1 Department of Immunology and Molecular Pathology, University College London,
Windeyer Institute, 46 Cleveland St, London W1T 2AH, UK
2 National Institute for Medical Research, Laboratory of Protein Structure, The
Ridgeway, Mill Hill, London NW7 1AA, UK
3 Hybrigenics, 3-5 Impasse Reille, 75014 Paris, France
4 Cancer Research UK Viral Oncology Group, Wolfson Institute for Biomedical
Research, University College London, Gower Street, London WC1E 6BT, UK
Author for correspondence (e-mail:
mary.collins{at}ucl.ac.uk)
Accepted 28 May 2003
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Summary |
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Key words: KSHV, vFLIP, IKK, Hsp90
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Introduction |
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Two roles have been proposed for vFLIP. By analogy with FLIP proteins
expressed by herpesvirus saimiri, equine herpesvirus and molluscum contagiosum
poxvirus, it has been suggested that vFLIP blocks Fas-mediated apoptosis
(Bertin et al., 1997;
Hu et al., 1997
;
Thome et al., 1997
). Indeed,
vFLIP inhibits procaspase-8 cleavage after Fas triggering
(Belanger et al., 2001
) and is
able to promote tumour growth when expressed in a Fas-sensitive B cell
lymphoma cell line (Djerbi et al.,
1999
). More recently, vFLIP protein has been implicated in the
activation of the transcription factor NF-
B. vFLIP can activate
NF-
B-driven reporter constructs in 293T cells
(Chaudhary et al., 1999
), and
also interacts with and activates the central kinase of the NF-
B
signalling pathway, I
B kinase (IKK) when ectopically expressed in a
non-small-cell lung carcinoma cell line
(Liu et al., 2002
).
Many signals for NF-B activation converge on the cytokine-inducible
protein kinase complex IKK. The complex contains two catalytic components,
IKK
and IKKß (also called IKK1 and IKK2)
(DiDonato et al., 1997
;
Mercurio et al., 1997
;
Zandi et al., 1997
;
Regnier et al., 1997
), and a
regulatory subunit, IKK
(Rothwarf
et al., 1998
) [also called NF-
B essential modulator (NEMO)
(Yamaoka et al., 1998
),
IKK-associated protein 1 (IKKAP1)
(Mercurio et al., 1999
) and
14.7-interacting protein (FIP-3) (Li et
al., 1999
)]. IKK
and IKKß are homologous proteins of
85 kDa and 87 kDa, respectively, with 50% sequence identity. IKK
is
necessary for activation of IKK
and IKKß
(Makris et al., 2000
);
heterodimers of IKK
and IKKß are bound by four IKK
molecules to form a large complex
(Tegethoff et al., 2003
).
Recently, the chaperone protein Hsp90 and a co-chaperone (Cdc37) have been
identified as additional components of the IKK complex
(Chen et al., 2002
).
KSHV infection is associated with three proliferative disorders in
immune-compromised patients: Kaposi's sarcoma (KS), primary effusion lymphoma
(PEL) (a proliferation of immature B cells) and a variant of multicentric
Castleman's disease (MCD) (Boshoff et al., 2002;
Cesarman et al., 1995;
Moore et al., 1996
;
Soulier et al., 1995
). In
KSHV-infected PEL cells, the NF-
B pathway is constitutively active
(Liu et al., 2002
;
Keller et al., 2000
) and the
cells undergo apoptosis when challenged with the inhibitor of
cytokine-inducible I
B
phosphorylation, Bay 11-7082
(Keller et al., 2000
). This
suggested a role for constitutive NF-
B activation in the survival of
these cells. Given the two contrasting roles previously assigned to vFLIP, we
set out to investigate which proteins interact with vFLIP in KSHV-infected PEL
cells.
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Materials and Methods |
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Cell lines and lentiviral transduction
The KSHV-infected PEL cell line, BC3, was grown in RPMI1640 with 10% foetal
calf serum (FCS), penicillin and streptomycin at 37°C in 5%
CO2. 293T cells were maintained in Dulbecco's modified Eagle's
medium with 10% FCS, penicillin and streptomycin at 37°C in 10%
CO2. Lentivirus encoding vFLIP and GFP or GFP alone was produced
using a transient transfection of 293T cells as described previously
(Neil et al., 2001;
Zufferey et al., 1997
). 293T
cells were transduced with each virus and the efficiency of cell transduction
measured by FACScan analysis of eGFP positive cells. Cells were treated with
0.5 µM geldanamycin (GA) (Calbiochem) dissolved in DMSO or an equal volume
of DMSO in serum-free medium for 16 hours.
