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Previous Article | Next Article 
Journal of Virology, November 2000, p. 10838-10845, Vol. 74, No. 22
Department of Microbiology-Immunology,
Northwestern University Medical School, Chicago, Illinois
606111; Program in Viral Oncogenesis
and Tumor Immunology, Department of Virology and Molecular Biology,
St. Jude Children's Research Hospital, Memphis, Tennessee
381052; and Department of Pathology,
University of Tennessee Health Sciences Center, Memphis, Tennessee
381633
Received 30 May 2000/Accepted 23 August 2000
Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is
expressed on the membranes of B lymphocytes and blocks B-cell receptor
(BCR) signaling in EBV-transformed B lymphocytes in vitro. The
phosphotyrosine motifs at positions 74 or 85 and 112 within the LMP2A
amino-terminal domain are essential for the LMP2A-mediated block of
B-cell signal transduction. In vivo studies indicate that LMP2A allows
B-cell survival in the absence of normal BCR signals. A possible role
for Akt in the LMP2A-mediated B-cell survival was investigated. The
protein kinase Akt is a crucial regulator of cell survival and is
activated within B lymphocytes upon BCR cross-linking. LMP2A expression
resulted in the constitutive phosphorylation of Akt, and this LMP2A
effect is dependent on phosphatidylinositol 3-kinase activity. In
addition, recruitment of Syk and Lyn protein tyrosine kinases (PTKs) to
tyrosines 74 or 85 and 112, respectively, are critical for
LMP2A-mediated Akt phosphorylation. However, the ability of LMP2A to
mediate a survival phenotype downstream of Akt could not be detected in
EBV-negative Akata cells. This would indicate that LMP2A is not
responsible for EBV-dependent Burkitt's lymphoma cell survival.
Epstein-Barr virus (EBV), a
gammaherpesvirus, causes infectious mononucleosis in normal adolescents
and persists in the B lymphocytes of a majority of adults. EBV is
potentially oncogenic, being etiologically linked to a variety of
hematopoietic diseases, such as African Burkitt's lymphoma (BL),
Hodgkin's disease, adult T-cell leukemia, and
lymphoproliferative diseases in immunocompromised individuals
(58). In addition, EBV is associated with two epithelial pathologies: nasopharyngeal carcinoma and oral hairy leukoplakia (for reviews, see references 39 and
58).
Primary human B lymphocytes infected in vitro with EBV become
immortalized, establishing lymphoblastoid cell lines (LCLs). These
EBV-transformed LCLs contain the EBV episome and express a restricted
set of latently encoded viral proteins: six nuclear proteins (EBNA-1,
EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, and EBNA-LP), three integral
membrane proteins (latent membrane protein 1 [LMP1], LMP2A, and
LMP2B), and two small RNAs (EBERs) (for reviews, see references
34 and 41). However, of the
proteins expressed during latency, messages of EBNA-1, EBERs, BARTs,
and LMP2A have been detected in peripheral B lymphocytes from
individuals harboring latent infections (12, 13, 17, 55,
68). In addition, the LMP2A transcript is consistently detected
in NPC and other EBV-related malignancies (for reviews, see references
39, 41, and 58). Thus, LMP2A may
have significant roles in vivo for viral persistence and EBV-related diseases.
LMP2A forms aggregates, which localize to the plasma
membrane of latently infected B cells. LMP2A contains a 119-amino-acid cytoplasmic amino terminus, 12 hydrophobic transmembrane domains, and a
27-amino-acid cytoplasmic carboxyl terminus (43, 61). The
amino-terminal domain includes eight tyrosine residues, two of which
form an immunoreceptor tyrosine-based motif (10, 57). In
addition, the amino-terminal domain has been shown to be tyrosine phosphorylated and necessary for LMP2A association with Src family protein tyrosine kinases (PTKs) and Syk PTK (8, 42).
Phosphorylated tyrosines provide potential binding sites for cellular
proteins containing Src homology 2 domains. Src homology 2 domains are noncatalytic domains that are conserved among cytoplasmic signaling molecules and bind to phosphotyrosine motifs (for a review, see reference 45). Studies have demonstrated the
requirement of LMP2A phosphotyrosine motifs at positions 74 or 85 and
112 to bind Syk and Lyn, respectively (25, 26).
