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© 1999 Macmillan Magazines Ltd

.................................................................NF-kB is a target of AKT inanti-apoptotic PDGF signallingJulia A. Romashkova & Sergei S. Makarov

Thurston Arthritis Research Center, and Center for In¯ammatory Disorders,University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599±

7280, USA

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The mechanisms of cell proliferation and transformation areintrinsically linked to the process of apoptosis: the default ofproliferating cells is to die unless speci®c survival signals areprovided1,2. Platelet-derived growth factor (PDGF) is a principalsurvival factor that inhibits apoptosis and promotes pro-liferation1, but the mechanisms mediating its anti-apoptoticproperties are not completely understood. Here we show thatthe transcription factor NF-kB3±5 is important in PDGF signal-ling. NF-kB transmits two signals: one is required for the induc-tion of proto-oncogene c-myc and proliferation, and the second,an anti-apoptotic signal, counterbalances c-Myc cytotoxicity. Wehave traced a putative pathway whereby PDGF activates NF-kBthrough Ras and phospatidylinositol-3-kinase (PI(3)K) to thePKB/Akt protein kinase and the IkB kinase (IKK); NF-kB thusappears to be a target of the anti-apoptotic Ras/PI(3)K/Aktpathway6,7. We show that, upon PDGF stimulation, Akt transientlyassociates in vivo with IKK and induces IKK activation. These®ndings establish a role for NF-kB in growth factor signallingand de®ne an anti-apoptotic Ras/PI(3)K/Akt/IKK/NF-kB path-way, thus linking anti-apoptotic signalling with transcriptionmachinery.

c-Myc is a central regulator of cell growth, death and differen-tiation. c-Myc is required for cell proliferation but, in the absenceof survival factors, it induces apoptosis1,8. PDGF inhibits c-Myc-induced apoptosis by activating the Ras/PI(3)K/Akt6,7 pathway, butthe downstream effectors of this pathway are poorly de®ned. Oneconsequence of PDGF treatment is the activation of NF-kB9. NF-kBcontrols the expression of numerous genes involved in in¯amma-tion, tumour development and immune responses3±5. In unstimu-lated cells, NF-kB is retained in the cytoplasm through aninteraction with inhibitory proteins known as IkB. The canonicalmechanism of NF-kB activation involves signal-inducible degrada-tion of IkB, which causes the translocation of NF-kB to the nucleusand transcription activation of targeted genes. As NF-kB hasbeen implicated in the regulation of apoptosis4, proliferation10

and c-Myc expression11, we investigated a role for NF-kB inPDGF signalling.

We used two types of primary ®broblast: normal human skin®broblasts (NHF) and rat ®broblast-like synoviocytes (FLS). PDGFtreatment induced NF-kB DNA-binding activity, which becameapparent after 1 h of stimulation, peaked at 3 h (Fig. 1a) anddeclined thereafter (data not shown). Supershift analysis hasshown that the induced NF-kB was a RelA/p50 heterodimer (datanot shown). The induction of NF-kB DNA-binding activity coin-cided with the degradation of IkBa (Fig. 1a). To examine the role ofNF-kB in mitogenic activity of PDGF, NF-kB activation wasarrested by infecting the cells with an adenoviral vector encodinga non-degradable (`super-repressor') form of IkBa (AdsrIkBa)12

(Fig. 1a). This inhibited DNA synthesis (Fig. 1b) and abrogatedthe induction of c-Myc message (data not shown) and protein(Fig. 1c). Thus, NF-kB activation is required for the expression ofc-Myc and proliferation.

To investigate the function of NF-kB in anti-apoptotic PDGFsignalling, exogenous c-Myc and super-repressor IkBa (srIkBa)were constitutively expressed in serum-deprived cells. To facilitate

detection, a reporter vector encoding green ¯uorescent protein(EGFP) was co-transfected with the c-Myc and srIkBa vectors. Inthe absence of PDGF, constitutive c-Myc expression caused wide-spread death (Fig. 2a); the transfected cells had a characteristicapoptotic appearance, being round and condensed (data notshown). As expected, PDGF rescued the cells from c-Myc-inducedapoptosis, but the co-expression of srIkBa abrogated the rescue(Fig. 2a). To minimize the in¯uence of variability in transfectionsand the observation error, we modi®ed the apoptotic assay. In amodi®ed experiment, we divided the transfected cells between twodishes and investigated the time course of cell viability. c-Myc-induced cell death was delayed and inhibited by PDGF (Fig. 2b, left),but the expression of srIkBa abrogated this protection (Fig. 2b,right). In the absence of c-Myc, the expression of srIkBa did notaffect cell viability regardless of the presence of PDGF (Fig. 2b,right). These data indicate that NF-kB activation is required for theanti-apoptotic activity of PDGF. To examine the anti-apoptoticeffects of NF-kB activation, exogenous NF-kB (p50 and RelAsubunits) was co-expressed with c-Myc in serum-deprived cells.Although p50 alone had no apparent effect, the expression of RelAalone or in a combination with p50 increased cell viability by 3 to 5-fold (Fig. 2c), indicating that NF-kB activation itself can inhibitc-Myc-induced apoptosis.

