ABSTRACT The Fas-associated death domain (FADD) adaptor protein FADD/Mort-1 is recruited by several members of the tumor necrosis factor receptor (TNFR) superfamily during cell death

activated via death receptors. Since most studies have focused on the interaction of FADD with plasma membrane proteins, FADD's subcellular location is thought to be confined to the

cytoplasm. In this report, we show for the first time that FADD is present in both the cytoplasm and the nucleus of cells, and that its nuclear localization relies on strong nuclear

localization and nuclear export signals (NLS and NES, respectively) that reside in the death-effector domain (DED) of the protein. Specifically, we found that a conserved basic KRK35

death-inducing efficacy of FADD reconstituted in FADD-deficient T cells. SIMILAR CONTENT BEING VIEWED BY OTHERS PTPN23-DEPENDENT ESCRT MACHINERY FUNCTIONS AS A CELL DEATH CHECKPOINT Article

Open access 28 November 2024 DECIPHERING DED ASSEMBLY MECHANISMS IN FADD-PROCASPASE-8-CFLIP COMPLEXES REGULATING APOPTOSIS Article Open access 06 May 2024 IKK-MEDIATED TRAF6 AND RIPK1

INTERACTION STIFLES CELL DEATH COMPLEX ASSEMBLY LEADING TO THE SUPPRESSION OF TNF-Α-INDUCED CELL DEATH Article 21 April 2023 INTRODUCTION Programmed cell death induced by the interaction of

cell surface death receptors with their respective extracellular ligands plays a central role in numerous life and death decisions ranging from embryonic development to cellular homeostasis.

In mammals, the intracellular signaling initiated by death receptors of the tumor necrosis factor receptor (TNFR) superfamily leads to the activation of hierarchically organized

intracellular signaling pathways that typically culminate in the death of cells bearing such receptors.1 Fas-associated death domain (FADD) is an adaptor death domain (DD)-containing protein

of approximately 25 kDa2 shared by several death receptors that couple death signals to the intracellular death machinery. The human protein consists of 208 amino acids arranged in 12

amphipathic helices functionally divided into six helices in the death-effector domain (DED), which is located at its NH2-terminus, and six helices that form the DD at the COOH-terminus.3

Mechanistically, DD-containing adaptor proteins like FADD or TNFR1-associated DD (TRADD) appear to be required for cell death signaling induced by DD-containing receptors such as Fas/CD95,

TNFR1, and TRAIL 1 and TRAIL 2 receptors.2,4,5 The best characterized signaling mechanism mediated by FADD is the formation of the death-inducing signal complex (DISC) in which, upon

activation of death receptors, FADD interacts with the intracellular DD of the receptors and/or other adaptor proteins such as TRADD,2,6,7 and the DED of FADD subsequently recruits and

activates caspase-8.8 The intervention of FADD in this mechanism is primordial to signal death because expression of a dominant-negative form of FADD consisting of the DD alone (DN-FADD)

impairs Fas and TNFR1 death signaling cascades.9 Moreover, FADD−/− knockout mice display profound defects in apoptotic pathways, particularly in the immune system.10 In addition to its

prominent role in cell death, FADD may have a role in cell-cycle control and proliferation of lymphoid cells,10,11,12,13 as well as embryonic development.14 FADD's mode of action is

assumed to take place in the cytoplasm, possibly because the majority of studies document the interaction of FADD with plasma membrane receptors and associated proteins. However, the

cellular biology and regulation of FADD, its dynamics, as well as the understanding of cellular processes governed by FADD, remain poorly understood. One level of the regulation of FADD in

mammalian cells was shown to occur via its phosphorylation at the serine-194 by a nonidentified 70-kDa cell-cycle-related kinase,15 but the biological significance of FADD phosphorylation is

still a conundrum since both forms of FADD can interact with activated death receptors. In this report, we provide new experimental evidence for the presence of FADD in the nucleus and the

existence of functional nuclear localization signals (NLSs) in the amino-acid sequence of FADD. We propose that the mechanism of action of FADD needs to be reconsidered as a result of its

nucleocytosolic localization. This concept will undoubtedly help to resolve questions on functions regulated by this signaling adaptor molecule. RESULTS AND DISCUSSION We have detected

surprising constitutive expression of FADD in both the cytoplasm and nuclear compartments in a spectrum of cell lines examined by confocal microscopy. HeLa, HEK 293, Jurkat, HTC, COS-1, and