In addition to PTKs associating with LMP2A, LMP2A has a dramatic effect
on B-cell signaling. In primary B lymphocytes, ligation of the B-cell
receptor (BCR) initially induces the up-regulation of protein tyrosine
phosphorylation by recruiting and activating Src family PTKs. This
event is followed by the recruitment of other tyrosine and
serine/threonine kinases and phosphatases, the hydrolysis of
phospholipids, activation of protein kinase C, mobilization of calcium,
and activation of nuclear transcription factors that are specific for
transcribing the BCR signal-specific genes required for mediating
clonal expansion, differentiation, and apoptosis (for a review, see
reference 18). In in vitro studies, the expression
of LMP2A blocks BCR-mediated signal transduction (47).
In vivo, LMP2A expression prevents BCR-negative B lymphocytes from undergoing apoptosis and enables these BCR-negative cells to
persist within peripheral lymphoid organs (9). This suggests that LMP2A can provide both a developmental signal and a survival signal to normal B cells.
B-cell survival can be promoted by activation of the serine-threonine
kinase Akt. Akt was identified as a proto-oncogene and is the cellular
homologue of a protein encoded in the genome of the retrovirus AKT8
(6). Furthermore, the deregulation of Akt activity has
been linked to tumorigenesis in humans (5, 64, 65). Upon BCR
activation, there is a rapid activation of the nonreceptor PTK,
phosphatidylinositol 3-kinase (PI3-K) (27). Activation
of PI3-K results in the production of phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3] and phosphatidylinositol 3,4-biphosphate [PtdIns(3,4)P2], which recruit Akt to the
plasma membrane by binding the pleckstrin homology domain in Akt.
Membrane-anchored Akt is subsequently phosphorylated on
Thr308 and Ser473 by
PtdIns(3,4,5)P3-dependent kinase-1 and
integrin-linked kinase/PtdIns(3,4,5)P3-dependent kinase-2, respectively (for reviews, see references
14 and 21). Phosphorylation of
both residues is required for maximal activation of Akt.
Akt is a multifunctional mediator of PI3-K involved in regulating
several cellular functions by interacting with a variety of downstream
substrates. Through various cytokines, growth factors, and chemokines,
Akt can deliver antiapoptotic signals through various mechanisms.
Interleukin-2-induced activation of Akt results in promotion of T-cell
survival by upregulating the expression of Bcl-2 and c-MYC
(1). Akt activation has also been found to block
apoptosis by inhibiting caspase activity in a variety of cell
types (11, 32, 50). Moreover, studies have demonstrated the
ability of Akt to induce phosphorylation of the BAD protein, preventing
its association with Bcl-2 and resulting in cell survival (16, 20,
49, 53). More specifically, Akt, which can be regulated by PTKs
Syk, Btk, and Lyn (15, 38), can inactivate glycogen synthase
kinase 3 (GSK-3) following BCR activation (28), which may
promote cell survival and activate nuclear gene transcription (4,
52).
Since previous studies indicated that LMP2A provides a B-cell survival
signal, we investigated the regulation of Akt by LMP2A. We demonstrate
that the expression of LMP2A leads to the constitutive phosphorylation
of Akt. Furthermore, we show that LMP2A mediates the constitutive
phosphorylation of Akt via PI3-K and requires the recruitment of Syk
and Lyn to mediate this effect. However, the ability of LMP2A to
mediate an overall cell survival effect via Akt could not be detected
in tissue culture or upon transfer of LMP2A-expressing cells to SCID mice.
Constitutive phosphorylation of Akt in EBV+
LMP2A+ LCLs.
Previous studies had indicated that LMP2A
mediates a survival signal in peripheral B cells (9). To
investigate a possible role of Akt in this LMP2A-mediated effect, the
phosphorylation state of Akt in cleared lysates from BCR-stimulated and
unstimulated EBV+ LMP2A+ and EBV+
LMP2A
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Latent Membrane Protein 2A-Mediated Effects on the
Phosphatidylinositol 3-Kinase/Akt Pathway
ABSTRACT
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LCLs was determined using the phospho-Akt Pathway
kit from Cell Signaling Technology (Fig.
