[CANCER RESEARCH 57, 615-619, February 15, 19971

Advances in Brief

CRADD, a Novel Human ApoptoticAdaptor Moleculefor Caspase-2,and FasL/Tumor Necrosis Factor Receptor-interacting Protein RIP'

Manzoor Ahmad, Srinivasa M. Srinivasula, Lijuan Wang, Robert V. Talanian, Gerald Litwack,Teresa Fernandes-Alnemri, and Emad S. Alnemri2

Centerfor ApoptosisResearchand the Kimmel Cancer Institute,JeffersonMedical College. Philadelphia, Pennsylvania19107 (M. A., S. M. S.. L W.. G. L, T F.A.. E. S.Al, andBASFBioresearch Corporation, Worcester, Massachusetts01605 [R. V. 1'.]

Abstract

FADD/MORT1 is a death domain (DD)-containing adapter/signalingmoleculethat interactswith the intracellularDD of FAS/APO-I (CD95)and tumor necrosisfactor receptor1 and the prodomainof caspase-8(MchS/MACH/FLICE). FADD engagementof caspase-8presumably activates this caspaseand leadsto apoptosi&Another DD-containing adaptsr/signaling molecule,CRADD, wasIdentified and wasshown to induceapoptosls CRADD has a dual-domain structure similar to that of FADD.It has an NH2-terminal caspase homology domain that interacts wIthcaspase-2and a COOH-terminal DD that interacts with RIP. CRADD Isconstitutively expressed in many tissues and thus could play a role inregulating apoptosis in mammalian cells.

Introduction

The cell surfacereceptorFAS/APO-l (CD95) belongsto a familyof transmembrane cytokine receptors that includes TNFR-l ,@TNFR-2, and the nerve growth factor receptor (for reviews, seeRefs.1 and 2). FAS/APO-I and TNFR-l trigger apoptosis in mammaliancells after binding to their respective natural ligands or specificagonistantibodies(3—5).These two receptorsshare significant homology, especially in their COOH-terminal cytoplasmic domain,which is also called the DD (6, 7). Recent evidence suggeststhatreceptor-ligand interaction causesreceptor aggregation and oligomerization of the DD (8—10).The apoptotic signal is thought to betransducedthroughinteractionof the oligomerizedreceptorDD witha set of cytoplasmicadaptor/signalingmolecules(9, 11—16).Theseadaptors couple the receptors to the upstream signal-transducingcaspases(17—20).Several candidate adaptor/signaling molecules thatinteract with the DD of FAS and TNFR-l have been identified,including FADDIMORT1, TRADD, and RIP (1 1—13,15). Significantly, all of these proteins have a dual-domain structure (an NH2-terminal domain and a COOH-terminal DD analogous to that of FASand TNFR-1; Ref. 21). Interestingly, the FADD NFI2-terminal domain, which is also called the DED, is related to the NH2-terminal

prodomain of the signal-transducing caspases-8 and -10 (Mch5IMACH/FLICE and Mch4; Refs. 17—20).Association of FADD DEDwith procaspase-8 is thought to be responsible for triggering theactivation of this proteaseby an as yet unknown mechanism (17—19).

Received1212196;accepted1/9/97.Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpage

charges.This article mustthereforebe herebymarkedadvertisementin accordancewith18 U.S.C. Section 1734 solely to indicate this fact.

I Suppoiled by Research Grants AG 13487 and Al 35035 from the NIH and in part byaresearchgrantfromIDUNPharmaceuticals.M.A. andS.M.S.contributedequallytothis work.

2 To whom requests for reprints should be addressed, at Kimmel Cancer Institute,

JeffersonMedical College, Bluemle Life SciencesBuilding, 233 South 10th Street,Philadelphia, PA 19107. Phone: (215) 503-4632; Fax: (215) 923-1098; E-mail:EAlnemti@lac.jci.tju.edu.

3 The abbreviations used are: TNFR, tumor necrosis factor receptor; DED, death

effectordomain;CD, caspasedomain;DD, deathdomain;GST,glutathione-S-transferase;EST,expressedsequencetag.