How does PDGF activate NF-kB? As the Ras/PI(3)K/Akt pathwayis critical in anti-apoptotic PDGF signalling, we examined thepossible role of this pathway. The expression of a dominant negative(DN) RasN17 mutant strongly inhibited PDGF-induced NF-kBactivation (Fig. 3a), indicating that Ras may be involved in NF-kBactivation. In anti-apoptotic PDGF signalling, PI(3)K is the proxi-mal downstream target of Ras13. Wortmannin and LY294002,speci®c inhibitors of PI(3)K, both inhibited NF-kB activation(Fig. 3b), indicating that PI(3)K may be involved in PDGF-inducedNF-kB activation. Importantly, neither DN Ras nor wortmannin

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86 NATURE | VOL 401 | 2 SEPTEMBER 1999 | www.nature.com

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Figure 1 The mitogenic function of NF-kB. FLS were infected with Ad vectors as indicated,

incubated for 48 h in a serum-free medium and stimulated with PDGF. a, Time course of

NF-kB activation. IkBa protein (top) and NF-kB DNA-binding activity (bottom) were

determined in the cytosolic and nuclear extracts. b, NF-kB activation is required for DNA

synthesis. [3H]Thymidine incorporation was determined after 48 h PDGF stimulation.

Mean values 6 s.e.m. are shown from three experiments performed in duplicate.

Identical results were obtained in NHF (data not shown). c, NF-kB activation is required for

c-Myc protein expression. c-Myc immunostaining was performed after 24 h PDGF

stimulation. Original magni®cation 200´.


affected the induction of NF-kB by tumour necrosis factor-a(TNFa) and 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulithat probably do not involve Ras and PI(3)K (Fig. 3c).

As Akt is a proximal downstream effector of the anti-apoptoticRas/PI(3)K pathway6,7,13, we next examined the involvement of Aktin NF-kB activation. To this end, we used a kinase-dead AktK179Mmutant14, which effectively inhibited the activation of Akt by PDGF(data not shown). The expression of AktK179M strongly inhibitedNF-kB reporter-gene expression, indicating that Akt is involvedin NF-kB activation (Fig. 3d). Similarly to the RasN17 and PI(3)Kinhibitors, AktK179M expression did not affect cell viability (datanot shown), thus ruling out the possibility that the inhibition ofNF-kB reporter-gene expression re¯ected a cytotoxic effect. Ourdata indicate that, in PDGF signalling, NF-kB is a target of the Ras/PI(3)/Akt pathway.

We next investigated the link between Akt and NF-kB. PDGFtreatment causes degradation of IkBa (Fig. 1a), which indicates thatIKK is involved. The inducible degradation of IkBa occurs throughseveral consecutive events, an initial step being the phosphorylationof IkBa by a multisubunit IKK complex, the signalsome5. Twocatalytic subunits of IKK, IKKa and IKKb, phosphorylate IkBa onresidues Ser 32 and Ser 36 and target IkBa to ubiquitination andproteasomal degradation5,15. To inhibit IKK, we used DN mutants ofthe IKKa and IKKb subunits. Whereas DN IKKa exerted onlyminimal (if any) inhibitory effect (data not shown), the expressionof DN IKKb strongly inhibited PDGF-induced NF-kB reporter-

gene expression (Fig. 3e). This prompted us to explore the causalrelationship between Akt and IKK in NF-kB activation. We foundthat the expression of a constitutively active myristylated Akt(myrAkt) alone was suf®cient for NF-kB activation (12-foldinduction in FLS (Fig. 3f) and 7-fold induction in NHF (data notshown)). The induction of NF-kB reporter by myrAkt was con-sistently observed in other cell types, including NIH 3T3 ®broblasts(data not shown). DN IKKb attenuated myrAkt-induced NF-kBactivation (Fig. 3f), indicating that IKK is a target of Akt in PDGFsignalling. To investigate the link between Akt and IKK, we assessedthe temporal relationship between these kinases. To assess thekinetics of Akt activation, we determined its phosphorylation onresidues Thr 308 and Ser 473, which is a prerequisite for the catalyticactivity of Akt13. Akt phosphorylation was apparent after 5 min ofstimulation, reached a peak at 60 min and declined thereafter(Fig. 4a). These kinetics overlapped with the time course of IKKactivation: the phosphorylation of IkBa on Ser 32 was evident30 min after PDGF stimulation and persisted for at least 2 h(Fig. 4b and data not shown). These data indicate that IKK might

