A Novel Enhancer of the Apaf1 Apoptosome Involved inCytochrome c-dependent Caspase Activation and Apoptosis*

Received for publication, July 17, 2000, and in revised form, December 8, 2000Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M006309200

Zhi-Liang Chu‡, Frederick Pio, Zhihua Xie, Kate Welsh, Maryla Krajewska, Stan Krajewski,Adam Godzik, and John C. Reed§

From the Burnham Institute, La Jolla, California 92037

Apaf1/CED4 family members play central roles in apo-ptosis regulation as activators of caspase family celldeath proteases. These proteins contain a nucleotide-binding (NB) self-oligomerization domain and a caspaserecruitment domain (CARD). A novel human proteinwas identified, NAC, that contains an NB domain andCARD. The CARD of NAC interacts selectively with theCARD domain of Apaf1, a caspase-activating proteinthat couples mitochondria-released cytochrome c (cyt-c)to activation of cytosolic caspases. Cyt-c-mediated acti-vation of caspases in cytosolic extracts and in cells isenhanced by overexpressing NAC and inhibited by re-ducing NAC using antisense/DNAzymes. Furthermore,association of NAC with Apaf1 is cyt c-inducible, result-ing in a mega-complex (>1 MDa) containing both NACand Apaf1 and correlating with enhanced recruitmentand proteolytic processing of pro-caspase-9. NAC alsocollaborates with Apaf1 in inducing caspase activationand apoptosis in intact cells, whereas fragments of NACrepresenting only the CARD or NB domain suppressApaf1-dependent apoptosis induction. NAC expressionin vivo is associated with terminal differentiation ofshort lived cells in epithelia and some other tissues. Theability of NAC to enhance Apaf1-apoptosome functionreveals a novel paradigm for apoptosis regulation.

CED4 family proteins constitute a unique family of caspase-activating molecules. The founding member of this family,CED4, was discovered in the nematode Caenorhabditis elegansin screens for genes that are essential for developmental pro-grammed cell death (1). CED4 contains an N-terminal CARD1

followed by an NB domain, the later containing classicalWalker A and B box motifs recognized as important in bindingnucleotide triphosphates. CED4 functions as an activator of the

caspase, CED3, in vitro and in vivo (2, 3). The NB domain ofCED4 oligomerizes in an ATP-dependent manner (4, 5),whereas the CARD binds a complementary N-terminal CARDfound in the zymogen proform of CED3 (6). Protease activationis thought to result from the induced proximity of CED3 zymo-gens bound to oligomerized CED4, where the weak intrinsicprotease activity of the proenzymes is sufficient for trans-pro-teolysis of closely juxtaposed pro-caspases (4, 7). Proteolyticcleavage of pro-CED3 then produces the large and small sub-units of the heterotetrameric, autonomously active enzyme.

The closest homologue of CED4 identified in humans andother mammals thus far is Apaf1 (apoptosis protease-activat-ing factor-1) (8). Similar to CED4, the Apaf1 protein contains aCARD, followed by an NB domain that shares significantamino acid sequence identity with the NB domains of CED4and a family of ATPases associated with pathogen resistance(R genes) in plants (3, 5, 9), thus constituting the NB-ARC(Apaf-1/R gene/CED4) domain family (also known as NACHTdomain). Unlike CED4, however, the NB-ARC domain of Apaf1is followed by multiple WD repeats. These WD domains par-ticipate in auto-repression of Apaf1, locking it into an inactive,unoligomerized state until bound by cyt-c. In response to mul-tiple cell death stimuli, changes in mitochondrial membranepermeability result in release of cyt-c into the cytosol, where itbinds and activates Apaf-1, thus coupling mitochondrial dam-age to a mechanism for caspase activation (10). Cyt-c, in con-junction with dATP or ATP, induces formation of a large Apaf1oligomer (estimated to be an octamer), via its NB-ARC domain,and exposes the CARD of Apaf1 for interactions with a com-plementary CARD found in the N-terminal prodomain of pro-caspase-9 (11–14). By the induced proximity method, juxta-posed pro-caspase-9 (pro-Casp9) zymogens then trans-proteolyze each other, generating the characteristic large andsmall subunits typical of activated caspases. Active caspase-9bound to oligomerized Apaf1 then directly cleaves and activatespro-caspase-3, an effector caspase that is responsible both forcleavage and activation of additional downstream caspases andfor direct cleavage of a variety of substrate proteins that com-mit the cell to an apoptotic demise. A close homologue of Apaf1has recently been identified in the fly, Drosophila melano-gaster, apparently operating as a caspase-activator via a simi-lar cyt-c-inducible mechanism (15–17). Gene ablation studiesin mice and flies indicate that Apaf1 plays a critical role inprogrammed cell death in certain tissues and in response tomany types of cell death stimuli in vivo (18).

In this report, we describe the identification and initial func-tional characterization of a novel regulator of Apaf1, which wehave termed NAC.

MATERIALS AND METHODS

cDNA Cloning and Plasmid Construction—The NAC cDNA sequencewas found using PSI-BLAST and the CARD sequence of CARD4/Nod1

* This work was supported by CaPCURE, National Institutes ofHealth Grants GM61694 and NS36821, and the United States Depart-ment of Defense. The costs of publication of this article were defrayed inpart by the payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 U.S.C. Section1734 solely to indicate this fact.

‡ Current address: Arena Pharmaceuticals, 6166 Nancy Ridge Dr.,San Diego, CA 92121.

§ To whom correspondence should be addressed: Burnham Institute,10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-646-3140; Fax:858-646-3194, E-mail: jreed@burnham.org.

