From the Cancer Research UK Institute for Cancer Studies, The University of Birmingham Medical School, Birmingham B15 2TT, United Kingdom
Received for publication, September 25, 2002, and in revised form, November 7, 2002
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ABSTRACT |
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The Epstein-Barr virus (EBV) latent membrane
protein 1 (LMP1) is an integral membrane protein that functions as a
constitutively activated member of the tumor necrosis factor
receptor family. Whereas LMP1 has been shown to activate the NF- Epstein-Barr virus
(EBV)1 is a ubiquitous human
herpesvirus associated with the development of both lymphoid and
epithelial tumors. As a common virus infection, EBV appears to have
evolved to exploit the process of B cell development to persist as a
lifelong asymptomatic infection. However, the virus can contribute to
oncogenesis as evidenced by its frequent detection in certain tumors,
namely Burkitt's lymphoma, post-transplant B cell lymphomas,
Hodgkin's disease, and nasopharyngeal carcinoma (NPC), and by its
unique ability to efficiently transform resting B cells in
vitro into permanently growing lymphoblastoid cell lines (1).
These transforming effects are associated with the restricted
expression of EBV genes such that only a subset of so-called latent
virus proteins are expressed in virus-infected tumors and in
lymphoblastoid cell lines. The full complement of eight latent genes
comprising six nuclear antigens (EBNAs) and two membrane proteins (LMP1
and LMP2) are expressed only in post-transplant B cell tumors and in
lymphoblastoid cell lines whereas different forms of latency are
manifest in Burkitt's lymphoma (EBNA1 only) and in Hodgkin's disease
and NPC (EBNA1, LMP1, and LMP2). These distinct forms of EBV latency
appear to be a vestige of the pattern of latent gene expression used by
the virus during the establishment of persistent infection within the B
cell pool (2). Key to the ability of EBV to efficiently colonize memory
B cells is the expression of LMP1 and LMP2 both of which provide
essential survival signals. The aberrant adoption of these forms of
latency can contribute to transformation as evidenced by expression of
LMP1 and LMP2 in NPC and Hodgkin's disease.
LMP1 is the major transforming protein of EBV behaving as a classical
oncogene in rodent fibroblast transformation assays and being essential
for EBV-induced B cell transformation in vitro (3, 4). LMP1
has pleiotropic effects when expressed in cells resulting in induction
of cell surface adhesion molecules and activation antigens (5),
up-regulation of anti-apoptotic proteins (Bcl-2, A20) (6, 7), and
stimulation of cytokine production (interleukin-6 and interleukin-8)
(8, 9). Recent studies have demonstrated that LMP1 functions as a
constitutively activated member of the tumor necrosis factor receptor
(TNFR) superfamily activating a number of signaling pathways in a
ligand-independent manner (10, 11). Functionally, LMP1 resembles CD40,
a member of the TNFR, and can partially substitute for CD40 in
vivo providing both growth and differentiation responses in B
cells (12).
The LMP1 protein is an integral membrane protein of 63 kDa and can be
subdivided into three domains: (a) a
NH2-terminal cytoplasmic tail (amino acids 1-23) that
tethers and orientates the LMP1 protein to the plasma membrane,
(b) six hydrophobic transmembrane loops that are involved in
self-aggregation and oligomerization (amino acids 24-186), and
(c) a long COOH-terminal cytoplasmic region (amino acids
187-386) that possesses most of the signaling activity of the
molecule. Two distinct functional domains referred to as COOH-terminal activation regions 1 and 2 (CTAR1 and CTAR2) have been
identified on the basis of their ability to activate the NF- The expression of LMP1 in NPC is associated with increased metastatic
spread, an effect that is also reflected in the ability of LMP1 to
induce increased cell motility and invasive growth when expressed in
epithelial cells in vitro (21-25). Thus it appears that the
transforming ability of LMP1 may be regulated by novel signaling
pathways that, in addition to the NF- The PI3K family of enzymes is activated by a wide range of
extracellular growth and mitogenic stimuli including ligands of the
TNFR family such as TNF- Cell Lines and the Establishment of Derivatives--
293RcRLMP1
cells carrying an ecdysone-regulatable LMP1 gene have been
described previously (9). 293RcRLMP1 cells were cultured in Dulbecco's
modified Eagle's medium containing 5% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin with 400 µg/ml zeocin, and 400 µg/ml G418. LMP1
expression was induced in 293RcLMP1 cells after the addition of 10 µM muristerone A (Invitrogen). HeLa cell clones
stably expressing LMP1 have been described previously (15). Cells were
cultured in Dulbecco's modified Eagle's medium containing 5% fetal
calf serum, 2 mM glutamine, 100 units/ml penicillin, and
100 µg/ml streptomycin supplemented with 0.5 µg/ml mycophenloic
acid, 20 µg/ml xanthine, and 10 µg/ml hypoxanthine. Rat-1 cells
carrying the gpt selectable marker or stably expressing LMP1 were
generated after transfection of Rat-1 cells with either pSV2gpt or the
LMP1 expression vector, pSV2MTLM. Rat-1 cells stably expressing a
chimeric RatCD2:LMP1 have been described previously (9).
Activation of the RatCD2:LMP1 Chimeric Protein in Rat-1
Cells--
106 cells seeded into each well of a 6-well
plate (Nunc) were serum-starved for 24 h prior to cross-linking.
Briefly, cells were treated with 1 µg/ml anti-RatCD2 (OX34) followed
by a 1/2000 dilution of rabbit anti-mouse IgG (Dako). At various times
after cross-linking, cells were recovered in lysis buffer and snap
frozen prior to analysis.
Microinjection of Swiss 3T3 Fibroblasts--
Swiss 3T3
fibroblasts were cultured in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. 5 × 103-104 cells were seeded onto each well of a
Teflon-coated microscope slide (Hendley-Essex) in 100 µl of medium.