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Figure 2 The anti-apoptotic function of NF-kB. a, NF-kB activation is required for the

anti-apoptotic activity of PDGF. Cell viability was assessed after 18 h incubation in a low-

serum medium. Bars represent mean values 6 s.e.m. from four experiments in FLS (left)

and one experiment in NHF (right). b, Time course of c-Myc-induced apoptosis in FLS in

the absence (left) or presence (right) of srIkBa. c, The overexpression of NF-kB inhibits c-

Myc-induced apoptosis. Cell viability was assessed after 18-h incubation in a low serum

medium. Bars represent mean values 6 s.e.m. from two experiments in each of FLS (left)

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Figure 3 NF-kB is a target of a Ras/PI(3)K/Akt/IKK pathway. Unless otherwise noted,

transfected cells (5 mg of each vector) were serum-starved for 48 h and stimulated with

PDGF for 18 h. Bars represent mean values 6 s.e.m. from n individual experiments

performed in duplicate. a, The involvement of Ras. PDGF-induced activation of 3xkBCAT

in individual experiments was in the range of 8- to 37- fold in FLS (n � 4) and 9- to 15-

fold in NHF (n � 3). b, The involvement of PI(3)K. Wortmannin (100 nM) and LY924002

(10 mM) were added 30 min before PDGF (FLS, n � 2; NHF; n � 1). c, Inhibitors of Ras/

PI(3)K do not affect TNFa- and PMA-induced NF-kB activation. FLS were stimulated with

TNFa (50 ng ml-1), PMA (50 ng ml-1) or PDGF for 6 h. Stippled bars: DN Ras-transfected

cells. Hatched bars: wortmannin-pretreated cells. TNFa-, PMA- and PDGF-induced wild-

type 3xkB CAT activation was 37-, 11- and 8-fold, respectively; these values were

accepted for 100% induction (solid bars) (FLS, n � 2). d, The involvement of Akt (FLS,

n � 2; NHF, n � 2). e, The involvement of IKK (FLS, n � 3). f, NF-kB activation by

myrAkt. FLS were transfected with 2.5 mg of each vector. Mean values 6 s.d. from a

single experiment performed in duplicate are shown. Identical data were obtained in NHF

(not shown).


be a direct target of Akt, an assumption reinforced by the observa-tion that PDGF-mediated phosphorylation of IkBa did not requirede novo protein synthesis (Fig. 4b and data not shown).

To assess the possible interaction between Akt and the IKKsignalsome in vivo, we immunoprecipitated endogenous Akt andanalysed the immune complexes for the presence of IKK using acombination of Akt and IKKa antibodies. The immunodetectionrevealed two bands corresponding to Akt (relative molecular mass�Mr� � 63K) and IKKa (Mr � 85K; Fig. 4c). These bands wereabsent when the immune complexes were precipitated by pread-sorbed Akt antibodies, indicating the speci®city of detection(Fig. 4c). Interestingly, the association of IKK and Akt was detectedonly in cells stimulated with PDGF for 1 h, not in unstimulated cellsor those stimulated for 4 h. Consistent with the data shown inFig. 4a, we found that the immunoprecipitated Akt was phosphoryl-ated at 1 h of stimulation, and, to a much lesser degree, at 4 h(Fig. 4c). Similar kinetics of Akt and IKK association were observedin NIH 3T3 ®broblasts (Fig. 4d). In these experiments, we detectedIKK in Akt immune complexes using an antibody against IKKb,another catalytic subunit of the IKK complex. Furthermore, theimmunoprecipitation of endogenous IKKb in PDGF-stimulatedNIH 3T3 cells brought down endogenous Akt (Fig. 4d). Combined,these observations show that, upon PDGF stimulation, Akt and IKKtransiently associate in vivo.

To determine whether this association was functional (inducedthe catalytic activity of IKK) NIH 3T3 cells were transfected withhaemagglutinin (HA)-tagged Akt vectors, stimulated with PDGFfor 1 h, immunoprecipitated with anti-HA antibodies and assessedfor IkB kinase activity. As a positive control, endogenous IKKactivity was determined in TNFa-stimulated untransfected cells.In cells transfected with wild-type (wt)Akt.HA, IKK activity wasdetected only in HA immune complexes from PDGF-treated cells,whereas myrAkt.HA-transfected cells contained IKK activity with-out stimulation (Fig. 4e). Conversely, IKK activity was undetectablein PDGF-stimulated cells transfected with the kinase-deadAktK179M.HA (Fig. 4f). The IKK activity correlated with thepresence of IKK protein (Fig. 4e). Notably, Akt was undetectablein IKK immune complexes from TNFa-stimulated cells (Fig. 4e).This indicates that, in contrast to PDGF, TNFa does not induce theassociation of Akt with IKK. Together, our data indicate that PDGF-stimulated activation of NF-kB is mediated by transient associationof Akt with IKK and the activation of IKK.