1, top panel). EBV+
LMP2A LCLs demonstrate an increase in Akt phosphorylation
upon BCR activation (Fig. 1, lanes 1 to 4), as expected. Whereas in
EBV+ LMP2A+ LCLs, there was a dramatic
phosphorylation of Akt in the absence of BCR activation, and the amount
of Akt phosphorylation was unchanged upon BCR stimulation (Fig. 1,
lanes 5 and 6). Therefore, LMP2A results in the constitutive
phosphorylation of Akt. Equal loading of Akt in each lane was verified
by immunoblotting with anti-Akt antibodies (Fig. 1, lower panel).
Whole-cell lysates from untreated and platelet-derived growth factor
treated NIH 3T3 cells were used to confirm the unphosphorylated and
phosphorylated forms of Akt (60-kDa protein) (data not shown).
View larger version (58K):
[in a new window]
FIG. 1.
Akt is constitutively phosphorylated in EBV+
LMP2A+ LCLs. EBV+ LMP2A LCLs
demonstrate an increase in Akt phosphorylation upon activation of the
BCR, as expected (lanes 1 through 4, top panel). EBV+
LMP2A+ LCLs indicate that Akt phosphorylation is present
without BCR activation and does not change upon activation of the BCR
(lanes 5 and 6, top panel). The amounts of protein loaded and Akt
expressed for each LCL were similar (bottom panel). EBV+
LMP2A+ cells and EBV+ LMP2A cells
(107) were left untreated or treated with anti-sIg for 5 min, lysed with 1.0% Triton X, and separated by sodium dodecyl
sulfate-9% polyacrylamide gel electrophoresis (SDS-9% PAGE).
Immobilon membranes were immunoblotted with phospho-specific Akt (top
panel) or Akt (bottom panel) polyclonal antibodies, incubated with
rabbit HRP-conjugated immunoglobulin G (IgG) antibody, and detected by
enhanced chemiluminescence (ECL). Protein standards are indicated at
the left in kilodaltons.
Wortmannin inhibits constitutive and induced phosphorylation of
Akt in EBV+ LMP2A+ LCLs.
In B lymphocytes,
induction of Akt phosphorylation is mediated through the PI3-K pathway
upon activation of the BCR (28, 38). Additionally, LMP2A has
been shown to induce the constitutive phosphorylation of the p85
subunit of PI3-K (46). To determine if LMP2A-mediated Akt
phosphorylation is PI3-K dependent, Akt phosphorylation was analyzed in
EBV+ LMP2A LCLs and EBV+
LMP2A+ LCLs following treatment with wortmannin. Wortmannin
is a microbial secondary metabolite found in a variety of fungal
species and is a potent and selective inhibitor of PI3-K
(54). Upon treatment of the EBV+
LMP2A+ and EBV+ LMP2A LCLs with
increasing concentrations of wortmannin (100, 200, 500, and 1,000 nM)
for 5 min at 37°C, Akt phosphorylation was lost (Fig.
2, lanes 3 to 6, and 8 to 11, upper
panel). The EBV+ LMP2A LCL was used as a
positive control to illustrate the inhibition of PI3-K activity by
wortmannin upon activation of the BCR (Fig. 2, lanes 1 to 6, upper panel). Thus, the constitutive phosphorylation of Akt by
LMP2A is mediated through the PI3-K pathway. This data would indicate
that PI3-K is constitutively active in LMP2A-expressing cells.
Treatment of LCLs with dimethyl sulfoxide alone did not have any effect
on the level of Akt phosphorylation or cell survival (data not shown).
|
Constitutive phosphorylation of Akt is abolished in
EBV+ LMP2AY74F LCLs and EBV+ LMP2AY112F
LCLs.
