IUBMB
Life, 56(7): 395–401, July 2004
Critical Review FADD and its Phosphorylation Jing Zhang, Dapeng Zhang and Zichun Hua The State Key Laboratory of Pharmaceutical Biotechnology, and Institute of Molecular and Cell Biology, Nanjing Universtiy, Nanjing 210093, China
Summary The adaptor protein FADD is essential for apoptosis induced by ‘death receptors’, mediating aggregation and autocatalytic activation of caspase-8. Surprisingly, FADD is also involved in regulating T and B cell development. Accumulating evidences now suggest that FADD and its phosphorylation have additional roles in controlling pathways of cellular activation and proliferation, while the kinase modifying FADD phosphorylation is still unidentified. The cellular localization of FADD may also contribute to define FADD’s role in apoptosis or proliferation. FADD may be a pivotal molecule which coupling the opposite cell processes of proliferation and apoptosis. FADD, probably modulated by phosphorylation, may function as a ‘cell renewal set point’ co-regulating proliferation and apoptosis in parallel. IUBMB Life, 56: 395–401, 2004 Keywords FADD; apoptosis; phosporylation; kinase; T cell activation; proliferation.
Lymphocyte homeostasis is a balance between lymphocyte proliferation and lymphocyte death. Apoptosis plays a prominent role in lymphocyte development and homeostasis. Many diseases are associated with either excessive or insufficient apoptosis, such as AIDS, cancer and autoimmunity (1). Death receptors (DR) belonging to the TNF receptor family have been thought to play an important role in regulation of immunity. They transmit apoptosis signals initiated by their specific ‘death ligands’ and are characterized by an intracellular death domain that serves to recruit adapter proteins such as FADD (Fas-associated death domain) and TRADD (2, 3). To date, eight DRs have been described: TNFR1 (CD120a), Fas (APO-1 or CD95), TRAMP (DR3, Apo3 or Wsl-1), TRAIL-R1 (DR4), TRAIL-R2 (DR5), DR6, NGFR and EDA-R. Among these, the best characterized is Received 22 April 2004; accepted 10 August 2004 Address correspondence to: Zichun Hua, Department of Biochemistry, Nanjing University. Tel: + 86-25-3592396-809. Fax: + 86-25-3324605. E-mail:
[email protected] ISSN 1521-6543 print/ISSN 1521-6551 online # 2004 IUBMB DOI: 10.1080/15216540400008929
Fas, which has been shown to play a crucial role in the immune system, in both immune cell-mediated cytotoxicity and down-regulation of immune responses. Following trimerization of Fas after ligation with its natural ligand FasL, apoptosis is initiated. Fas clustering recruits the FADD adapter protein and forms the death-inducing signaling complex (DISC), causing the activation of caspase-8. Caspase-8, in turn, activates the downstream caspases, such as caspase-3, culminating in apoptosis (see Fig. 1.). FADD (also known as MORT-1) is best known as a cytoplasmic adaptor protein bridging activated death receptor with initiator caspase (4). Fas and FADD associate through a conserved motif known as the death domain (DD), which is found in both the Fas cytoplasmic tail and the FADD protein. In turn, FADD can recruit caspases, notably caspase-8, through a second element known as the death effector domain (DED), present in FADD and in the predomain of the caspase. FADD is critical for signaling from Fas and certain other members of the TNF-R family. It may mediate apoptotic signaling of all the death domain receptors (5 – 7). FADDdeficient thymocytes do not undergo apoptosis following Fas triggering (5). FADD also seems to be involved in apoptosis induction by other death receptors, demonstrated by overexpression of C-FADD (C-terminal half of FADD which lacks the death effector domain, 91 – 201aa for mouse FADD) which inhibits TNF-R1 and DR3-induced apoptosis (8, 9). FADD is not only involved in death receptor signaling of apoptosis. Recently it has become clear that FADD, especially its phosphorylation, is involved in lymphocyte proliferation and activation as well as apoptosis (10, 11). Furthermore, in contrast to death receptor-deficient mice, FADD7/ 7 mice die in utero, suggesting a role for FADD in embryonic development (5, 6). Here we summarize the present state of knowledge about FADD and review the seemingly apposing role of FADD in regulating apoptosis and cell activation and proliferation in relation to T-cell homeostasis.