After overnight attachment, the medium was replaced with serum-free
medium and the cells incubated for an additional 24 h. Cells were
microinjected with pSG5-based expression vectors containing wild-type
LMP1 (pSG5-LMP1), or mutant LMP1 proteins carrying a defective CTAR1
domain (pSG5-LMP1AAA), defective CTAR2 domain
(pSG5-LMP1378Stop), or defective CTAR1 and CTAR2 domains
(pSG5-LMP1AAA/378Stop). These mutants have been described
previously (9). For certain experiments, cells were co-microinjected
with wild-type LMP1 (pSG5-LMP1) and expression vectors containing
dominant negative forms of Cdc42 (pRK5-Myc-N17Cdc42), Rac
(pRK5-Myc-N17Rac), or Rho (pRK5-Myc-N17Rho) (kind gifts from Professor
Alan Hall, Ludwig Institute for Cancer Research, London, UK).
Expression vectors containing a constitutively active PI3K
(p110CAAX) or a dominant-negative p85 were kind gifts from Dr.
Julian Downward, ICRF, London, UK. p110CAAX encodes the
catalytic subunit of type IA PI3K linked to a CAAX membrane
targeting motif of Ras. Dominant-negative p85 encodes a wild-type p85
regulatory subunit deleted for the amino-terminal Src homology 2 domain.
To evaluate PI3K activation in microinjected cells, pSG5-based LMP1
expression vectors were co-microinjected with an expression vector
carrying a green fluorescent protein (GFP)-tagged GRP1 PH domain fusion
protein (29). Between 3 and 5 h after microinjection, cells were
fixed in 4% paraformaldehyde and visualized under UV fluorescence for
evidence of membrane targeting of the GFP tag. In each experiment, at
least 200 cells were microinjected; each experiment was performed at
least three times.
Immunostaining of Microinjected Cells for LMP1 and
Actin--
Between 3 and 5 h after microinjection, cells were
fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton
X-100. Cells were immunostained for LMP1 expression using the
LMP1-specific monoclonal antibody OTT22C (a kind gift from Dr. J. Middledorp, Netherlands) and Oregon Green-conjugated goat anti-mouse
IgG (Molecular Probes). Filamentous actin was visualized using a 1/400
dilution of TRITC-labeled phalloidin (Sigma).
Immunoprecipitations and Western Blotting--
Subconfluent
cultures were lysed in an Nonidet P-40-based lysis buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 0.1 mM sodium orthovanadate, 10% glycerol, 1% Nonidet P-40, 5 mM EDTA, 10 µg/ml aprotinin, and 1 mM
phenylmethylsulfonyl fluoride). 400 µg of cleared total cell lysate
was immunoprecipitated with either 15 µl of anti-p85 (Serotec) or 25 µl of anti-LMP1 (CS1-4). Samples were incubated at 4 °C for
2 h, and immune complexes captured after a further 2-h incubation
with protein A-agarose (Sigma). After 4 washes in cold lysis buffer,
the beads were resuspended in 50 µl of gel sample buffer and boiled
for 5 min. Immunoprecipitated proteins were separated on 7.5%
polyacrylamide gels and proteins transferred to nitrocellulose. After
blocking, membranes were probed with either a monoclonal antibody to
LMP1 (S12), or a rabbit polyclonal antibody to p85 (Upstate
Biotechnology). Bound antibody was detected using anti-mouse
horseradish peroxidase-conjugated secondary antibody and visualized by
enhanced chemiluminescence (Amersham Biosciences).
PI3K and Akt Kinase Assays--
PI3K activity was assayed as
described (30). Briefly, cells were serum-starved for 24 h prior
to lysis in an Nonidet P-40-based buffer (25 mM HEPES, pH
7.5, 150 mM NaCl, 0.1 mM sodium orthovanadate, 10 glycerol, 1% Nonidet P-40, 5 mM EDTA, 100 units/ml
aprotinin, and 1 mM phenylmethylsulfonyl fluoride). In some
cases, cells were stimulated with 100 ng/ml insulin-like growth
factor-1 or 10% fetal calf serum for the indicated times (5-10 min).
Cell lysates were immunoprecipitated with anti-p85 monoclonal antibody U13 (Serotec). Protein A-Sepharose immune complexes were washed twice
with phosphate-buffered saline containing 1% Nonidet P-40, once with
phosphate-buffered saline, once with 0.1 M Tris-HCl, pH
7.5, containing 0.5 M LiCl, once with distilled water, and once with 25 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EDTA. The beads were suspended in 50 µl of 25 mM HEPES, pH 7.5, 100 mM NaCl, 0.5 mM EGTA, and 10 µg of phosphatidylinositol (Sigma),
sonicated for 5 min at 20 °C, and preincubated at 20 °C for 10 min. Twenty µCi of [
Akt kinase assays were carried out according to the manufacturers
instructions (New England Biolabs). Briefly, 200 µg of total cell
lysate was immunoprecipitated overnight with immobilized mouse anti-Akt
IgG. After washing in two changes of lysis buffer and three changes of
kinase assay buffer, the beads were resuspended in kinase assay buffer
containing 1 µg of GSK3 peptide substrate and 10 µM
ATP. After incubation at 30 °C for 30 min, the reaction was stopped
with the addition of 20 µl of gel sample buffer. Supernatants were
resolved by SDS-PAGE and proteins transferred to nitrocellulose. After
blocking, membranes were probed with a phospho-GSK3 specific antibody
(Cell Signaling Technology). Bound antibody was detected using
anti-mouse horseradish peroxidase-conjugated secondary antibody and
visualized by enhanced chemiluminescence (Amersham Biosciences).
Cell Viability and Apoptosis Assays--
Cell viability was
measured using the AlamarBlueTM Assay (Serotec). Briefly,
cells were plated out at 5 × 103 cells/well in
triplicate on a 96-well plate and left overnight. Cell viability was
analyzed at 72 h post-treatment with or without 20 µM LY294002. AlamarBlueTM was added
aseptically at 10% of the culture volume, and the cells were incubated
for 4 h at 37 °C. Fluorescence was measured with excitation
wavelength at 560 nm and emission wavelength at 590 nm. For detection
of apoptosis, cells were stained with acridine orange. Vector control
or LMP1-expressing clones were seeded into each well of a 6-well plate
at a concentration of 5 × 105 cells per well. 24 h later, medium was replaced with serum-free medium containing 20 µM LY294002 or Me2SO vehicle control. Cells were cultured for an additional 72 h after which time they were recovered as single cell suspensions. Cells were examined for evidence
of apoptosis after incubation of cell suspensions with 5 µg/ml
acridine orange followed by microscopic analysis.
