letters to nature

................................................................. detection, a reporter vector encoding green ¯uorescent protein

NF-kB is a target of AKT in (EGFP) was co-transfected with the c-Myc and srIkBa vectors. In
the absence of PDGF, constitutive c-Myc expression caused wide-
anti-apoptotic PDGF signalling spread death (Fig. 2a); the transfected cells had a characteristic
apoptotic appearance, being round and condensed (data not
Julia A. Romashkova & Sergei S. Makarov shown). As expected, PDGF rescued the cells from c-Myc-induced
apoptosis, but the co-expression of srIkBa abrogated the rescue
Thurston Arthritis Research Center, and Center for In¯ammatory Disorders, (Fig. 2a). To minimize the in¯uence of variability in transfections
University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599± and the observation error, we modi®ed the apoptotic assay. In a
7280, USA modi®ed experiment, we divided the transfected cells between two
.............................................................................................................................................. dishes and investigated the time course of cell viability. c-Myc-
The mechanisms of cell proliferation and transformation are induced cell death was delayed and inhibited by PDGF (Fig. 2b, left),
intrinsically linked to the process of apoptosis: the default of but the expression of srIkBa abrogated this protection (Fig. 2b,
proliferating cells is to die unless speci®c survival signals are right). In the absence of c-Myc, the expression of srIkBa did not
provided1,2. Platelet-derived growth factor (PDGF) is a principal affect cell viability regardless of the presence of PDGF (Fig. 2b,
survival factor that inhibits apoptosis and promotes pro- right). These data indicate that NF-kB activation is required for the
liferation1, but the mechanisms mediating its anti-apoptotic anti-apoptotic activity of PDGF. To examine the anti-apoptotic
properties are not completely understood. Here we show that effects of NF-kB activation, exogenous NF-kB (p50 and RelA
the transcription factor NF-kB3±5 is important in PDGF signal- subunits) was co-expressed with c-Myc in serum-deprived cells.
ling. NF-kB transmits two signals: one is required for the induc- Although p50 alone had no apparent effect, the expression of RelA
tion of proto-oncogene c-myc and proliferation, and the second, alone or in a combination with p50 increased cell viability by 3 to 5-
an anti-apoptotic signal, counterbalances c-Myc cytotoxicity. We fold (Fig. 2c), indicating that NF-kB activation itself can inhibit
have traced a putative pathway whereby PDGF activates NF-kB c-Myc-induced apoptosis.
through Ras and phospatidylinositol-3-kinase (PI(3)K) to the How does PDGF activate NF-kB? As the Ras/PI(3)K/Akt pathway
PKB/Akt protein kinase and the IkB kinase (IKK); NF-kB thus is critical in anti-apoptotic PDGF signalling, we examined the
appears to be a target of the anti-apoptotic Ras/PI(3)K/Akt possible role of this pathway. The expression of a dominant negative
pathway6,7. We show that, upon PDGF stimulation, Akt transiently (DN) RasN17 mutant strongly inhibited PDGF-induced NF-kB
associates in vivo with IKK and induces IKK activation. These activation (Fig. 3a), indicating that Ras may be involved in NF-kB
®ndings establish a role for NF-kB in growth factor signalling activation. In anti-apoptotic PDGF signalling, PI(3)K is the proxi-
and de®ne an anti-apoptotic Ras/PI(3)K/Akt/IKK/NF-kB path- mal downstream target of Ras13. Wortmannin and LY294002,
way, thus linking anti-apoptotic signalling with transcription speci®c inhibitors of PI(3)K, both inhibited NF-kB activation
machinery. (Fig. 3b), indicating that PI(3)K may be involved in PDGF-induced
NF-kB activation. Importantly, neither DN Ras nor wortmannin
c-Myc is a central regulator of cell growth, death and differen-
tiation. c-Myc is required for cell proliferation but, in the absence
[3H] Thymidine incorporation,
a b 16 – PDGF
of survival factors, it induces apoptosis1,8. PDGF inhibits c-Myc- 14 + PDGF
induced apoptosis by activating the Ras/PI(3)K/Akt6,7 pathway, but fold induction 12
the downstream effectors of this pathway are poorly de®ned. One IκBα 10
P <0.005
consequence of PDGF treatment is the activation of NF-kB9. NF-kB 8
controls the expression of numerous genes involved in in¯amma- RelA/p50
6
tion, tumour development and immune responses3±5. In unstimu- PDGF (h): 0 1 2 3 3 4
lated cells, NF-kB is retained in the cytoplasm through an AdCMV AdsrIκBα 2
interaction with inhibitory proteins known as IkB. The canonical 0
mechanism of NF-kB activation involves signal-inducible degrada- AdCMV AdsrIκBα
tion of IkB, which causes the translocation of NF-kB to the nucleus c
–PDGF + PDGF + PDGF
and transcription activation of targeted genes. As NF-kB has
been implicated in the regulation of apoptosis4, proliferation10
and c-Myc expression11, we investigated a role for NF-kB in
PDGF signalling.
We used two types of primary ®broblast: normal human skin
®broblasts (NHF) and rat ®broblast-like synoviocytes (FLS). PDGF
treatment induced NF-kB DNA-binding activity, which became
apparent after 1 h of stimulation, peaked at 3 h (Fig. 1a) and
declined thereafter (data not shown). Supershift analysis has
shown that the induced NF-kB was a RelA/p50 heterodimer (data
not shown). The induction of NF-kB DNA-binding activity coin-
cided with the degradation of IkBa (Fig. 1a). To examine the role of AdCMV AdsrIκBα
NF-kB in mitogenic activity of PDGF, NF-kB activation was
arrested by infecting the cells with an adenoviral vector encoding Figure 1 The mitogenic function of NF-kB. FLS were infected with Ad vectors as indicated,
a non-degradable (`super-repressor') form of IkBa (AdsrIkBa)12 incubated for 48 h in a serum-free medium and stimulated with PDGF. a, Time course of
(Fig. 1a). This inhibited DNA synthesis (Fig. 1b) and abrogated NF-kB activation. IkBa protein (top) and NF-kB DNA-binding activity (bottom) were
the induction of c-Myc message (data not shown) and protein determined in the cytosolic and nuclear extracts. b, NF-kB activation is required for DNA
(Fig. 1c). Thus, NF-kB activation is required for the expression of synthesis. [3H]Thymidine incorporation was determined after 48 h PDGF stimulation.
c-Myc and proliferation. Mean values 6 s.e.m. are shown from three experiments performed in duplicate.
To investigate the function of NF-kB in anti-apoptotic PDGF Identical results were obtained in NHF (data not shown). c, NF-kB activation is required for
signalling, exogenous c-Myc and super-repressor IkBa (srIkBa) c-Myc protein expression. c-Myc immunostaining was performed after 24 h PDGF
were constitutively expressed in serum-deprived cells. To facilitate stimulation. Original magni®cation 200´.