Previous studies have shown that LMP2A function is
dependent upon tyrosines at positions 74 or 85 and 112. Specifically,
tyrosines 74 or 85 and 112 are required for LMP2A to bind Syk and
Lyn, respectively, allowing LMP2A to regulate their
activity (25, 26). Furthermore, Syk and Lyn PTKs
have been found to affect Akt activity in BCR-activated B
lymphocytes (38). Therefore, Akt phosphorylation in
EBV+ LMP2AY74F LCLs, EBV+ LMP2AY85F LCLs,
and EBV+ LMP2AY112F LCLs was analyzed (Fig.
3). EBV+ LMP2AY74F LCLs
and EBV+ LMP2AY112F LCLs showed a loss of
constitutive Akt phosphorylation (Fig. 3, lanes 1 and 9, upper panel),
resulting in the restoration of BCR regulation of Akt phosphorylation.
The loss of tyrosine 85 resulted in only a partial loss of constitutive
Akt phosphorylation (Fig. 3, lane 5, upper panel). Upon activation of
the BCR in these tyrosine point mutant LCLs, Akt phosphorylation levels
increased as shown previously in EBV+ LMP2A
LCLs (Fig. 3, lanes 2, 6, and 10, upper panel). Interestingly, the
EBV+ LMP2AY112F LCL showed a reduction of Akt
phosphorylation upon BCR stimulation, which is consistently observed in
LCLs containing this mutation. The significance of this finding is
currently being investigated.
|
Expression of LMP2A in EBV Akata cells.
LCLs
express LMP1, which has been reported to mimic CD40 receptor-mediated
signaling and to upregulate Bcl-2 expression, resulting in cell
survival (22, 29, 31). Furthermore, previous studies have
not been able to show any difference in cell survival between EBV+ LMP2A+ LCLs and EBV+
LMP2A LCLs (44, 63). This may be because the
LMP1 signal is dominant, and any LMP2A-mediated survival signal would
be concealed as a result. Thus, to investigate the mechanism by which
LMP2A may mediate the survival of B lymphocytes via the Akt pathway,
EBV Akata BL cells expressing LMP2A were derived as
previously described (30). The Akata cell line contains a
characteristic t(8;14) translocation that results in deregulation of
the proto-oncogene c-MYC (66). Although the
parental Akata BL cells are EBV+, upon serial passage in
culture, a small percentage of cells spontaneously lose the EBV genome
(62). Comparative studies have indicated that whereas
EBV+ Akata cells are modestly resistant to
apoptotic stimuli and are tumorigenic, the EBV
cells have lost these characteristics (36, 60). Since LMP2A is expressed in the absence of LMP1 within the primary B-cell reservoir
of latent EBV in vivo (17, 55, 68), EBV Akata
cells may provide an appropriate cell system with which to assess the
effect of LMP2A within B cells in the absence of LMP1.
Akata cells stably expressing LMP2A or containing
vector alone were generated, and LMP2A expression was verified by a Western blot (24) (Fig. 4A).
LMP2A expression is evident in the EBV+ LMP2A+
LCL positive control (Fig. 4A, lane 7, upper panel), whereas no LMP2A
was detected in the vector control EBV Akata cells, as
expected (Fig. 4A, lane 6, upper panel). All of the
EBV/LMP2A+ Akata cells expressed LMP2A at or
near the levels of an EBV+ LMP2A+ LCL (Fig. 4A,
lanes 1 through 5, upper panel). Previous studies have shown that LMP2A
function is dependent upon the protein being phosphorylated (26,
46). Thus, the phosphorylation state of LMP2A in the
EBV/LMP2A+ Akata cells was determined by
equally dividing LMP2A immunoprecipitations and immunoblotting with an
HRP-linked antiphosphotyrosine antibody or an LMP2A antibody (Fig. 4B)
as previously described (25). LMP2A was constitutively
phosphorylated in all representative EBV/LMP2A+ Akata cells, as shown in the
control EBV+ LMP2A+ LCL lane. The amount of
LMP2A immunoprecipitated from each cell line was approximately the same
(Fig. 4B, lower panel).
|
EBV/LMP2A+ Akata cells constitutively
phosphorylate Akt.
To verify that LMP2A mediates the constitutive
phosphorylation of Akt in Akata cells, cell lysates were generated from
EBV/LMP2A+ and EBV/vector
control Akata cells following BCR activation and subjected to
immunoblotting with phospho-specific Akt antibodies.