Once caspase-8is activated, it can potentially activate the remainingdownstream executioner caspases(CPP32, Mch2, and Mch3) througha protease amplification cascade(19, 22).

In this paper, we report the identification and molecular cloning ofan adaptor/signalingmolecule structurally related to FADD. ItsCOOH-terminal domain is significantly related to the COOH-terminalDD of FADD, TNFR-l, and RIP. However, its NH2-terminal domainis significantly related to the prodomain of human caspase-2(ICH-l;Ref. 23) and caspase-9 (mch6IICE-LAP6; Refs. 24 and 25) and to theprodomain of the nematode caspase CED-3 (26). This protein caninteractwith caspase-2andRIP and is thusnamedCRADD4 (caspaseand RIP adaptor with death domain). CRADD could play a role in theFASITNFR apoptoticpathway by recruitingcaspase-2to this pathway.

Materials and Methods

cDNA Cloning.CRADD wasclonedfroma humanJurkatUni-ZAPXRcDNA library (27)throughaPCRapproach.Briefly, theprimaryamplificationwas done using vector-specific (T3) primer and a primer (GCCACAGTTCAAGTCCTC) derivedfrom the 3' sequenceof humanEST clone 24032(GenBankaccessionnumberr37937).The primary PCRproductswere thensubjectedto a secondaryamplificationwith primers(ATGGAGCCAGAGACAAACandACCFGCCTGTAATGTGATG)derivedfromthe5' sequenceofhuman EST clone 24032 (GenBank accession number t78285). The PCRproduct was cloned into the SmaI site of pBluescript II KS + vector and thensequenced.The openreadingframeof CRADD wassubclonedin-frameintoBamHI/XhoI-cut pET2I(b), and a T7-tagged CRADD was generated from thepET-CRADD plasmid by PCR and subcloned into the mammalian expressionvectorpcDNA-3 at the EcoRVsite.

TheDD of RIPwasalsoclonedfrom thehumanJurkatUni-ZAP XR eDNAlibrary using a PCR-based approach. The primary amplification was doneusing vector-specific (T3) primer and a primer (CTGAATGCFCTGAGGCAG)derivedfromthe3' nontranslatedsequenceof humanRIP (GenBankaccessionnumberu25994).The final PCRproductwasobtainedusingnestedprimers (GATAATACCACTAGTCTG and UAG11'CTGGCTGACGTAAATC) derivedfrom the openreadingframeof humanRIP sequence.ThePCRproductwasclonedinto theSmaIsiteof pBluescnptII KS+ vectorandthe bluntedBamHI siteof pET28(c).

NorthernBlot Analysis.Tissuedistributionanalysisof CRADD mRNAwasperformedon Northernblots preparedby Clontechcontaining2 @gñanepolyadenylatedRNA. A radioactiveCRADD eDNA probewaspreparedusingthe random-primed DNA-labeling method in the presence of [a-32P]dCTP.The blots were hybridized, washed, and then visualized by autoradiography.

GST Fusion PrOteinsand in Vitro Binding Assay. The open readingframeof CRADDwassubclonedinto thebacterialexpressionvectorpGEX-2Tin-framewith an NH2-terminalGST tag.To produceGST-CRADDDD andGST-CRADDCD, CRADD eDNA wasdigestedwith BamHI,which cleavesbetweenthe CD and DD coding regions,and the two fragmentswere subcloned in-frame into pGEX-2T vector. The GST fusion proteinswere cxpressed in DH5a bacteria and then immobilized on glutathione-Sepharose.

4 The sequence reported in this paper has been deposited in the GenBank database

(accession number U84388).

