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) recep- tor family act via a common set of sig- naling molecules to regulate cell viabil- ity 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 TNF 2. Several lines of evi- dence 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 inde- pendently 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 destruc- th, 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 vi- ability 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, acti- vation of distinct signaling pathways.

Death induction without protein synthesis The induction of cell death by TNF

can occur in cells whose protein syn- thesis has been fully blocked. This im- plies that the induced death is brought about by molecules that pre-exist in a latent form in the ceil. It also implies that death-triggering receptors can acti- vate this latent machinery through a protein-interaction cascade.

Recent findings indicate that death induction by TNF (and CD95) involves a group of proteases, the caspases (or ICE/CED3 proteases), which also play a central role in other apoptotic pro- cesses (for review, see Ref. 5). These proteases are indeed latent in the living cell and are activated during the death process. The stimulated receptors re- cruk a caspase family member, whose

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 protein- protein 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 prob- ably serves other functions (for review, see Ref. 8). Three of these proteins, MORTI/FADD 9,1°, TRADD n 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 re- spective adapter molecules: the DD of CD120a binds to that of TRADD and the DD in CD95 binds to that in MORT1/ FADD. In addition, the DD of MORT1/FADD can bind to the DD of TRADD, and the DD of RIP to the DDs of both TRADD and MORTI/FADD I~.

These associations be- tween DDs occur as a conse- quence of receptor-ligand binding ~4 and seem to in- volve electrostatic interac- tions. NMR spectroscopy of the DD of CD95 confirms that this region, which comprises a series of antiparalle| amphi- pathic o~=helices, has many exposed charged residues ~5.

Death effector (MORT) domain interactions. MORTI/FADD is recruited to activated CD95 molecules in association with a member of the ICE/ CED3 protease family, cas- pase 8 0VlACH(~/FLICE/ Mch5) 7. It seems likely that the caspase 8-MORT1/FADD complex is also recruited to activated CDI20a mol- ecules, and associates with them indirectly, through the

FRONTLi ES binding of the MORT/FADD DD to the DD of c, . . . . . . . . . . ~ ~,;oa-as~ocmted TRADD. Bind- ing 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 proteo- lyric 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 incom- plete protease region have negative- dominant 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 full- length variant, thus enhancing its re- cruitment to the receptors&

Another recently described caspase, Mch4 (caspase 10), which probably acts similarly to caspase 8, also contains the

TNF

C D 1 2 0 a ~ (p55 TNF receptor) 1

( ~ Protein synthesis- Protein synthesis- dependent independent protective cytotoxic mechanisms mechanisms

\

Protein synthesis blocking agents; viruses

Cell death

Figure 1 Activation by tumor necrosis factor (TNF) of both cyto- toxic and protective mechanisms. Studies of how the cytocidai activity of TNF is affected by agents that block protein synthesis as well as other studies led to the hypothesis that this activity is controlled by a balance between two opposing TNF effects: activation of protein- synthesis-independent cytotoxic mechanisms, and in- duced synthesis of proteins that can block the cyto- toxic mechanisms. The marked sensitization of cells to TNF cytotoxicity by protein-synthesis-blocking agents and certain viruses might reflect suppression of the synthesis or activity of these proteins, t:',us 'blocking the blockage' of the TNF-induced cytotoxic activity.

Copyright © 1997, Elsevier Science Ltd. All rights reserved. 0968-0004/97/$1700 Pil: S0968-0004(97)01015-3 107

CD95 CD120a CD120b (Fas/Apo-1), (p55 TNF receptor) (p75 TNF receptor)

I t i

, TRADD

"',,, "-... - , ' [l......:::::=

(MACH/FLICE) II ."ll U,l

i ,"!I m i Processing of l ] RAIDD [ other caspases I I =' and cleavage of [,] Caspase 2 death r~ (ICH-1) substrates ]] Synthesis of

| [~ protective U proteins

CELL DEATH

~)i ~/I17C/:7: ̧ :~!: i : i i i ~

J

TRAF2

I

. . . . . . ~ NIK "~ . . . . (MAPKKK)

,, I | !

V

NF-KB activation

¢ . ¢ ¢ ¢ ¢

CDw121a (ILI-R[)

c

0RAK, ,TRAF6)

Rgure 2 Schematic illustration of the known proteins and interacting motifs that take part in the induction of cell death and of resistance to it by the TNF receptors (CD120a and CD120b), by Fas/Apo-1 (CD120a) and by the type I interleukin 1 (IL-1) receptor (CDw121a). Cell-death induction occurs by recruitment of caspases (caspase 8, and perhaps also caspase 2) to the receptors through protein-protein bindings that involve homophilic interactions of death domain and caspase prodomain motifs. Cellular resistance to TNF cytotoxicity involves induced synthesis of some protective proteins via the transcription factor NF-KB. Activation of NF-KB involves the adapter proteins TRAF2 and TRAF6 and the serine/threonine protein kinases, NIK and IRAK. Motifs indicated in the figure are: the cysteine- rich extracellular-domain motif that defines the TNF/NGF family (pale green); the immunoglobulin faro- ily motif from the extracellular domain of CDw121a (purple loops); the death domain (red); the death effector domain, or 'MORT' domain (the caspase 8 prodomain motif) (yellow); the ICH-1/caspase 2 prodomain motif (star); the ICE/CED3 protease sequence motif (blue outline); TRAF carboxyl terminus (purple); TRAF amino terminus (light purple); serine/threonine kinase motif (red outline).