EBV/LMP2A+ Akata cells demonstrated a
constitutive phosphorylation of Akt (Fig.
5, lanes 1, 4, and 7, upper panel)
comparable to that seen in LCLs (Fig. 1). In addition, the level of Akt
phosphorylation did not change upon stimulation of the BCR for 2 or 5 min (Fig. 5). This data also confirmed that it is LMP2A that regulates
the phosphorylation of Akt in the absence of BCR activation in
EBV-infected B lymphocytes.
|
/LMP2A+ and EBV/vector Akata
cells were treated with wortmannin or left untreated, and
immunoblotting was performed using phospho-specific Akt antibodies. Treatment of cells with wortmannin blocked the constitutive
phosphorylation of Akt in EBV/LMP2A+ Akata
cells (Fig. 6, lanes 3 and 7, upper
panel). Activation of the BCR following wortmannin treatment did not
restore Akt phosphorylation in those cells, confirming the irreversible
effect of wortmannin in blocking PI3-K activity. The
EBV/vector Akata cell was used as a control (Fig. 6,
lanes 9 to 12, upper panel). Thus, LMP2A appears to be functionally
equivalent with respect to Akt phosphorylation when expressed in either
an LCL or a BL background.
|
LMP2A does not promote tumorigenicity or enhance survival in Akata
BL cells.
In light of our observation that LMP2A expression
induced phosphorylation of Akt in EBV Akata BL cells, we
next addressed whether this effect of LMP2A contributes to the enhanced
survival and tumorigenic potential of EBV+ Akata cells
(36, 60). Although these EBV-dependent properties have been
attributed to the EBER RNAs (35), we have found that relative to EBV infection, the EBERs only partially restore tumorigenic potential to EBV Akata cells and do so independently of
an effect on cell survival (59). Since LMP2A expression has
been detected in both EBV+ Akata cells (36) and
a majority of BL biopsies analyzed in one study (67), it
seemed likely that LMP2A, through its activation of Akt, may contribute
to the tumorigenic potential of Akata BL cells by promoting cell
survival. To test this, the tumorigenic potential of three
EBV Akata lines expressing LMP2A was evaluated in SCID
mice as previously described (59). As shown in Table
1, expression of LMP2A in EBV Akata cells did not increase their tumorigenic
potential. Although two of the LMP2A-expressing lines induced
tumors in one of five mice injected, these tumors arose very late in
the assay, at 19 and 20 weeks postinjection, in contrast to the 5-week
latency period for generation of tumors derived from
EBV+ Akata cells. Furthermore, neither of these tumors
expressed detectable LMP2A. Additionally, we observed no ability
of LMP2A expression to either up-regulate Bcl-2 expression or
down-regulate c-MYC levels (data not shown), as previously reported for
EBV infection (36, 60). Thus, LMP2A is not responsible for
these previously noted effects of EBV infection in Akata cells.
|
LMP2A does not enhance cell survival in vitro.
To determine
whether LMP2A promotes a cell survival signal via the Akt pathway,
EBV/LMP2A+ and EBV/vector Akata
cells were subjected to apoptotic stimuli (serum deprivation or
UV irradiation) and monitored as previously described (36).
Previous studies have shown that the EBV Akata cell line
is susceptible to the induction of apoptosis by serum
deprivation and UV treatment (36, 60). As expected, the
EBV/vector Akata cell (B7) showed a substantial decrease
in the percentage of metabolic activity from 100 to less than 10 when
cells were exposed to decreasing serum concentrations (10 to 0%) (Fig.
7A). The B6 and G7
EBV/LMP2A+ Akata cell lines
demonstrated a similar decrease in metabolic activity. UV
treatment of EBV/LMP2A+ Akata cells G7 and F6
also showed no difference in the percentage of metabolic activity
compared to the EBV/vector Akata clone, B7. Thus, despite
the fact that LMP2A induced constitutive phosphorylation of Akt, we
found no evidence that this translates to increased cell survival
within BL cells.
|
B-lymphoma cell line, the phosphorylation of p85
results in phosphorylation and activation of the kinase subunit of
PI3-K, p110 (37). These observations strongly suggest that
LMP2A expression may result in the constitutive activation of PI3-K. In
vivo studies have previously shown that LMP2A provides a survival
signal to normal B cells (9). Since PI3-K is an important
regulator of Akt, whose activation is important for cell survival, we
investigated whether LMP2A may activate the PI3-K-Akt signaling pathway.
