5 Adaptor Proteins in Death Receptor Signaling

Chapter 5 / Death Signal Adaptors 93 5 Adaptor Proteins in Death Receptor Signaling Nien-Jung Chen, PhD and Wen-Chen Yeh, MD, PhD SUMMARY Signal transduction induced by death receptors belonging to the tumor necrosis factor receptor (TNFR) superfamily has been an area of intensive research for the past several years. The major advances arising from these studies have been the characterization of critical signal-transducing adaptor molecules and the delineation of parallel but opposing signaling pathways, some inducing apoptosis and others promoting cell survival. An imbalance in favor of either apoptosis or cell survival can have disastrous pathological consequences, including cancer, autoimmunity, or immune deficiency. Many adaptor proteins have been reported in the literature to be involved in death receptor signaling. In this chapter, we will focus on molecules whose functions have been investigated by multiple approaches, particularly gene targeting in mice and ex vivo biochemical studies. By validating or clarifying the function of each adaptor, we hope to construct a blueprint of the various signaling channels triggered by death receptors, providing a foundation for further scientific investigations and practical therapeutic designs. INTRODUCTION Cancer biologists and oncologists have struggled for years to devise ways of eradicat- ing cancer cells while sparing normal ones. One breakthrough that has emerged during the past decade has been the investigation of the molecular mechanisms of apoptosis (1). Apoptosis is a critical physiological process that is subject to intricate regulation. Indeed, many cancers arise from the dysregulation of apoptotic or antiapoptotic signals, and such dysregulation is often attributable to mutation or altered expression of specific molecules (1,2). The elucidation of the nature of the individual signaling proteins in pathways leading to apoptosis or antiapoptosis has become a central issue in cancer biology as well as in tissue development and immune system regulation. From: Cancer Drug Discovery and Development: Death Receptors in Cancer Therapy Edited by: W. S. El-Deiry © Humana Press Inc., Totowa, NJ 93 94 Chen and Yeh Some of the most important apoptotic signaling pathways are those induced by engagement of the death receptors (DRs). The death receptors are a subset of ligand- specific cell-surface receptors belonging to the TNFR superfamily, and are characterized by the presence of a motif called the death domain (DD) in their cytoplasmic tails (3). It is these death domains that confer the ability to induce apoptosis on the death receptors. Apoptosis is triggered when the death receptors are engaged by specific death factors, ligands such as Fas ligand (FasL) (4), TNF (5,6), TNF-like molecule 1A (TL1A) (7), and TNF-related apoptosis-inducing ligand (TRAIL) (8). The death factors are not only toxic to many transformed cell types in vitro but also play important roles in the regulation of immune responses. The physiological and clinical relevance of the death receptor family thus makes its study very compelling. Over the past 8 yr, the apoptosis signaling pathways induced by stimulation of various death receptors, including TNFR1 (9,10), Fas (CD95) (11,12), the TRAIL receptors (DR4 and DR5) (13–16), DR3 (17), and DR6 (18), have come under intense investigation. There are two basic models of death receptor-induced apoptosis signaling cascades: one exemplified by the engagement of Fas and the other by the engagement of TNFR1 (Fig. 1). In the first two sections of this chapter, we will discuss the adaptor proteins that are involved in Fas and TNFR1 signaling. We start with an overview of each model system and move to a detailed description of validated and putative functions of selected adaptors. A special emphasis will be placed on information gained from studies of gene- targeted “knockout mice.” In the third section, we will discuss signaling pathways triggered by other death receptors that share features with our model systems but also contain some unique adaptor proteins. In the final section, we will discuss perspectives on ques- tions in death receptor signaling that remain to be answered and on the knowledge that can potentially be garnered from studies of new adaptor proteins. STIMULATION OF FAS TRIGGERS A “SUPERHIGHWAY” APOPTOTIC SIGNAL Engagement of Fas triggers a swift and efficient apoptotic signal. The first event following the binding of the death factor FasL to Fas is the direct recruitment of Fas- associated death domain protein (FADD) (19,20) to the cytoplasmic tail of Fas. As we shall see in the following sections, FADD is the common adaptor protein upon which almost all death receptor signaling pathways converge (21) (Fig. 1). FADD binds to Fas through the interaction of their homologous death domains, an event that unmasks the N-terminal death-effector domain (DED) of FADD. The DED allows FADD to then recruit caspase-8 (also called FLICE) (22,23) to the growing complex of proteins, which is now