DED motif 16, as does a phosphoprotein of unknown function, called PEA15 (Refs 6, 7). The detailed structure of this motif and the way in which it prompts protein-protein imeractions are still unknown.

a~vati~. Studies of other cas- pases have shown that they become activated as a consequence of their proteolytlc processing. This occurs in

108

caspase=substrate sites (downstream of aspartate residues), allowing the pro- teases to become self-activated and to activate one another. The active pro- tease contains two fragments of the protease precursor that associate non- covalently. It is not knm~ whether the activation of caspase 8, foiiowing its recruitment to stimulated CD95 or CDl20a, also depends on its proteolytic

TIBS 22 - APRIL 1997

processing (by its own activ- ity 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 process- ing of various other caspases following the recruitment of caspase 8 (Refs 6, 7). Such processing is probably medi- ated by caspase 8 itself ~7.

An additional possible route for caspase activation by re- ceptors of the TNF/NGF family was recently suggested by the discovery of a new 'death adapter protein', RAIDD. This protein contains a carboxy- terminal 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 re- cruit it to CDI20a through se- quential interactions of RAIDD, RIP, TRADD and CD120a. The contribution of this pathway to CD120a and CD95 cytotoxic- ity 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 agents, they can become vul- nerable to the cytocidal et- fect. This change might de- pend largely on the timing of protein synthesis inhibition: cells exposed to TNF before exposure to protein synthesis inhibitors might not be so sensitive to the cytocidai ef- fect as cells in which protein synthesis was prevented from the first moment of exposure to TNE This dependence on timing implies that TNF itself can induce the synthesis of

proteins that protect cells from its own O'NF's) cytotoxicit~.

A significant advance in understand- ing this self-control mechanism ca~ae with the finding that NF-KB, a transcrip- tion factor activated by TNF, can pro- vide cells with resistance against TNF cytotoxidty. Moreover, blocking the function of NF-KB is shown to result in marked sensitization of cells to the

TIBS 22 ~ APRil. 1997

cytocidal effect of TNF ~9-22. These find- ings confirm that proteins induced by TNF play a major role ~n restricting TNF- induced 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-KB and enhance cellular resistance to TNF cytotoxicity, apparently via NF-KB- induced proteins 23.

The Ng.KB-a©t!vating oasoade. NF-~B con- sists of a homo- or heterodimer of DNA- binding proteins related to the proto- oncogene c-Rel. In most cells, it exists in a latent, cytoplasmicaily localized state, bound to inhibitory proteins (col- lectively called IKB) that mask its nu- clear localization signal. Cytokines that activate NF-KB, such as TNF and IL-I, do so by inducing phosphoryRation of h<B. This phosphorylation targets IKB for degradation by proteasomes (for review, see Ref. 24).

As in the case of death induction by CD120a, initiation of NF-KB activation by this receptor involves TRADD, which is recruited to the receptor by DD-DD interactions. However, further events in the NF-KB-inducing cascade invo!:,e a member of another adapter protek'. ~am- ily, TRAF2, which binds to the region upstream of the DD in TRADD as well as to a region upstream of the DD in RIP, through the motif shared by this family - the TRAF domain 2S,26. A number of re- ceptors of the TNF/NGF family, including the second TNF receptor, CD120b (the p75 TNF receptor), interact directly with TRAF2 and, like CD120a, can stimulate NF-KB by involving this adapter protein (for example, see Ref. 27). In addition, the activation of NF-~B by CDwl21a (the It-1 type I receptor) involves a member of the TRAF family, TRAF6, which inter- acts with the receptor through an inter- mediate protein, IRAK 2s.

A recently described serine/threonine protein kinase, NIK, which binds to TRAF2, activates NF-KB and seems to be a necessary component of an NF-KB- activating cascade common to TNF and IL-I. NIK shows high sequence homology to several kinases that act within mitogeu- activated protein (MAP) kinase cascades, specifically to those that act as MAP kinase klnase kinases (MAPKKKs), and might function similarly within a novel MAP kinase cascade. Overproduction of NIK in cells results in a marked increase of their resistance to TNF cytotoxicity,

FRONTLINES whereas synthesis of a ki- nase-deficient mutant in- creases their sensitivity z~.

The questions ahead Maior advances have been

~ade in the past year to- wards elucidation of mecha- nisms 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 signal- ing activRies are involved in the cytocidal effect arid in cellular resistance to it? How is the balance between sig- naling for the opposing effects regu- lated? How do different members of the receptor family differ in their triggering of these effects? With regard to distal events in these cascades, the gaps in our knowledge are even greater. A few of the substrate proteins cleaved by the activated caspases have been identi- fied, as have several proteins whose induction by TNF provides some resist- ance to its cytocidal activity (e.g. the mitochondrially localized manganese superoxide dismutase). The main death substrates and protective proteins, how- ever, may still have to be found. There is also a long way to go before the role of each of the proteins within the apop- totic program is fully clarified. At least some of the distal events in death induc- tion by receptors of the TNF/NGF family are shared with other apoptotic pro- cesses. Further exploration of these dis- tal events is therefore likely to broaden our general understanding of ceil death 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., Switzer- land; and from the Israeli Ministry of Arts and Sciences.

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Apoptosis cartoon reproduced with k!nd permission from Paolo Ruggiero, Domp(~ Research Centre, L'Aquila, Italy.

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DAVID WALLACH

Department of Membrane Research and Biophysics, The Weizmann Institute of Science, Rehovot, 76100, Israel.

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