LMP1 Interacts with p85, the Regulatory Subunit of PI3K--
The
recent demonstration that a number of TNFR family members such as CD40,
TNFR1, and TRANCE-R engage the PI3K/Akt signaling pathway prompted us
to investigate whether LMP1 could also activate this pathway. For both
CD40 and the TRANCE receptor, the regulatory subunit of PI3K, p85, is
recruited to the activated receptor complex after receptor ligation
(31, 32). Given that LMP1 behaves as a constitutively activated TNF
receptor, it was reasoned that PI3K would associate with LMP1 in a
constitutive manner. Thus immunoprecipitation experiments were
performed in HeLa cells stably expressing LMP1 to determine whether
PI3K formed part of the LMP1 signaling complex (Fig.
1). LMP1 was efficiently
immunoprecipitated from HeLa cells (Fig. 1A) and these
immunoprecipitates were found to contain a significant proportion of
p85 when probed with a specific p85 antibody (Fig. 1B). The
reciprocal analysis using p85 immunoprecipitates confirmed the
interaction with LMP1 (Fig. 1D) and demonstrated a
diminished ability to immunoprecipitate p85 in LMP1-expressing cells
(Fig. 1C). This may result from the LMP1-p85
interaction reducing the efficiency of p85 LMP1 Expression Results in Constitutive Activation of the
PI3K/Akt Pathway--
To directly examine the ability of
LMP1 to activate PI3K, the basal level of PI3K activity was measured in
three independent HeLa clones stably expressing LMP1. These cells were
serum-starved for 24 h and the ability of immunoprecipitated PI3K
(p85 immunoprecipitates) to phosphorylate PI, assayed by lipid kinase
assay. As shown in Fig. 2A,
the basal levels of PI3K activity were up to 2-fold greater in
LMP1-expressing clones compared with two vector control transfectants. The elevated PI3K activity in LMP1-expressing cells correlated with
activation of Akt as determined using a phosphospecific antibody that
recognizes Akt phosphorylated on serine 473 (Fig. 2B). To confirm that LMP1 expression resulted in activation of Akt, in vitro kinase assays were performed using Akt immunoprecipitates and a GSK3 peptide as substrate. These assays demonstrated
significantly higher levels of basal Akt activity in LMP1-expressing
HeLa cells compared with vector controls and this activity was
inhibited by the LY290042 compound confirming the dependence of this
effect on PI3K (Fig. 2C). Identical results were obtained
using A431 cells stably expressing LMP1 (data not shown).
Transient Induction of LMP1 Expression Results in Activation of the
PI3K/Akt Pathway--
The constitutively active nature of
LMP1 can obscure the impact of this protein on cell signaling pathways.
To overcome this problem, an ecdysone-inducible system was generated to
provide regulatable expression of LMP1. In this system, binding of the steroid hormone Muristerone A to a hybrid ecdysone-modified retinoic acid receptor allows the subsequent activation of an
ecdysone-responsive target gene. HEK 293 cells carrying the pVgRXR
plasmid (293EcR), which encodes the receptor subunits, and pIND-LMP1,
which contains the LMP1 cDNA under the control of
ecdysone-responsive elements, were generated. A tightly regulatable
clone was identified that demonstrated induction of LMP1 expression
after 6 h of Muristerone A treatment with steadily increasing LMP1
levels up to 48 h post-treatment and no LMP1 expression in cells
not treated with the hormone (Fig. 3A). This induction of LMP1
expression correlated temporally with increased PI3K activity which at
6-48 h post-treatment was 2-3-fold higher than that observed in
non-LMP1 expressing cells (Fig. 3B). Consistent with this
increase in PI3K activity, induction of LMP1 resulted in
phosphorylation of p85, which was evident as early as 3 h after
induction and remained elevated at 12 h (Fig. 3C). Likewise, Akt activity as determined by phosphorylation at serine 473 was induced after 3 h and increased steadily over 24 h in concert with PI3K activity (Fig. 3D). To rule out the
possibility that hormone treatment of 293 cells resulted in PI3K/Akt
activation, parental 293 RXR cells lacking pIND LMP1 were serum-starved
prior to treatment with 10 µM Muristerone A. Muristerone
treatment of these cells failed to induce either PI3K activity or Akt
phosphorylation (data not shown).
Membrane Oligomerization of LMP1 Is Required for PI3K
Activation--
To determine whether membrane oligomerization of LMP1
is an essential requirement for PI3K/Akt activation, we used of an
expression system in which a chimeric RatCD2-LMP1 chimera can be
induced to signal after the addition of antibodies to CD2 and
subsequent cross-linking with anti-mouse IgG (9). As the chimeric
molecule lacks the amino terminus and transmembrane spanning regions of LMP1, this approach also allowed us to assess the contribution of the
cytosolic COOH terminus to LMP1-induced PI3K/Akt activation. Treatment
of Rat-1 cells stably expressing the Rat CD2-LMP1 chimera with the
anti-CD2 mAb (OX34) and cross-linking anti-mouse immunoglobulin G
induced Akt phosphorylation within 30 min and this continued to
increase for up to 24 h (Fig.
4A). Consistent with the
increase in Akt activity, an antibody specific for the phosphorylated
form of GSK-3 (p-GSK3) demonstrated phosphorylation of this Akt
substrate after CD2 engagement (Fig. 4B) and this effect was
further confirmed by in vitro kinase assays (Fig.
4C). Pretreatment of Rat CD2LMP1-expressing cells with
LY294002 significantly reduced Akt phosphorylation induced by CD2
engagement (Fig. 4D). To rule out the possibility that
antibody treatment alone of cells could contribute to PI3K/Akt activation, a neomycin-resistant Rat-1 cell clone, Rat-1 neo clone 2, was incubated in the presence of the Rat CD2 monoclonal antibody and
subsequent cross-linking with ant-mouse IgG. Unlike the Rat CD2LMP1-expressing cells, treatment of the Rat-1 neo cells with anti-CD2 and anti-mouse IgG failed to induce either PI3K activity or
Akt phosphorylation (data not shown). These data demonstrate that LMP1
oligomerization is required for activation of the PI3K/Akt pathway and
that this activation is mediated by the LMP1 cytoplasmic domain.