86
© 1999 Macmillan Magazines Ltd NATURE | VOL 401 | 2 SEPTEMBER 1999 | www.nature.com

affected the induction of NF-kB by tumour necrosis factor-a
(TNFa) and 12-O-tetradecanoylphorbol-13-acetate (TPA), stimuli
that probably do not involve Ras and PI(3)K (Fig. 3c).
As Akt is a proximal downstream effector of the anti-apoptotic
Ras/PI(3)K pathway6,7,13, we next examined the involvement of Akt
in NF-kB activation. To this end, we used a kinase-dead AktK179M
mutant14, which effectively inhibited the activation of Akt by PDGF
(data not shown). The expression of AktK179M strongly inhibited
NF-kB reporter-gene expression, indicating that Akt is involved
in NF-kB activation (Fig. 3d). Similarly to the RasN17 and PI(3)K
inhibitors, AktK179M expression did not affect cell viability (data
not shown), thus ruling out the possibility that the inhibition of
NF-kB reporter-gene expression re¯ected a cytotoxic effect. Our
data 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. PDGF
treatment causes degradation of IkBa (Fig. 1a), which indicates that
IKK is involved. The inducible degradation of IkBa occurs through
several consecutive events, an initial step being the phosphorylation
of IkBa by a multisubunit IKK complex, the signalsome5. Two
catalytic subunits of IKK, IKKa and IKKb, phosphorylate IkBa on
residues Ser 32 and Ser 36 and target IkBa to ubiquitination and
letters to nature
gene expression (Fig. 3e). This prompted us to explore the causal
relationship between Akt and IKK in NF-kB activation. We found
that the expression of a constitutively active myristylated Akt
(myrAkt) alone was suf®cient for NF-kB activation (12-fold
induction in FLS (Fig. 3f) and 7-fold induction in NHF (data not
shown)). 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-kB
activation (Fig. 3f), indicating that IKK is a target of Akt in PDGF
signalling. To investigate the link between Akt and IKK, we assessed
the temporal relationship between these kinases. To assess the
kinetics of Akt activation, we determined its phosphorylation on
residues Thr 308 and Ser 473, which is a prerequisite for the catalytic
activity of Akt13. Akt phosphorylation was apparent after 5 min of
stimulation, reached a peak at 60 min and declined thereafter
(Fig. 4a). These kinetics overlapped with the time course of IKK
activation: the phosphorylation of IkBa on Ser 32 was evident
30 min after PDGF stimulation and persisted for at least 2 h
(Fig. 4b and data not shown). These data indicate that IKK might
a FLS b
NHF
100 100

