Modifications and intracellular trafficking of FADD/MORT1 and caspase-8 after stimulation of T lymphocytes

The adaptor protein FADD/MORT1 is essential for apoptosis induced by 'death receptors', such as Fas (APO-1/CD95), mediating aggregation and autocatalytic activation of caspase-8. Perhaps surprisingly, FADD and caspase-8 are also critical for mitogen-induced proliferation of T lymphocytes. We generated novel monoclonal antibodies specific for mouse FADD and caspase-8 to investigate whether cellular responses, apoptosis or proliferation, might be explained by differences in post-translational modification and subcellular localisation of these proteins. During both apoptosis signalling and mitogenic activation, FADD and caspase-8 aggregated in multiprotein complexes and formed caps at the plasma membrane but they did not colocalise with lipid rafts. Interestingly, mitogenic stimulation, but not Fas ligation, induced a unique post-translational modification of FADD. These different modifications may determine whether FADD and caspase-8 induce cell death or proliferation.     

Phosphorylation of FADD at serine 194 by CKIalpha regulates its nonapoptotic activities

FADD is essential for death receptor (DR)-induced apoptosis. However, it is also critical for cell cycle progression and proliferation, activities that are regulated by phosphorylation of its C-terminal Ser194, which has also been implicated in sensitizing cancer cells to chemotherapeutic drugs and in regulating FADD's intracellular localization. We now demonstrate that casein kinase Ialpha (CKIalpha) phosphorylates FADD at Ser194 both in vitro and in vivo. FADD-CKIalpha association regulates the subcellular localization of FADD, and phosphorylated FADD was found to colocalize with CKIalpha on the spindle poles in metaphase. Inhibition of CKIalpha diminished FADD phosphorylation, prevented the ability of Taxol to arrest cells in mitosis, and blocked mitogen-induced proliferation of mouse splenocytes. In contrast, a low level of cycling splenocytes from mice expressing FADD with a mutated phosphorylation site was insensitive to CKI inhibition. These data suggest that phosphorylation of FADD by CKI is a crucial event during mitosis.     

FADD/MORT1, a signal transducer that can promote cell death or cell growth.

FADD/MORT1 is a cytosolic adaptor protein which is critical for signalling from CD95 (Fas/APO-1) and certain other members of the tumour necrosis factor receptor (TNF-R) family (called 'death receptors'). Two protein interaction domains have been identified in FADD/MORT1. The C-terminal 'death domain' is needed for recruitment of FADD/MORT1 to ligated 'death receptors' and the N-terminal 'death effector domain' mediates oligomerisation and activation of caspase-8 zymogens. Caspase-8 activates other cysteine proteases by cleavage and this starts a proteolytic cascade which constitutes the 'point of no return' in apoptosis signalling. Experiments in mice lacking FADD/MORT1 function proved that this adaptor is required for CD95- and TNF-RI-transduced cell death but is dispensable for other pathways to apoptosis. Surprisingly, FADD/MORT1 is also essential for mitogen-induced proliferation of T-lymphocytes. Therapeutic activation of FADD/MORT1 function may be used to kill unwanted cells in cancer or autoimmunity and its suppression may help prevent cell death in certain degenerative disorders.     

c-FLICE inhibitory protein expression inhibits T-cell activation

Cellular FLICE-inhibitory protein (c-FLIP) inhibits death receptor-mediated apoptosis by specific interaction with FADD and procaspase-8, and may thus interfere with activation events mediated by FADD and caspase-8. Recent studies, however, suggest that c-FLIP also transmits activation signals. The role of c-FLIP on T-cell activation was examined here using several transgenic mice with variable c-FLIP expression. In all c-FLIP-transgenic mice, Fas-mediated apoptosis and in vitro activation-induced T-cell death were suppressed, and T-cell proliferation and IL-2 production were inhibited. c-FLIP transgene also promoted in vivo thymocyte death. Higher c-FLIP transgene expression was correlated with a more profound suppression of T-cell activation and a prominent disturbance in mature thymocyte development. There was no evidence of increased activation and proliferation in all c-FLIP-transgenic T cells examined. Instead, suppression of T-cell activation in c-FLIP-transgenic T cells could be a combinatory effect of FADD/caspase-8-dependent signals and c-FLIP-specific activities.     