The CTAR1 Domain of LMP1 Is Responsible for PI3K
Activation--
To identify the domain within the LMP1 cytoplasmic
tail that is responsible for PI3K/Akt activation, we made use of an
expression vector, pPH-GRP1-GFP, comprising GFP fused to the PH domain
of the GRP1 protein. The PH domain of GRP1 is a specific receptor for
3'-phosphorylated phosphoinositide lipids (phosphatidylinositol 1,4,5-trisphosphate (PIP3) and phosphatidylinositol 3,4,-bisphosphate (PI3-4P2)) generated as a consequence of PI3K activity and, as a
GFP fusion, can be used to monitor the PI3K activity at the cellular
level (29). Swiss 3T3 cells co-microinjected with pSG5 and pPH-GRP1-GFP
showed a diffuse cytoplasmic distribution (Fig. 5A), whereas cells
co-microinjected with a constitutively activated form of the catalytic
subunit of PI3K, p110CAAX, showed clear plasma membrane
association resulting from translocation of the GFP-tagged fusion
protein to sites of PI3K activity at the plasma membrane (Fig.
5B). Co-microinjection of wild-type LMP1 resulted in
translocation of GRP1-GFP to the plasma membrane confirming that the
expression of wild-type LMP1 resulted in 3'-phosphoinositide generation
in cell membranes (Fig. 5C). To identify the domain of LMP1
required for PI3K activation, co-microinjection experiments were
performed with LMP1 mutants defective for the CTAR1 and CTAR2 effector
domains. Co-expression of the CTAR1 mutant pSG5LMP1AAA did
not result in GRP1-GFP translocation to the cell membrane implicating
CTAR1 as the domain of LMP1 responsible for this response (Fig.
5D). This was confirmed with the CTAR2 mutant,
pSG5LMP1378Stop, where co-expression resulted in
translocation of the GFP-tagged GRP1 fusion protein to the plasma
membrane (Fig. 5E). The CTAR1/CTAR2 double mutant
pSG5LMP1AAA/378Stop failed to induce GRP1-GFP translocation
consistent with the role of CTAR1 in this effect (data not shown).
LMP1-induced Actin Polymerization Requires PI3K Activity and Is
Regulated by the Small Rho GTPases--
In addition to activating
downstream targets such as Akt, 3-phosphoinositide-dependent
protein kinase 1 (PDK1), and protein kinase C, PI3K also plays a
key role in regulating actin cytoskeletal organization through
activation of the small Rho GTPases. The lipid products generated by
PI3K regulate the activity of a number of guanine nucleotide exchange
factors for the small Rho GTPases. The recent demonstration that LMP1
induces filopodia and actin stress-fiber formation (33) prompted us to
examine whether the induction of PI3K by LMP1 resulted in actin
polymerization. In agreement with our previous findings, microinjection
of LMP1 resulted in robust actin stress-fiber formation (Fig.
6, A and B).
Co-microinjection of LMP1 with dominant-negative Rho (N17Rho),
completely inhibited actin stress-fiber formation (Fig. 6, C
and D), demonstrating the requirement for Rho in this
response. Similarly, co-microinjection of dominant negative Cdc42
(N17Cdc42) almost completely abrogated LMP1-induced actin stress-fiber
formation (Fig. 6, E and F) whereas dominant-negative Rac (N17Rac) partially blocked this effect (Fig. 6,
G and H).
To establish a role for PI3K in LMP1-mediated actin stress-fiber
formation, two approaches were employed. First, LMP1 was microinjected
into cells that were then treated with the LY294002 compound. Compared
with cells microinjected with LMP1 alone (see Fig. 6A),
cells treated with LY294002 showed very little stress-fiber formation
(Fig. 7B) although there was
clear expression of the LMP1 protein (Fig. 7A). In other
experiments LMP1 was co-microinjected with a dominant-negative form of
p85 ( Role of LMP1-induced PI3K Activity in Morphological Transformation
and Cell Survival--
Previous work demonstrating that PI3K
activation is responsible for the morphological transformation of Rat-1
fibroblasts (34) prompted us to examine whether PI3K activation plays a role in the morphological transformation of Rat-1 fibroblasts by LMP1.
Vector control and LMP1-expressing Rat-1 cells were cultured in the
presence or absence of 20 µM LY294002 for 18-24 h, and the morphology of the cells subsequently examined by phase microscopy. Whereas treatment of vector control cells with LY294002 was associated with slight growth inhibition (Fig. 9,
A and B), it was not associated with an
alteration in cell morphology. In marked contrast, treatment of
LMP1-expressing cells with LY294002 resulted in a profound alteration
in cell morphology (Fig. 9D). Whereas control untreated LMP1
expressing cells cultured showed evidence of reduced contact inhibition
and cell-rounding (Fig. 9C), cells cultured in the presence
of 20 µM LY294002 showed a clear-cut reversal of the transformed phenotype, with cells re-acquiring a flatter morphology (Fig. 9D).
To investigate the contribution of PI3K activation to cell survival,
vector control and LMP1-expressing Rat-1 cells were cultured in the
presence or absence of LY294002 under serum-free conditions. Whereas
vector control clones showed only minimal evidence of cell death after
treatment (Fig. 10, A and
C), prolonged treatment of LMP1-expressing cells with 20 µM LY294002 resulted in a profound decrease in cell
viability (Fig. 10, B and D). To investigate this phenomenon further, a panel of vector control and LMP1-expressing clones were treated with increasing concentrations of LY294002, and the
effects on cell survival after serum withdrawal were analyzed in a cell
viability assay. Increasing concentrations of LY294002 resulted in a
20-50% decrease in cell number in vector control clones, attributable
to the effects of PI3K inhibition on cell proliferation (Fig.