A549 cells were immunostained with a monoclonal IgG1 directed against the DD of FADD or with a polyclonal antibody that recognizes the phosphorylated form of FADD (data not shown, and Figure

1A). These cell lines examined exhibited FADD concentrated in the nucleus of the cells (Figure 1A, panels a,b,d,f). In contrast, we observed that in Jurkat T-lymphocytes approximately 50%

of the cell population harbored FADD in the nucleus, whereas the other 50% displayed FADD in the cytoplasm (Figure 1Ac). The use of this monoclonal anti-FADD has been validated and well

documented in specific studies for its ability to recognize (1) native and denatured FADD, (2) FADD recruited in the DISC of Fas-activated cells, and (3) the dominant-negative form of FADD

termed DN-FADD.16,17,18,19 The presence of FADD in the nucleus was confirmed by the examination of the expression of FADD in protein samples from nuclear and cytosolic fractions extracted

from HeLa cells (Figure 1B) in which FADD exhibited a nuclear/cytosolic pattern of expression similar to that found for transcriptional activators and other nuclear proteins.20 Since its

discovery, FADD has been reported to exert its function primarily by signaling at the plasma membrane;2,21 therefore, our finding that FADD displays nuclear localization was unprecedented.

Examination of the primary amino-acid sequence of full-length FADD (Figure 1C) led us to the identification of one putative NLS located in Helix 3 of the DED. The NLS of FADD corresponded to

a highly conserved short stretch of three basic amino acids KRK35 (Figure 2) that resembled a functional monopartite NLS of other well-characterized nuclear proteins such as Myc.22 To

define whether the KRK35 motif of FADD was a functional NLS, a mutant form of FADD (NLS Mutant-FADD) was generated by replacing the KRK35 with the sequence AAA35 through site-directed

mutagenesis. HeLa cells were transfected with either wild-type (WT)-FADD or NLS Mutant-FADD, and were immunostained with FADD- and Alexa Fluor594-conjugated antibodies. Overexpression of

full-length FADD in HeLa cells resulted in the appearance of spread filament-like structures as observed by confocal microscopy (Figure 3A), similar to those reported by Siegel _et al._23

that were attributed to the formation of death-effector filaments characterizing the overexpression of most DEDcontaining proteins. In most cells overexpressing FADD, the fluorescence was no

longer concentrated in the nucleus (Figure 3Aa) compared to our observations for the endogenous FADD (Figure 1A), perhaps suggesting that the formation of filamentous structures may be an

artefact of overexpression. Nonetheless, expression of the NLS Mutant-FADD in the cells led to a strong exclusion from the nucleus (Figure 3Ab) and formation of less apparent, filament-like

structures. These data suggest that the basic KRK35 sequence in the DED of FADD is required for FADD's subcellular distribution and its nuclear localization. To determine whether the

presence of FADD in the nucleus is dependent on nucleocytosolic trafficking, we scrutinized the primary sequence of FADD for putative leucine-rich motifs with conserved spacing and

hydrophobicity that are characteristic of nuclear proteins. Analysis of the primary amino-acid sequence of FADD revealed the existence of one consensus [LX2–3LX2–3LXL; see Nakielny and

Dreyfuss24 for review) leucine-rich nuclear export signal (NES) located in the DED of human FADD formed by the sequence LTELKFLCL28 (Figure 2), which conforms to a _rev_-type consensus NES

as that found in HIV-1 Rev.25 Using site-directed mutagenesis we eliminated the leucine-rich motif of FADD, and generated an NES Mutant-FADD mutant harboring the amino-acid sequence

ATEAKFACA28. Consistent with the hypothesis that FADD is transported to the nucleus, HeLa cells transfected with the NES Mutant-FADD showed significant retention of FADD in the nucleus

despite overexpressing this protein (Figure 3Ac). This observation was in marked contrast to the pattern observed with overexpressed WT-FADD or the NLS Mutant FADD (Figures 3Aa and Ab,

respectively), supporting the premise that FADD is shuttled into the nucleus, and that the NES sequence in FADD is functional and necessary for FADD's nuclear export. To validate and

extend these results, green-fluorescent protein (GFP)- tagged FADD was generated by linking GFP to the N-terminus of FADD to assess the subcellular distribution of GFP fusion proteins in

living cells by localizing direct fluorescence from the GFP moiety. Expression of GFP-FADD in HeLa cells led to a heterogeneous pattern of cytosolic and nuclear fluorescence among cells

(Figure 3Bb), while both HEK 293 and A549 cells displayed a significant amount of green fluorescence in the cytoplasm (Figure 3Cb and Cf, respectively). It should be noted that the fusion of

