ⁄ CD95)-associated proteinsform a death-inducing signaling complex (DISC) withthe receptor. EMBO J 14, 5579–5588.20 Scaffidi C, Volkland J, Blomberg I, Hoffmann I, Kr amme rPH & Peter ME (2000) Phosphorylation of FADD ⁄MORT1 at serine 194 and association with a 70-kDa cellcycle-regulated protein kinase. J Immunol 164, 1236–1242.21 Alappat EC, Feig C, Boyerinas B, Volkland J, SamuelsM, Murmann AE, Thorburn A, Kidd VJ, SlaughterCA, Osborn SL et al. (2005) Phosphorylation of FADDat serine 194 by CKIalpha regulates its nonapoptoticactivities. Mol Cell 19, 321–332.22 Alappat EC, Volkland J & Peter ME (2003) Cell cycleeffects by C-FADD depend on its C-terminal phosphor-ylation site. J Biol Chem 278, 41585–41588.23 Faustino RS, Cheung P, Richard MN, Dibrov E,Kneesch AL, Deniset JF, Chahine MN, Lee K, Black-wood D & Pierce GN (2008) Ceramide regulation ofnuclear protein import. J Lipid Res 49, 654–662.24 Grullich C, Sullards MC, Fuks Z, Merrill AH &Kolesnick R (2000) CD95(Fas ⁄ APO-1) signals ceramidegeneration independent of the effector stage of apopto-sis. J Biol Chem 275, 8650–8656.25 Grassme H, Jekle A, Riehle A, Schwarz H, Berger J,Sandhoff K, Kolesnick R & Gulbins E (2001) CD95signaling via ceramide-rich membrane rafts. J BiolChem 276, 20589–20596.26 Wagenknecht B, Roth W, Gulbins E, Wolburg H &Weller M (2001) C2-ceramide signaling in glioma cells:synergistic enhancement of CD95-mediated, caspase-dependent apoptosis. Cell Death Differ 8, 595–602.27 Kabra NH, Kang C, Hsing LC, Zhang J & Winoto A(2001) T cell-specific FADD-deficient mice: FADD isrequired for early T cell development. Proc Natl AcadSci USA 98, 6307–6312.28 Bender LM, Morgan MJ, Thomas LR, Liu ZG &Thorburn A (2005) The adaptor protein TRADD acti-vates distinct mechanisms of apoptosis from the nucleusand the cytoplasm. Cell Death Differ 12, 473–481.N. Fo¨ger et al. FADD trafficking and CD95 signalingFEBS Journal 276 (2009) 4256–4265 ª 2009 The Authors Journal compilation ª 2009 FEBS 4265 . Subcellular compartmentalization of FADD as a new level of regulation in death receptor signaling Niko Fo¨ger1, Silvia Bulfone-Paus1, Andrew. independent of CD95internalization, formation of the death- inducing signaling complex, andcaspase-8 activation. In contrast to nuclear export of FADD, its
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