1 The abbreviations used are: CARD, caspase recruitment domain;LRR, leucine-rich repeat; EGFP, enhanced green fluorescent protein;PBS, phosphate-buffered saline; IP, immunoprecipitation; IVT, in vitrotranslated; PAGE, polyacrylamide gel electrophoresis; aa, amino acids;HA, hemagglutinin; cyt-c, cytochrome c; pro-Casp9, pro-caspase-9;ZVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ke-tone; NB, nucleotide binding; Ac-DEVD-AFC, acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl-coumarin; DAPI, 49,6-diamidino-2-phenylin-dole; GST, glutathione S-transferase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 12, Issue of March 23, pp. 9239–9245, 2001© 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 9239

by guest on October 7, 2020

http://ww

w.jbc.org/

Dow

nloaded from

as query. This search revealed homology with a predicted protein ofEST clone KIAA0926 in the Kazusa DNA Research Institute braingenomic data base. Jurkat total RNA was reverse-transcribed to cDNAswith Moloney murine leukemia virus reverse transcriptase (Strat-agene) and random hexanucleotide primers. Three overlapping cDNAfragments of NAC were amplified using Turbo Pfu DNA polymerase(Stratagene) and three sets of oligonucleotide primers as follows: set 1,59-CCGAATTCACCATGGCTGGCGGAGCCTGGGGC-39 (forward) and59-CCGCTCGAGTCAACAGAGGGTTGTGGTGGTCTTG-39 (reverse);set 2, 59-CCCGAATTCGAACCTCGCATAGTCATACTGC-39 (forward)and 59-GTCCCACAACAGAATTCAATCTCAACGGTC-39 (reverse);, andset 3, 59-TGTGATGAGAGAAGCGGTGAC-39 (forward) and 59-CCGCT-CGAGCAAAGAAGGGTCAGCCAAAGC-39 (reverse). The resultantcDNA fragments were ligated into mammalian expression vectorpcDNA3-Myc. From these overlapping cDNA fragments, full-lengthNAC cDNA was assembled in pcDNA3-Myc and pcDNA3-HA at EcoRIand XhoI cloning sites. The nucleotide sequence of the assembled full-length NAC was confirmed by DNA sequencing analysis. The regionsencoding the CARD or the NB domain (amino acids 329–547) werepolymerase chain reaction-amplified from Jurkat cDNA using primerset 3 and the primers 59- CCCGAATTCGAACCTCGCATAGTCATACT-GC-39 (forward) and CCGCTCGAGTCAACAGAGGGTTGTGGTGGTC-TTG-39 (reverse), respectively. The resultant polymerase chain reactionfragments were digested with EcoRI and XhoI and ligated intopcDNA3-Myc and into vector pGEX-4T1 for GST fusion proteinproduction.

Antibodies—Polyclonal antisera were generated in rabbits using key-hole limpet hemocyanin- and ovalbumin-conjugated (Pierce) syntheticpeptides with sequences corresponding to residues aa 161–180 (Bur241)or aa 1058–1077 (Bur242) of NAC. Mouse monoclonal antibody recog-nizing human APAF1 was purchased from R & D Systems (Minneapo-lis, MN). Epitope-specific antibodies for FLAG, HA, or Myc tag wereobtained from Sigma, Roche Molecular Biochemicals, and Santa CruzBiotechnology, respectively.

DNAzymes—An anti-NAC DNAzyme oligonucleotide was designedby the method of Joyce (19), targeting the translation initiation regionof NAC mRNA and containing 2-O-methylnucleosides at the 59-end andan inverted thymidine at the 39-end for nuclease resistance. The se-quences of the catalytic (AS) and control (C) noncatalytic oligonucleo-tides are as follows: AS, 59-(2-O-MeC2-O-MeCAGCCAGGCTAGCTA-CAACGACTCTGTCC-InvT)-39, and (C, 59-(2-O-MeC2-O-MeCAGCCA-GGCTACCTACAACGACTCTGTCC-InvT)-39, respectively (OperonTechnologies).

Immunohistochemistry—Normal human tissues for immunohisto-chemistry analysis were obtained from biopsy and autopsy specimens,fixed in Bouin’s solution (Sigma), and embedded in paraffin. Tissuesections were immunostained using a diaminobenzidine-based detec-tion method employing the Envision-Plus-horseradish peroxide system(Dako). Nuclei were counterstained with hematoxylin.

Coimmunoprecipitation and Immunoblotting Assays—For immuno-precipitation and immunoblotting analyses, cells were lysed in eitherbuffer A (142.4 mM KCl, 5 mM MgCl2, 10 mM HEPES (pH 7.4), 0.5 mM

EGTA, 1 mM EDTA, and 0.2% Nonidet P-40 for cytoplasmic extracts),buffer B (20 mM HEPES (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, and 1 mM

EDTA, for hypotonic lysis), or ELB (50 mM HEPES (pH 7.4), 250 mM

NaCl, 5 mM EDTA, and 0.4% Nonidet P-40, for whole cell extracts), allsupplemented with 1 mM dithiothreitol, 12.5 mM b-glycerol phosphate,1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 13 proteaseinhibitor mix (Roche Molecular Biochemicals). Cell lysates were clari-fied by centrifugation and subjected to immunoprecipitation using spe-cific antibodies and protein G or A beads. Immune complexes wereresolved in SDS-PAGE gels, transferred to nitrocellulose membranes,and immunoblotted with antibodies followed by detection using ECL(Amersham Pharmacia Biotech) (20).

In Vitro Protein Interaction Assays—GST fusion proteins were ex-pressed from pGEX-4T1 in XL-1-blue Escherichia coli cells (Stratagene)and affinity-purified using GSH-Sepharose. Purified GST fusion pro-teins (0.1–0.5 mg) immobilized on 10–15 ml of GSH-Sepharose beadswere incubated with 1 mg/ml bovine serum albumin in 100 ml of bufferA for 30 min at 25 °C. The beads were then incubated overnight at 4 °Cwith 1 ml of rabbit reticulocyte lysates (TnT-lysate; Promega) contain-ing 35S-labeled, IVT proteins in 100 ml of buffer A supplemented with0.5 mg/ml bovine serum albumin. Proteins on beads were washed fourtimes in 500 ml of buffer A, followed by boiling in 20 ml of Laemmli-SDSsample buffer, SDS-PAGE and detection by fluorography.