10E). In contrast, Rat-1 cells expressing LMP1 were clearly
more sensitive to PI3K inhibition, with cell numbers being reduced by
85-90%. To confirm that the cytotoxicity observed after LY294002
treatment was because of apoptosis, suspensions of control and
LY294002-treated cells were suspended in a solution containing 5 µg/ml acridine orange. This protocol has been used extensively to
differentiate between viable and apoptotic cells. Vector control and
LMP1-expressing clones cultured for 72 h in the absence of serum
showed little evidence of apoptosis with the majority of cells
displaying intense nuclear staining defining dense nuclear chromatin
(Fig. 11, A and
C). This was in marked contrast to LY294002-treated cells
where the condensed chromatin characteristic of apoptosis was observed
(Fig. 11, B and D). Compared with a
representative vector control clone (Fig. 11B), LY294002 treated LMP1-expressing cells displayed significantly higher numbers of
apoptotic cells (Fig. 11D).
We have shown that LMP1 can activate the PI3K/Akt pathway and that
this is responsible for some of the phenotypic effects of LMP1
associated with cell transformation. This result is not surprising
given the similarities between LMP1 signaling and that elicited from
the TNFR family, members of which can activate PI3K. Thus, TNF- An understanding of the signaling capacity of LMP1 is key to defining
its role in EBV-induced oncogenesis and in identifying pathways that
may be of general significance in the transformation process. As the
only EBV-encoded protein with the characteristics of a classical
oncogene, the signaling pathways engaged by this molecule are clearly
crucial in effecting B cell transformation and in inducing a plethora
of phenotypic effects relevant to cellular transformation. Whereas
previous work has focused on the contribution of the NF- The ability of LMP1 to activate PI3K helps to explain a number of the
phenotypic consequences of its expression in different cellular
environments. PI3K plays a key role in regulating actin cytoskeletal
organization and cell shape remodeling by regulating the activity of
the small Rho GTPases (40, 41). We recently reported that LMP1 induces
filopodial extensions associated with lamellopodia and stress fibers in
Swiss 3T3 cells (33). In this report we show that LMP1 can induce
robust actin stress-fiber formation that is dependent on the small Rho
GTPases and on PI3K. Through the use of a panel of LMP1 mutants, we
have identified CTAR1 as the region of LMP1 essential for actin
stress-fiber formation and this correlates with the ability of CTAR1 to
mediate PI3K activation. In our earlier study (33) we demonstrated that
Cdc42-induced filopodia formation was mediated through the
transmembrane spanning regions of LMP1 whereas here, Rho-mediated
stress-fiber formation requires CTAR1-generated signals. Although,
Cdc42-induced filopodia formation was not investigated in this current
study, the possibility that LMP1 may activate both Cdc42 and Rac/Rho
independently through distinct mechanisms clearly warrants further
examination. An important consequence of these effects on actin
filament remodeling and cell shape change is the ability of LMP1 to
induce morphological transformation in Rat-1 cells. Interesting, this
phenotype could be reversed by the LY294002 PI3K inhibitor suggesting
that PI3K-generated signals are an important component in LMP1-mediated
cell transformation. Future studies will elucidate the precise
mechanisms involved in this process and the relative contributions of
the small Rho GTPases. The significance of these observations for the
function of LMP1 in EBV-infected epithelial cells and B cells is worthy of consideration. Previous studies have demonstrated that LMP1 expression in NPC is associated with more advanced tumors and with
increased metastatic spread (22, 24, 25). These observations concur
with in vitro data showing that LMP1 induces morphological transformation as well as a more motile and invasive phenotype in
Madin-Darby canine kidney cells
(23).2 Interestingly these
effects of LMP1 were mapped to the CTAR1 region that we have now shown
to be responsible for PI3K activation. The activation and migration of
B cells also requires small Rho GTPase-dependent
cytoskeletal changes that are crucial for the formation of germinal
centers (42, 43). Thus the ability of LMP1 to induce actin
reorganization is likely to contribute to the establishment of EBV
persistence in the memory B cell pool.
The role of Akt in cellular growth transformation is now clearly
established (44). Akt has emerged as a critical signaling molecule that
regulates a variety of cellular processes including cell growth,
proliferation, and apoptosis. Although Akt is implicated in the
regulation of various metabolic events, it is its role in regulating
aspects of cell survival that has received most attention. Akt targets
and inactivates a number of pro-apoptotic molecules associated with
the induction of apoptosis (45). These include the pro-apoptotic Bcl-2
family member, Bad, caspase 9, and GSK3 among others (46). The ability
of Akt to modulate these key effector molecules explains how cytokines
and growth factors are able to promote cell survival in response to
growth factor or serum withdrawal. The recent finding that Akt is able
to induce NF- Given the profound effects of activating PI3K on cell survival and
proliferation, it is not surprising that a diverse range of viruses
have evolved to target this pathway for the efficient promotion of
virus infection and replication. This is particularly evident for tumor
viruses where oncoproteins such as polyoma middle T antigen, SV40 large
T antigen, and the KSHV-encoded GPCR have all been shown to activate
the PI3K/Akt pathway. Indeed, PI3K activity was first discovered in a
complex with polyoma middle T antigen and the c-src tyrosine
kinase (49). That PI3K and Akt activation are critical for polyoma
middle T antigen-mediated cellular transformation is borne out by the
findings that mutation of the putative p85 binding site in polyoma
middle T antigen abrogates its ability to transform rodent fibroblast
cell lines and to protect from apoptosis induced by serum withdrawal
(50, 51). Unlike these other oncoproteins, LMP1 does not possess
intrinsic tyrosine kinase activity nor has it been shown to associate
with any known tyrosine kinase(s). Thus, the mechanism by which LMP1
recruits and activates PI3K is clearly a focus for future work as is a more detailed examination of the functional contribution of PI3K activation to LMP1-induced transformation.