Induction, %

Induction, %
proteasomal degradation5,15. To inhibit IKK, we used DN mutants of 80 80
the IKKa and IKKb subunits. Whereas DN IKKa exerted only 60 60
minimal (if any) inhibitory effect (data not shown), the expression 40 40
of DN IKKb strongly inhibited PDGF-induced NF-kB reporter- 20 20
0 0
a FLS + PDGF NHF κB reporter: wt mut wt κB reporter: wt mut wt wt
_ PDGF
PDGF: + + + PDGF: + + + +
120 Inhibitor: – – RasN17 Inhibitor: – – Wort LY
120
P <0.005 P <0.05
Viability, %

100 100
80 80 c d
60 60 100 100
Induction, %

Induction, %
40 40
80 80
20 20
0 60 60
0
c-Myc: – + + c-Myc: – + + 40 40
srlκBα: – – + srlκBα: – – + 20 20
0 0
b – srlκBα + srlκBα
TNFα PMA PDGF κB reporter: wt mut wt
+ PDGF Wortmannin PDGF: + + +
– PDGF Inhibitor: – – Akt K179M
RasN17
100 + c-Myc 100
80 – c-Myc 80 e f
Viability, %

60 60 14
100
Fold induction

12
Induction, %

40 40
80 10
20 20 8
60
0 0 6
0 10 18 24 h 0 10 18 24 h 40
4
20 2
+ PDGF 0 0
c FLS NHF κB reporter: wt
_ PDGF κB reporter: wt mut wt mut wt wt
PDGF: + + + myrAkt: _ + + +
P <0.005 Inhibitor: – – IKKβ (K44A) Inhibitor: _ _ _ IKKβ (K44A)
100 P <0.005 100
80 80
Viability, %

P <0.005 P <0.05 Figure 3 NF-kB is a target of a Ras/PI(3)K/Akt/IKK pathway. Unless otherwise noted,
60 60
transfected cells (5 mg of each vector) were serum-starved for 48 h and stimulated with
40 40
PDGF for 18 h. Bars represent mean values 6 s.e.m. from n individual experiments
20 20
performed in duplicate. a, The involvement of Ras. PDGF-induced activation of 3xkBCAT
0 0
c-Myc: + c-Myc: +
in individual experiments was in the range of 8- to 37- fold in FLS (n ˆ 4) and 9- to 15-
+ + + + + + + +
p50: – p50: –
fold in NHF (n ˆ 3). b, The involvement of PI(3)K. Wortmannin (100 nM) and LY924002
– + – + – + – +
RelA: – RelA: – (10 mM) were added 30 min before PDGF (FLS, n ˆ 2; NHF; n ˆ 1). c, Inhibitors of Ras/
– – + + – – + +
PI(3)K do not affect TNFa- and PMA-induced NF-kB activation. FLS were stimulated with
Figure 2 The anti-apoptotic function of NF-kB. a, NF-kB activation is required for the TNFa (50 ng ml-1), PMA (50 ng ml-1) or PDGF for 6 h. Stippled bars: DN Ras-transfected
anti-apoptotic activity of PDGF. Cell viability was assessed after 18 h incubation in a low- cells. Hatched bars: wortmannin-pretreated cells. TNFa-, PMA- and PDGF-induced wild-
serum medium. Bars represent mean values 6 s.e.m. from four experiments in FLS (left) type 3xkB CAT activation was 37-, 11- and 8-fold, respectively; these values were
and one experiment in NHF (right). b, Time course of c-Myc-induced apoptosis in FLS in accepted for 100% induction (solid bars) (FLS, n ˆ 2). d, The involvement of Akt (FLS,
the absence (left) or presence (right) of srIkBa. c, The overexpression of NF-kB inhibits c- n ˆ 2; NHF, n ˆ 2). e, The involvement of IKK (FLS, n ˆ 3). f, NF-kB activation by
Myc-induced apoptosis. Cell viability was assessed after 18-h incubation in a low serum myrAkt. FLS were transfected with 2.5 mg of each vector. Mean values 6 s.d. from a
medium. Bars represent mean values 6 s.e.m. from two experiments in each of FLS (left) single experiment performed in duplicate are shown. Identical data were obtained in NHF
and NHF (right). (not shown).