Gel-sieve Chromatography Analysis—Cytosolic extracts were pre-pared using buffer B (above) as described (21) and incubated (1.5 mg)with cyt-c (10 mM) and dATP (1 mM) for 10 min at 30 °C, and then 100

mM ZVAD-fmk was added. The treated protein lysates were immedi-ately fractionated by using a Superose-6 HR 10/30 gel filtration columnin elution buffer containing 50 mM Tris (pH 7.4), 100 mM KCl, 1.5 mM

MgCl2, 1 mM EDTA, and 1 mM dithiothreitol. Column fractions (0.5 ml)were analyzed for NAC and Apaf1 by SDS-PAGE, followed byimmunoblotting.

Caspase Assays—Cytosolic extracts were prepared in hypotonicbuffer B as described (21) and incubated (10 mg) with various concen-trations of cyt-c and 1 mM dATP in Caspase buffer (21) for 30 min at30 °C. Caspase substrate Ac-DEVD-AFC (100 mM) (Calbiochem) wasthen added, and protease activity was measured continuously by mon-itoring the release of fluorigenic AFC at 37 °C. Alternatively, trans-fected cells were directly lysed in Caspase Lysis buffer (10 mM HEPES(pH 7.4), 25 mM NaCl, 0.25% Triton X-100, and 1 mM EDTA), normal-ized for protein content, and monitored for cleavage of Ac-DEVD-AFCas described (21). Processing of IVT 35S-labeled pro-caspase-9 in cyto-solic extracts was monitored by SDS-PAGE (20).

Apoptosis Assays—Cells were transfected with pEGFP (CLON-TECH) and effector plasmids using SuperFect transfection reagents(Qiagen) as indicated. After culturing 1.5 days in media containingreduced serum (0.1% fetal bovine serum), floating and adherent cells(recovered by trypsinization) were pooled, and cells were fixed in 3.7%formaldehyde/PBS, stained with 1 mg/ml 49,6-diamidino-2-phenylindole(DAPI), and the percentage of GFP-positive cells with apoptotic mor-phology (nuclear fragmentation, chromatin condensation) was deter-mined by fluorescence microscopy (20, 21).

Molecular Modeling—A three-dimensional model of the CARD do-main of NAC was generated using the MODELLER program, essen-tially as described (22), based on the structures of the CARDs of Apaf1,pro-Casp9, and Raidd (23, 24).

RESULTS AND DISCUSSION

NAC-encoding cDNAs were obtained by reverse tran-scriptase-polymerase chain reaction, revealing a continuousopen reading frame encoding a 1473-amino acid protein (Fig.1). The predicted NAC protein contains an NB domain, fol-lowed by leucine-rich repeats (LRR), and a CARD domain.Thus, unlike Apaf1/CED4 family proteins that also containCARD and NB domains, the CARD domain of NAC is located atits C rather than N terminus. The NB domain of NAC containsclassical Walker A and B boxes indicative of ATP-binding pro-teins and is most similar in amino acid sequence to the NBdomain of Nod1/CARD4 (29%) (25, 26), followed by humanAPAF1 (17%), the Drosophila Apaf1 homologue (12%), and theC. elegans CED4 protein (12%) (Fig. 1C). Moreover, recombi-nant NAC NB domain was observed to bind ATP and to self-associate in an ATP-dependent manner in vitro (not shown).The CARD domain of NAC shares 21, 19, and 8% amino acididentity with the CARD domains of Nod1/CARD4, huApaf1,and CED4, respectively (Fig. 1D). The NAC CARD sequencewas readily threaded onto the structures of other CARDs usingthe MODELLER program (Fig. 1E), suggesting conservation ofthe 6 a-helical fold typical of these domains (23). The LRRs ofNAC are reminiscent of Nod1/CARD4 and plant stress-re-sponse (R) proteins, which also contain LRRs. In NB-contain-ing plant R proteins, the LRRs function as interaction motifsfor pathogen responses (9), suggesting a possible role of thesestructures in linking NAC to specific signaling pathways. Ad-ditional NAC cDNAs were obtained that presumably representalternative mRNA splicing products that encode shorter pro-teins lacking 31- or 45-amino acid segments (or both) locatedbetween the LLR and CARD (Fig. 1).

NAC mRNAs were widely expressed in human tissues, withhighest levels found in blood leukocytes, thymus, spleen, andheart (Fig. 2A). Antisera were raised against synthetic NACpeptides and confirmed to bind specifically the NAC protein byimmunoblotting and immunoprecipitation assays (Fig. 2B, andnot shown). NAC protein was detected in several adult humantissues, with highest levels in kidney, brain, and epidermisamong the tissues examined (Fig. 2C). A smaller anti-NACimmunoreactive band was detected in thymus lysates, which

Enhancer of Apaf1 Involved in Cyt c-dependent Caspase Activation9240

by guest on October 7, 2020

http://ww

w.jbc.org/

Dow

nloaded from

FIG. 1. Sequence analysis of NAC. A,schematic representation showing do-main structure of human NAC. The NBdomain (aa 329–547, filled box), theleucine-rich repeats (aa 808–947, filledbars), and the CARD (aa 1373–1473, dot-ted box) are depicted. Hatched boxes indi-cate sequences derived from two alterna-tively spliced exons. B, predicted aminoacid sequences of human NAC. The posi-tions for the P-loop (Walker A) andWalker B of NB domain are indicated.The amino acids sequences of LRR re-peats and CARD are underlined and inbold letters, respectively. Italic letters in-dicate sequences for the alternativelyspliced exons. C, alignment of the NB do-main of NAC (aa 329–547) with NB do-mains of Nod1/CARD4 (aa 197–408),Apaf1 (aa 138–355), and C. elegans CED4(aa 154–374). Alignment was conductedusing ClustalW. Identical and similar res-idues are shown in black and gray shades,respectively. Positions of P-loop andWalker B sequences are indicated. D,alignment of CARDs of NAC (aa 1373–1465), Nod1/CARD4 (aa 15–104), Apaf1(aa 1–89), and CED4 (aa 2–89). Identicaland similar residues are shown in blackand gray shading, respectively. E, three-dimensional model of NAC CARD do-main, showing predicted 6 a-helixes (la-beled H1–H6).

