Cell death induction by TNF: a matter of self control

TIBS 22 - APRIL 1997

Ceil death iad ction by TNF: a matter o ,df contro Members of the tumor necrosis factor (TNF)/nerve growth factor (NGF) receptor family act via a common set of signaling molecules to regulate cell viability and differentiation and, as has been known for 30 years, cell death. Two of the best-studied receptors in this family are CD95 (Fas/Apol), which plays a major role in T-cell-mediated toxicity ~, and the p55 TNF receptor, CD120a, which binds the cytokine TNF2. Several lines of evidence suggest that the killing of cells by TNF is regulated by two opposing kinds of TNF activities: (1) activation of some cytotoxic mechanisms that occur independently of protein synthesis, and (2) stimulation of mechanisms that protect cells from death, and that depend on protein synthesis. It is suggested that this balance between induced destructh,e and protective effects might account for the observed ability of TNF to act selectively, destroying both diseased (virus-infected and transformed) cells and cells treated with protein synthesis inhibitors, while hardly affecting the viability of normal cells (see Refs 3, 4 and lit. cit. therein; Fig. 1). Recent findings on the signaling mechanisms of the TNF/NGF-receptor family confirm tlus hypothetical model, and suggest that these two antagonistic functions reflect, at least in part, activation of distinct signaling pathways.

FRONTLi ES binding of the MORT/FADD DD to the DD of ~c,. . ~,;oa-as~ocmted ........ TRADD. Binding of MORT1/FADD to caspase 8 involves a shared sequence motif, the 'death effector domain' (DED) or 'MORT domain', found in the region upstream of the DD in MORT!, e~d found in duplicate in the 'prodomain' (the region upstream of the proteolyric moiety) of caspase 8. Caspase 8 exists in multiple splice variants that share the DED motif, but differ in their carboxy-terminal regions. In addition to binding to MORT1/FADD, the different variants also self-associate and bind to each other through their DEDs. Variants containing an incomplete protease region have negativedominant effects on the function of the full-length protease. Variants that lack this protease region altogether, however, can augment cytotoxicity by virtue of their ability to interact with the fulllength variant, thus enhancing its recruitment to the receptors& Another recently described caspase, Mch4 (caspase 10), which probably acts similarly to caspase 8, also contains the

function (the protease is apparently activated following its recruitment) is required for death induction °,7 via a series of protein-protein interactions, and discussed below (Fig. 2). Death domain interactions. The death domain (DD) is a conserved proteinprotein interaction sequence motif of about 90amino acids, initially noticed in the intracellular domains of CD120a and CD95. Its presence is necessary and sufficient for death induction by these receptors. Th~ motif is also found in a variety of other proteins, where it probably serves other functions (for review, see Ref. 8). Three of these proteins, MORTI/FADD9,1°, TRADDn and RIP~2, act as adapter proteins in both the CD120a and the CD95 death-inducing cascades. The DDs of CD120a and CD95 can self-associate and also bind to the DDs in their reTNF spective adapter molecules: the DD of CD120a binds to CD120a~ that of TRADD and the DD in (p55 TNF receptor) 1 CD95 binds to that in MORT1/ FADD. In addition, the DD of MORT1/FADD can bind to ( ~ Protein synthesisthe DD of TRADD, and the Protein synthesisdependent DD of RIP to the DDs of both independent protective TRADD and MORTI/FADDI~. cytotoxic mechanisms mechanisms These associations be\ tween DDs occur as a consequence of receptor-ligand binding ~4 and seem to inProtein synthesis Death induction without protein synthesis volve electrostatic interacblocking agents; The induction of cell death by TNF tions. NMR spectroscopy of viruses can occur in cells whose protein syn- the DD of CD95 confirms that thesis has been fully blocked. This im- this region, which comprises plies that the induced death is brought a series of antiparalle| amphiCell death about by molecules that pre-exist in a pathic o~=helices, has many latent form in the ceil. It also implies exposed charged residues ~5. Death effector (MORT) domain that death-triggering receptors can actiFigure 1 vate this latent machinery through a interactions. MORTI/FADD is Activation by tumor necrosis factor (TNF) of both cytorecruited to activated CD95 protein-interaction cascade. toxic and protective mechanisms. Studies of how the Recent findings indicate that death molecules in association cytocidai activity of TNF is affected by agents that block protein synthesis as well as other studies led to the induction by TNF (and CD95) involves a with a member of the ICE/ hypothesis that this activity is controlled by a balance group of proteases, the caspases (or CED3 protease family, casICE/CED3 proteases), which also play a pase 8 0VlACH(~/FLICE/ between two opposingTNFeffects: activation of proteinsynthesis-independentcytotoxic mechanisms, and incentral role in other apoptotic pro- Mch5) 7. It seems likely that duced synthesis of proteins that can block the cytocesses (for review, see Ref. 5). These the caspase 8-MORT1/FADD toxic mechanisms. The marked sensitization of cells proteases are indeed latent in the living complex is also recruited to TNF cytotoxicityby protein-synthesis-blockingagents cell and are activated during the death to activated CDI20a moland certain viruses might reflect suppression of the synthesis or activity of these proteins, t:',us 'blocking process. The stimulated receptors re- ecules, and associates with the blockage' of the TNF-inducedcytotoxic activity. cruk a caspase family member, whose them indirectly, through the 107 Copyright© 1997,ElsevierScienceLtd.Allrightsreserved.0968-0004/97/$1700 Pil:S0968-0004(97)01015-3