Apoptotic potential of Fas-associated death domain on regulation of cell death regulatory protein cFLIP and death receptor mediated apoptosis in HEK 293T cells

Fas-associated death domain (FADD) is a common adaptor molecule which plays an important role in transduction of death receptor mediated apoptosis. The FADD provides DED motif for binding to both procaspase-8 and cFLIP molecules which executes death receptor mediated apoptosis. Dysregulated expression of FADD and cFLIP may contribute to inhibition of apoptosis and promote cell survival in cancer. Moreover elevated intracellular level of cFLIP competitively excludes the binding of procaspase-8 to the death effector domain (DED) of FADD at the DISC to block the activation of death receptor signaling required for apoptosis. Increasing evidence shows that defects in FADD protein expression are associated with progression of malignancies and resistance to apoptosis. Therefore, improved expression and function of FADD may provide new paradigms for regulation of cell proliferation and survival in cancer. In the present study, we have examined the potential of FADD in induction of apoptosis by overexpression of FADD in HEK 293T cells and validated further its consequences on the expression of pro and anti-apoptotic proteins besides initiation of death receptor mediated signaling. We have found deficient expression of FADD and elevated expression of cFLIP(L) in HEK 293T cells. Our results demonstrate that over expression of FADD attenuates the expression of anti-apoptotic protein cFLIP and activates the cascade of extrinsic caspases to execution of apoptosis in HEK 293T cells.     

Phosphorylation of FADD/ MORT1 at serine 194 and association with a 70-kDa cell cycle-regulated protein kinase

The adapter molecule Fas-associated death domain protein (FADD)/mediator of receptor-induced toxicity-1 (MORT1) is essential for signal transduction of the apoptosis-inducing receptor CD95 (APO-1/Fas) as it connects the activated receptor with the effector caspase-8. FADD also plays a role in embryonic development and the cell cycle reentry of T cells. FADD is phosphorylated at serine residues. We now show that phosphorylation exclusively occurs at serine 194. The phosphorylation of FADD was found to correlate with the cell cycle. In cells arrested at the G2/M boundary with nocodazole, FADD was quantitatively phosphorylated, whereas only nonphosphorylated FADD was found in cells arrested in G1/S with hydroxyurea. In this context, we have identified a 70-kDa cell cycle-regulated kinase that specifically binds to the C-terminal half of FADD. Because CD95-mediated apoptosis is independent of the cell cycle, phosphorylation of FADD may regulate its apoptosis-independent functions.     

The role of death effector domain-containing proteins in acute oxidative cell injury in hepatocytes

Apoptosis is a mechanism that regulates hepatic tissue homeostasis and contributes to both acute and chronic injury in liver disease. The apoptotic signaling cascade involves activation of the death-inducing signaling complex (DISC) and subsequent recruitment of proteins containing death effector domains (DED), which regulate downstream effector molecules. Prominent among these are the Fas-associated death domain (FADD) and the cellular caspase 8-like inhibitory protein (cFLIP), and alterations in these proteins can lead to severe disruption of physiological processes, including acute liver failure or hepatocellular carcinoma. Their role in cell signaling events independent of the DISC remains undetermined. Oxidative stress can cause cell injury from direct effects on molecules or by activating intracellular signaling pathways including the mitogen-activated protein kinases (MAPKs). In this context, prolonged activation of the cJun N-terminal kinase (JNK)/AP-1/cJun signaling pathway promotes hepatocellular apoptosis, whereas activation of the extracellular signal-regulated kinase (Erk) exerts protection. We investigated the roles of FADD and cFLIP in acute oxidant stress induced by the superoxide generator menadione in hepatocytes. Menadione resulted in dose-dependent predominantly necrotic cell death. Hepatocytes expressing a truncated, dominant-negative FADD protein were partially protected, whereas cFLIP-deficient hepatocytes displayed increased cell death from menadione. In parallel, Erk phosphorylation was enhanced in hepatocytes expressing dnFADD and decreased in cFLIP-deficient hepatocytes. Hepatocyte injury was accompanied by increased release of proapoptotic factors and increased JNK/cJun activation. Thus, FADD and cFLIP contribute to the regulation of cell death from acute oxidant stress in hepatocytes involving MAPK signaling. This implies that DED-containing proteins are involved in the regulation of cellular survival beyond their role in cell death receptor-ligand-mediated apoptosis.     