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Figure 1. A model for apoptosis signaling by Fas and FADD-mediated cell cycle. Clustering of Fas upon binding of FasL recruits FADD/MORT1, which is a bipartite molecule with a death effector domain (DED) at the amino terminus and a death domain (DD) at the carboxyl terminus. FADD binds to Fas (via a DD – DD interaction) and recruits caspase 8 to the receptor via DED-DED interaction (4). The aggregation of Fas, FADD and caspase-8 has been named the death-inducing signaling complex (DISC) and results in the proteolytic autoactivation of caspase-8 and the formation of the active caspase 8 enzyme complex, which is a tetramer composed of p18 and p10. Subsequently, activated caspase 8 cleaves other caspases, resulting in efficient cell death. Another complex of X’ induced by FADD is crucial for the cell cycle (11). Unphosphorylated FADD protein binds to an intracellular protein X and forms a machinery (some other proteins, such as caspase 8 and FLIP, may also be involved in the complex), which promotes the cell cycle progression from G1 to S stage. Phosphorylation of FADD like FADD (S191D) leads to dissociation of the FADD-X complex. The mutant FADD (S191D) protein fails to bind protein X at all phases of the cell cycle, leading to an inactive X protein and subsequent failure of the cell cycle progression (11).
STRUCTURE AND PHOSPHORYLATION SITE OF FADD The human FADD gene is composed of two exons separated by a 2-kb intron and is located on chromosome 11q13.3 (12). This region of the genome is amplified in certain breast adenocarcinomas and has also been recognized as a diabetes susceptibility locus. It is therefore possible that mutations in this gene play a role in
tumourigenesis or autoimmunity. Northern blot and in situ hybridization analysis showed that FADD mRNA is expressed in all adult and embryonic tissue in both mice and human (4, 6). The human FADD protein contains 208 amino acids. The mouse FADD is a 205-amino-acids protein that predicted protein sequence shows 68% identity and shares 80% overall amino acid similarity with the sequence of described human
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FADD (13). The conservation is particularly high in two regions, the C-terminal ‘death domain’ and the N-terminal ‘death effector domain’, which are involved in homotypic protein – protein interaction. Human FADD contains two serine clusters, one located at the N and one at the C terminus of the molecular. Phosphorylation of FADD occurs only at a C-terminal serine cluster and is located at serine 194 (14), while mouse FADD is phosphorylated at both serine and threonine residues and the major phosphorylation occurs at the serine residues revealed by 2-D analysis (13). One of the phosphorylation sites of mouse FADD is serine 191, an amino acid that is equivalent to serine 194 of human FADD. This position is outside the
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FADD death effector- and death domains, but it is the key to its role in the regulation of growth and proliferation (11). A point mutant of C-FADD changing serine 194 into alanine was no longer phosphorylated in an in vitro kinase assay and was also no target for the kinase in vivo (14). Both human and murine FADD are specifically phosphorylated at a single serine residue, suggesting that the site and the function of this serine-specific phosphorylation are conserved between both species. Alignment of FADD proteins from different mammalian species shows a remarkable conservation of serine 191 (mouse) or 194 (human) and its surrounding amino acids (Fig. 2). Interestingly, while the death effector- and death domains are highly conserved across all species examined, the region
Figure 2. The conserved functional domains and phosphorylation sites of FADDs in seven different species. The alignment was generated using T-coffee and Clustalw and was curated manually. Identical residues were shaded and the threshold for identity shading is 85%. The residue numbers are based on the corresponding amino acid residue position in the mouse FADD protein. Death effector domain and death domain are indicated in the boxes at the top of the sequences. The known phosphorylation site, mouse Ser 191 (equivalent to Ser194 of human FADD), and other two putative ones, Ser 187, Ser 202, are indicated by asterisks to highlight their conservation in mammals. Two regions: NLS, nuclear localization signal and NES, nuclear export signal, are also indicated. Most of the FADD protein sequences were obtained from NCBI or Ensembl. The pig and zebrafish FADD sequences were assembled from the EST database.
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containing serine191 or 194 is absent from zebrafish, drosophila, and anopheles FADD proteins (see Fig. 2). These data suggest that the C-terminal region of the mammalian FADD may represent a domain distinct from its more conserved (and primitive) apoptotic region and this novel domain was acquired by mammals and required during evolution.