TIBS 22 - APRIL 1997

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processing (by its own activity or through effects of other proteases), or occurs merely by interdigitation of several protease molecules upon the binding of their prodomains to MORTi/FADD. There is clear evidence, though, Ior processing of various other caspases following the recruitment of caspase 8 (Refs 6, 7). Such processing is probably mediated by caspase 8 itself ~7. An additional possible route for caspase activation by receptors of the TNF/NGF family was recently suggested by the discovery of a new 'death adapter protein', RAIDD. This protein contains a carboxyterminal DD that binds to the DI) of RIP and also contains an amino-terminal sequence homologous to that of the pmdom~in of ICH-1 (caspase 2). It can bind ICH-1 and recruit it to CDI20a through sequential interactions of RAIDD, RIP, TRADD and CD120a. The contribution of this pathway to CD120a and CD95 cytotoxicity is still unknown ~. A protein synthesis-dependent protective cascade if cells that are normally

resistant to killing by either CDl20a or CD95 are exposed to protein synthesis blocking DEATH agents, they can become vulRgure 2 nerable to the cytocidal etSchematic illustration of the known proteins and interacting motifs that take part in the induction of fect. This change might decell death and of resistance to it by the TNF receptors (CD120a and CD120b), by Fas/Apo-1 pend largely on the timing of (CD120a) and by the type I interleukin 1 (IL-1) receptor (CDw121a). Cell-death induction occurs by protein synthesis inhibition: recruitment of caspases (caspase 8, and perhaps also caspase 2) to the receptors through cells exposed to TNF before protein-protein bindings that involve homophilic interactions of death domain and caspase prodomain exposure to protein synthesis motifs. Cellular resistance to TNF cytotoxicity involves induced synthesis of some protective proteins via inhibitors might not be so the transcription factor NF-KB.Activation of NF-KBinvolves the adapter proteins TRAF2 and TRAF6and the serine/threonine protein kinases, NIK and IRAK. Motifs indicated in the figure are: the cysteinesensitive to the cytocidai efrich extracellular-domain motif that defines the TNF/NGF family (pale green); the immunoglobulin farofect as cells in which protein ily motif from the extracellular domain of CDw121a (purple loops); the death domain (red); the death synthesis was prevented from effector domain, or 'MORT' domain (the caspase 8 prodomain motif) (yellow); the ICH-1/caspase 2 the first moment of exposure prodomainmotif (star); the ICE/CED3 protease sequence motif (blue outline); TRAFcarboxylterminus to TNE This dependence on (purple);TRAFamino terminus (light purple); serine/threonine kinase motif (red outline). timing implies that TNF itself can induce the synthesis of DED motif 16, as does a phosphoprotein caspase=substrate sites (downstream of proteins that protect cells from its own of unknown function, called PEA15 aspartate residues), allowing the pro- O'NF's) cytotoxicit~. (Refs 6, 7). The detailed structure of teases to become self-activated and to A significant advance in understandthis motif and the way in which it activate one another. The active pro- ing this self-control mechanism ca~ae prompts protein-protein imeractions tease contains two fragments of the with the finding that NF-KB, a transcripare still unknown. protease precursor that associate non- tion factor activated by TNF, can proa~vati~. Studies of other cas- covalently. It is not k n m ~ whether vide cells with resistance against TNF pases have shown that they become the activation of caspase 8, foiiowing cytotoxidty. Moreover, blocking the activated as a consequence of their its recruitment to stimulated CD95 or function of NF-KB is shown to result in proteolytlc processing. This occurs in CDl20a, also depends on its proteolytic marked sensitization of cells to the