Large-scale immunoprecipitation
Anti-vFLIP 6/14 rat monoclonal antibody
(Low et al., 2001) and control
rat IgG were covalently coupled to NHS-activated Sepharose 4B resin
(Amersham). 1x1010 BC3 cells were washed in PBS and incubated
for 30 minutes at 4°C in 10 ml lysis buffer [20 mM Tris-HCl pH 7.5, 150 mM
NaCl, 0.2% NP-40, 10% glycerol, 1 mM PMSF and protease inhibitor cocktail
(Roche)]. The lysate was centrifuged at 16,000 g for 10
minutes and then the cytoplasmic extract was divided equally between vFLIP and
control resins for incubation at 4°C for 2 hours. The resin was washed
three times in lysis buffer with 500 mM NaCl and 100 µl SDS-PAGE sample
buffer lacking ß-mercaptoethanol was added to elute immunoprecipitated
proteins. The sample buffer was removed from the resin and
ß-mercaptoethanol was added and the samples were heated to 95°C for 4
minutes. The samples were divided 9:1 between two 12% SDS-PAGE gels. The gel
containing 90% of the sample was stained with a Colloidal Blue Coomassie
staining kit (Invitrogen). The gel containing 10% of the sample was stained
using silver.
In-gel digest
Protein bands of interest were excised from the Coomassie stained gel and
extracted with 200 mM ammonium bicarbonate / 50% acetonitrile, reduced with 20
mM DTT and then alkylated in 5 mM iodoacetamide and dehydrated. The gel slices
were swollen in a minimal volume of 2 ng µl-1 trypsin (Promega)
in 5 mM ammonium bicarbonate for in-gel digestion. Peptide mass fingerprinting
was performed using a Reflex III time-of-flight mass spectrometer (Bruker
Daltonik) with a nitrogen laser and a Scout-384 probe, to obtain positive ion
mass spectra of digested protein with pulsed ion extraction in reflectron
mode. An accelerating voltage of 26 kV was used with detector bias gating set
to 2 kV and mass cut-off of m/z = 650. Matrix surfaces were
prepared using recrystallised -cyano-4-hydroxycinammic acid and
nitrocellulose using the fast evaporation method (Vorm et al., 1994). 0.4
µl of digestion supernatant was deposited on the matrix surface and allowed
to dry prior to desalting with water. Peptide mass fingerprints thus obtained
were searched against the non-redundant protein database of the National
Centre for Biotechnology Information (NCBI) using the program MASCOT
(Perkins et al., 1999
).
Yeast two-hybrid interaction
Proteins interacting with vFLIP were identified using high throughput
yeast-two-hybrid analysis at Hybrigenics (Paris). The vFLIP bait was
constructed as a LexA, C-terminal fusion in the pB27 plasmid derived from the
original pBTM116 (Vojtek et al., 1995). To generate an expression library, a
randomly primed cDNA library from human placenta poly(A+) RNA was constructed
and inserted into the pP6 plasmid derived from pACT2
(Rain et al., 2001). The
library was then transformed into yeast and 107 independent yeast
colonies were collected, pooled and stored at -80°C in aliquots. The
screen was performed to ensure that at least 5x107
interactions were tested. The mating protocol has been described elsewhere
(Fromont-Racine et al., 2002
).
The screening conditions were optimized for vFLIP bait using a test screen
before performing the full-size screening. For all the selected clones, LacZ
activity was measured in a semiquantitative XGal overlay assay. The prey
fragments of the positive clones were amplified by PCR, analysed on agarose
gel, and sequenced at their 5' and 3' junctions on a PE3700
sequencer. The resulting sequences were then used to identify the
corresponding gene in the GenBank database (NCBI) using an automated Blast
analysis procedure. Clones obtained many times in different screens against
the same libraries were discounted as false positives.
