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Journal of Virology, June 2001, p. 5711-5718, Vol. 75, No. 12
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.12.5711-5718.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
PY Motifs of Epstein-Barr Virus LMP2A Regulate
Protein Stability and Phosphorylation of LMP2A-Associated
Proteins
Masato
Ikeda,
Akiko
Ikeda, and
Richard
Longnecker*
Department of Microbiology-Immunology,
Northwestern University Medical School, Chicago, Illinois 60611
Received 21 December 2000/Accepted 22 March 2001
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ABSTRACT |
Latent membrane protein 2A (LMP2A) is expressed in latent
Epstein-Barr virus (EBV) infection. We have demonstrated that Nedd4 family ubiquitin-protein ligases (E3s), AIP4, WWP2/AIP2, and Nedd4, bind specifically to two PY motifs present within the LMP2A
amino-terminal domain. In this study, LMP2A PY motif mutant viruses
were constructed to investigate the role of the LMP2A PY motifs. AIP4
was found to specifically associate with the LMP2A PY motifs in
EBV-transformed lymphoblastoid cell lines (LCLs), extending our
original observation to EBV-infected cells. Mutation of both of the
LMP2A PY motifs resulted in an absence of binding of AIP4 to LMP2A,
which resulted in an increase in the expression of Lyn and the
constitutive hyperphosphorylation of LMP2A and an unknown 120-kDa
protein. In addition, there was a modest increase in the constitutive
phosphorylation of Syk and an unidentified 60-kDa protein. These
results indicate that the PY motifs contained within LMP2A are
important in regulating phosphorylation in EBV-infected LCLs, likely
through the regulation of Lyn activity by specifically targeting the
degradation of Lyn by ubiquination by Nedd4 family E3s. Despite
differences between PY motif mutant LCLs and wild-type LCLs, the PY
motif mutants still exhibited a block in B-cell receptor (BCR) signal
transduction as measured by the induction of tyrosine phosphorylation
and BZLF1 expression following BCR activation. EBV-transformed LCLs
with mutations in the PY motifs were not different from wild-type LCLs
in serum-dependent cell growth. Protein stability of LMP1, which
colocalizes with LMP2A, was not affected by the LMP2A-associated E3s.
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TEXT |
Epstein-Barr virus (EBV) is a
potentially oncogenic herpesvirus persisting in B lymphocytes of most
adult humans (for reviews, see references 15, 16, and
26). B lymphocytes infected with EBV in vitro are
immortalized and are termed lymphoblastoid cell lines (LCLs), and they
provide a model of in vivo latent infection (15, 16, 26).
These EBV-transformed LCLs express a restricted set of
latency-associated viral products, including six nuclear proteins
(EBNAs), three integral membrane proteins (latent membrane protein 1 [LMP1], LMP2A, and LMP2B), and two small RNAs (EBERs) (15,
16). However, EBNA1, LMP1, and LMP2A are the proteins consistently detected in nasopharyngeal carcinoma tumor biopsies and
EBV-related malignancies (for reviews, see references 1, 26, and 30). The LMP2A mRNA is also consistently
detected by PCR analysis in peripheral B lymphocytes from humans with
latent EBV infections (23-25, 31). Thus, LMP2A is
suspected of having an important role in vivo for EBV latency and
persistence (16).
In primary B lymphocytes, B-cell receptor (BCR) activation leads to
signal transduction cascades, including the recruitment and activation
of cellular protein tyrosine kinases (PTKs) (5, 8). The
Src family PTKs, Lyn, Fyn, and Blk, are activated following BCR
stimulation, which is followed by activation and binding of the Syk PTK
to the BCR through an interaction of Syk SH2 domains with the
immunoreceptor tyrosine-based activation motifs contained within BCR
(J. C. Cambier, Letter, Immunol. Today, 16:110, 1995). Interestingly, both the Src family PTKs and the Syk PTK are also associated with LMP2A (11, 12).
LMP2A is expressed in aggregates in the plasma membrane of latently
infected B cells (18). Most of the anti-phosphotyrosine reactivity within these B cells is associated with LMP2A aggregates (17, 18). The Src family Lyn PTK binds to tyrosine 112, whereas the Syk PTK binds to the LMP2A immunoreceptor tyrosine-based
activation motif present at tyrosines 74 and 85 of the LMP2A
amino-terminal domain via SH2-phosphotyrosine interactions (11,
12). The association of LMP2A with the Src family and the Syk
PTKs is essential for the LMP2A-mediated block of BCR signal
transduction observed in EBV-immortalized LCLs grown in culture
(11, 12). Thus, the amino terminus of LMP2A appears to act
as a functional decoy for BCR-associated proteins, which results in the
down-modulation of BCR-mediated signal transduction.
Also contained within the amino-terminal domain of LMP2A are two PY
motifs (PPPPY). These motifs are conserved in clinical EBV isolates and
in the EBV-related herpesvirus papio (4, 9). PY motifs
interact with WW domains through a consensus sequence of xPPxY
(6). Our previous studies indicated that the LMP2A PY
motifs specifically associate with Nedd4 family ubiquitin-protein ligases, such as AIP4, WWP2/AIP2, and Nedd4 (14). The
ubiquitin proteolytic pathway plays a crucial role in the degradation
of short-lived and regulatory proteins important in various cellular processes (7, 13). The ubiquitin-protein ligase (E3)
facilitates the sequential transfer of ubiquitin to target proteins.