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1 MEARDKOVLRSLRLELGAEV LVEGLVLOYLY@EGILTENH IQEINAOTTG

51 LRKTMLLLDI LPSRGPXAFD TILDSLQEFP WVREKLKKAREEMITDLPAG

101 DRLTGIPSHI LNSSPSDRQI NQLAOKLGPEWEPMVLSLGLSQTDIYR@KA

151 NHPHNVQSQVVEAIrIRWRQRTGKQ@TFQSLBNGLRAVEVDPSLLLBMLE

CLONING OF AN ADAFFOR MOLECULE FOR CASPASE-2AND RIP

A

B @ø CD

Fig. I. Amino acid sequenceof human CRADDand its homology to other proteins.A, predictedamino acid sequenceof human CRADD. The NH2-terminalCD homologyregionandthe COOH-termimi DD homologyregionaresingle-anddoubleunderlined, respectively. B, a schematic illustratingthedual-domainstructureof CRADD. TheCD domainof CRADDisequivalentto theDEDdomainof FADD. C,colinearalignmentof theCRADD CDregion with the prodomains of caspase-2.caspase-9,and CED-3 and the COOH terminus of humaninhibitor of apoptosis IAP-l. Asterisks, the criticalL27 and G65 of CED-3 that are conserved inCRADD andthe otherproteins.D, colinearalignmentoftheDDofCRADDwiththeDDsofFADD,TNFR-l, andRIP.Amino acidsthatareidenticalinat least three of the sequencesare shaded.Theaminoacid residuesarenumberedto the left.

*

*

C@RADD

caapase-2caapase-9

cED—3hIAP—1

@RADDcaspase-2ca.spa.se-9

cED—3hIAP—i.

D@RADD

hFADDTNFR—1

RIP

@DD

TNFR—1RIP

Labeled interacting proteins were prepared by in vitro transcription and translation in the presenceof [35S]methionineusingPromega'sT7-couplediranscription/translation TNT kit according to the manufacturer's recommendations.After translation,equalamountsof thetranslationreactionsweredilutedto 100 @lin GST binding buffer [50 mi@iIris-Cl (pH 7.6), 120 msi NaCI, and0.5%Brij supplementedwith proteaseinhibitors]andallowedto incubatewiththevariousGSTfusionproteinson ice for 2—3h. Thebeadswerewashed3—4timeswith thesamebufferandboiledin SDSsamplebuffer.Theproteinswerethenresolvedon a 10%SDSgel andvisualizedby autoradiography.

Transfections and Histochemical Detection. Humanembryonickidney293 cells and MCF-7 cells were transiently transfected using the Lipo

fectAMINE method.Approximately2 X l0@'cellsweretransfectedwith 0.25@.tgof thepcDNA3.lIHisIlacZ vector(Invitrogen)and1 @gof CRADD eDNA

cloned in pcDNA3, using LipofectAMlNE (Life Technologies) according tothe manufacturer's recommendations. After 36—40h, expression of the lacZgeneproduct in cells was detectedusing a f3-galactosidasestainingkit (Invitrogen).Cellswerevisualizedunderthemicroscope,andbluecells (healthyandapoptotic)werecounted.Thepercentageof deadcellswasexpressedasthemeanpercentageof blue apoptoticcells asa fraction of the total numberofblue cells counted.

Cleavage Assays. Baculovirus p35 and human caspaseswere 35S-labeledin vitro using Promega's TNT kit as described above. Two pi of the translation

reactions were incubated with purified recombinant caspase-2(100 ng) in ICEbuffer [25 mMHEPES,I mtviEDTA, 5 mMDIT, and0.1%3-[(3-cholamido

propyl)dimethylamino]-l-propanesulfonate(pH 7.5)] in a final volumeof 10s.d.The reactionwas incubatedat 37°Cfor I h and thenanalyzedby TricineSDS-PAGEandautoradiography.

Results and Discussion

Identification and Cloning of Human CRADD. Recent evidencesuggests that the prodomain of caspasescould mediate their interac

1.35kb

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169 QRFGKQ@YQ4HNc@. .VEVDPSJJLLEMLE150 NTEKE!@vA4vG@s . .C@I .@VADLVQ402 RTPPP@LEI@GRV@D . . J1DLLCc@.EDIEE339 REGIK@VGdAQM.HOCSRIDLLSS!IYVSc@

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Fig. 2. CRADD expression. Human tissue Northern blots (Clontech) containing 2 @gofpolyadenylatedRNAwereprobedwithradioactiveCRADDeDNA,exposedtoKodakX-ray film, andphotographed.PBL, peripheralblood leukocyte.