Our study indicates that, in PDGF signalling, proliferative andpro-apoptotic (c-Myc induction) and anti-apoptotic signals bifur-cate at the level of NF-kB (Fig. 5). Consistent with the `dual-signal'hypothesis postulating coupling of the proliferation pathways withthose of cell death1,2, NF-kB has a dual role: it mediates theinduction of pro-apoptotic c-Myc, and delivers an anti-apoptoticsignal counterbalancing c-Myc cytotoxicity, conceivably by induc-ing the expression of a protective gene (or multiple genes). Giventhe roles of ras, myc and akt in cancer, our study has evidentrami®cations for understanding the mechanisms of cell transforma-tion. It may explain why the ability of Ras to transform cells dependson NF-kB activation16. Oncogenic Ras activates two independentexecutive mechanisms: an anti-apoptotic PI(3)K/Akt pathway and apro-apoptotic Raf/MAPK pathway6,17. Our results indicate thatinhibition of NF-kB might block anti-apoptotic Akt signallingwithout interfering with the pro-apoptotic pathway. Our observa-tions also indicate a novel role for NF-kB in rheumatoid arthritis.One feature of this disease is aggressive hyperplasia of the synoviumassociated with overexpression of c-Myc and activation of NF-kB18.As PDGF is a principal growth factor in rheumatoid arthritis FLScells, NF-kB activation may contribute to synovial hyperplasia bypromoting proliferation and inhibiting c-Myc-induced apoptosis.Our data identify NF-kB as target of the anti-apoptotic Ras/PI(3)K/Akt pathway. Several putative targets of this pathway have beenproposed, among them Bad19,20, caspase-921 and the transcriptionfactor Forkhead22,23. As all these targets represent proteins withintrinsic pro-apoptotic properties, Akt appeared to be a negativeregulator of the cell-death machinery. Our data indicate that Aktcan also promote survival by activating anti-apoptotic NF-kBsignalling. The interplay of these mechanisms in the anti-apoptoticactivities of Akt13 has yet to be determined. The mechanism wherebyPDGF activates NF-kB also warrants further investigation. Ourobservations indicate that this mechanism involves consecutiveactivation of Akt and IKK, which is consistent with the observedphosphorylation and degradation of IkBa and induction of NF-kBDNA-binding activity. However, under certain circumstances, PI(3)Kcan activate NF-kB through a mechanism that does not involve IkBdegradation24. There is also evidence that Akt can activate NF-kBtranscriptional function through an alternative IkB-independent

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a b

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Figure 4 Akt associates with IKK in vivo and induces IKK activation. a, Akt activation. FLS

lysates were consecutively immunodetected with phospho-Akt (Ser 473) (top) and total

Akt (bottom) antibodies. NIH 3T3 lysates were from the antibody manufacturer. b, IkBa

phosphorylation. FLS lysates were consecutively immunodetected with p-IkBa (Ser 32)

(top) and total IkBa (bottom) antibodies. CHX, cycloheximide. c, The association of Akt

and IKK in FLS. Akt immune complexes were consecutively immunodetected with primary

Akt and IKKa (top) and p-Akt (bottom) antibodies. Mr, relative molecular mass. d, The

association of Akt and IKK in NIH 3T3 cells. Akt and IKKb immune complexes were

immunodetected with primary Akt and IKKb antibodies. e, Akt activates IKK. HA.Akt and

IKKa immune complexes were assayed for IKK activity (top). The 35K GST-IkBa band is

shown. The blot was consecutively immunodetected with IKKa (middle) and Akt (bottom)

antibodies. f, Catalytic activity of Akt is required for IKK activation. IKK activity and Akt

protein were assessed as in e.