GFP with FADD yields a protein of approximately 52 kDa, considerably higher than the molecular size of endogenous FADD present in the cells, which may compromise trafficking mechanisms of

this overexpressed fusion protein. However, in every cell type examined for the expression of GFP-NLS Mutant-FADD, all the green-fluorescent cells exhibited profound cytoplasmic staining and

a loss of nuclear fluorescence (Figures 3Bc, 3Cc and Cg) compared with their respective controls, supporting the notion that a KRK35 cluster defines a functional NLS necessary for the

normal shuttling of FADD to the nucleus. In contrast, confocal analyses of cells transfected with GFP-NES Mutant-FADD (Figures 3Bd, 3Cd and Ch) showed a prominent increase in the intensity

of green fluorescence in the nucleus. Thus, GFP-NES Mutant-FADD is actively transported into the nucleus in the absence of this unique leucine-rich sequence. Overexpression of FADD in cells

has been reported to induce apoptosis owing to FADD oligomerization and the association of FADD molecules with plasma membrane death receptors in the absence of death receptor ligand.26

Although overexpression of FADD in HeLa, HEK 293, and A549 cells induced cell death, the percentage of death detected at the time of data acquisition (24 h post-transfection) was negligible,

in contrast to the 80% cell death found in Jurkat T- cells after 24 h of cell transfection with FADD (data not shown). In lymphoid cells, the interaction of FADD with death receptors

constitutes an important step of the immune response.27 Since induction of lymphocyte apoptosis through cell death receptors is a physiologically well-characterized model of apoptosis, we

examined whether the mutational changes that we introduced in FADD exerted any influence in FADD's ability to trigger cell death. For these experiments, we used the FADD-deficient

Jurkat T-cell line,28 which is resistant to apoptosis induced by the Fas/CD95 receptor. Since oligomerization of FADD following overexpression has been reported to induce both apoptosis and

necrosis,26 we quantified apoptotic cell death by measuring cell shrinkage, a distinctive feature of apoptotic cell death.29 Cell shrinkage was examined by fluorescence-activated cell

sorting (FACS) analyses of the forward-scatter light in the gated green-fluorescent cells expressing the GFP-FADD proteins that were treated with or without anti-Fas. The expression of the

various GFP constructs in FADD-deficient cells led to the same pattern of subcellular distribution as observed for HeLa, HEK 293, and A549 cells (not shown). As expected, expression of

GFP-WT-FADD-induced a significant increase in the amount of apoptotic cell shrinkage (Figure 3D). Upon treatment with anti-Fas for 1 h, the percentage of apoptosis detected in GFP-WT-FADD

transfected cells increased slightly compared to the untreated cell population (Figure 3D). This result suggested that the FADD-transfected cells were insensitive to exogenous Fas receptor

activation, probably as a result of the saturating concentration of FADD molecules present in the cells. Similarly, the percentage of apoptosis found in GFP-NLS Mutant-FADD-transfected cells

was independent of Fas activation (Figure 3D). In contrast, reconstitution of the FADD-deficient cells with GFP-NES Mutant-FADD significantly reduced the apoptosis. This effect probably

reflects the fact that the GFP-NES Mutant-FADD is secluded to a significant extent in the nucleus (Figure 3Bd, and 3Cd and Ch). Alternatively, it is plausible that the presence of FADD in

the nucleus may activate survival mechanisms. Our investigation suggests that many questions regarding the mechanisms that control the intracellular transport and biology of FADD remain

unsolved. However, our findings unveil a novel level in the regulation of FADD. The observation that in a homogenous Jurkat cell population, individual cells can be segregated into either

cytosolic or nuclear FADD (Figure 1Ac) raises the intriguing question as to which factors determine the subcellular localization of FADD in a given cell and whether this localization

predetermines sensitivity to death receptor stimulation, specifically in light of the asynchronous nature of the cell death process. Recently, it has been shown that TRADD can be shuttled to

the nucleus as well, although TRADD's constitutive expression is mainly localized in the cytoplasm and Golgi apparatus, and the regulation of the nucleocytosolic transport remains

unknown.30 The study of the mechanistic basis of FADD's presence in the nucleus may lead to new avenues for the role of FADD in the nucleus. EXPERIMENTAL METHODS CELL CULTURE CONDITIONS