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CLONING OF AN ADAPTOR MOLECULE FOR CASPASE-2AND RIP

additional adaptor molecules, we searched the GenBank EST database for sequenceswith homology to the prodomain of caspase-2orcaspase-9.The sequenceof an EST clone (clone 24032) from a humaninfant brain eDNA library had significant homology to the prodomain

of caspase-2.The DNA sequenceof thiscloneoverlapsperfectlywithsequencesof several other clones in the EST data base. Using the 5'and 3' sequenceinformationderivedfrom this EST clone andothers,the open reading frame was verified and then cloned by PCR. Theopen reading frame encodes a protein of 199 amino acids (Fig. IA)that exhibits a dual-domain structure related to FADD (Fig. IB).

CRADD Has CD and DD Homology Regions A BLAST searchof the GenBank/European Molecular Biology Laboratory data baserevealed that the NH2 terminus of CRADD (residues 1—78)sharesignificant homology with the prodomains of caspase-2 (residues15—91;31% identity), caspase-9 (residues 1—79;28% identity), andCED-3 (residues2—78;24% identity; Fig. 1C). The CD homologyregion of CRADD also has significant homology to the COOHterminus of human inhibitor of apoptosis IAP-l (residues 440—516;

27% identity). Interestingly, residues L27 and G65 of CED-3, whichwhen mutated result in loss of function (26), are aligned and conserved in CRADD and the other proteins (Fig. lC). This suggeststhatthese residues could also be critical for CRADD function. TheBLAST search also revealed that the COOH terminus of CRADD

(residues 121—199)contains a DD homology region related to the DDof FADD, TNFR-l, and RIP (Fig. 1D). The identity of the DD ofCRADD with that of FADD is 23%, and the identity of the DD ofCRADD with that of TNFR-1 or RIP is —20%.

Tissue Distribution of CRADD. To determine the tissue distribution of CRADD mRNA, various tissue mRNA sampleswere subjected to Northern blot analysis. Expression of the CRADD mRNAwas detected in most adult tissues and in the fetal liver (Fig. 2). Thehighest expression was observed in the thymus, testes,and fetal liver.The size of the CRADD mRNA is approximately 1.35 kb, which is

B

:@

Fig. 4. Induction of apoptosis in cells transfected withCRADD and @-galactosidaseexpression plasmids. Human embryonic kidney 293 cells were transfected, fixed, and thenstainedto visualize @-galactosidaseactivity. A and B, tworepresentativefields of cells transfectedwith (3-galactosidaseplasmid alone. C and D, two representativefields of cellstransfectedwith CRADD and(3-galactosidaseplasmids.

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Fig. 3. Specificinteractionsof GST-CRADD,GST-CRADDCD, andGST-CRADDDD with in vitro-translatedcaspase-2andthe DD of RIP.[35SJMethionine-labeledcaspase-2or the DD of RIP was precipitated with GST (Lane 1), GST-CRADD CD (Lane2), GST-CRADDDD (Lane3), or GST-CRADD(Lane4) fusionproteinsimmobilizedonglutathione-Sepharose beads.The bound proteins were then analyzed by SDS-PAGE andautoradiography.

tion with adaptormoleculesinvolvedin cell death-signalingpathways(17—19).The prodomains of caspase-8 and caspase-lO contain two

tandemregionsof homologywith the NH2-terminal DED of FADD(17—19).Caspase-8 is recruited to the FAS/tumor necrosis factor deathpathway through interaction of its prodomain with the DED of FADD(17, 18). Other caspaseswith long prodomains such as caspase-2orcaspase-9 could also be recruited to death-signaling complexesthrough interaction with other novel adaptor molecules. To identify

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Table I Quantitative analysis of the apoptotic activity of CRADD

Human embryonic kidney 293 cells were cotransfected with CRADD or mutantCRADD and f3-galactosidase cDNAs and then fixed, stained, and counted as described in“Materialsand Methods. The data represent the percentage of apoptotic cells ±SD.