PDGF Ras NF-κB

anti-apoptotic gene(s)

c-Myc

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apoptosisPI(3)K Akt IKK

Figure 5 NF-kB in PDGF signalling. PDGF activates the Ras/PI(3)K/Akt pathway. Activated

Akt transiently associates with IKK and induces the activation of IKK and NF-kB. NF-kB

transmits a signal required for the induction of c-Myc expression and proliferation, and an

anti-apoptotic signal that inhibits c-Myc cytotoxicity.


mechanism, by modulating the transcriptional potential of RelA (M.Mayo, L. Madrid and A. Baldwin, personal communication). Thecontribution of these pathways remains to be clari®ed. Another issueis the role for the atypical isoforms of protein kinase C (PKC) andreactive oxygen intermediates (ROI), both of which have beenimplicated in mitogenic signalling25,26 and NF-kB activation3,4. Asthe atypical forms of PKC can associate with both Akt27 and IKK28,this indicates possible cooperation between PKC and Akt. Together,our ®ndings indicate that the association of Akt and IKK mayrepresent a distinct channel for NF-kB activation, similar to thoseprovided by NIK and MEKK1 in TNFa and IL-1 signalling5,29. M

MethodsReagents.

Unless otherwise noted, human recombinant PDGF-BB (Gibco BRL) was used at a ®nalconcentration of 50 ng ml-1. Plasmids wtAkt.HA, AktK179M.HA and myrAkt.HA weregifts from P. Tsichlis; RasN17 was a gift from C. Der; and IKKa (K44A) and IKKb (K44A)were gifts from J. Woronicz. The CMV RelA, CMV p50, CMV srIkBa, CMV c-Myc and3xkBCAT vectors were gifts from A. Baldwin. The EGFP vector was from Promega. Allplasmids were puri®ed from bacterial endotoxin using Detoxigel (Pierce). Adenoviralinfections were performed as described12.

Cell culture.

We obtained primary FLS from rats with streptococcal cell wall (SCW)-induced arthritis30.The cells were maintained in RPMI 1640/15% FBS and used at passages 5±18. Primaryforeskin NHF were a gift from W. Kaufmann. The cells were maintained in RPMI 1640/10% FBS and used at passages 12±20.

Proliferation assay.

Ad-infected FLS (104 cells per well) were starved for 48 h and stimulated with PDGF foranother 48 h. [3H]thymidine (0.5 mCi per well) was added during the last 24 h. Inindividual experiments, incorporated radioactivity in PDGF-stimulated AdCMV-infectedcells was in the range of 3,000 to 6,000 c.p.m.

NF-kB assays.

We determined NF-kB DNA-binding activity in FLS using electromobility-shift assay(EMSA) as described12. The speci®city of binding was determined by competition with acold probe and by supershift analysis with anti-p50 and anti-RelA antibodies. To assessNF-kB reporter-gene expression, cells (105 cells per 100 mm dish) were transfected withwild-type or mutant 3xkBCAT reporter vectors and corresponding DN inhibitors. Unlessotherwise noted, each transfection contained 180 mg of SuperFect reagent (Quiagen) and5 mg of each reporter and expression vector. The amount of DNA was kept constant byaddition of an empty CMV plasmid. CAT activity was determined using the Quan-T-CATkit (Amersham) and normalized on total protein. In some experiments, we used 3xkBLUC reporters; the luciferase assays were in good agreement with CAT assays.

Apoptotic assays.

Cells were co-transfected in a complete medium with a reporter EGFP along with c-Mycand srIkBa expression vectors (2 mg of each DNA per 100 mm dish). Attached ¯uorescentcells were counted in three randomly chosen ®elds after 18 h incubation in a low serum(0.1% FBS) medium. In each experiment, at least 400 ¯uorescent cells were scored inPDGF-treated control (EGFP only) cells; these numbers were accepted for 100% viability.Alternatively, transfected FLS were divided between two dishes, allowed to adhere for 24 hand incubated in the low serum medium. Three randomly chosen observation ®elds weremarked on the dishes, and the numbers of attached ¯uorescent cells were counted atindicated time points. To assess the in¯uence of NF-kB overexpression on c-Mycapoptosis, EGFP and c-Myc were co-transfected with p50 and RelA expression vectors(1.5 mg of each DNA per 100 mm dish), and the viability was assessed after 18 h incubationin low serum medium. At least 250 ¯uorescent cells were scored in PDGF-treated controlcells (EGFP plus c-Myc) (accepted for 100% viability).

Immunodetection.