AND TRANSFECTIONS Jurkat- and FADD-deficient T cells were cultured as previously described.17,28 HeLa cells were grown as monolayer cultures in Joklik's minimum essential medium and

were harvested with Versene as previously described.31 Human embryonic kidney HEK 293 cells were grown in Eagle's minimum essential medium (EMEM) containing 10% (v/v) heat-inactivated

fetal calf serum, 2 mM glutamine, 100 U penicillin/ml, and 100 U streptomycin/ml. Human lung carcinoma A549 cell line was cultured in a equal dilution (v/v) of F12 nutrient mixture and EMEM

medium supplemented with 5% (v/v) heat-inactivated fetal calf serum, 2 mM glutamine, 100 U penicillin ml, and 100 U streptomycin/ml. FADD-deficient cells were transiently transfected by

electroporation with a BTX ECM 600 electroporator (Genotronics, Inc., San Diego, CA, USA). A total of 10 million exponentially growing cells were harvested, resuspended in 400 _μ_l of

complete medium in disposable 0.4 cm gap BTX cuvettes (Genotronics, Inc.), and mixed with 10 _μ_g of each of the plasmids. Electroporation was performed by setting the BTX ECM 600

electroporator at 260 V, 1040 _μ_F, and 720 Ω. After transfection, cells were immediately placed in six-well plates containing 6 ml of complete medium per well, and were used 18 h after

transfection. Transfection efficiency was routinely measured by FACS and was determined to be between 25 and 40%. For apoptosis analyses, the green-fluorescent populations of cells were

gated for each of the groups. Adherent cell cultures were plated 24 h prior to transfection and were allowed to grow to 50–70% confluence. Cells were washed with Opti-MEM (Life Technologies,

Inc.) and transfected using Fugene-6 (Roche, Germany) according to the manufacturer's recommendations. The precipitates were incubated with cells in Opti-MEM medium, which was replaced

by complete medium after 4 h of transfection. Cells were maintained at 37°C and 5% CO2, 95% air until analyses. PREPARATION OF DNA CONSTRUCTS AND SITE-DIRECTED MUTAGENESIS pcDNA3 vector

containing the full length of FADD was kindly provided by Dr. A Strasser (Melbourne, Australia). To generate GFP-tagged FADD, we excised the cDNA of FADD from the pcDNA3-FADD vector through

digestion with _Bam_H1 and _Kpn_1 and subcloned into the multiple cloning site of _Bam_H1/_Kpn_1-digested pEGFP-C2 (BD Biosciences Clontech, Palo Alto, CA, USA). Plasmid DNA was prepared by

using Qiagen Maxiprep kits (Valencia, CA, USA). Site-specific mutagenesis was introduced by PCR with the QuikChange kit (Stratagene, La Jolla, CA, USA) by following the manufacturer's

instructions and using pcDNA3-FADD or GFP-FADD as templates. Oligonucleotides were individually designed following the QuikChange manufacturer's recommendations and were synthesized by

Integrated DNA Tech. (Coralville, IA, USA). Mutations were verified by PCR with the BigDye™ Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) and analysis were

performed at the NIEHS Sequencing Core (Research Triangle Park, NC, USA). For elimination of the putative NLS, KRK35 was mutated to AAA35 by using the oligonucleotide sequence 5′ GC CTC GGG

CGC GTG GGC GCG GCC GCG CTG GAG CGC GTG CAG AG 3′ and its reverse complementary. This mutant is designated NLS Mutant FADD. For destruction of the sequence LTELKFLCL, leucines were

mutagenized to alanines by using the primer 5′CG AGC AGC GAG GCG ACC GAG GCC AAG TTC GCA TGC GCC GGG CGC GTG GG3′ and its reverse complementary. This mutant is designated NES Mutant FADD.

CELL FRACTIONATION AND WESTERN BLOT ANALYSES Cytoplasmic and nuclear extracts were prepared by standard methods with a fractionation kit (Biovision, CA, USA), and 20 _μ_g of each denatured

protein sample was analyzed by Western blot as previously described.17 Antibodies for tubulin (Upstate Biotechnology, NY, USA) and lamin B (Zymed, CA, USA) were used as positive controls for

the cytosolic and nuclear fractions, respectively. Results were confirmed with commercially available fractionated nuclear extracts (4C Biotech, Belgium; not shown). IMMUNOSTAINING AND

CONFOCAL MICROSCOPY HeLa, HEK 293, and A549 cells were plated and transiently transfected on glass-bottom culture dishes (MatTek Corporation, Ashland, MA, USA) for live imaging, and on

two-well Lab-Tek chamber slides (Nalge Nunc International, Naperville, IL, USA) for immunostaining. To prepare cells for immunofluorescence, monolayers of HeLa, HEK 293, A549, and pelleted