A dominant negative Fas-associated death domain protein mutant inhibits proliferation and leads to impaired calcium mobilization in both T-cells and fibroblasts

Death domain-containing members of the tumor necrosis factor (TNF) receptor family ("death receptors") can induce apoptosis upon stimulation by their natural ligands or by agonistic antibodies. Activated death receptors recruit death domain adapter proteins like Fas-associated death domain protein (FADD), and this ultimately leads to proteolytic activation of the caspase cascade and cell death. Recently, FADD has also been implicated in the regulation of proliferation; functional inhibition of FADD results in p53-dependent impairment of proliferation in activated T-cells. In this study we have further analyzed T-cells derived from transgenic mice expressing a dominant negative FADD mutant (FADD DN) under control of the lck promoter in vitro so as to identify the signaling pathways that become engaged upon T-cell receptor stimulation and that are regulated by death receptors. FADD DN expression inhibits T-cell proliferation, both at the G(0) --> S transition and in the G(1) phase of continuously proliferating cells. We observe a decrease in the release of calcium from intracellular stores after T-cell receptor stimulation, whereas influx of extracellular calcium seems to be unaffected. FADD DN-expressing fibroblasts show a similarly inhibited cell growth and impaired calcium mobilization indicating that the modulation of proliferation and calcium response by death receptors is not cell type-specific.     

Regulation of protein kinase C inactivation by Fas-associated protein with death domain

Protein kinase C (PKC) plays important roles in diverse cellular processes. PKC has been implicated in regulating Fas-associated protein with death domain (FADD), an important adaptor protein involved in regulating death receptor-mediated apoptosis. FADD also plays an important role in non-apoptosis processes. The functional interaction of PKC and FADD in non-apoptotic processes has not been examined. In this study, we show that FADD is involved in maintaining the phosphorylation of the turn motif and hydrophobic motif in the activated conventional PKC (cPKC). A phosphoryl-mimicking mutation (S191D) in FADD (FADD-D) abolished the function of FADD in the facilitation of the turn motif and hydrophobic motif dephosphorylation of cPKC, suggesting that phosphorylation of Ser-191 negatively regulates FADD. We show that FADD interacts with PP2A, which is a major phosphatase involved in dephosphorylation of activated cPKC and FADD deficiency abolished PP2A mediated dephosphorylation of cPKC. We show that FADD deficiency leads to increased stability and activity of cPKC, which, in turn, promotes cytoskeleton reorganization, cell motility, and chemotaxis. Collectively, these results reveal a novel function of FADD in a non-apoptotic process by modulating cPKC dephosphorylation, stability, and signaling termination.     

The requirements for Fas-associated death domain signaling in mature T cell activation and survival

Fas-associated death domain (FADD) is a death domain containing cytoplasmic adapter molecule required for the induction of apoptosis by death receptors. Paradoxically, FADD also plays a crucial role in the development and proliferation of T cells. Using T cells from mice expressing a dominant negative form of FADD (FADDdd), activation with anti-TCR Ab and costimulation or exogenous cytokines is profoundly diminished. This is also seen in wild-type primary T cells transduced with the same transgene, demonstrating that FADD signaling is required in normally differentiated T cells. The defective proliferation does not appear to be related to the early events associated with TCR stimulation. Rather, with a block in FADD signaling, stimulated T cells exhibit a high rate of cell death corresponding to the initiation of cell division. Although CD4 T cells exhibit a moderate deficiency, this effect is most profound in CD8 T cells. In vivo, the extent of this defective accumulation is most apparent; lymphocytic choriomenigitis virus-infected FADDdd-expressing mice completely fail to mount an Ag-specific response. These results show that, in a highly regulated fashion, FADD, and most likely caspases, can transduce either a signal for survival or one that leads directly to apoptosis and that the balance between these opposing outcomes is crucial to adaptive immunity.     

Emerging Roles for the Death Adaptor FADD in Death Receptor Avidity and Cell Cycle Regulation

The Fas-associated death domain protein FADD is best known as an adaptor protein that senses a signal received at a death receptor and nucleates the assembly of the death-inducing signaling complex. Recent work reveals unexpected properties for this signaling protein, suggesting new roles for FADD in apoptotic signaling and in non-apoptotic functions linked to chemical modification of the FADD C-terminus. These new studies suggest novel types of high valency complexes may form in the plasma membrane and in the nucleus, raising intriguing questions as to how FADD senses the environment and responds to different signaling inputs to promote a biochemical response. In particular, we discuss the role of FADD in death receptor avidity and examine the relationship between FADD phosphorylation and subcellular localization with respect to various biological functions. Since FADD serves to modulate both apoptosis and cell cycle progression, these new findings promote the concept that differential complex assembly dictates disparate cellular processes mediated by this adaptor molecule.     