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

© 1999 Macmillan Magazines Ltd 87
letters to nature
be a direct target of Akt, an assumption reinforced by the observa-
tion that PDGF-mediated phosphorylation of IkBa did not require
de novo protein synthesis (Fig. 4b and data not shown).
To assess the possible interaction between Akt and the IKK
signalsome in vivo, we immunoprecipitated endogenous Akt and
analysed the immune complexes for the presence of IKK using a
combination of Akt and IKKa antibodies. The immunodetection
revealed two bands corresponding to Akt (relative molecular mass
…M r † ˆ 63K) and IKKa (M r ˆ 85K; Fig. 4c). These bands were
absent 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 detected
only in cells stimulated with PDGF for 1 h, not in unstimulated cells
or those stimulated for 4 h. Consistent with the data shown in
Fig. 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 observed
in NIH 3T3 ®broblasts (Fig. 4d). In these experiments, we detected
IKK in Akt immune complexes using an antibody against IKKb,
another catalytic subunit of the IKK complex. Furthermore, the
immunoprecipitation of endogenous IKKb in PDGF-stimulated
NIH 3T3 cells brought down endogenous Akt (Fig. 4d). Combined,
these observations show that, upon PDGF stimulation, Akt and IKK
transiently associate in vivo.
a b
FLS NIH 3T3 PDGF – CHX + CHX
(min): 0 30 60 0 30 60
PDGF
(min): 0 5 15 30 60 120 20 0
p-Akt
e
Akt
IP: α-HA
To determine whether this association was functional (induced
the catalytic activity of IKK) NIH 3T3 cells were transfected with
haemagglutinin (HA)-tagged Akt vectors, stimulated with PDGF
for 1 h, immunoprecipitated with anti-HA antibodies and assessed
for IkB kinase activity. As a positive control, endogenous IKK
activity was determined in TNFa-stimulated untransfected cells.
In cells transfected with wild-type (wt)Akt.HA, IKK activity was
detected 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 undetectable
in PDGF-stimulated cells transfected with the kinase-dead
AktK179M.HA (Fig. 4f). The IKK activity correlated with the
presence of IKK protein (Fig. 4e). Notably, Akt was undetectable
in IKK immune complexes from TNFa-stimulated cells (Fig. 4e).
This indicates that, in contrast to PDGF, TNFa does not induce the
association of Akt with IKK. Together, our data indicate that PDGF-
stimulated activation of NF-kB is mediated by transient association
of Akt with IKK and the activation of IKK.
Our study indicates that, in PDGF signalling, proliferative and
pro-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 with
those of cell death1,2, NF-kB has a dual role: it mediates the
induction of pro-apoptotic c-Myc, and delivers an anti-apoptotic
signal counterbalancing c-Myc cytotoxicity, conceivably by induc-
ing the expression of a protective gene (or multiple genes). Given
the roles of ras, myc and akt in cancer, our study has evident
p-IκBα
rami®cations for understanding the mechanisms of cell transforma-
IκBα
tion. It may explain why the ability of Ras to transform cells depends
on NF-kB activation16. Oncogenic Ras activates two independent
αIKKα executive mechanisms: an anti-apoptotic PI(3)K/Akt pathway and a
pro-apoptotic Raf/MAPK pathway6,17. Our results indicate that
+T Fα
α