CLONING OF AN ADAFFOR MOLECULE FOR CA5PASE-2 AND RIP

incubated with in vitro-translated [35S]methionine-labeled caspaseproenzymes, and the reaction products were analyzed by SDS-PAGEand autoradiography.Mature caspase-2was able to processits ownprecursor proenzyme but not other procaspases (Fig. 5). Maturecaspase-2 was also able to cleave baculovirus p35 (Fig. 5C). Thissuggests that caspase-2 has a very narrow specificity and that itprefers a cleavage site with a DQXD@ G sequence.This sequenceispresent in procaspase-2 and p35 but not in the other known procaspasesexamined. These data suggest that CRADD could recruitcaspase-2,which could then execute the apoptotic program by itselfwithout directly processing/activatingother caspases.Alternatively,

A TX ICE-30 @PP32Mch2caspase-4 caspase.-1 caspase-3 caspase-6

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B Mch3 Mch4 Mch5 Mch6caspase-7 cupase-1O caspase-8 caspase-9

Caspase@@2— + + — + — +

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C ICH-1 P35caspase-2 ___________

Caspase-2— + — +

pro-p20

__..-25kDap1o-*@@ ..@-10kDa

Fig. 5. Protease activity of caspase-2 toward other caspases. 35S-labeled procaspases orbaculovirusp35 was incubatedwith (+) or without (—)purified caspase-2(100 ng/reaction)for 1 h at 37'C. The reactionproductswere then analyzedby Tricine-SDSPAGE and autoradiography. A, activity of caspase-2 toward proenzymes of caspase-4(TX), caspase-l (!CE-30), caspase-3 (CPP32/Yama/Apopain), and caspase-6 (Mch2). B,activity of caspase-2toward proenzymes of caspase-7 (Mch3, ICE-LAP3, CMH-l),caspase-lO(Mch4),caspase-8(Mch5/MACH/FLICE),andcaspase-9(Mchó/ICE-LAP6).C, activity of caspase-2toward procaspase-2(ICH-I) and p35. The cleavageproductsofprocaspase-2and p35 are indicated.p35 has an NH2-terminal1'7-His6 tag, thus the10-Wa productseemslargerthan 10kDa.

% ± SD(No of cells counted)

17±2.8(771)76± 1.9(894)27 ±3.5(730)26 ±2.8 (450)

eDNA

fJ-galactosidaseCRADDCRADD (27L-F)CRADD (65G-R)

consistent with the size of the eDNA clone obtained by PCR from thehuman Jurkat T-lymphocyte library.

In Vitro Interaction of CRADD with Caspase-2 and RIP. Toidentify interacting partners of CRADD, in vitro-translated caspase-2,caspase-9,FADD, TNFR-l, and the DD of RIP were precipitated withvarious GST fusion proteins immobilized on glutathione-Sepharose

resin. As shown in Fig. 3, caspase-2 specifically associated withGST-CRADD (Lane 4) and GST-CRADD CD (Lane 2), which contains the CRADD CD homology region. Caspase-2 did not interactwith GST or with GST-CRADD DD, which contains the DD but lacksthe CD homology region. No interaction was detected between

CRADD and caspase-9,FADD, or TNFR-l (data not shown). Interestingly, RIP DD specifically associated with GST-CRADD andGST-CRADD DD but not with GST or GST-CRADD CD (Fig. 3).These observations suggest that CRADD is an adaptor molecule for

caspase-2and RIP.Overexpression of CRADD Induces Apoptosis Overexpression

of FADD, which is an adaptor molecule for caspase-8 and FAS/TNFR- 1, induces apoptosis in mammalian cells ( 11, 12), most likelyby activating caspase-8. To study the functional role of CRADD inmammalian cells, human embryonic kidney 293 cells were transientlycotransfected with two expression plasmids encoding CRADD or