Enhancer of Apaf1 Involved in Cyt c-dependent Caspase Activation 9241

by guest on October 7, 2020

http://ww

w.jbc.org/

Dow

nloaded from

remains to be characterized (Fig. 2C). Differences in NACmRNA and protein expression in some tissues suggest thepossibility of post-transcriptional regulation (Fig. 2, A and C).

Immunohistochemical analysis of the in vivo patterns ofNAC expression in adult human tissues demonstrated associ-ation with differentiation in stratified epithelia of the skin,esophagus, intestine, and cervix, as well as in the prostategland where differentiated luminal secretory cells were NAC-immunopositive and undifferentiated basal cells were immu-nonegative (Fig. 2D and not shown). Differentiated macro-phages and granulocytes were also strongly NAC-immunopositive (3–4 intensity/on 0–4 scale), whereas theirbone marrow precursors were immunonegative (not shown). Inthe testis, NAC immunointensity also increased in concert withthe differentiation program from spermatocytes (negative), tospermatids (0–1 intensity/on 0–4 scale), to spermatozoa (1–2intensity), reaching highest intensity in the residual bodiesthat represent cellular remnants (2–3 intensity) (not shown).Thus, NAC expression is associated with differentiation ofsome types of short lived cells in vivo.

In vitro binding experiments were performed using a glutathi-one S-transferase (GST) fusion protein containing the CARD ofNAC. The CARD of NAC bound efficiently to itself and alsointeracted selectively in vitro with the CARDs of Apaf1, Nod1,and CED4 but not with the CARDs of Bcl10, pro-Casp9, pro-caspase-1, pro-caspase-2, pro-caspase-11, Raidd, or Cardiak(RIP2) (Fig. 2A and not shown). The ability of NAC to interactwith itself, Apaf1, Nod1, and CED4 in cells was confirmed bycoimmunoprecipitation of epitope-tagged proteins from tran-siently transfected HEK293 cells (Fig. 2B and not shown). Theendogenous ;160-kDa NAC protein could also be coimmunopre-cipitated with endogenous Apaf1 (but not Nod1) from cells that

intrinsically express NAC, using anti-NAC antisera (Fig. 2D).Interestingly, compared with full-length Apaf1, NAC coim-

munoprecipitated more efficiently with a truncation mutant ofApaf1 which lacks the WD repeats that normally maintain thisprotein in an auto-repressed state (14), suggesting that “acti-vated” Apaf1 interacts preferentially with NAC (Fig. 2B). Con-sistent with this observation, association of full-length Apaf1with NAC was inducible by stimuli such as staurosporine,which trigger cyt-c release and result in Apaf1 activation (27).Before exposure to death stimuli, relatively little NAC wascoimmunoprecipitated with Apaf1, but within 10 min NAC/Apaf1 complexes were readily detected (Fig. 3E).

Since Apaf1 is known to form complexes with pro-Casp9 (10),we examined the effects of NAC on Apaf1/pro-Casp9 interac-tions by coimmunoprecipitation. Overexpression of NAC bytransient transfection in HEK293 cells increased the relativeamount of Apaf1 that coimmunoprecipitated with pro-Casp9(Fig. 3C), suggesting that NAC enhances rather than inhibitsinteractions of these proteins. These coimmunoprecipitationexperiments also hinted that NAC may exist in a complexsimultaneously with Apaf1 and pro-Casp9, since NAC wasrecovered in anti-Casp9 immunoprecipitates when Apaf1 wascoexpressed (Fig. 3C), even though NAC cannot directly bindpro-Casp9 (Fig. 3A and not shown) and was not recovered inanti-Casp9 immune complexes when Apaf1 was not coex-pressed (Fig. 3B).

Cyt-c induces formation of a large holoenzyme complex(apoptosome) containing multiple Apaf1 and Casp9 moleculesin an estimated 8:8 stoichiometry (12). The apoptosome can bemonitored by gel-sieve chromatography of cyt-c-stimulated cy-tosolic extracts and typically migrates at ;700 kDa (28). How-ever, a larger apoptosome of .1 MDa has been reported in

FIG. 2. NAC expression in human tissues. A, Northern analysis. A 32P-labeled partial NAC cDNA was hybridized to filter-immobilizedpoly(A)1 RNA from various tissues (1 mg/lane) (CLONTECH) and visualized by x-ray autoradiography (upper panel). The same RNA blot wassubsequently hybridized with a human b2actin cDNA probe, controlling for RNA loading (bottom panel). PBL, peripheral blood leukocytes. B,validation of anti-NAC antibody. 293T cells were transfected with plasmids encoding either Myc-tagged full-length (FL) NAC (which contains thesequence (aa 161–180) recognized by anti-NAC peptide antiserum) or Myc-CARD (containing only the C-terminal CARD domain of NAC). Lysatesfrom transfected cells were analyzed by immunoblotting 2 days later, probing blots with anti-Myc (left panel) or anti-NAC (Bur241) (right panel)antibodies. C, NAC protein expression in adult human tissues. Whole cell lysates (50 mg) from the indicated human cells were analyzed bySDS-PAGE/immunoblotting using anti-NAC (Bur241) antiserum with ECL-based detection. Dark and open arrowheads indicate full-length NACprotein and a smaller NAC fragment (seen only in thymus) possibly arising by protease cleavage or alternative mRNA splicing. D, immunohis-tochemistry was used to analyze NAC expression in human tissues. Examples provided illustrate induction of NAC expression during epithelialdifferentiation in skin (epidermis) stained with anti-NAC antisera (Bur241) in the absence (a) and presence (b) of the competing immunogenicpeptide, and in esophagus (c) and uterine cervix (d). Similar results were obtained using two different anti-peptide antisera.