Gel filtration
2x107-2x108 cells were incubated in lysis
buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 mM EGTA, 1 mM DTT, 0.2% NP-40,
5% glycerol, 1 mM Na3V04, 10 mM ß-glycerophosphate,
5 mM NaF, 1 mM PMSF and protease inhibitor cocktail) for 30 minutes at
4°C. The extract was centrifuged at 100,000 g for 1 hour
at 4°C. 100 µl of the supernatant was loaded on a Superose 6 PC 3.2/30
column (Amersham) previously equilibrated in Buffer B (25 mM Tris-HCl pH 7.6,
150 mM NaCl, 0.2% NP-40, 5% glycerol). The fractionation was performed using
an LKB:µSeparation unit (Amersham) controlled using Smart Manager 5.1
software. The flow rate of the column was maintained at 40 µl
min-1 and 22 fractions of 100 µl each were collected. 25 µl
of each fraction were separated by SDS-PAGE gel for immunoblot, whereas 50
µl of each fraction was used for kinase assays. The column was calibrated
in Buffer B using protein standards: thyroglobulin (669 kDa), ferritin (440
kDa) and catalase (232 kDa) (Amersham).
Small-scale immunoprecipitation, GST pull down and
immunoblotting
Cytoplasmic extracts from transfected 293T cells were incubated either with
1.5 µg of vFLIP antibody and 20 µl protein-G/Sepharose (Sigma) or with
GST-IKK truncation mutants pre-bound to glutathione Sepharose 4B
(Amersham) for 2 hours at 4°C. The complexes were washed three times in
wash buffer [20 mM Tris-HCl pH 7.5, 500 mM NaCl, 0.2% NP-40, 10% glycerol, 1
mM PMSF and protease inhibitor cocktail (Roche)]. Proteins were separated by
electrophoresis on a 12% SDS-polyacrylamide gel then transferred to Hybond ECL
nitrocellulose membranes (Amersham) for immunoblot analysis. Blots were
incubated overnight at 4°C in blocking solution (PBS containing 5% low-fat
milk and 0.1% Tween 20) and then incubated with primary antibody for 1 hour.
Primary antibodies: anti-vFLIP 6/14 antibody (1:100 dilution), anti-Xpress
(1:5000 dilution) (Invitrogen 46-0528), anti-IKK
rabbit polyclonal
(1:1000 dilution) (Cell Signalling Technology 2682), anti-IKKß goat
polyclonal (1:200 dilution) [Santa Cruz (SC)-7330], anti-IKK
rabbit
polyclonal (1:200 dilution) (SC-8330) and anti-vCyclin rat monoclonal (1:100
dilution) (gift from S. Mittnacht, Institute of Cancer Research, London, UK).
Bound antibodies were detected with peroxidase-conjugated secondary antibodies
(1:2000 dilution) and visualized using electrochemical luminescence (ECL)
(Amersham).
IB
kinase assay
For kinase assays, 50 µl of each column fraction or 100-200 µg of
cytoplasmic extract were incubated for 2 hours at 4°C with 1.5 µg
antibody and protein-G/Sepharose. For kinase assays using anti-IKKß
antibody, an additional pre-clearance step of 1 hour at 4°C with 1.5 µg
normal rabbit serum and 20 µl protein-G/Sepharose was included. Immune
complexes were washed three times in 0.5 ml high salt buffer (25 mM Tris-HCl
pH 7.6, 500 mM NaCl, 1 mM EGTA, 1 mM DTT, 0.2% NP-40, 5% glycerol, 1 mM
Na3VO4, 10 mM ß-glycerophosphate, 5 mM NaF, 1 mM
PMSF and protease inhibitor cocktail). Immune complexes were then washed a
further two times in kinase wash buffer (20 mM HEPES pH 7.6, 50 mM NaCl, 20 mM
ß-glycerophosphate, 0.5 mM DTT, 1 mM PMSF) before 40 µl kinase
reaction buffer (20 mM HEPES pH 7.6, 50 mM NaCl, 10 mM MgCl, 2 mM DTT, 20
µM ATP, 0.1 mM Na2VO4 and protease inhibitor
cocktail) was added. 0.5 µl of P32--ATP and 1 µg of
wild-type I
B
_1-54 or mutant I
B
_1-54 (S32A/S36A)
GST fusion protein was added to each reaction, which were incubated at
30°C for 30 minutes and then stopped by the addition of SDS-PAGE sample
buffer. The samples were separated by 12% SDS-PAGE and radiolabelled
phosphoproteins were visualized by autoradiography.