This analysis demonstrated that LMP2A mediates the constitutive
phosphorylation of Akt both in EBV-transformed LCLs and
LMP2A-expressing EBV BL cells. BCR activation of
EBV+ LMP2A+ LCLs and
EBV/LMP2A+ Akata cells did not significantly
alter the level of Akt phosphorylation. Use of the PI3-K inhibitor
wortmannin indicated that the observed Akt phosphorylation was mediated
through the PI3-K pathway. In addition, tyrosines 74 or 85 and 112, which are necessary for LMP2A function in blocking BCR signaling, were
critical for LMP2A to mediate the constitutive phosphorylation of Akt.
However, we were unable to demonstrate any growth advantage for B cells
expressing LMP2A compared to control cells.
The serine-threonine kinase Akt has emerged as a major target for PI3-K
(14) and is stimulated in response to BCR activation (3, 38). Both Syk and Btk have been proven necessary for maximal Akt activation (15, 38), whereas Lyn may serve as a
negative regulator of Akt activity. Phosphorylation of Akt on threonine308 and serine473 is essential for its
activation. This is mediated by PDK-1 and PDK-2, respectively (2,
19). Besides PI3-K, several PTKs are required for modulating Akt
function. Both Syk and Lyn have been found to play important roles in
LMP2A function. LMP2A functional studies have shown that the
immunoreceptor tyrosine-based motif-like tyrosines 74 and 85 in the
amino-terminal domain of LMP2A are necessary for recruiting Syk PTK,
resulting in a constitutively phosphorylated and activated kinase
(25) which may result in the activation of Akt. In addition,
Syk has also been found to associate with PI3-K through the adapter
protein c-cbl (51). It is possible that LMP2A uses its
association with Syk to constitutively activate the PI3-K-Akt pathway;
however, how LMP2A exerts its effects on PI3-K is currently not known.
Previous studies have also shown that LMP2A phosphorylation and the
LMP2A-mediated block on BCR signaling are dependent on the binding of
the Src family kinase Lyn to the tyrosine 112 in the amino-terminal
domain of LMP2A (26). Moreover, by binding Lyn, LMP2A is
able to down-modulate Lyn kinase activity and direct the
ubiquitin-dependent degradation of Lyn through its association with WW
domain-containing Nedd4-like ubiquitin-protein ligases (30).
Previous studies have demonstrated the role of Akt in preventing
apoptosis (23, 32, 33). Akt can mediate this
function by inhibiting BAD, a death-promoting member of the Bcl-2
family (49), by preventing proteolytic activation of caspase
9 (11), and by inhibiting the induction of
proapoptotic gene expression through its phosphorylation of
members of the Forkhead family of transcription factors
(7). It is somewhat surprising, therefore, that
LMP2A-induced phosphorylation of Akt in EBV Akata BL
cells had no detectable effect on cell survival. Previous work from our
laboratory and others has indicated that Akata cells that have lost the
EBV genome are highly sensitive, relative to their EBV+
counterparts, to apoptotic stimuli, such as serum deprivation, cycloheximide, and hypoxia (36, 60). This has been
attributed to the ability of EBV to modestly up-regulate Bcl-2
expression in these cells and to induce a repression of c-MYC levels
under growth-limiting conditions. Presumably this ability to inhibit apoptosis contributes to the EBV-dependent tumorigenic
potential of Akata cells (36, 60). Indeed, we have shown
recently that enforced expression of Bcl-2 in EBV Akata
cells does promote BL cell survival and as a likely result enhances
tumorigenic potential (Ruf et al., unpublished observation). Thus, the
failure of LMP2A to enhance the survival of EBV Akata
cells is not due to an inherent inability of these cells to respond to
antiapoptotic factors. The failure of LMP2A to enhance survival
of EBV Akata cells in the face of proapoptotic
stimuli or to increase their tumorigenic potential suggests that if
LMP2A-induced phosphorylation of Akt does provide a survival signal,
this signal alone is not sufficient to protect Akata BL cells from
apoptosis. This is consistent with the earlier observation that
BCR-mediated activation of Akt is not sufficient to promote cell
survival in the EBV BL cell line RAMOS (28).