For c-Myc immunostaining we used primary c-Myc antibodies (sc-764, Santa CruzBiotechnology) (1:200 dilution) and a secondary HRP-conjugated antibody. For westernblotting, lysates were resolved on SDS±PAGE and transferred to nitrocellulose. Total andphosphorylated IkBa were detected with primary IkBa antibodies (Rockland Inc.)(1:1000) and p-IkBa (Ser 32) antibodies (1:1000) (New England Biolabs). Whereindicated, cells were pre-treated for 1 h with cycloheximide (CHX) (10 mg ml-1); toprevent degradation of phospho-IkBa, proteasomal inhibitor MG132 (5 mM) was addedwith CHX. Total and phosphorylated Akt were detected with primary Akt (1:1000) and p-Akt (Ser 473) (1:1000) antibodies (New England Biolabs). IKKa was detected using eithergoat polyclonal antibodies against the carboxy-terminus of IKKa (1:1000) (sc-7120, SantaCruz Biotechnology) or monoclonal antibodies against full-length IKKa (1.5 mg ml-1)(Pharmingen). IKKb was detected using antibodies against its C-terminus (1:400) (a giftfrom A. Manning). ECL detection was performed using an ECL kit (Du Pont NEN).

Immunoprecipitation.

Cells were lysed in a lysis buffer (5 mM HEPES pH 7.4, 150 mM NaCl, 1% Triton X-100,10 mM glycerol, 1 mM EDTA, 2 mM Na3VO4, 5 mM phenylmethylsulphonil ¯uoride(PMSF), 5 mg ml-1 aprotinin, 5 mg ml-1 leupeptine and 5 mg ml-1 pepstatine). EndogenousAkt was immunoprecipitated by 1-h incubation with an anti-Akt antibody (NEB) (1 mgper 1 mg of protein) and 50 ml of protein A Sepharose (Pharmacia). Where indicated,the antibody was preadsorbed with an Akt peptide (a gift from T. Davis). Endogenous IKKwas immunoprecipitated by 1-h incubation with 1 mg of an antibody against the C-terminus of IKKb and 50 ml of protein A Sepharose. Immune complexes were washed ®vetimes with lysis buffer, boiled and subjected to SDS±PAGE.

IkB kinase assay.

NIH 3T3 cells were transiently transfected with HA-tagged Akt vectors, and starved byincubating in a 0.5% FBS medium for 48 h, followed by an overnight incubation in aserum-free medium. Cells were stimulated for 1 h with PDGF (100 ng ml-1) and immu-noprecipitated with HA antibodies (Y-11, Santa Cruz Biochemicals (SCB)) (1 mg per 1 mgcell protein). Where indicated, the antibody was pre-adsorbed with an HA peptide (SCB).In control reactions, serum-starved untransfected cells were stimulated for 10 min withTNFa (100 ng ml-1) and cell lysates were immunoprecipitated with IKKa monoclonalantibodies (Pharmingen) (2 mg per 1 mg cell protein). In vitro IkB kinase activity wasperformed with recombinant GST-IkBa (1±54) as a substrate, as described15. Reactionproducts were separated by 10% SDS±PAGE, transferred to nitrocellulose and visualizedwith a phosphoimager. The blots were subsequently immunodetected for the presence ofAkt and IKK proteins.

Statistics.

P values of comparisons were calculated using a paired two-tailed t-test.

Received 6 May; accepted 21 June 1999.

1. Harrington, E. A., Bennett, M. R., Fanidi, A. & Evan, G. I. c-Myc-induced apoptosis in ®broblasts is

inhibited by speci®c cytokines. EMBO J. 13, 3286±3295 (1994).

2. Evan, G. CancerÐa matter of life and cell death. Int. J. Cancer 71, 709±711 (1997).

3. Baldwin, A. S. Jr. The NF-kB and I-kB proteins: new discoveries and insights. Annu. Rev. Immunol. 14,

649±683 (1996).

4. Yin Foo, S. & Nolan, G. P. NF-kappaB to the rescue: RELS, apoptosis and cellular transformation.

Trends Genet. 15, 229±235 (1999).

5. Mercurio, F. & Manning, A. M. Multiple signals converging on NF-kappaB. Curr. Opin. Cell Biol. 11,

226±232 (1999).

6. Kauffmann-Zeh, A. et al. Suppression of c-Myc-induced apoptosis by Ras signalling through PI(3)K

and PKB. Nature 385, 544±548 (1997).

7. Kennedy, S. G. et al. The PI 3-kinase/Akt signaling pathway delivers an anti-apoptotic signal. Genes

Dev. 11, 701±713 (1997).

8. Evan, G. I. et al. Induction of apoptosis in ®broblasts by c-myc protein. Cell 69, 119±128 (1992).

9. Olashaw, N. E., Kowalik, T. F., Huang, E. S. & Pledger, W. J. Induction of NF-kB-like activity by

platelet-derived growth factor in mouse ®broblasts. Mol. Biol. Cell 3, 1131±1139 (1992).

10. Hinz, M. et al. NF-kB function in growth control: regulation of cyclin D1 expression an dG0/G1-to-S-

phase transition. Mol. Cell. Biol. 19, 2690±2698 (1999).