PDGF: – + + –
NF

Blocking inhibition of NF-kB might block anti-apoptotic Akt signalling

N

– – + –
–T

peptide:
c FLS IKK assay without interfering with the pro-apoptotic pathway. Our observa-
IP: α-Akt GST-IκBα tions also indicate a novel role for NF-kB in rheumatoid arthritis.
PDGF (h): 0 1 4 1 ID: α-Akt+
Mr ID: α-IKKα One feature of this disease is aggressive hyperplasia of the synovium
Blocking – – – + α-IKKα
97K IKKα associated with overexpression of c-Myc and activation of NF-kB18.
peptide: 66K As PDGF is a principal growth factor in rheumatoid arthritis FLS
97K IKKα
97K ID: α-Akt cells, NF-kB activation may contribute to synovial hyperplasia by
66K Akt
IgG
66K Akt promoting proliferation and inhibiting c-Myc-induced apoptosis.
46K IgG
Mr Our data identify NF-kB as target of the anti-apoptotic Ras/PI(3)K/
97K
ID: α-p-Akt
wtAkt.HA myrAkt.HA
Akt pathway. Several putative targets of this pathway have been
66K p-Akt proposed, among them Bad19,20, caspase-921 and the transcription
46K f IP: α-HA factor Forkhead22,23. As all these targets represent proteins with
PDGF: + + + + intrinsic pro-apoptotic properties, Akt appeared to be a negative
d
Blocking
peptide: – + – +
regulator of the cell-death machinery. Our data indicate that Akt
NIH 3T3 ID: α-Akt+ 46K
IKK assay
can also promote survival by activating anti-apoptotic NF-kB
IP: α-Akt α-IKKβ α-IKKβ 30K
GST-IκBα
signalling. The interplay of these mechanisms in the anti-apoptotic
97K IKKβ ID: α-Akt
activities of Akt13 has yet to be determined. The mechanism whereby
66K Akt 66K PDGF activates NF-kB also warrants further investigation. Our
Akt
46K IgG IgG observations indicate that this mechanism involves consecutive
46K
PDGF (h): 0 1 4 1 activation of Akt and IKK, which is consistent with the observed
AktK179M.HA wtAkt.HA phosphorylation and degradation of IkBa and induction of NF-kB
DNA-binding activity. However, under certain circumstances, PI(3)K
Figure 4 Akt associates with IKK in vivo and induces IKK activation. a, Akt activation. FLS can activate NF-kB through a mechanism that does not involve IkB
lysates were consecutively immunodetected with phospho-Akt (Ser 473) (top) and total degradation24. There is also evidence that Akt can activate NF-kB
Akt (bottom) antibodies. NIH 3T3 lysates were from the antibody manufacturer. b, IkBa transcriptional function through an alternative IkB-independent
phosphorylation. FLS lysates were consecutively immunodetected with p-IkBa (Ser 32)
(top) and total IkBa (bottom) antibodies. CHX, cycloheximide. c, The association of Akt anti-apoptotic gene(s)
and IKK in FLS. Akt immune complexes were consecutively immunodetected with primary PDGF Ras PI(3)K Akt IKK NF-κB
c-Myc apoptosis
Akt and IKKa (top) and p-Akt (bottom) antibodies. Mr, relative molecular mass. d, The
proliferation
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 Figure 5 NF-kB in PDGF signalling. PDGF activates the Ras/PI(3)K/Akt pathway. Activated
shown. The blot was consecutively immunodetected with IKKa (middle) and Akt (bottom) Akt transiently associates with IKK and induces the activation of IKK and NF-kB. NF-kB
antibodies. f, Catalytic activity of Akt is required for IKK activation. IKK activity and Akt transmits a signal required for the induction of c-Myc expression and proliferation, and an
protein were assessed as in e. anti-apoptotic signal that inhibits c-Myc cytotoxicity.

88
© 1999 Macmillan Magazines Ltd NATURE | VOL 401 | 2 SEPTEMBER 1999 | www.nature.com