Hepatocyte Fas-associating death domain protein/mediator of receptor-induced toxicity (FADD/MORT1) levels increase in response to pro-apoptotic stimuli

We examined the regulation of Fas-associating death domain (FADD) protein as an important adaptor molecule in apoptosis signaling and hypothesized that the regulation of FADD could contribute to hepatocyte death. FADD/mediator of receptor-induced toxicity (MORT1) is required for activation of several signaling pathways of cell death. In this study we report the interesting and unexpected result that actinomycin D increased the expression of FADD protein, and we demonstrate that other cellular stresses like ultraviolet irradiation or heat shock could also increase FADD levels in hepatocytes. In cells treated with actinomycin D, FADD levels were elevated homogeneously in the cytoplasm. The increase in cytoplasmic FADD protein by actinomycin D or FADD overexpression alone both correlated with cell death, and specific antisense inhibition of FADD expression consistently diminished approximately 30% of the cell death induced by actinomycin D. These data indicate that FADD protein expression can increase rapidly in hepatocytes exposed to broadly cytotoxic agents.     

Ultraviolet irradiation increases FADD protein in apoptotic human keratinocytes

Ultraviolet irradiation (UV) can induce keratinocyte apoptosis by activating death receptors that recruit the intracellular adaptor molecule FADD/MORT1 (Fas-associating death domain protein/mediator of receptor-induced toxicity). We hypothesized that UV could alter FADD expression levels to augment UV-induced keratinocyte apoptosis. In a dose-dependent manner UV B irradiation increased the expression of FADD protein in a human keratinocyte cell line (CCD-1106) with a corresponding increase in caspase-8 cleavage and cellular apoptosis. FADD overexpression induced cell death in 80% of cells compared with 10% spontaneous cell death in controls. Inhibition of FADD protein by adenoviral expression of anti-sense FADD reduced keratinocyte apoptosis. Regulation of FADD expression by UV may serve to enhance death receptor-mediated keratinocyte death.     

Calmodulin mediates Fas-induced FADD-independent survival signaling in pancreatic cancer cells via activation of Src-extracellular signal-regulated kinase (ERK)

Pancreatic cancer remains a devastating malignancy with a poor prognosis and is largely resistant to current therapies. To understand the resistance of pancreatic tumors to Fas death receptor-induced apoptosis, we investigated the molecular mechanisms of Fas-activated survival signaling in pancreatic cancer cells. We found that knockdown of the Fas-associated protein with death domain (FADD), the adaptor that mediates downstream signaling upon Fas activation, rendered Fas-sensitive MiaPaCa-2 and BxPC-3 pancreatic cells resistant to Fas-induced apoptosis. By contrast, Fas activation promoted the survival of the FADD knockdown MiaPaCa-2 and BxPC-3 cells in a concentration-dependent manner. The pharmacological inhibitor of ERK, PD98059, abrogated Fas-promoted cell survival in FADD knockdown MiaPaCa-2 and BxPC-3 cells. Furthermore, increased phosphorylation of Src was demonstrated to mediate Fas-induced ERK activation and cell survival. Immunoprecipitation of Fas in the FADD knockdown cells identified the presence of increased calmodulin, Src, and phosphorylated Src in the Fas-associated protein complex upon Fas activation. Trifluoperazine, a calmodulin antagonist, inhibited Fas-induced recruitment of calmodulin, Src, and phosphorylated Src. Consistently, trifluoperazine blocked Fas-promoted cell survival. A direct interaction of calmodulin and Src and their binding site were identified with recombinant proteins. These results support an essential role of calmodulin in mediating Fas-induced FADD-independent activation of Src-ERK signaling pathways, which promote survival signaling in pancreatic cancer cells. Understanding the molecular mechanisms responsible for the resistance of pancreatic cells to apoptosis induced by Fas-death receptor signaling may provide molecular insights into designing novel therapies to treat pancreatic tumors.     