Enhancer of Apaf1 Involved in Cyt c-dependent Caspase Activation9242

by guest on October 7, 2020

http://ww

w.jbc.org/

Dow

nloaded from

some types of cells, suggesting that other proteins may associ-ate with the Apaf1-Casp9 complex (29). In cyt-c-stimulatedextracts from HEK293 cells, which contain relative little en-dogenous NAC, as determined by immunoblotting (not shown),a single Apaf1-containing apoptosome of ;700 kDa was ob-served by gel-sieve chromatography (Fig. 2F). However, in ex-tracts from NAC-transfected HEK293 cells, two Apaf1-contain-ing apoptosomes were evident, including a large .1 MDacomplex that contained both Apaf1 and NAC (as determined bycoimmunoprecipitation analysis of the column fractions inwhich Apaf1 and NAC coeluted) (Fig. 2F). Similarly, in cellsthat contain relative high levels of endogenous NAC, such asJurkat T-cells, the endogenous NAC and Apaf1 molecules coe-luted in gel-sieve experiments, revealing two apoptosomeswhere greater amounts of NAC were present in the larger of

these multiprotein complexes (Fig. 2F, top). In extracts lackingcyt-c treatment, Apaf1 eluted as a monomer, whereas NAC wasspread over multiple fractions without a clear elution peak (notshown). Also, NAC did not coelute in gel-sieve chromatographyexperiments with Nod1 using untreated or cyt-c-stimulatedextracts,2 confirming that NAC associates selectively with theApaf1 apoptosome.

To explore whether the Apaf1-containing apoptosome withwhich NAC associates displays caspase activity, endogenousNAC was immunoprecipitated from control- and cyt-c-stimu-lated Jurkat cell extracts, and associated caspase activity wasmeasured based on cleavage of the fluorigenic caspase-sub-

2 Z.-L. Chu, F. Pio, Z. Xie, K. Welsh, M. Krajewska, S. Krajewski, A.Godzik, and J. C. Reed, unpublished observations.

FIG. 3. NAC interacts with Apaf1 apoptosome. A, CARD-CARD interactions. IVT 35S-labeled CARDs of NAC, Apaf1, CED4, and pro-Casp9,or full-length Bcl10 were incubated with immobilized GST (lane 2) or GST fusion containing the CARD of NAC (NAC-CARD) (lane 3). One-tenthof input IVT 35S-labeled proteins was also loaded directly into gels (lane 1). B, full-length NAC forms complexes with Apaf1, CED4, and itself.Human 293T cells were cultured in 6-well plates and were transiently transfected with expression plasmids (pcDNA3, 1 mg each) encodingepitope-tagged (HA or FLAG tag) full-length HA-NAC, full-length FLAG-Apaf1, HA-Apaf1 lacking the WD repeats (HA-Apaf1(DWD)), HA-CED4,or an inactive form of HA-tagged pro-Casp9 harboring a catalytic site mutation (C287A) (20) in the presence (1) or absence (2) of pcDNA3 (1 mg)encoding Myc-tagged full-length NAC. Immunoprecipitations IP) were performed 1 day later using either a mouse monoclonal antibody to Myc(lanes 4 and 6) or a control mouse IgG (Cntl) (lane 5). Immune complexes were resolved by SDS-PAGE and analyzed by immunoblotting usinganti-HA or anti-FLAG antibodies. Lysates derived from each transfection (10% of immunoprecipitation input) were loaded directly in gels ascontrols (lanes 1–3). These interaction results were further confirmed reciprocally by probing Myc-NAC in immune complexes derived from theindicated target proteins (data not shown). C, NAC enhances Apaf1/pro-Casp9 interaction. 293T cells were transfected with FLAG-Apaf1 incombination with FLAG-pro-Casp9 (C287A) (lanes 2 and 3) and Myc-NAC (lane 3). Transfected cells were cultured in the presence of ZVAD-fmk(100 mM) for 24 h and then lysed under hypotonic, detergent-free conditions in the presence of 50 mM ZVAD-fmk. Cell lysates were subjected toimmunoprecipitation using a rabbit polyclonal anti-human caspase-9 antibody (35). The resulting immune complexes were fractionated bySDS-PAGE, transferred to nitrocellulose membranes, and subsequently probed for the presence of FLAG-Apaf1 and FLAG-pro-Casp9 usinganti-FLAG antibody (upper panel), and for NAC using anti-Myc antibody (lower panel). Immunoblot analysis of lysates from transfected cellsconfirmed production of equivalent amounts of FLAG-Apaf1 in all samples (not shown). D, endogenous NAC associates with endogenous Apaf1.Cytoplasmic extracts (500 mg of protein in 0.3 ml) derived from Jurkat cells were treated with cyt-c (10 mM) and dATP (1 mM) in the presence of50 mM ZVAD-fmk and subjected to immunoprecipitation (IP) with anti-NAC antisera (Bur241) in the absence (lane 1) or presence (lane 2) of 10 mgof competing immunogenic peptide (aa 161–180). The resulting immune complexes were analyzed by SDS-PAGE/immunoblotting with antibodiesagainst NAC (Bur242) (top) and Apaf1 (bottom). Molecular mass markers are indicated in kDa. E, induction of NAC/Apaf1 association bystaurosporine (STS). Jurkat cells (107) were treated with staurosporine for the indicated times before preparing lysates (250 mg of total protein)and performing immunoprecipitations with monoclonal anti-Apaf1 antibody and protein G beads overnight at 4 °C. Immune complexes wereanalyzed by SDS-PAGE/immunoblotting using anti-NAC (Bur241) (top) and anti-Apaf1 (bottom) antibodies. Immunoblot analysis of lysates (lanes1–4) confirmed that staurosporine treatment had no effect on total NAC protein levels under these conditions (not shown). F, NAC associates withApaf1 apoptosome. Top panel, cytoplasmic extracts (1.5 mg) prepared from Jurkat cells were treated with cyt-c and dATP for 10 min at 30 °C, andthen 100 mM ZVAD-fmk was added, and the samples were placed on ice before fractionation on a Superose-6 gel filtration column. Column fractionswere assayed by SDS-PAGE/immunoblotting for Apaf1 and NAC, using specific antibodies. The positions of molecular mass markers and the voidvolume fraction are indicated. Bottom panels, lysates prepared from 293T cells transfected (Transf.) with plasmids encoding FLAG-Apaf1 andMyc-NAC or FLAGApaf1 alone were analyzed by gel-seive chromatography, as above. Column fractions were analyzed by SDS-PAGE/immuno-blotting using anti-FLAG and anti-Myc antibodies. Apoptosome complexes derived from epitopically expressed Apaf1 or NAC were probed usingepitope-specific antibodies (FLAG or Myc). Fractions in which Apaf1 and NAC coeluted were pooled and immunoprecipitated with anti-FLAGantibodies to recover Apaf1 or with an IgG control antibody (Cntl), and the resulting immune complexes were immunoblotted for NAC usinganti-Myc epitope antibody.