/3-galactosidase at a 1:4 ratio of @-galactosidaseplasmid to CRADD

plasmid. Histochemical examination of @3-galactosidase-expressingcells (blue cells) revealed that cells transfected with /3-galactosidaseand CRADD expression plasmids had morphological changes typicalof adherentcells undergoing apoptosis (Fig. 4C and D). Most bluecells were intensely stained, round, and shrunken, with typical membrane blebbing and loss of adherence. In contrast, cells transfectedwith the f3-galactosidaseexpression plasmid alone seemed healthy,with a predominantly uniform cytoplasmic (3-galactosidasestainingpattern (Fig. 4A and B). Similar results were obtained with the humanbreast adenocarcinoma cell line MCF-7 (data not shown). Quantitativeanalysis of these results revealed that —76± 1.9% of blue cellscotransfected with CRADD and @3-galactosidaseexhibited morphological changes typical of apoptosis (Table 1). Approximately,17 ±2.8% of blue cells transfected with the f3-galactosidaseexpression plasmid exhibited such a phenotype. Interestingly, site-directed

mutagenesis of L27 to F or G65 to R resulted in the loss of CRADDapoptotic activity (Table I). These data clearly demonstrate thatCRADD is an apoptosis-inducingprotein and that, like CED-3, L27and G65 are important for its apoptotic activity. This also suggeststhat the CD domain of CRADD is necessary for its activity.

Caspase-2 Does Not Activate Other Known Caspases. Recently,we demonstrated that mature caspase-8 (MchS) or caspase-lO (Mch4)

could activate all known caspases(22). Thus, clustering of caspase-8and/or caspase-lO by association of their prodomains with FADDcould trigger their activation. This could initiate a proteolytic cascadeleading to activation of the remaining caspases.By analogy, clusteringof procaspase-2 by association with CRADD could trigger its activation and initiation of a proteolytic cascade.To determine the activityof caspase-2toward the other known caspases,mature caspase-2was

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CLONING OF AN ADAPTOR MOLECULE FOR CA5PASE-2 AND RIP

Camonis,J. H., andWallach,D. Self-associationof the “deathdomains―of thep55tumor necrosisfactor(TNF) receptorandFas/APO1promptssignalingfor TNF andFas/APOIeffects.J. Biol. Chem.,270: 387—391,1995.

11. Boldin, M. P., Varfolomeev, E. E, Pancer, Z., Malt, I. L., Camonis, J. H., andWallach, D. A novel protein that interactswith the deathdomain of Fas/APO1containsa sequencemotif related to the death domain. J. Biol. Chem., 270: 7795—7798, 1995.

12. Chinnaiyan, A. M., O'Rourke, K., Tewari, M., and Dials, V. M. FADD, a novel death

domain-containingprotein, interactswith the death domain of Fas and initiatesapoptosis.Cell, 81: 505—512,1995.

13. Hsu, H., Xiong, J., and Goeddel, D. V. The TNF receptor 1-associated proteinTRADD signals cell death and NFKB activation. Cell, 81: 495-504, 1995.

14. Hsu, H., Shu, H-B., Pan, M-G., and Goeddel, D. V. TRADD-TRAF2 and TRADDFADD interactionsdefinetwo distinct‘NPreceptor-Isignaltransductionpathways.Cell, 84: 299—308,1996.

15. Stanger, B. Z., Leder, P., Lee, T-H., 1Gm, H., and Seed, B. RIP: a novel protein

containinga deathdomainthat interactswith Fas/APO-l (CD95)in yeastandcausescell death.Cell, 81: 513—523,1995.

16. Hsu, H., Huang, J., Shu, H. B., Baichwal, V., and Goeddel, D. V. TNF-dependentrecruitmentof the protein kinaseRIP to the TNF receptor-I signaling complex.Immunity,4: 387—396,1996.

17. Boldin, M. P., Goncharov, T. M., Goltsev, Y. V., and Wallach, D. Involvement ofMACH, a novelMORTI/FADD-interactingprotcase,in Fas/Apo-l-andTNF receptor-inducedcell death.Cell, 85: 803—815,1996.

18. Muzio, M., Chinnaiyan,A. M., Kischkel,F. C., O'Rourke,K., Shevchenko,A., Ni,J., Scaffidi,C.,Bretz,J. D.,Thang,M.,Gentz,R.,Mann,M.,Krammer,P. H.,Peter,M. E., andDixit.V. M.FLICE,a novelFADD-homologousICFJCED-3-likeproteaxe,is recruitedto CD95 (Fas/Apo-l) death-inducingsignalingcomplex.Cell, 85:817—827,1996.