A dominant interfering mutant of FADD/MORT1 enhances deletion of autoreactive thymocytes and inhibits proliferation of mature T lymphocytes.

Members of the tumour necrosis factor receptor family that contain a death domain have pleiotropic activities. They induce apoptosis via interaction with intracellular FADD/MORT1 and trigger cell growth or differentiation via TRADD and TRAF molecules. The impact of FADD/MORT1-transduced signals on T lymphocyte development was investigated in transgenic mice expressing a dominant negative mutant protein, FADD-DN. Unexpectedly, FADD-DN enhanced negative selection of self-reactive thymic lymphocytes and inhibited T cell activation by increasing apoptosis. Thus signalling through FADD/MORT1 does not lead exclusively to cell death, but under certain circumstances can promote cell survival and proliferation.     

FADD deficiency impairs early hematopoiesis in the bone marrow

Signal transduction mediated by Fas-associated death domain protein (FADD) represents a paradigm of coregulation of apoptosis and cellular proliferation. During apoptotic signaling induced by death receptors including Fas, FADD is required for the recruitment and activation of caspase 8. In addition, a death receptor-independent function of FADD is essential for embryogenesis. In previous studies, FADD deficiency in embryonic stem cells resulted in a complete lack of B cells and dramatically reduced T cell numbers, as shown by Rag1(-/-) blastocyst complementation assays. However, T-specific FADD-deficient mice contained normal numbers of thymocytes and slightly reduced peripheral T cell numbers, whereas B cell-specific deletion of FADD led to increased peripheral B cell numbers. It remains undetermined what impact an FADD deficiency has on hematopoietic stem cells and progenitors. The current study analyzed the effect of simultaneous deletion of FADD in multiple cell types, including bone marrow cells, by using the IFN-inducible Mx1-cre transgene. The resulting FADD mutant mice did not develop lymphoproliferation diseases, unlike Fas-deficient mice. Instead, a time-dependent depletion of peripheral FADD-deficient lymphocytes was observed. In the bone marrow, a lack of FADD led to a dramatic decrease in the hematopoietic stem cells and progenitor-enriched population. Furthermore, FADD-deficient bone marrow cells were defective in their ability to generate lymphoid, myeloid, and erythroid cells. Thus, the results revealed a temporal requirement for FADD. Although dispensable during lymphopoiesis post lineage commitment, FADD plays a critical role in early hematopoietic stages in the bone marrow.     

FADD adaptor in cancer

FADD (Fas Associated protein with Death Domain) is a key adaptor molecule transmitting the death signal mediated by death receptors. In addition, this multiple functional protein is implicated in survival/proliferation and cell cycle progression. FADD functions are regulated via cellular sublocalization, protein phosphorylation, and inhibitory molecules. In the present review, we focus on the role of the FADD adaptor in cancer. Increasing evidence shows that defects in FADD protein expression are associated with tumor progression both in mice and humans. Better knowledge of the mechanisms leading to regulation of FADD functions will improve understanding of tumor growth and the immune escape mechanisms, and could open a new field for therapeutic interventions.     

A Fas-associated death domain protein/caspase-8-signaling axis promotes S-phase entry and maintains S6 kinase activity in T cells responding to IL-2

Fas-associated death domain protein (FADD) constitutes an essential component of TNFR-induced apoptotic signaling. Paradoxically, FADD has also been shown to be crucial for lymphocyte development and activation. In this study, we report that FADD is necessary for long-term maintenance of S6 kinase (S6K) activity. S6 phosphorylation at serines 240 and 244 was only observed after long-term stimulation of wild-type cells, roughly corresponding to the time before S-phase entry, and was poorly induced in T cells expressing a dominantly interfering form of FADD (FADDdd), viral FLIP, or possessing a deficiency in caspase-8. Defects in S6K1 phosphorylation were also observed. However, defective S6K1 phosphorylation was not a consequence of a wholesale defect in mammalian target of rapamycin function, because 4E-BP1 phosphorylation following T cell activation was unaffected by FADDdd expression. Although cyclin D3 up-regulation and retinoblastoma hypophosphorylation occurred normally in FADDdd T cells, cyclin E expression and cyclin-dependent kinase 2 activation were markedly impaired in FADDdd T cells. These results demonstrate that a FADD/caspase-8-signaling axis promotes T cell cycle progression and sustained S6K activity.