B
and mitogen-activated protein kinase pathways, these effects alone are
unable to account for the profound oncogenic properties of LMP1. Here
we show that LMP1 can activate phosphatidylinositol 3-kinase
(PI3K), a lipid kinase responsible for activating a diverse range of
cellular processes in response to extracellular stimuli. LMP1 was found to stimulate PI3K activity inducing phosphorylation and subsequent activation of Akt, a downstream target of PI3K responsible for promoting cell survival. Treatment of LMP1-expressing cells with the
PI3K inhibitor LY294002 resulted in decreased cell survival. The tumor
necrosis factor receptor-associated factor-binding domain of LMP1 was
found to be responsible for PI3K activation. The ability of LMP1 to
induce actin stress-fiber formation, a Rho GTPase-mediated phenomenon,
was also dependent on PI3K activation. These data implicate PI3K
activation in many of the LMP1-induced phenotypic effects associated
with transformation and suggests that this pathway contributes both to
the oncogenicity of this molecule and its role in the establishment of
persistent EBV infection.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B
transcription factor pathway (13). This effect contributes to the many
phenotypic consequences of LMP1 expression including the induction of
various anti-apoptotic and cytokine genes. LMP1 is also able to engage
the mitogen-activated protein kinase cascade resulting in
activation of extracellular-regulated protein kinase, c-Jun
NH2-terminal kinase, and p38 and to stimulate the
JAK/signal transducers and activators of transcription pathway (9,
14-17). Many of these effects result from the ability of
TNFR-associated factors (TRAFs) to interact either directly with CTAR1
or indirectly via the death domain protein TRADD to CTAR2 (1). The
binding of TRAFs to the multimerized cytoplasmic tails of LMP1 provides a platform for the assembly and activation of upstream signaling molecules including the NIK and Tpl-2 mitogen-activated protein kinase
kinase kinases (18, 19). The precise mechanisms responsible for signal
initiation from these multiprotein complexes remain unknown. The region
between CTAR1 and CTAR2 (so-called CTAR3) has been suggested to be
responsible for the JAK/signal transducers and activators of
transcription pathway although other data refute this finding and
deletion of this region has no effect of the efficiency of B cell
transformation (17, 20).
B and mitogen-activated protein
kinase cascades, may account for the ability of this molecule to
influence cell motility and to provide a profound survival advantage.
One pathway that fulfils these criteria is that mediated by
phosphatidylinositol 3-kinase (PI3K), which via generation of specific
phospholipids, activates a diverse range of cellular processes
including cell growth, motility, adhesion, and survival (26).
and CD40, thus suggesting that LMP1 may
also target this pathway (27, 28). PI3K-generated phospholipids are
responsible for activating the Akt (PKB) kinase thereby promoting cell
survival and for regulating the Rho family of small GTPases resulting
in effects on the actin cytoskeleton and on cell signaling. Here we
demonstrate that LMP1 can activate the PI3K/Akt pathway and that this
effect is responsible for LMP1-induced actin polymerization, morphological transformation, and also contributes to cell survival. Furthermore, we show that the region of LMP1 previously identified as
being essential for EBV-induced B cell transformation, CTAR1, is also
responsible for PI3K activation.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP and MgCl2
(final concentration, 10 mM) were added, and samples were
further incubated for 20 min at 20 °C. The reaction was stopped by
addition of chloroform/methanol, 11.6 M HCl (50:100:1),
phospholipids were extracted with chloroform, and the organic phase was
washed with methanol, 1 M HCl (1:1). Reaction products were
concentrated in vacuo, dissolved in chloroform, spotted on
Silica Gel-60 plates (Merck) impregnated with 1 potassium oxalate, and
resolved by chromatography in chloroform/methanol, 28%
ammonia/water (43:38:5:7) for 45 min. Phosphorylated products were
detected by Image Analyzer (Amersham Biosciences) and then
exposed on x-ray films (Kodak).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
antibody binding as total
cellular levels of p85 were unaltered in LMP1-expressing cells (data
not shown).
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Fig. 1.
The p85 regulatory subunit of PI3K associates
with LMP1 in vivo. A, LMP1
immunoprecipitated from HeLa LMP1 clones with the CS1-4 antibody is
detected with the LMP1-specific monoclonal antibody, S12. B,
probing of the same immune complexes with a polyclonal antisera to p85
demonstrated the presence of p85 in LMP1 immune complexes.
C, p85 immunoprecipitated with the U13 monoclonal antibody
confirmed reduced expression of p85 in LMP1 expressing cells.
D, reciprocal immunoprecipitations confirmed the association
of LMP1 with p85 in p85 immunoprecipitates.
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Fig. 2.
Stable expression of LMP1 results in elevated
levels of PI3K activity and increased phosphorylation of Akt.
A, total cell lysates from representative vector control or
LMP1 expressing HeLa cell clones were analyzed for basal PI3K activity.
PI3K activity in p85 immunoprecipitates was analyzed in
vitro by lipid kinase assay using phosphatidylinositol as a
substrate (*, average of duplicate determinations). B, total
cell lysates from the same panel of clones were analyzed for evidence
of Akt phosphorylation after immunoblotting with a rabbit polyclonal
antibody specific for the Ser-473 phosphorylated form of Akt.
C, Akt kinase activity was assessed in a representative
vector control and LMP1-expressing clones after immunoprecipitation of
Akt and in vitro kinase assay using a GSK-3 peptide
substrate. Akt kinase activity was assessed in the presence or absence
of 20 µM LY294002, a selective inhibitor of PI3K.
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Fig. 3.
PI3K activity and Akt phosphorylation
coincide temporally with the induction of LMP1 in 293 cells. 293 RcR cells carrying an ecdysone-regulatable LMP1 gene were
serum-starved for 24 h prior to incubation with 10 µM Muristerone A to induce expression of LMP1. At
selected time points, cell lysates were prepared and assayed for LMP1
expression, after immunoblotting with the CS1-4 pool of antibodies
specific for LMP1 (A), PI3K activity by in vitro
lipid kinase assay (B), p85 phosphorylation, after
immunoprecipitation of p85 and subsequent blotting with a
phosphotyrosine-specific antibody (C), and phosphorylation
of Akt after immunoblotting with a rabbit polyclonal antibody specific
for the serine 473-phosphorylated form of Akt (D).
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Fig. 4.
LMP1-induced activation of PI3K/Akt requires
the cytosolic COOH terminus of LMP1. Rat-1 cells expressing a
CD2-LMP1 chimera were serum-starved for 24 h prior to Rat CD2
cross-linking with mouse anti-CD2 and rabbit anti-mouse IgG. Extracts
from cell lysates were blotted for phosphorylated Akt using a rabbit
polyclonal antibody specific for the Ser473-phosphorylated
form of Akt (A), and phosphorylated GSK-3 (pGSK-3) using a
mouse monoclonal antibody specific for the phosphorylated form of GSK-3
(B). C, in vitro kinase assays were
performed on immunoprecipitated Akt from the same panel of lysates.