19. Fernandes-Alnemri,T., Armstrong. R., Krebs, J., Srinivasula, S. M., Wang, L.,Bullrich, F.,Fritz, L., Trapani,J.A., Tomaselli,K. J.,Litwack,G.,andAlnemri,E. S.In vitro activation of CPP32 and mch3 by McM, a novel human apoptotic cysteineproteasecontaining two FADD-like domains.Proc. NatI. Acad. Sci. USA, 93:7464—7469,1996.

20. Alnemri, E. S., Livingston, D. J., Nicholson, D. W., Salvesen, G., Thornberry, N. A.,Wong,W. W., andYuan,J.HumanICE/CED-3proteasenomenclature.Cell. 87: 171,1996.

21. Cleveland, J. L., and lIsle, J. N. Contenders in FasIJl'NF death signaling. Cell, 81:479—482,1995.

22. Srinivasula, S. M., Abroad, M., Fernandes-Alnemri, T., Litwack, G., and Alnemri,E. S. Molecular orderingof theFas-apoptoticpathway:the Fas/APO-1 proteaseMchSis a CrmA-inhibitableproteasethat activatesmultiple Ced-3IICE-likecysteineproteases.Proc.Natl. Acad.Sci. USA, 93: 14486—14491,1996.

23. Wang, L., Miura, M., Bergeron, L., Thu. H., and Yuan, J. Ich-l, an lce/ced-3-relatedgene,encodesbothpositiveandnegativeregulatorsof programmedcelldeath.Cell,78:739—750,1994.

24. Srinivasula, S. M., Fernandes-Alnemri, 1., Zangrilli, J., Robertson, N., Armstrong,R.C.,Wang,L.,Trapani,J. A.,Tomaselli,K.J., Litwack,G.,andAlnemri,E S.TheCed-3/ICE-likehomologMch6andthe lamincleavingenzymeMch2aaresubstratesfor theapoptoticmediatorCPP32.J.Biol.Chem.,271:27099—27106,1996.

25. Dean, H., Orth, K., Chinnaiyan, A., Poirier. 0., Froeich, C. J., He, W., and Dixit,V. M. ICE-LAP6, a novel memberof the ICE/ced-3 genefamily, is activatedby thecytotoxicT-cell proteasegranzymeB. J. Biol. Chem.,271: 16720—16724,1996.

26.Yuan,J.,Shaham,S.,Ledoux,S.,Ellis,H.M.,andHorvitz,H.R.TheC.eleganscelldeath gene ced-3 encodes a protein similar to mammalian interleukin-l(3-convertingenzyme.Cell,75:641—652,1993.

27. Fernandes-Alnemri, T., Litwack, G., and Alnemri, E. S. CPP32, a novel humanapoptoticproteinwithhomologytoCaenorhabditiseleganscelldeathproteinCed-3and mammalianinterleukin-l(3-convertingenzyme.J. Biol. Chem., 269: 30761—30764,1994.

619

there could be still another target caspasedownstream of caspase-2capable of initiating the proteasecascade.It is also possible thatCRADD may associate with and trigger the activation of another asyet unknown caspase.

In conclusion, we have identified and characterized a novel apoptotic adaptor molecule named CRADD. CRADD has a dual-domain

structure similar to FADD and it might be a component of theFAS/TNFR-l apoptotic signaling complex. Its ability to interact withthe DD of RIP and the prodomainof caspase-2suggeststhat it couldplay a role in the FASITNFR-1 apoptotic pathway by recruitingcaspase-2. CRADD could also associate with the DD of other unidentifled signaling/adaptor molecules or TNFR family members. ThisCRADD pathway might be a tissue-specific pathway complementaryto the FAS!FNFR-1-FADD-caspase-8 pathway.The identificationofCRADD addsanother adaptor molecule to the FAS/TNFR-l apoptoticsignaling complex. The targeted disruption of the CRADD gene willallow a greater understanding of its role in the death-signaling pathways of mammalian cells.

Note Added in Proof

In theJanuary2, 1997issueof thejournal Nature,DuanandDixit reportedthe identification of a death adaptormolecule that is nearly identical toCRADD. They reported the finding that this protein can interact with RIP andICH-l, whichisconsistentwithourresults.

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