mechanism, by modulating the transcriptional potential of RelA (M.
Mayo, L. Madrid and A. Baldwin, personal communication). The
contribution of these pathways remains to be clari®ed. Another issue
is the role for the atypical isoforms of protein kinase C (PKC) and
reactive oxygen intermediates (ROI), both of which have been
implicated in mitogenic signalling25,26 and NF-kB activation3,4. As
the 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 may
represent a distinct channel for NF-kB activation, similar to those
provided by NIK and MEKK1 in TNFa and IL-1 signalling5,29. M
Methods
Reagents.
Unless otherwise noted, human recombinant PDGF-BB (Gibco BRL) was used at a ®nal
concentration of 50 ng ml-1. Plasmids wtAkt.HA, AktK179M.HA and myrAkt.HA were
gifts 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 and
3xkBCAT vectors were gifts from A. Baldwin. The EGFP vector was from Promega. All
plasmids were puri®ed from bacterial endotoxin using Detoxigel (Pierce). Adenoviral
infections were performed as described12.
Cell culture.
30
We obtained primary FLS from rats with streptococcal cell wall (SCW)-induced arthritis .
The cells were maintained in RPMI 1640/15% FBS and used at passages 5±18. Primary
foreskin 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 for
another 48 h. [3H]thymidine (0.5 mCi per well) was added during the last 24 h. In
individual experiments, incorporated radioactivity in PDGF-stimulated AdCMV-infected
cells 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 a
cold probe and by supershift analysis with anti-p50 and anti-RelA antibodies. To assess
NF-kB reporter-gene expression, cells (105 cells per 100 mm dish) were transfected with
wild-type or mutant 3xkBCAT reporter vectors and corresponding DN inhibitors. Unless
otherwise noted, each transfection contained 180 mg of SuperFect reagent (Quiagen) and
5 mg of each reporter and expression vector. The amount of DNA was kept constant by
addition of an empty CMV plasmid. CAT activity was determined using the Quan-T-CAT
kit (Amersham) and normalized on total protein. In some experiments, we used 3xkB
LUC 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-Myc
and srIkBa expression vectors (2 mg of each DNA per 100 mm dish). Attached ¯uorescent
cells 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 in
PDGF-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 h
and incubated in the low serum medium. Three randomly chosen observation ®elds were
marked on the dishes, and the numbers of attached ¯uorescent cells were counted at
indicated time points. To assess the in¯uence of NF-kB overexpression on c-Myc
apoptosis, 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 incubation
in low serum medium. At least 250 ¯uorescent cells were scored in PDGF-treated control
cells (EGFP plus c-Myc) (accepted for 100% viability).
Immunodetection.
For c-Myc immunostaining we used primary c-Myc antibodies (sc-764, Santa Cruz
Biotechnology) (1:200 dilution) and a secondary HRP-conjugated antibody. For western
blotting, lysates were resolved on SDS±PAGE and transferred to nitrocellulose. Total and
phosphorylated IkBa were detected with primary IkBa antibodies (Rockland Inc.)
(1:1000) and p-IkBa (Ser 32) antibodies (1:1000) (New England Biolabs). Where
indicated, cells were pre-treated for 1 h with cycloheximide (CHX) (10 mg ml-1); to
prevent degradation of phospho-IkBa, proteasomal inhibitor MG132 (5 mM) was added
with 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 either
goat polyclonal antibodies against the carboxy-terminus of IKKa (1:1000) (sc-7120, Santa
Cruz 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 gift
from A. Manning). ECL detection was performed using an ECL kit (Du Pont NEN).
NATURE | VOL 401 | 2 SEPTEMBER 1999 | www.nature.com
© 1999 Macmillan Magazines Ltd
letters to nature
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). Endogenous
Akt was immunoprecipitated by 1-h incubation with an anti-Akt antibody (NEB) (1 mg
per 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 IKK
was 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 ®ve
times 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 by
incubating in a 0.5% FBS medium for 48 h, followed by an overnight incubation in a
serum-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 mg
cell 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 with
TNFa (100 ng ml-1) and cell lysates were immunoprecipitated with IKKa monoclonal
antibodies (Pharmingen) (2 mg per 1 mg cell protein). In vitro IkB kinase activity was
performed with recombinant GST-IkBa (1±54) as a substrate, as described15. Reaction
products were separated by 10% SDS±PAGE, transferred to nitrocellulose and visualized
with a phosphoimager. The blots were subsequently immunodetected for the presence of
Akt 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).
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protein, non-tagged GFP gave no change in ¯uorescence or loss
Proc. Natl Acad. Sci. USA 95, 9067±9069 (1998). of protein when incubated under similar conditions with ClpA,
30. Makarov, S. S. et al. Suppression of experimental arthritis by gene transfer of interleukin 1 receptor ClpP and ATP (results not shown). We conclude that even the stable
antagonist cDNA. Proc. Natl Acad. Sci. USA 93, 402±406 (1996).
b-barrel of GFP can be destabilized and degraded by the ClpAP
machinery when recruited to it as a result of the addition of the ssrA
Acknowledgements sequence.
We thank P. Tsichlis, J. Woronicz, A. Manning, F. Mercurio, C. Der, T. Davis, W. Kaufmann, To test whether we could detect unfolding of GFP11 as a step
A. Danilkovitch and A. Baldwin for sharing reagents; A. Baldwin, C. Der and P. Cohen for
distinct from the degradation carried out by ClpAP, we incubated
stimulating discussions; C. Bradham for assistance in kinase assays; J. Watson for editorial
assistance; and members of the S.M. laboratory, A. Miagkov and D. Kovalenko for GFP11 with stoichiometric amounts of ClpA on its own. In the
technical assistance. This work was supported by grants from the National Institutes of presence of ATP, but not of ATP-gS, there was a small decrease
Health and the Arthritis Foundation. (20%) in ¯uorescence intensity (Fig. 2a), indicating that some
Correspondence and requests for materials should be addressed to S.M. (e-mail: fraction of the input GFP11 molecules could be unfolded. The
smak@med.unc.edu). observed effect was small, however, compared with the effect of
adding a stoichiometric amount of ClpA and ClpP, which caused a
complete loss of ¯uorescence as a result of the total proteolytic
degradation of the added GFP11 (Fig. 2a, and results not shown).
................................................................. We reasoned that, if GFP11 molecules unfolded by ClpA were
released from it and then refolded, establishing an equilibrium
Globalunfolding of asubstrate protein between unfolding and refolding, then preventing refolding should
reveal the unfolding action of ClpA, producing a larger drop in
by the Hsp100 chaperone ClpA ¯uorescence. To prevent refolding, we added a GroEL `trap' mutant
(D87K, in which an aspartate residue at position 87 is replaced by a
Eilika 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, USA
³ 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 chaperone
family, forms hexameric rings that bind to the free ends of the
double-ring serine protease ClpP (refs 1, 2). ClpA directs the ATP-
dependent degradation of substrate proteins bearing speci®c
sequences3±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 eukaryotic
protein, Hsp104, can dissociate low-order aggregates11. ClpA has
been proposed to destabilize protein structure, allowing passage
of proteolysis substrates through a central channel into the ClpP
proteolytic cylinder12±14. Here we test the action of ClpA on a
stable monomeric protein, the green ¯uorescent protein GFP,
onto which has been added an 11-amino-acid carboxy-terminal
recognition peptide, which is responsible for recruiting truncated
proteins to ClpAP for degradation5,15. Fluorescence studies both
with and without a `trap' version of the chaperonin GroEL, which
binds non-native forms of GFP16, and hydrogen-exchange experi-
ments directly demonstrate that ClpA can unfold stable, native
proteins in the presence of ATP.