Enhancer of Apaf1 Involved in Cyt c-dependent Caspase Activation 9243

by guest on October 7, 2020

http://ww

w.jbc.org/

Dow

nloaded from

strate Ac-DEVD-AFC (21). These experiments revealed thatNAC is associated with active caspases after but not beforecyt-c stimulation (Fig. 4A). Control immunoprecipitates lackedsignificant caspase activity, confirming the specificity of theseresults. Although activities of different size apoptosomes canvary depending on salt concentrations, use of detergents, andthe presence of endogenous inhibitors (X-chromosone-linkedinhibitor of apoptosis protein and second mitochondrial activa-tor of caspases) (29),2 these findings argue that the largerapoptosome containing NAC is active.

In HEK293 cells, which contain little endogenous NAC, over-expression of NAC by transient transfection promotes forma-tion predominantly of the larger .1-MDa apoptosome uponcyt-c stimulation, with relatively little of the smaller ;700-kDaapoptosome present (Fig. 3F). To contrast the function of Apaf1in the presence and absence of NAC, therefore, we compared

cyt-c-induced proteolytic processing of pro-Casp9 and activa-tion of downstream caspases (Ac-DEVD-AFC cleavage) in ex-tracts prepared from control- and NAC-transfected HEK293cells (Fig. 4B). Extracts containing elevated NAC displayedincreased processing of pro-Casp9 and greater cyt-c-inducedactivation of downstream caspases, suggesting that NAC en-hances Apaf1 activity. Conversely, reducing NAC protein levelsusing antisense expression plasmids (not shown) or antisense/DNAzyme oligonucleotides decreased the ability of cyt-c toactivate caspases in cell extracts in vitro. Fig. 4C, for example,shows experiments performed using Jurkat cells, which con-tain relatively higher levels of endogenous NAC, demonstrat-ing DNAzyme-mediated ablation of NAC protein without con-comitant changes in the levels of Apaf1. Extracts preparedfrom anti-NAC DNAzyme-treated Jurkat cells were less sensi-tive to cyt-c compared with control oligonucleotide-treated

FIG. 4. NAC modulates Apaf1 function. A, NAC associates with active caspases after cyt-c stimulation. Jurkat cytoplasmic extracts weretreated with cyt-c/dATP for 30 min at 30 °C (a, b and c) or left untreated (d), and then subjected to immunoprecipitation with anti-NAC antiserum(Bur241) (a, b, and d) in the presence (b) or absence (a and d) of competing immunogenic peptide or the corresponding preimmune sera (c). Theresulting immune complexes were washed extensively and then monitored for DEVDase activity, expressing results as relative fluorescent units(RFU) released from Ac-DEVD-AFC per mg of protein input for immunoprecipitation (data representative of three independent experiments). B,NAC enhances cyt-c-induced pro-Casp9 processing and activation of downstream caspases. 293T cells were transfected with empty pcDNA3(CNTL) or pcDNA3 encoding NAC (10 mg of DNA) in 10-cm plates. Cytoplasmic extracts were prepared from transfected cells after 24 h. Cell lysates(10 mg) were incubated with 35S-labeled pro-Casp9 in the presence or absence of cyt-c (10 mM) and dATP (1 mM) (cyt-c) at 30 °C for 1 h and thenanalyzed by SDS-PAGE/fluorography (top). Alternatively, various concentrations of cyt-c were added to extracts without IVT pro-Casp9 (bottom),and caspase activity was measured in 10-mg aliquots after incubation at 30 °C for 0.5 h by monitoring release of AFC from caspase substrateAc-DEVD-AFC (expressed as relative fluorescent units (RFU) per mg protein per min). C, reductions in NAC decrease sensitivity to cyt-c. JurkatT cells were subjected to two sequential lipofections (at 0 and 1 day) with either catalytically active anti-NAC DNAzyme (AS) or inactive controloligonucleotide (C) (20 mM final concentration). Whole cell extracts were prepared from an aliquot of the cells, normalized for protein content (50mg), and analyzed by SDS-PAGE/immunoblotting using anti-NAC and anti-Apaf1 antibodies (top). Alternatively, detergent-free cytoplasmicextracts were prepared and treated either with 10 mM cyt-c and 1 mM dATP (cyt-c) or with 10 ng of granzyme B (Grz B), and DEVDase activity wasmeasured at 0, 10, 20, and 30 min (mean 6 S.E., n 5 3) (bottom). D, NAC collaborates with Apaf1 and pro-Casp9 in inducing caspase activation.Cytoplasmic extracts were prepared from 293T at 1 day following transfection with plasmids encoding the following: (a) pro-Casp9 (50 ng DNA),Apaf1 (50 ng), and NAC (2 mg); (b) pro-Casp9 and Apaf1; or (c) pro-Casp9 alone (all transfections normalized for 2.5 mg of total DNA using pcDNA3)and assayed for caspase activity by addition of 100 mM Ac-DEVD-AFC to cell lysates and monitoring AFC release (RFU/mg lysate) over time.Although not shown, negligible caspase activity was detected in lysates of cells transfected individually with Apaf1 or NAC or with the combinationof NAC and Apaf1 (in the absence of pro-Casp9). E, NAC enhances apoptosis induced by staurosporine and by Apaf1. 293T cells were transfectedwith pEGFP (0.1 mg) and plasmids encoding pro-Casp9 (0.05 mg), Apaf1 (0.05 or 2.0 mg), or NAC (0.5 or 2 mg), as indicated. Total DNA input wasnormalized with empty pcDNA3. Transfected cells were cultured in media containing 0.1% fetal bovine serum for 1.5 days (1st to 7th columns) ortreated with staurosporine (STS) at the indicated concentration for 3 h (8th to 13th columns) prior to fixing and staining with DAPI (black bars 5with NAC; white bars 5 without NAC). The % GFP-positive apoptotic cells (exhibiting nuclear fragmentation and chromatin condensation) wasdetermined by fluorescence microscopy (mean 6 S.E., n 5 3). Immunoblot analysis of replicate transfections confirmed production of plasmid-encoded proteins and demonstrated that NAC and Apaf1 did not affect each other’s expression (not shown). F, dominant-negative mutants of NACinhibit apoptosis induction by Apaf1 and staurosporine (STS) but not Fas. 293T cells were transfected with 0.1 mg of pEGFP DNA and either 0.5or 2 mg of plasmids encoding the CARD or NB domain of NAC, together with plasmids encoding pro-Casp9 (50 ng) and Apaf1 (1 mg) (left) or Fas(0.3 mg) (right). Alternatively, cells were treated with 1 mM staurosporine for 5 h (middle). Cells were fixed and stained with DAPI, enumeratingthe percentage of apoptotic GFP-positive cells (mean 6 S.E., n 5 3). Immunoblot analysis confirmed production of the CARD and NB domains ofNAC and demonstrated no affect on levels of Apaf1 or pro-Casp9 (not shown).