THE RELATED KINASE FOR PHOSPHORYLATION OF FADD Regulation of protein activity by phosphorylation is a common mechanism used for a variety of signal transduction pathways. It is well known that phosphorylated and unphosphorylated FADD interact equally well with Fas (11). Recent reports suggest Fas may also be phosphorylated in vivo (15). Using a series of GST-murine Fas variants, novel proteins, including kinases, were identified that associated specifically with the membrane-proximal, cytoplasmic tail of Fas but not with the death domain. One of these kinases (about 43-kD) phosphorylates FADD (15). As similar finding has been reported by Tschopp’s group, they identified a 130-kD kinase that induces FADD phosphorylation and inhibits Fasmediated Jun NH2-terminal kinase activation, which is designated Fas-interacting serine/threonine kinase/homeodomain-interacting protein kinase (FIST/HIPK3) as a novel Fasinteracting protein (16). Moreover, it was also found that protein kinase C (PKC)z interacts with FADD in vivo and that PKCz immunoextracts prepared from KG1a cells are able to directly phosphorylate FADD in vitro (17). PKC exerts a protective function against Fas death pathway and contribute to the lack of DISC formation. So it is hypothesized that PKCz may regulate DISC formation by influencing FADD phosphorylation status which facilitates caspase-8 inhibition and subsequent Fas resistance. Human FADD was reported to be highly phosphorylated specifically in G2/M phases and binds to be a yet-to-be identified G2/M-specific kinase, whereas FADD phosphorylation was low in G1/S (14, 18). Many cell cycle-regulated protein kinases phosphorylate in a proline-directed manner (19). Serine 194 is part of a Met-Ser-Pro motifs that presents the most conserved region at the C terminus between human and murine FADD (Fig. 2). However, classical cell cycleregulating kinases such as members of the cdc and cdk family which are active at the G2/M boundary could not be responsible for the FADD phosphorylation. In addition, the amount of phosphorylated FADD did not increase by treatment with PMA, that implying not the protein kinase C was not responsible for this modification (14). Furthermore, recombinant casein kinase II also failed to phosphorylate FADD in vitro. An unknown cell cycle-regulated kinase of 70 kDa has been reported to phosphorylate FADD somewhere between G2 and M phase, and up to now it is still unidentified (14).
In summary, the kinase that is responsible for FADD has not been identified, further studies are necessary to identify the FADD kinase and characterize its function in the signaling pathway.
THE CELLULAR LOCALIZATION OF FADD FADD is known mainly for its death receptor adaptor function at the cell surface. Thus, it is widely assumed that FADD is primarily a cytoplasmic protein and its role solely for cytoplasmic DISC. Recent evidences indicate that FADD primarily resides in the nucleus and appears to shuttle between nucleus and cytoplasm. Its nuclear localization has newly been reported relying on strong nuclear localization and nuclear export signals (NLS and NES, respectively) that reside in the death effector domain of the protein (20). The revealed NLS and NES are highly conserved between FADD proteins from various species (Fig. 2). Another paper also evidently supports the role of NLS and NES motifs in nuclear localization in that mutation of the conserved phenylalanine at the 25th amino acid residue, which is located inside NES, to glycine or deletion of N-terminal DED domain which containing the NLS and NES will alter FADD’s nuclear distribution, and normally wild type FADD primarily localizes to the nucleus (21). In addition to the NLS and NES motifs, it was also revealed that FADD’s accumulation in the nucleus and exporting to the cytoplasm requires the phosphorylation site Ser-194 in several adherent cell lines (21). The role of Ser-194 phosphorylation in determining the localization of FADD depends on the interaction with the nucleocytoplasmic shuttling protein exportin-5 (21). This concept will undoubtedly help to resolve questions on functions regulated by FADD. In addition to its well-established role in transduction of apoptotic signals, FADD may also play a role in regulating genome surveillance and perhaps in other as yet unidentified cellular processes. But the subcellular localization of FADD remains controversial. The report by O’Reilly et al shows that FADD is localized in the cytoplasm but not the nucleus in the cells tested, such as mouse L929 fibroblasts, human Hela cells and so on (22).
THE PROLIFERATIVE ROLE OF FADD AND ITS PHOSPHORYLATION Recently, a number of investigations have focused on the role for FADD in T cell activation and development. It was found that T-lymphocytes lacking FADD could not proliferate upon stimulation with antigen or mitogens, such as antiCD3/anti-CD28 and concanavalin A stimulation (5, 10, 23). Moreover, no B-lymphocytes were generated in the absent of FADD (5). In reconstitution assays, FADD seems to function downstream of interactions between interleukin-2 (IL-2) and the IL-2 receptor (IL-2R), and its deficiency in T cells produces a phenotype similar to IL-2R-deficiet mice, including