D, to establish a role for PI3K in LMP1-mediated Akt
activation, Rat-1 CD2:LMP1 cells were incubated in the presence or
absence of 20 µM LY294002 and, 60 min after
cross-linking, assayed for Akt phosphorylation by Western blotting
using a rabbit polyclonal antibody specific for the
Ser473-phosphorylated form of Akt.
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Fig. 5.
LMP1-mediated PI3K activation is mediated
through the CTAR1 domain of LMP1. To assay PI3K activity in
vivo, quiescent 3T3 fibroblasts were co-microinjected with a
GRP1-PH-GFP fusion protein and pSG5 (A), p110CAAX
(B), wild-type LMP1 (C), LMP1AAA
(D), or LMP1378stop (E). 3-5 h after
microinjection, cells were fixed in 4% paraformaldehyde, and evidence
of PI3K activation (3'-phosphoinositide generation) as demonstrated
by membrane targeting of the GFP-PH-GRP1 fusion protein was established
for each protein. A representative photomicrograph is shown for each
plasmid. In each case, between 200 and 250 cells were microinjected per
experiment. Bar represents 30 µm.
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Fig. 6.
LMP1-mediated stress-fiber formation is
mediated through the small Rho GTPases. To investigate the role of
the small Rho GTPases in LMP1-mediated actin stress-fiber formation,
quiescent Swiss 3T3 fibroblasts were co-microinjected with LMP1 and
empty vector (A and B) or dominant negative forms
of Rho (C and D), Rac (E and
F), and Cdc42 (G and H). LMP1
expression (A, C, E, and G)
was visualized after fixing and staining with the CS1-4 pool of
antibodies and Oregon Green-conjugated anti-mouse IgG, whereas actin
stress-fiber formation was visualized after incubation with
rhodamine-conjugated phalloidin (B, D,
F, and H). A representative photomicrograph is shown
for each plasmid. In each case, between 200 and 250 cells were
microinjected per experiment. Bar represents 30 µm.
Np85) that is deleted for the amino-terminal Src homology 2 domain and thus competes for binding of the catalytic p110 subunit.
Co-microinjection of LMP1 with
Np85 resulted in complete inhibition
of actin stress-fiber formation (Fig. 7, C and
D). Consistent with this observation, co-microinjection of
LMP1 with N17Ras also inhibited LMP1-mediated actin stress-fiber
formation (Fig. 7, E and F) presumably via the
ability of N17Ras to sequester p85-p110 complexes rendering them
unavailable for recruitment to activated receptors. Unlike the
wild-type LMP1 protein (Fig. 8,
A and B), microinjection of the CTAR1-deletion
mutant pSG5LMP1AAA failed to induce actin stress-fiber
formation (Fig. 8, C and D), whereas
microinjection of the CTAR2 mutant, pSG5LMP1378Stop,
resulted in robust stress-fiber formation (Fig. 8, E and
F). This result is consistent with the ability of CTAR1 to
stimulate PI3K activity thus substantiating the role of PI3K in
LMP1-induced stress-fiber formation. Microinjection of the CTAR1/CTAR2
double mutant pSG5LMP1AAA/378Stop failed to induce actin
stress-fiber formation (Fig. 8, G and H)
demonstrating that the amino terminus, transmembrane spanning regions,
and the repeat region between CTAR1 and CTAR2 do not contribute to this
effect.
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Fig. 7.
LMP1-mediated stress-fiber formation requires
PI3K activity. To investigate the role of PI3K in LMP1-mediated
actin stress-fiber formation, quiescent Swiss 3T3 fibroblasts were
co-microinjected with LMP1 and empty vector (pSG5) (A and
B), a dominant negative form of p85, Np85 (C
and D), or a dominant-negative form of Ras, N17Ras
(E and F). In A and B,
cells were treated with 20 µM LY294002, prior to fixation
4-5 h post-microinjection. LMP1 expression (A,
C, and E) was visualized after fixing and
staining with the CS1-4 pool of antibodies and Oregon Green-conjugated
anti-mouse IgG, whereas actin stress-fiber formation was visualized
after incubation with rhodamine-conjugated phalloidin (B,
D, and F). A representative photomicrograph is
shown for each plasmid. In each case, between 200 and 250 cells were
microinjected per experiment. Bar represents 30 µm.
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Fig. 8.
LMP1-mediated actin stress-fiber formation
maps to the CTAR1 domain of LMP1. To determine the domain of LMP1
required for LMP1-mediated actin stress-fiber formation, quiescent
Swiss 3T3 fibroblasts were co-microinjected with LMP1 (A and
B) and a panel of LMP1 mutants defective for CTAR1
(C and D), CTAR1 (E and F),
or both CTAR1 and CTAR2 (G and H). LMP1
expression (A, C, E, and G) was visualized after
fixing and staining with the CS1-4 pool of antibodies and Oregon
Green-conjugated anti-mouse IgG, whereas actin stress-fiber formation
was visualized after incubation with rhodamine-conjugated phalloidin
(B, D, F, and H). A representative
photomicrograph is shown for each plasmid. In each case, between 200 and 250 cells were microinjected per experiment. Bar
represents 30 µm.
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Fig. 9.
LMP1-induced transformation of Rat-1
fibroblasts requires PI3K activity. Vector control (A
and B) or LMP1-expressing (C and D)
clones were incubated in the presence (A and C)
or absence (B and D) of the PI3K inhibitor
LY294002 for 48 h, and cell morphology recorded by phase-contrast
microscopy. Magnification ×400.
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Fig. 10.
Prolonged exposure of LMP1-expressing Rat-1
cells to LY294002 results in cytotoxicity. Representative vector
control (A and C) or LMP1-expressing clones
(B and D) were switched to serum-free medium
containing vehicle alone (A and B) or 20 µM LY294002 (C and D). 72 h
after exposure, cell viability was examined by phase-contrast
microscopy. Magnification ×200. E, vector control and
LMP1-expressing Rat-1 cells were switched to serum-free medium
containing increasing concentrations of the PI3K inhibitor LY294002,
72 h later, cell viability was determined by Alamar
BlueTM assay. Data are represented as the means of
triplicate determinations with S.E.