Enhancer of Apaf1 Involved in Cyt c-dependent Caspase Activation9244

by guest on October 7, 2020

http://ww

w.jbc.org/

Dow

nloaded from

cells, in terms of caspase activation. In contrast, sensitivity togranzyme B-mediated caspase activation was not affected, con-firming a specific defect in the cyt-c pathway which depends onApaf1 for caspase activation. Further evidence that NAC reg-ulates the cyt-c/Apaf1-dependent activation of caspases in cellextracts was obtained by affinity preabsorption of NAC fromextracts using a GST fusion protein containing the CARD ofNAC and by expression in cells of a dominant-negative frag-ment of NAC consisting of only the CARD domain (not shown).

Consistent with experiments involving cell extracts, NACalso collaborated with Apaf1 in inducing caspase activation andapoptosis in intact cells. In transient transfection experimentsusing HEK293 (Fig. 4, D and E) or other cell lines (not shown),overexpression of NAC by itself (not shown) or in combinationwith pro-Casp9 had little effect on caspase activation or apo-ptosis. In contrast, overexpressing NAC together with Apaf1and pro-Casp9 resulted in synergistic increases in activation ofcaspases and induction of apoptosis, as determined from co-transfections that employed suboptimal amounts of Apaf1-en-coding plasmid (Fig. 4, D and E). Overexpression of NAC alsosensitized cells to suboptimal concentrations of apoptosis in-ducers such as staurosporine, which triggers apoptosis throughan Apaf1-dependent mechanism (30), but not by anti-Fas an-tibody which utilizes an Apaf1-independent pathway (Fig. 4Eand not shown). Immunoblotting experiments demonstratedthat the enhanced sensitivity of NAC-transfected cells to stau-rosporine was not due to differences in the levels of Apaf1 orpro-Casp9 proteins produced in cells (not shown).

Whereas full-length NAC enhanced apoptosis and caspaseactivation induced by overexpressing Apaf1 or by treatment ofcells with Apaf1-dependent apoptotic stimuli (Fig. 4, D and E),fragments of NAC containing only the CARD or NB domainhad the opposite effect, interfering with apoptosis induced bycoexpression of Apaf1/pro-Casp9 and by Apaf1-dependent stim-uli such as staurosporine without affecting Apaf1-independentpathways activated by Fas (Fig. 4F). Again, immunoblottingexperiments demonstrated that these reductions in Apaf1-de-pendent apoptosis caused by these dominant-negative frag-ments of NAC were not secondary to effects on levels of theApaf1 or pro-Casp9 proteins (not shown).

Although some CARD-containing proteins, including Nod1/CARD-4 (25, 26) and Bcl10/mE10 (31, 32), reportedly induceNF2kB activation, NAC did not induce NF-kB, when overex-pressed in cells (data not shown).

Cells of various tissues vary in their sensitivity to cyt-c-induced activation of caspases, a finding that cannot be ac-counted for by differences in the levels of Apaf1 protein ordownstream caspases (33, 34). The discovery of NAC suggestsa mechanism for fine-tuning Apaf1 function, based on whetherNAC is expressed and perhaps on whether NAC interacts withunidentified proteins via its LRR or other domains. Apaf1/CED4 family proteins directly bind CARD-containing caspases,promoting protease activation upon oligomerization by bring-ing the suboptimally active pro-enzymes into close proximity,allowing them to trans-process each other (4, 12–14). AlthoughNAC enhances cyt-c-mediated pro-Casp9 processing, it doesnot directly bind this caspase (nor caspases-1, -2, -6, -7, -8, -10,

or -11).2 Rather, interactions of NAC with Apaf1 facilitateApaf1-mediated activation of pro-Casp9, thus revealing a newparadigm for apoptosis regulation. The precise mechanism bywhich NAC enhances Apaf1 function remains to be elucidated.

Acknowledgments—We thank E. Alnemri for caspase-9 and Apaf1cDNAs; G. Joyce for DNAzyme design; H. Bettendorf for technicalassistance; S. Matsuzawa, H. Chan, H. Zhang, and S. Kitada for dis-cussions; and R. Cornell for manuscript preparation.