Jurkat cells were rinsed in phosphate-buffered saline (PBS) and fixed for 10 min at room temperature in freshly prepared 4% (w/v) paraformaldehyde (Sigma, St. Louis, MO, USA). Cells were

then permeabilized with 0.2% Triton X-100 and treated with 4% BSA–PBS solution as a blocking reagent. Cells were exposed to mouse anti-FADD (BD Biosciences-Pharmingen, CA, USA), or rabbit

antiphospho-ser194-FADD (Cell Signaling Technologies, Beverly, MA, USA) diluted 1 : 50 and 1 : 300, respectively, in a 1% BSA–PBS solution and were incubated overnight at 4°C. After three

washes with PBS, Fluor594®-conjugated anti-mouse or FITC-conjugated anti-rabbit (Molecular Probes, Eugene, OR, USA) were diluted in 1% BSA–PBS and added to the cells for 1 h. After three

washes with PBS, the upper chamber portions of the slides were removed, and the fixed cells were mounted and preserved with Prolong Anti-Fade (Molecular Probes). Negative controls for

staining consisting of fixed cells exposed to either Alexa-Fluor594®-conjugated IgG or FITC-conjugated were done to confirm specificity. For live GFP-tagged imaging, cells were analyzed 18 h

post-transfection, and the DNA-specific fluorochrome Hoechst 33342 (Molecular Probes) was occasionally added to the cells to visualize nuclei. Images from immunostaining and live imaging

were collected by using a Zeiss inverted laser scanning confocal microscope LSM 410 UV (Zeiss, Thornwood, NY, USA) with a C-Apo × 40 water-immersion objective. An excitation wave length of

488 nm with an LP 505 nm emission filter was used to detect GFP and FITC, and a wavelength of 568 nm with an LP 590 nm emission filter was used to detect Alexa Fluor 594. Images were

analyzed with the LSM-510 Image Browser Software. FLUORESCENCE-ACTIVATED CELL SORTING ANALYSIS FADD-deficient cells transfected with the pEGF-C2 empty vector, WT-GFP FADD, NLS Mutant-FADD or

NES Mutant-FADD-transfected cells were treated 18 h post-transfection with or without 500 ng / ml of anti-human Fas IgM CH-11 for 1 h. Flow-cytometric analyses for changes in cell volume

were examined on a Becton Dickinson FACSort using CellQuest (BD Biosciences) software through forward-scatter light analyses of cells, as reported previously.32 For examination of the

GFP-positive cell populations, wave lengths of 488 and 530 nm were used for excitation and emission, respectively. To ensure that the amount of cell death was quantified on the positively

transfected cell population exclusively, we determined analyses by gating on the GFP-positive cell population. Results were analyzed with Student's _t_-test for statistical

significance. ABBREVIATIONS * TNFR: tumor necrosis factor receptor * NLS: nuclear localization signal * NES: nuclear export signal * DED: death-effector domain * DD: death domain REFERENCES

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275: 19609–19619 Article  CAS  Google Scholar  Download references AUTHOR INFORMATION Author notes * M Gómez-Angelats Present address: Almirall Research Centre, Cardener 68-74, 08024,

Barcelona, Spain AUTHORS AND AFFILIATIONS * The Laboratory of Signal Transduction, Molecular Endocrinology Group, National Institute of Environmental Health Sciences, National Institutes of

Health, 111 Alexander Drive, Research Triangle Park, 27709, NC, USA M Gómez-Angelats & J A Cidlowski Authors * M Gómez-Angelats View author publications You can also search for this

author inPubMed Google Scholar * J A Cidlowski View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to J A Cidlowski.

ADDITIONAL INFORMATION Edited by G Melino RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Gómez-Angelats, M., Cidlowski, J. Molecular evidence for the

nuclear localization of FADD. _Cell Death Differ_ 10, 791–797 (2003). https://doi.org/10.1038/sj.cdd.4401237 Download citation * Received: 12 November 2002 * Revised: 10 January 2003 *

Accepted: 12 February 2003 * Published: 18 June 2003 * Issue Date: 01 July 2003 * DOI: https://doi.org/10.1038/sj.cdd.4401237 SHARE THIS ARTICLE Anyone you share the following link with will

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content-sharing initiative KEYWORDS * FADD/Mort-1 * FAS * nuclear localization signal * nuclear export signal