Cell viability assays
The viability of BC3 populations was measured directly by haemocytometry.
For annexin-V/propidium-iodide binding assays, 106 cells were
washed once in cold PBS before staining with TACSTM AnnexinV-FITC
Apoptosis detection kit (R&D Systems) and analysis with a FACSCaliber
using CellQuest software (Becton Dickinson, Franklin Lakes, NJ).
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Results |
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Endogenous vFLIP is associated with an activated IKK complex
To identify proteins interacting with the endogenous vFLIP in cells
infected with KSHV, we purified vFLIP from BC3 PEL cells by
immunoprecipitation. Proteins that coimmunoprecipitated with vFLIP but were
not precipitated by a control rat antibody were excised and identified by mass
spectrometry. Fig. 2 shows that
five proteins including vFLIP were identified in the vFLIP lane but not in the
control lane. All five proteins were clear matches with high Mascot scores
(Perkins et al., 1999). Three
of these proteins were identified as the core components (IKK
,
IKKß and IKK
) of IKK. The band containing IKK
was also
found to contain the chaperone protein, Hsp90, which has recently been
identified as an additional component of the IKK complex
(Chen et al., 2002
).
|
Fig. 3A shows that all the
soluble vFLIP in BC3 cells is present in a high molecular weight protein
complex. The three components of the IKK complex (IKK, IKKß and
IKK
) eluted from the Superose 6 column in the same fractions as vFLIP.
The Superose 6 fractions were also analysed for I
B
kinase
activity associated with vFLIP (Fig.
3A, bottom). Kinase activity was found in fractions 4-7, with the
major peak in fraction 5, identical to the distribution of vFLIP and IKK.
Fig. 3B demonstrates the
specificity of the kinase assay. Immune complexes precipitated using an
isotype-matched control antibody did not have an associated kinase activity
and the vFLIP immune complex was not able to phosphorylate a mutant
GST-I
B
containing point mutations at the two IKK targets in
I
B
, S32A and S36A. Fig.
3C shows that all detectable vFLIP in BC3 cell lysate is
associated with IKK
, because immunoprecipitation with an
anti-IKK
antibody depleted vFLIP from cell lysate, but did not affect
I
B
.
|
Activity of the vFLIP-IKK complex depends upon Hsp90
In KSHV-infected B cells, other viral or cellular proteins might co-operate
with vFLIP to activate IKK. To investigate whether vFLIP expressed at a
similar level to that in BC3 cells was sufficient to activate the IKK complex,
we transduced 293T cells with a lentiviral vector expressing both vFLIP and
GFP. Fig. 4A shows that
IKK and activated IKK were associated with vFLIP in the transduced 293T
cells. Anti-vFLIP antibody also co-immunoprecipitated IKK
and
IKK
in these cells (data not shown).
|
To investigate the role of Hsp90 in the vFLIP-IKK complex, we used the
nucleotide analogue GA, which inhibits the function of Hsp90
(Whitesell et al., 1994). We
found no change in the size of the vFLIP-IKK complex
(Fig. 4B) or the inactive IKK
complex in control 293T cells (Fig.
4C), or on the levels of IKK
, IKKß or IKK
expression (data not shown) upon GA treatment. We did observe vFLIP in lower
fractions in both control and GA-treated cells, and attribute this to vFLIP
being in excess of the IKK components. However, IKK activity associated with
vFLIP in GA treated cells was significantly reduced
(Fig. 4B). The activity of the
vFLIP-IKK complex is therefore dependent on Hsp90.
GA kills PEL cells
We then examined whether GA could inhibit IKK activity and cause death of
KSHV-infected BC3 cells. Fig.
5A shows that 0.5 µM GA inhibited activity of the vFLIP-IKK
complex in BC3 cells. This concentration of GA also caused a loss in viability
of BC3 cells: after 48 hours, 72% of GA-treated cells were dead, compared with
35% of the control BC3 cells treated with DMSO in serum-free medium
(Fig. 5B). Cell death induced
by GA might be either apoptosis or necrosis, because the dying cells stained
with Annexin-V (Koopman et al.,
1994), which identifies cells that have lost phosphatidylserine
polarity in the plasma membrane, and with propidium iodide, which detects loss
in plasma membrane integrity (Fig.