called the “death-inducing signaling complex” (DISC) (24). The interaction of the DISC with caspase-8 activates the latter, possibly by auto-proteolytic processing (25), and activated caspase-8 in turn triggers the caspase cascade. Caspase-8 either directly activates execution caspases (26), or cleaves Bid (BH3 interacting domain death agonist) which leads to activation of the mitochondrial apoptotic pathway (27). The involvement of FLICE-associated huge protein (FLASH) (28), a protein that binds to and activates caspase-8, will be discussed in another chapter of this book. One of the controls of apoptotic signaling takes effect at the level of caspase-8. Recruitment of caspase-8 to the DISC can be inhibited by cellular FLICE-inhibitory protein (c-FLIP) (29), a protein that plays a crucial role in keeping Fas-mediated apoptosis in check. Chapter 5 / Death Signal Adaptors 95 Fig. 1. Signaling pathways modulated by Fas (left) and TNFR1 (right). Several other molecules have been implicated in minor pathways of death receptor- mediated apoptosis. It has recently been proposed that an alternative apoptotic pathway can be triggered by Fas via direct recruitment of death domain-associated protein (DAXX) (30). DAXX activates apoptosis signal-regulated kinase 1 (ASK1) which in turn activates the downstream c-Jun N-terminal kinase (JNK) pathway (31). Others have reported that Fas engagement may trigger necrosis in a process that requires the recruitment of receptor-interacting protein ( RIP) (32) and FADD (33). Finally, association of RIP with RIP-associated ICH-1/CED-3-homologous protein (RAIDD) (34,35) followed by recruitment of caspase-2 has been implicated in death receptor-induced apoptotic signaling. In the following sub-sections, we will discuss most of the adaptor proteins mentioned above with the exception of RIP (which will be discussed in the section headed “Signaling by TNFR1 Triggers Both Apoptotic and Antiapoptotic Pathways”). FADD The majority of genetic and biochemical studies addressing FADD function have provided evidence that this molecule is not only essential for Fas-mediated apoptosis but also plays a key role in almost all death receptor-induced apoptosis (19–21). In addition, FADD is required for a recently described pathway of T-cell necrosis that is mediated by 96 Chen and Yeh Fas or TRAIL but is independent of caspase-8 (33). Paradoxically, FADD is also required for embryonic cell survival, particularly at the stage of heart ventricular development (36). The precise function of FADD in embryogenesis remains to be determined. At the cellular level, FADD-deficient T-cells exhibit a defect in T-cell receptor (TCR)-mediated proliferation and deregulation of the cell-cycle machinery (37,38). The involvement of FADD in heart development and T-cell proliferation implies functions for this molecule in addition to its role as a common proapoptotic adaptor for death receptor signaling, and further suggests that death receptor functions may extend beyond inducing cell death. Caspase-8 and c-FLIP Caspase-8 is the key initiator caspase acting downstream of FADD during apoptosis induced by Fas and other death receptors (22,23,39). Not surprisingly, caspase-8-deficient cells are highly resistant to Fas- and death receptor-mediated apoptosis. Interest- ingly, caspase-8-knockout mice die during embryogenesis and exhibit a heart defect similar to that observed in FADD-deficient embryos (40). Caspase-8-deficient T-cells also show defects in TCR-mediated proliferation. However, unlike FADD-deficient T-cells, caspase-8-deficient T-cells have a normal cell cycle. Curiously, caspase-8-deficient T-cells stimulated via their TCRs fail to expand due to a paradoxical increase in cell death (41). The function of c-FLIP as an inhibitor of caspase-8 recruitment (29) led researchers to assume that c-FLIP-deficient mice would exhibit phenotypes opposite to those of FADD- or caspase-8-knockout mice. Indeed, cells lacking c-FLIP become highly sensi- tive to apoptosis induced by FasL as well as by other death factors such as TNF and TRAIL (V. Wong and W-C. Yeh, unpublished results). However, embryos lacking c-FLIP unexpectedly show a defect in heart development analogous to that in FADD- or caspase-8-knockouts (42). The mystery is deepened by the observation that the devel- oping heart tissues of FADD- or c-FLIP-deficient embryos show normal apoptosis in vivo. With respect to T lymphocytes, c-FLIP may play a role in responses to TCR engagement, since T-cells overexpressing c-FLIP show enhanced proliferation in response to TCR stimulation (43). Taken together, these results imply that FADD, caspase-8 and c-FLIP function in the cytoplasm as a block and are involved in signaling pathways in addition to death receptor-mediated apoptosis. These interactions may be cooperative or antagonistic in nature, and may depend on other players present in each unique signaling context. Bid Bid is a proapoptotic Bcl-2 family member that is recruited and cleaved by caspase-8 (44).

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