11. Kessler, D. J., Duyao, M. P., Spicer, D. B. & Sonenshein, G. E. NF-kB-like factors mediate interleukin 1

induction of c-myc gene transcription in ®broblasts. J. Exp. Med. 176, 787±792 (1992).

12. Miagkov, A. V. et al. NF-kB activation provides the potential link between in¯ammation and

hyperplasia in the arthritic joint. Proc. Natl Acad. Sci. USA 95, 13859±13864 (1998).

13. Downward, J. Mechanisms and consequences of activation of protein kinase B/Akt. Curr. Opin. Cell

Biol. 10, 262±267 (1998).

14. Franke, T. F. et al. The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-

activated phosphatidylinositol 3-kinase. Cell 81, 727±736 (1995).

15. Mercurio, F. et al. IKK-1 and IKK-2: cytokine-activated IkB kinases essential for NF-kB activation.

Science 278, 860±866 (1997).

16. Mayo, M. W. et al. Requirement of NF-kB activation to suppress p53-independent apoptosis induced

by oncogenic Ras. Science 278, 1812±1815 (1997).

17. Marte, B. M., Rodriguez-Viciana, P., Wennstrom, S. Warne, P. H. & Downward, J. R-Ras can activate

the phosphoinositide 3-kinase but not the MAP kinase arm of the Ras effector pathways. Curr. Biol. 7,

63±70 (1997).

18. Firestein, G. S. & Manning, A. M. Signal transduction and transcription factors in rheumatic disease.

Arthritis Rheum. 42, 609±621 (1999).

19. Datta, S. R. et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death

machinery. Cell 91, 231±241 (1997).

20. del Peso, L., Gonzalez-Garcia, M., Page, C., Herrera, R. & Nunez, G. Interleukin-3-induced

phosphorylation of BAD through the protein kinase Akt. Science 278, 687±689 (1997).

21. Cardone, M. H. et al. Regulation of cell death protease caspase-9 by phosphorylation. Science 282,

1318±1321 (1998).

22. Brunet, A. et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead

transcription factor. Cell 96, 857±868 (1999).

23. Kops, G. J. P. L. et al. Direct control of the Forkhead transcription factor AFX by protein kinase B.

Nature 398, 630±634 (1999).

24. Beraud, C., Henzel, W. J. & Baeuerle, P. A. Involvement of regulatory and catalytic subunits of

phosphoinositide 3-kinase in NF-kB activation. Proc. Natl Acad. Sci. USA 96, 429±434 (1999).

25. Berra, E. et al. Protein kinase C-z isoform is critical for mitogenic signal transduction. Cell 74, 555±

563 (1993).

26. Sundaresan, M., Yu, Z.-X., Ferrans, V. J., Irani, K. & Finkel, T. Requirement for generation of H2O2 for

platelet-derived growth factor signal transduction. Science 270, 296±299 (1995).

27. Konishi, H., Shinomura, T., Kuroda, S., Ono, Y. & Kikkawa, U. Molecular cloning of rat RAC protein

kinase a and b and their association with protein kinase C z. Biochem. Biophys. Res. Commun. 205,

817±825 (1994).

letters to nature

NATURE | VOL 401 | 2 SEPTEMBER 1999 | www.nature.com 89


28. Lallena, M. J., Diaz-Meco, M. T., Bren, G., Paya, C. V. & Moscat, J. Activation of kB kinase beta by

protein kinase C isoforms. Mol. Cell. Biol. 19, 2180±2188 (1999).

29. Karin, M. & Delhase, M. JNK or IKK, AP-1 or NF-kB, which are the targets for MEK kinase 1 action?

Proc. Natl Acad. Sci. USA 95, 9067±9069 (1998).

30. Makarov, S. S. et al. Suppression of experimental arthritis by gene transfer of interleukin 1 receptor

antagonist cDNA. Proc. Natl Acad. Sci. USA 93, 402±406 (1996).

Acknowledgements

We thank P. Tsichlis, J. Woronicz, A. Manning, F. Mercurio, C. Der, T. Davis, W. Kaufmann,A. Danilkovitch and A. Baldwin for sharing reagents; A. Baldwin, C. Der and P. Cohen forstimulating discussions; C. Bradham for assistance in kinase assays; J. Watson for editorialassistance; and members of the S.M. laboratory, A. Miagkov and D. Kovalenko fortechnical assistance. This work was supported by grants from the National Institutes ofHealth and the Arthritis Foundation.

Correspondence and requests for materials should be addressed to S.M. (e-mail:[email protected]).