5C,D).
|
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Discussion |
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We also demonstrated that vFLIP directly contacts IKK, which is
analogous to the function of the Tax protein of human T-cell leukaemia virus
type 1 (HTLV-1). Transformation of T cells by HTLV-1 is mediated by the
regulatory protein Tax, which stimulates expression of various genes regulated
by NF-
B (Sun and Ballard,
1999
). Tax has been shown to stimulate IKK activity
(Chu et al., 1998
;
Geleziunas et al., 1998
;
Uhlik et al., 1998
;
Yin et al., 1998
) by binding
directly to IKK
(Chu et al.,
1999
; Harhaj et al., 1999; Jin
et al., 1999
; Xiao et al.,
2000
). IKK
is predicted to contain five major coiled-coil
domains (Rothwarf et al.,
1998
; Sun et al.,
2000
), of which the second and fifth from the N-terminus contain
leucine zipper motifs (LZ1 and LZ2, respectively)
(Fig. 1). Deletions of LZ1
abolish the binding of Tax to IKK
, whereas mutants lacking LZ2 show
reduced Tax-IKK
interaction (Xiao
et al., 2001
). By contrast, our data demonstrate that a region of
IKK
including CCR3 and CCR4, between amino acids 150 and 272, is
crucial for vFLIP interaction. This shows that the structurally unrelated
viral proteins Tax and vFLIP have evolved distinct mechanisms to bind
IKK
and thereby activate IKK.
Constitutive activation of NF-B is a common feature of viruses that
transform lymphoid cells. Among the gammaherpesviruses, the latent membrane
protein 1 (LMP-1) of Epstein-Barr virus activates the NF-
B pathway by
TRADD and TRAF recruitment to its cytoplasmic tail
(Farrell, 1998
). K15, the
LMP-1 homologue encoded by KSHV, can interact with TRAFs
(Glenn et al., 1999
) but its
role in NF-
B activation in KSHV-infected cells remains unclear. Orf74
of KSHV encodes a constitutively active chemokine receptor homologue that
activates NF-
B (Schwarz et al., 2001) but Orf74 is not latently
expressed in KSHV-infected PEL cells (Chiou
et al., 2002
). However, the K1 transmembrane protein is expressed
in PEL cells and has been implicated in NF-
B activation by transgenic
mouse experiments (Prakash et al.,
2002
). KSHV might therefore use multiple, possibly cooperative,
strategies to activate NF-
B in different target cells and at various
points in the viral life cycle. It is intriguing that a third
gammaherpesvirus, herpesvirus saimiri, activates NF-
B by co-operative
action of two saimiri-specific transforming proteins, Tip and StpC
(Lee et al., 1999
; Merlo et
al., 2001; Yoon et al.,
1997
).
In addition to the IKK subunits, we found Hsp90 associated with vFLIP in
BC3 cells. This is consistent with a previous report that Hsp90 and a
co-chaperone, Cdc37, are additional components of the IKK complex
(Chen et al., 2002). This
previous study demonstrated that the Hsp90 inhibitor GA prevented both
TNF-induced membrane recruitment of the IKK complex to TNF-R1 and TNF-induced
IKK activation (Chen et al.,
2002
). GA also inhibited activity of the vFLIP-IKK complex,
although we did not observe the dissociation of IKK
from the IKK
complex reported by Chen et al. (Chen et
al., 2002
). Consistent with the inhibition of vFLIP-IKK activity,
GA also induced death of BC3 cells. This suggests that vFLIP activation of IKK
is crucial in the maintenance of BC3 cell survival. vFLIP activation of the
NF-
B pathway has also been shown to inhibit apoptosis when vFLIP was
ectopically expressed in a human leukaemic cell line
(Sun et al., 2003
). GA
analogues are promising anticancer agents because Hsp90 is crucial for
maintaining the function of several oncogenic proteins (Maloney et al., 2002).
Our data suggest that they might also be effective in the treatment of
KSHV-related malignancies.
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Acknowledgments |
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Footnotes |
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