We ®rst tested whether GFP, a very stable monomeric protein (it

is resistant to denaturation by 6M urea, for example; Fig. 1a), could
be degraded by the ClpAP machinery once recruited to it as a result
of the attachment of an 11-residue ssrA peptide to the GFP
terminus. Appendage of the peptide did not interfere with the
¯uorescent properties or stability of the GFP portion: the tagged Figure 1 GFP11, a derivative bearing an 11-amino-acid C-terminal ssrA tag recognized
protein (GFP11) gave the same ¯uorescence quantum yield and by ClpA, exhibits the same stability to denaturants as untagged GFP, and is degraded by
emission spectrum as non-tagged GFP (not shown) and was as ClpA/ClpP in the presence of ATP. a, Time course of ¯uorescence intensity changes of
stable to denaturant (Fig. 1a). When incubated with catalytic GFP11 and GFP after dilution to 600 nM in 6 M urea at 23 8C (top), 6 M guanidine-HCl at
amounts of ClpA and ClpP in the presence of ATP, however, 23 8C (middle), or 6 M guanidine-HCl at 37 8C (bottom). b, Time-course of the
GFP11 was completely degraded, as evidenced both by loss of ¯uorescence intensity change of 10 mM GFP11 incubated with 100 nM ClpA, 50 nM ClpP
¯uorescence (Fig. 1b) and by the disappearance of the protein on and 10 mM ATP. Excitation and emission wavelengths were 400 and 510 nm,
SDS±polyacrylamide gel electrophoresis (Fig. 1c). The kinetics of respectively. c, SDS±PAGE analysis of aliquots of the reaction used in b, taken at the
the two measurements were similar, preventing the resolution of an times indicated.

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