By contrast, recent work has indicated that LMP2A is able to promote
cell survival in epithelial cells (61a). However, transgenic
mice in which LMP2A is expressed in the basal layer of the epithelia do
not demonstrate any cell survival or tumorigenic phenotype
(40). Thus, any survival signal induced by LMP2A in BL cells
is unlikely to be sufficient to block apoptosis induced as a
consequence of the deregulation of c-MYC expression.
Another major downstream target of Akt is GSK-3, a constitutively
active serine-threonine kinase whose activity is inhibited by Akt.
Studies have primarily focused on insulin receptor-mediated signaling
activation of Akt resulting in a reduction in GSK-3 activity
(69). One of the major cellular processes regulated by GSK-3
is the subcellular localization of the NF-ATc transcription factor.
GSK-3 phosphorylates NF-ATc, resulting in its rapid export from the
nucleus (4). Recently, it has been demonstrated that activation of the BCR results in the activation of the
PI3-K-Akt-GSK-3 pathway (28). Induction of the BCR also
causes an increase in intracellular Ca2+ concentrations,
leading to the activation of the Ca2+-dependent phosphatase
calcineurin, which dephosphorylates NF-ATc (56). LMP2A
blocks calcium mobilization upon BCR activation (48). Thus,
LMP2A may use the PI3-K-Akt pathway to block the phosphorylation of
NF-ATc by GSK-3, resulting in transcriptional activation. This
would allow LMP2A to control B-cell gene transcription directly
while preventing the activation of Ca2+-dependent signaling pathways.
In summary, we have provided evidence that LMP2A promotes the
constitutive phosphorylation of Akt via the PI3-K pathway. In addition,
the activation of the PI3-K-Akt pathway by LMP2A does not promote
survival of BL cells grown in culture or models of BL tumorigenesis in
vivo. Since the PI3-K-Akt-GSK-3 pathway can regulate a variety of
cellular processes, in addition to cell survival, such as cell cycle
progression and transcription, LMP2A may be using this signal to
regulate different aspects of B-cell signal transduction. Future
studies which delineate the mechanism by which LMP2A is able to take
advantage of normal B cell processes should help define the strategy
EBV uses to persist in the human host.
| |
ACKNOWLEDGMENTS |
|---|
We thank the members of the Longnecker and Spear laboratories for providing invaluable advice. A special thanks goes to Luz Longan for producing the LMP2A retrovirus.
R.S. was supported by a minority research supplement from the National Institutes of Health. I.K.R. was supported by PHS grant T32-AI07372. J.S. is supported by USPHS grant CA73544 and Cancer Center Support (CORE) grant CA21765 from the National Cancer Institute and by the American Lebanese-Syrian Associated Charities (ALSAC). R.L. is a scholar of the Leukemia and Lymphoma Society of America. R.L. is supported by Public Health Service grants CA62234 and CA73507 from the National Cancer Institute and DE13127 from the National Institute of Dental and Craniofacial Research.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Microbiology-Immunology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. Phone: (312) 503-0467. Fax: (312) 503-1339. E-mail: r-longnecker{at}northwestern.edu.
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This article has been cited by other articles:
), BCR stimulated (+), and/or treated with 100, 200, 500, and 1,000 nM concentrations of wortmannin (WN). The cells were lysed,
separated by SDS-PAGE, immunoblotted with phospho-specific Akt (top
panel) or Akt (bottom panel) antibodies, and detected by ECL. Protein
standards are indicated at the left in kilodaltons.
, B6 (LMP2A+ (A) or G7
(LMP2A+) (B); 
, G7 (LMP2A+) (A) or F6
(LMP2A+) (B); 
, B7 (LMP2A