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.................................................................Globalunfoldingofasubstrateproteinby the Hsp100 chaperone ClpAEilika U. Weber-Ban*, Brian G. Reid*², Andrew D. Miranker³& Arthur L. Horwich*²

* Department of Genetics and ² Howard Hughes Medical Institute,

Yale University School of Medicine, Boyer Center, 295 Congress Avenue,

New Haven, Connecticut 06510, USA³ Department of Molecular Biophysics and Biochemistry, Yale University,260 Whitney Avenue, New Haven, Connecticut 06520, USA

..............................................................................................................................................

The bacterial protein ClpA, a member of the Hsp100 chaperonefamily, forms hexameric rings that bind to the free ends of thedouble-ring serine protease ClpP (refs 1, 2). ClpA directs the ATP-dependent degradation of substrate proteins bearing speci®csequences3±5, much as the 19S ATPase `cap' of eukaryotic protea-somes functions in the degradation of ubiquitinated proteins6±8.In isolation, ClpA and its relative ClpX can mediate the disas-sembly of oligomeric proteins9,10; another similar eukaryoticprotein, Hsp104, can dissociate low-order aggregates11. ClpA hasbeen proposed to destabilize protein structure, allowing passageof proteolysis substrates through a central channel into the ClpPproteolytic cylinder12±14. Here we test the action of ClpA on astable monomeric protein, the green ¯uorescent protein GFP,onto which has been added an 11-amino-acid carboxy-terminalrecognition peptide, which is responsible for recruiting truncatedproteins to ClpAP for degradation5,15. Fluorescence studies bothwith and without a `trap' version of the chaperonin GroEL, whichbinds non-native forms of GFP16, and hydrogen-exchange experi-ments directly demonstrate that ClpA can unfold stable, nativeproteins in the presence of ATP.

We ®rst tested whether GFP, a very stable monomeric protein (itis resistant to denaturation by 6M urea, for example; Fig. 1a), couldbe degraded by the ClpAP machinery once recruited to it as a resultof the attachment of an 11-residue ssrA peptide to the GFPterminus. Appendage of the peptide did not interfere with the¯uorescent properties or stability of the GFP portion: the taggedprotein (GFP11) gave the same ¯uorescence quantum yield andemission spectrum as non-tagged GFP (not shown) and was asstable to denaturant (Fig. 1a). When incubated with catalyticamounts of ClpA and ClpP in the presence of ATP, however,GFP11 was completely degraded, as evidenced both by loss of¯uorescence (Fig. 1b) and by the disappearance of the protein onSDS±polyacrylamide gel electrophoresis (Fig. 1c). The kinetics ofthe two measurements were similar, preventing the resolution of an

unfolding action from degradation. In contrast with the taggedprotein, non-tagged GFP gave no change in ¯uorescence or lossof protein when incubated under similar conditions with ClpA,ClpP and ATP (results not shown). We conclude that even the stableb-barrel of GFP can be destabilized and degraded by the ClpAPmachinery when recruited to it as a result of the addition of the ssrAsequence.

To test whether we could detect unfolding of GFP11 as a stepdistinct from the degradation carried out by ClpAP, we incubatedGFP11 with stoichiometric amounts of ClpA on its own. In thepresence of ATP, but not of ATP-gS, there was a small decrease(20%) in ¯uorescence intensity (Fig. 2a), indicating that somefraction of the input GFP11 molecules could be unfolded. Theobserved effect was small, however, compared with the effect ofadding a stoichiometric amount of ClpA and ClpP, which caused acomplete loss of ¯uorescence as a result of the total proteolyticdegradation of the added GFP11 (Fig. 2a, and results not shown).

We reasoned that, if GFP11 molecules unfolded by ClpA werereleased from it and then refolded, establishing an equilibriumbetween unfolding and refolding, then preventing refolding shouldreveal the unfolding action of ClpA, producing a larger drop in¯uorescence. To prevent refolding, we added a GroEL `trap' mutant(D87K, in which an aspartate residue at position 87 is replaced by a

Figure 1 GFP11, a derivative bearing an 11-amino-acid C-terminal ssrA tag recognized

by ClpA, exhibits the same stability to denaturants as untagged GFP, and is degraded by

ClpA/ClpP in the presence of ATP. a, Time course of ¯uorescence intensity changes of

GFP11 and GFP after dilution to 600 nM in 6 M urea at 23 8C (top), 6 M guanidine-HCl at

23 8C (middle), or 6 M guanidine-HCl at 37 8C (bottom). b, Time-course of the

¯uorescence intensity change of 10 mM GFP11 incubated with 100 nM ClpA, 50 nM ClpP

and 10 mM ATP. Excitation and emission wavelengths were 400 and 510 nm,

respectively. c, SDS±PAGE analysis of aliquots of the reaction used in b, taken at the

times indicated.

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