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Fig. 11.
Apoptosis is induced after prolonged
exposure of LMP1-expressing Rat-1 cells to LY294002. Vector
control (A and B) and LMP1-expressing clones
(C and D) were cultured in the absence
(A and C), or presence (B and
D) of 20 µM LY294002. Evidence of apoptosis
was determined 72 h later after incubation with 5 µg/ml acridine
orange. Arrows denote apoptotic cells with characteristic
condensed nuclear chromatin.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
has
been shown to activate PI3K and Akt and this effect was reported to be
required for activation of NF-
B (35, 36). Inhibition of PI3K was
found to potentiate TNF-
-induced apoptosis, an effect reminiscent of
that observed in this study when LMP1-expressing cells were treated
with the LY294002 inhibitor. LMP1 most closely resembles an activated
CD40 in its phenotypic effects and LMP1 can partially correct the B
cell development defect in CD40-deficient mice (12). It has thus been
suggested that this ability to mimic CD40 is required for EBV to
efficiently colonize the B cell pool and establish persistence in the
healthy host (2). Previous studies have demonstrated that CD40 ligation
can activate PI3K and Akt in B cells and that this effect is important
for both cell proliferation and survival (27, 32). Mice deficient in the p85 subunit of PI3K are severely impaired in B cell development with reduced proliferative responses to CD40 ligation (37, 38). Taken
together these data highlight the role of the PI3K pathway in B cell
growth and suggest that the ability of LMP1 to activate this pathway
contributes to EBV persistence in B cells.
B and
mitogen-activated protein kinase pathways to LMP1-induced effects, we
now demonstrate that the PI3K/Akt pathway is also activated by LMP1 and
that this may account for a some of the more profound oncogenic
properties of the protein. This observation supports our previous work
suggesting that certain aspects of LMP1 behavior in epithelial cells
resemble those induced by an activated ras gene that also
functions via activation of PI3K (39). Using co-immunoprecipitation
experiments we have demonstrated that LMP1 associates with the p85
regulatory subunit of PI3K, thus showing that PI3K is recruited to, and
forms part of, an LMP1 signaling complex. That this association results
in PI3K activation is evidenced by increased basal lipid kinase
activity in cells stably expressing LMP1 and by the ability of
inducible LMP1 expression to also stimulate PI3K activity. Although
PI3K associates with the LMP1 signaling complex, the exact nature of this interaction is currently unknown. The finding that a
dominant-negative form of p85, deleted for the amino-terminal Src
homology 2 domain, is able to block PI3K-mediated effects in Swiss 3T3
fibroblasts suggests that the LMP1-p85 association may be mediated
through the amino-terminal Src homology 2 domain. Interestingly, unlike most growth factor receptors, LMP1 lacks putative YXXL
motifs within its carboxyl terminus suggesting that the LMP1-p85
interaction may be mediated through a novel p85 domain, or is
facilitated indirectly through an as yet unidentified adaptor
protein(s). Use of the chimeric CD2-LMP1-(192-386) molecule
demonstrated that the cytosolic carboxyl terminus of LMP1 and not the
amino terminus and transmembrane spanning regions was responsible for
PI3K and Akt activation. To further define the LMP1 domains responsible for PI3K activation, a GFP-tagged GRP1 fusion protein that has high
affinity and specificity for phosphatidylinositol 3,4,5-triphosphate was used in microinjection studies (29). This approach showed that the
CTAR1 domain of LMP1 is responsible for mediating PI3K activation. This
finding identifies the TRAF binding region of LMP1 as the mediator of
PI3K activation and suggests that PI3K recruitment to LMP1 is mediated
through a TRAF molecule or an as yet unidentified adaptor protein.
B activation by directly phosphorylating and activating
I-
B kinase suggests that in addition to directly inactivating
pro-apoptotic effector molecules, Akt may contribute to the suppression
of apoptosis by activating NF-
B. Although this phenomenon appears to
be cell type-specific, it points to the ability of Akt to provide an
additional level of protection from apoptotic stimuli through the
activation of NF-
B. Our findings that PI3K inhibition results in
robust apoptosis in LMP1-expressing cells implies that LMP1-generated signals activate signal transduction pathways that generate
pro-apoptotic signals and that the PI3K/Akt pathway serves to
counteract this effect. The recent demonstration that PI3K negatively
regulates the activity of c-Jun NH2-terminal kinase and
signal transducers and activators of transcription (47) points to a
role for these factors in LMP1-associated cytotoxicity. It is possible
that PI3K/Akt plays a role in LMP1-mediated NF-
B activation and that
the apoptosis observed in LY294002-treated cells is a result of NF-
B
down-regulation. However, our preliminary work indicates that LY294002
treatment does not affect the ability of LMP1 to induce NF-
B in both
Rat-1 fibroblasts or in HeLa cells stably expressing LMP1 (data not shown). These data suggest that in addition to the ability of LMP1 to
up-regulate Bcl-2 in B cells (6), Akt may provide another anti-apoptotic pathway activated by LMP1 that contributes to the survival of EBV-infected cells. This possibility is supported by recent
work demonstrating that PI3K plays a role in both the survival and
proliferation of EBV-transformed B cells (48). These studies also
identify PI3K as an attractive target for the development of novel
approaches for the treatment of EBV-associated tumors.
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ACKNOWLEDGEMENT |
---|
We thank Professor Mike Wakelam for help and advice.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from Cancer Research UK and by a Medical Research Council Career Development Award (to A. G. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 44-121-414-4491;
Fax: 44-121-414-5376; E-mail: L.S.Young@bham.ac.uk.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M209840200
2 C. W. Dawson, G. Tramountanis, A. G. Eliopoulos, and L. S. Young, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: EBV, Epstein-Barr virus; PI3K, phosphatidylinositol 3-kinase; NPC, nasopharyngeal carcinoma; TNFR, tumor necrosis factor receptor; TRAF, tumor necrosis factor receptor-associated factors; GFP, green fluorescent protein; TRITC, tetramethylrhodamine isothiocyanate.
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