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a defect in T cell proliferation, an increased proportion of CD69 + T cells and an age-dependent disappearance of thymocytes (24). According to published observations that FADD(S191D) has defective cell proliferation but normal apoptosis (11) and a dominant negative mutant of FADD (FADD-DN, containing amino acid residues 80 – 208) transgenic mice has both impaired apoptosis and T cell proliferation (25), the proliferative effect of FADD is likely to be independent of caspase activity. Analysis of T cell-specific FADD-knockout and FADD7/7?RAG-17/ 7 complementation mice showed that FADD deficiency in the immune system leads to impaired proliferation following activation and inhibition of the proliferation stage in the CD47CD87 T cell compartment during thymocyte development (5). Similarly, the impact of FADD-transduced signals on T lymphocyte development was investigated in transgenic mice expressing a dominant negative mutant protein FADD-DN (25 – 27). FADD-DN lacks the N-terminal death effector domain and thereby blocks those TNF receptor family members that use FADD as an adapter. FADD-DN expression enhances deletion of autoreactive thymocytes and inhibits proliferation of mature T lymphocytes, blocking both quiescent cells from entering the cell cycle (the G0?S transition) and the proliferation of continuously cycling T cells in G1 (25). Furthermore, it has been observed that FADD-DN exerts a similar growth-inhibitory effect on fibroblasts, indicating that this effect is not limited to T cells and so is likely to act on more general, cell type-independent proliferation pathway (27). Lately, the ability of FADD to promote cell cycle progression of T cell was found to depend on the phosphorylation of Ser-194. Ser-194 is located outside the domains at the tail of FADD that has thus far not been well studied. Recent data on transgenic mutant FADD mice shows that regulation of FADD phosphorylation at Ser191 (the corresponding serine in mouse FADD) is crucial for cell cycle progression (11). Mice bearing the Asp mutation FADD (S191D) are runted and anemic and display splenomegaly. Apoptosis is unimpaired in these mice both FADD (S191A) and FADD (S191D), but the FADD (S191D) exhibit many immune developmental problems indicative of proliferative defects. How to explain the striking similarities of the immune phenotype between FADD(S191D) and FADD7/7?RAG-17/ 7 mice, and comparatively normal phenotype of FADD(S191A) mice, and the ability of FADD-DN to block cell proliferation? Only a single amino acid change results in dramatic effects in growth and hematopoiesis. Hua et al. propose a hypothetical model for illustrating the importance of FADD (11). In this model, the unphosphorylated FADD protein binds to an intracellular protein X and acquires an activity required for the G1 to S stage (Fig. 1) and FADD-X complex probably mainly exist in nucleus according to recent finding that wild type FADD primarily localizes to the nucleus (33). Phosphorylation of FADD leads to dissociate the FADD-X complex. In FADD
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(S191A) mice, the residual phosphorylation on threonine in FADD might help FADD to dissociate with X during G2/M phases and to regulate cell cycle in a relatively normal manner. Consistent with it, phosphorylation of endogenous FADD increases in the time of G2/M arrest and C-FADD with a S194E mutation mimicking phosphorylated C-FADD is more susceptible to a Taxol-induced G2/M arrest than C-FADD S194A (28). For FADD-DN, its overexpression leads to sequestration of protein X from the endogenous normal FADD protein, resulting in inhibition of cell growth. Constant phosphorylation of FADD, as mimicked by FADD (S191D) or FADD S194E, will lead to cell cycle arrest at G2/M phases and could not form FADD-X complex which is indispensable for G1 to S phase entry. Notably, Caspase-8 and c-FLIP-deficient mice exhibited a similar embryonic phenotype to FADD7/ 7 mice (29, 30). These mice all died around day 10.5 of embryogenesis, with apparent heart defects. Inactivation of caspase-8 leads to diminished T cell proliferation and lymphocyte activation defects shown in immunodeficient patients with an inherited caspase-8 mutation (31), and c-FLIPL overexpression results in increased T cell proliferation and production of IL-2 in transgenic mice (32). As a supplement to the above model, caspase-8 and c-FLIP might also be involved in the cell-cycle regulating complex formed by FADD and its complex is different from the DISC complex in the apoptotic event (Fig. 1). It thus suggests that the formation of this cell proliferation related-complex probably depends on the protein modification, such as phosphorylation, sumolation or ubiquitination etc. For these molecules, the alteration in protein will have potentially parallel effects on the opposing processes of proliferation and apoptosis. During both apoptosis signaling and mitogenic activation, FADD and caspase-8 aggregated in multiprotein complexes and formed caps at the plasma membrane. Interestingly, mitogenic stimulation, but not Fas ligation, induced a unique post-translational modification of FADD (22). These different modifications may determine whether FADD and caspase-8 induce cell death or proliferation. It is noteworthy that the first report indicating that phosphorylated FADD plays an essential role in the mechanisms of amplifications of chemotherapy-induced apoptosis, which clearly shows that FADD phosphorylation at 194 serine affects functions both upstream and downstream of the MEKK1/MKK7/JNK1 pathway and is closely associated with chemosensitivity in prostate cancer cells (33). The expression level of FADD, the subcellular localization, its post-translational modifications and the regulation of its function may coordinately control its ostensibly opposing processes. The precise mechanism of FADD in growth and proliferation remain to be elucidated. Further work is necessary to dissect FADD’s exact molecular role in cell cycle progression, its signal transduction pathway in this process and how FADD balances the process of apoptosis and proliferation.
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ACKNOWLEDGEMENTS This work is supported by the Tang Family Foundation and by the Chinese National Nature Science Foundation (30270291, 30330530) and the Ministry of Education of China (TRAPOYT and 20030284040) to Zi-Chun Hua.
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