REFERENCES

1. Yuan, J. Y., and Horvitz, H. R. (1990) Dev. Biol. 138, 33–412. Seshagiri, S., and Miller, L. (1997) Curr. Biol. 7, 455–4603. Chinnaiyan, A., Chaudhary, D., O’Rourke, K., Koonin, E., and Dixit, V. (1997)

Nature 388, 728–7294. Yang, X., Chang, H., and Baltimore, D. (1998) Science 281, 1355–13575. Jaroszewski, L., Rychlewski, L., Reed, J. C., and Godzik, A. (2000) Proteins 39,

197–2036. Chinnaiyan, A. M., O’Rourke, K., Lane, B. R., and Dixit, V. M. (1997) Science

275, 1122–11267. Salvesen, G. S., and Dixit, V. M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96,

10964–109678. Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90,

405–4139. Kobe, B., and Deisenhofer, J. (1995) Curr. Opin. Struct. Biol. 5, 409–416

10. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S., Ahmad, M., Alnemri, E.,and Wang, X. (1997) Cell 91, 479–489

11. Srinivasula, S. M., Ahmad, M., Fernandes-Alnemri, T., and Alnemri, E. S.(1998) Mol. Cell 1, 949–957

12. Zou, H., Li, Y., Liu, X., and Wang, X. (1999) J. Biol. Chem. 274, 11549–1155613. Saleh, A., Srinivasula, S., Acharya, S., Fishel, R., and Alnemri, E. (1999)

J. Biol. Chem. 274, 17941–1794514. Adrain, C., Slee, E. A., Harte, M. T., and Martin, S. J. (1999) J. Biol. Chem.

274, 20855–2086015. Rodriguez, A., Oliver, H., Zou, H., Chen, P., Wang, X., and Abrams, J. (1999)

Nat. Cell Biol. 1, 272–27916. Zhou, L., Song, Z., Tittel, J., and Steller, H. (1999) Mol. Cell 4, 745–75517. Kanuka, H., Sawamoto, K., Inohara, N., Matsuno, K., Okano, H., and Miura,

M. (1999) Mol. Cell 4, 757–76918. Cecconi, F., Alvarez-Bolado, G., Meyer, B. I., Roth, K. A., and Gruss, P. (1998)

Cell 94, 727–73719. Santoro, S. W., and Joyce, G. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94,

4262–426620. Cardone, M., Roy, N., Stennicke, H., Salvesen, G., Franke, T., Stanbridge, E.,

Frisch, S., and Reed, J. (1998) Science 282, 1318–132121. Deveraux, Q. L., Takahashi, R., Salvesen, G. S., and Reed, J. C. (1997) Nature

388, 300–30422. Schendel, S., Azimov, R., Pawlowski, K., Godzik, A., Kagan, B., and Reed, J.

(1999) J. Biol. Chem. 274, 21932–2193623. Chou, J., Matsuo, H., Duan, H., and Wagner, G. (1998) Cell 94, 171–18024. Qin, H., Srinivasula, S., Wu, G., Fernandes-Alnemri, T., Alnemri, E., and Shi,

Y. (1999) Nature 399, 549–55725. Bertin, J., Nir, W.-J., Fischer, C., Tayber, O., Errada, P., Grant, J., Keilty, J.,

Gosselin, M., Robison, K., Wong, G., Glucksmann, M., and DiStefano, P.(1999) J. Biol. Chem. 274, 12955–12958

26. Inohara, N., Koseki, T., Del Peso, L., Hu, Y., Yee, C., Chen, S., Carrio, R.,Merino, J., Liu, D., Ni, J., and Nunez, G. (1999) J. Biol. Chem. 274,14560–14567

27. Yang, J., Liu, X., Bhalla, K., Kim, C. N., Ibrado, A. M., Cai, J., Peng, I.-I.,Jones, D. P., and Wang, X. (1997) Science 275, 1129–1132

28. Cain, K., Brown, D. G., Langlais, C., and Cohen, G. M. (1999) J. Biol. Chem.274, 22686–22692

29. Cain, K., Bratton, S. B., Langlais, C., Walker, G., Brown, D. G., Sun, X. M., andCohen, G. M. (2000) J. Biol. Chem. 275, 6067–6070

30. Yoshida, H., Kong, Y. Y., Yoshida, R., Elia, A. J., Hakem, A., Hakem, R.,Penninger, J. M., and Mak, T. W. (1998) Cell 94, 739–750

31. Willis, T., Jadayel, D., Peng, H., Perry, A., Rauf-Abdul, M., Price, H., Karran,L., Majekodunmi, O., Wlodarska, I., Pan, L., Crook, T., Hamoudi, R.,Isaacson, P., and Dyer, M. (1999) Cell 96, 35–45

32. Yan, M., Lee, J., Schilbach, S., Goddard, A., and Dixit, V. (1999) J. Biol. Chem.275, 10287–10292

33. Burgess, D., Svensson, M., Dandrea, T., Groulund, K., Hammarquist, F.,Orrenius, S., and Cotgreave, I. (1999) Oncogene 6, 256–261

34. Deshmukh, M., and Johnson, J. E. (1998) Neuron 21, 695–70535. Krajewski, S., Krajewska, M., Ellerby, L. M., Welsch, K., Xie, Z., Deveraux,

Q. L., Salvesen, G. S., Bredesen, D. E., Rosenthal, R. E., Fiskum, G., andReed, J. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 5752–5757

Enhancer of Apaf1 Involved in Cyt c-dependent Caspase Activation 9245

by guest on October 7, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Krajewski, Adam Godzik and John C. ReedZhi-Liang Chu, Frederick Pio, Zhihua Xie, Kate Welsh, Maryla Krajewska, Stan

Caspase Activation and Apoptosis -dependentcA Novel Enhancer of the Apaf1 Apoptosome Involved in Cytochrome

doi: 10.1074/jbc.M006309200 originally published online December 11, 20002001, 276:9239-9245.J. Biol. Chem. 

  10.1074/jbc.M006309200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/276/12/9239.full.html#ref-list-1

This article cites 35 references, 15 of which can be accessed free at

by guest on October 7, 2020

http://ww

w.jbc.org/

Dow

nloaded from