CELL

108

FRONTLINES

TIBS 22 ~ APRil. 1997

cytocidal effect of TNF~9-22. These findings confirm that proteins induced by TNF play a major role ~n restricting TNFinduced death. They also point to NF-KB activation by TNF as a major route of induction of these protective proteins. It is notable that inter]eukin l 0L-l), a cytoldne that shares many activities wRh TNF, even though it binds to a distinct receptor, and compounds that stimulate protein kinase C, also activate NF-KBand enhance cellular resistance to TNF cytotoxicity, apparently via NF-KBinduced proteins 23. The Ng.KB-a©t!vatingoasoade. NF-~B consists of a homo- or heterodimer of DNAbinding proteins related to the protooncogene c-Rel. In most cells, it exists in a latent, cytoplasmicaily localized state, bound to inhibitory proteins (collectively called IKB) that mask its nuclear localization signal. Cytokines that activate NF-KB,such as TNF and IL-I, do so by inducing phosphoryRation of h
whereas synthesis of a kinase-deficient mutant increases their sensitivity z~. The questions ahead Maior advances have been ~ade in the past year towards elucidation of mechanisms that participate in the initiation and control of death by the TNF]NGF receptor famil>: Nevertheless, many questions have yet to be answered. What other signalApoptosis cartoon reproduced with k!nd permission ing activRies are involved in from Paolo Ruggiero, Domp(~ Research Centre, the cytocidal effect arid in L'Aquila, Italy. cellular resistance to it? How is the balance between sig5 Kumar, S. (1995) Trends Biochem. Sci. 20, naling for the opposing effects regu198-202 lated? How do different members of the 6 Boldin, M. P., Goncharov, T. M., receptor family differ in their triggering Goltsev, Y. V. and Waliach, D. (1996) Cell 85, of these effects? With regard to distal 803--815 7 Muzio, M. et al. (1996) Ceil 85, 817-827 events in these cascades, the gaps in 8 Feinstein, E. et al. (1995) Trends Biochem. Sci. our knowledge are even greater. A few 29, 342-344 of the substrate proteins cleaved by the 9 Boldin, M. P. et aL (1995) J. BioL Chem. 270, activated caspases have been identi7795-7798 fied, as have several proteins whose 10 Chinna!yan,A. M., O'Rourke, K., Tewari, M. and Dixit, V. M. (1995) Cell 81, 505-512 induction by TNF provides some resist11 Hsu, H., Xiong, J. and Goeddei, D. V. (1995) ance to its cytocidal activity (e.g. the Cell 81, 495-504 mitochondrially localized manganese 12 5tanger, B. Z. et al. (1995) Cell 81, 513-523 superoxide dismutase). The main death 13 Varfolomeev, E. E., Boidin, M. P., Goncharov, T. M. and Wallach, D. (1996) substrates and protective proteins, howJ. Exp. Med. 183, 1-5 ever, may still have to be found. There 14 Kischkel, F. C, et aL (1995) EMBOJ. 14, is also a long way to go before the role 5579-5588 of each of the proteins within the apop- 15 Huang, B. et aL (1996) Nature 384, totic program is fully clarified. At least 16 638-641 Fernandes-Alnemfi,T. et al. (1996) Proc. Natl. some of the distal events in death inducAcad. Sci. U. S. A. 93, 7464-7469 tion by receptors of the TNF/NGF family 17 Sdnivasula, S. M. et al. (1996) Proc. Natl. Acad. Sci. U. 5. A. 93, 14486-14491 are shared with other apoptotic pro18 Duan, H. and Dixit, V. M. (1997) Nature 385, cesses. Further exploration of these dis86-89 tal events is therefore likely to broaden 19 Beg, A. A. and Baltimore, D. (1996) Science 274, 782-784 our general understanding of ceil death 20 Wang, C-Y., Mayo, M. W. and Baldwin, A. S., Jr and its regulation.

A©knowledgements The author wishes to thank M. Boldin, N. Malinin, T. Goncharov, E. Varfolomeev, A. Kovalenko, Y. Goltsev and I. Mett for their contribution. Research in the author's laboratory was supported by grants from inter-Lab Ltd, Ness Ziona, israel; from Ares Trading S.A., Switzerland; and from the Israeli Ministry of Arts and Sciences.

References 1 Nagata, S. and Golstein, P. (1995) Science 267, 1449-1456 2 Vandenabeele,P., Declercq, W., Beyaert, R. and Rers, W. (1995) Trends Cell Biol. 5, 392-400 3 Hahn, T. et aL (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3814-3818 4 Nophar, Y., Holtmann, H., 8er, R. and Wallach, D. (1988) J. Immunol. 140, 3456-3460

(1996) Science 274, 784-787 21 Van Antwerp, D. J. et aL (1996) Science 274, 787-789 22 Liu, Z-G., Hsu, H., Goeddel, D. V. and Karin, M. (1996) Cell 87, 565-576 23 Holtmann, H. and Wallach, D. (1987) J. Immunot 139, 1161-1167 24 Miyamoto, S. and Verma, I. M. (1995) Adv. Cancer Res. 66, 255-292 25 Hsu, H., Shu, H-B., Pan, M-G. and Goeddel, D. V. (1996) Cell 84, 299-308 26 Takeuchi, M., Rothe, M. and Goeddel, D. V. (1996) J. Biol. Chem. 271, 19935-19942 27 Rothe, M., Sarma, V., Dixit, V. M. and Goeddel, D. V. (1995) Science 269, 1424-1427 28 Cao, Z. etal. (1996) Nature 383, 443-446 29 Malinin, N. L., Boldin, M. P., Kovalenko, A. V. and Wallach, D. Nature (in press)

DAVID WALLACH Department of MembraneResearchand Biophysics, The Weizmann Institute of Science, Rehovot,76100, Israel.

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