Different E3s are important in determining the selectivity of
ubiquitin-mediated protein degradation. Proteins ligated to
polyubiquitin chains are usually degraded by the 26S proteasome in an
ATP-dependent manner. Nedd4 family E3s may ubiquitinate LMP2A and
LMP2A-associated proteins through the interaction of the LMP2A PY motif
and the WW domain of the Nedd4 family E3s. Indeed, the rapid turnover of LMP2A is observed in LMP2A-expressing BJAB cell lines
(14). Furthermore, Lyn is ubiquinated and there is a
reduction and rapid turnover in the Lyn protein in LMP2A-expressing
cell lines (14, 32), suggesting that LMP2A and Lyn may be
degraded through the ubiquitin-proteasome pathway.
In this study, the LMP2A PY mutations examined in previous in vitro
studies (14) were incorporated into the EBV genome. LCLs
containing these mutations were isolated to investigate the functional
importance of the LMP2A PY motifs. Each LMP2A PY motif, PY1 (56 to 60 amino acids [aa]) and PY2 (97 to 101 aa), was changed from PPPPY to
PAAPY by PCR-mediated mutagenesis (Fig. 1A) using a previously
described strategy (11). Recombinant viruses were used to
infect purified B lymphocytes in culture to generate EBV-infected LCLs
(11). Multiple LCLs transformed by the PY motif mutants were identified and are characterized in this study.
AIP4 binds to the LMP2A PY motifs in vivo.
Previous studies
using the amino terminus of LMP2A expressed in bacteria demonstrated
that each of the LMP2A PY motifs is sufficient for the in vitro
association of LMP2A with WW domain-containing E3s, such as AIP4,
WWP2/AIP2, and Nedd4 (14, 19). Of these E3s, AIP4, which
bound LMP2A in cells of B-cell origin, was the most abundant E3
(14). Therefore, to verify the in vivo interaction of
LMP2A PY motifs with the Nedd4 family E3s, the binding of AIP4 to LMP2A
was investigated in wild-type LCLs and PY1PY2 LCLs. Rabbit polyclonal
sera directed against AIP4 was derived using a glutathione S-transferase-fusion protein containing the unique domain
of AIP4 (100 to 285 aa). The antisera demonstrated specific recognition of the AIP4-glutathione S-transferase fusion protein and an
epitope-tagged version of AIP4, as well as AIP4 in Triton X-100 lysates
from BJAB cells and LCLs (data not shown). Using these antisera, the interaction of AIP4 with LMP2A was investigated by immunoprecipitation of cell lysates from wild-type-infected LCLs and PY1PY2 LCLs using anti-LMP2A antibody followed by immunoblotting with the AIP4 antiserum (Fig. 1B). AIP4 was detected only in the
LMP2A immunoprecipitates in wild-type-infected LCLs, whereas it was not
detected in the PY1PY2 LCL or in BJAB cells which do not contain LMP2A
(Fig. 1B). Therefore, the two PPPPY sequences contained within the
LMP2A amino terminus are functional PY motifs for in vivo AIP4 binding.
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FIG. 1.
Site-directed mutation of the LMP2A PY motifs and
association of LMP2A and AIP4 in LMP2A PY1PY2 LCLs. (A) The first 119 amino acids of the LMP2A amino-terminal domain is shown schematically,
with each PY motif indicated by a box. PY motifs were mutated from
PPPPY to PAAPY, which was previously shown to block binding of the WW
domain-containing Nedd4 family ubiquitin E3s to LMP2A
(15). These mutations were made to determine the function
of the LMP2A PY motifs. A PvuII site was incorporated at
the mutation site to allow for identification of LCLs containing the
LMP2A point mutation. The restriction site addition was silent with
regard to the LMP2A amino acid sequence. (B) Triton X-100 lysates from
BJAB cells and LCLs (10 7 cells) were immunoprecipitated
(IP) with anti-LMP2A (lanes 1 to 3) or anti-AIP4 (lanes 4 to 6)
antibody. Precipitated proteins were separated on sodium dodecyl
sulfate-7.5% polyacrylamide gel electrophoresis gels and
immunoblotted with anti-AIP4 antibody. The position of AIP4 is
indicated by an arrow. Molecular mass standards (in kilodaltons) are
indicated at the left.
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Protein levels of LMP2A and Lyn in LMP2A PY1PY2 LCLs.
Previous
studies had demonstrated that the expression of LMP2A in EBV-negative
BJAB cells led to the rapid turnover of the Lyn PTK, whereas the
protein level of the Syk PTK and tubulin was unchanged
(14). In addition, the LMP2A protein was also rapidly
turned over when compared to Syk and tubulin (14). Thus, protein levels of LMP2A, Lyn, Syk, and tubulin were investigated by
immunoblottings in wild-type-infected LCLs and LCLs infected with the
LMP2A PY1PY2 mutant. When compared to wild-type-infected LCLs, there
was approximately a twofold increase, as determined by densitometry, in
the protein level of LMP2A in the PY1PY2 mutant-infected LCLs (Fig.
2A). However, LMP2A protein levels tended
to be inconsistent and some mutant LCLs demonstrated similar levels of
LMP2A protein when compared to the wild type (data not shown).
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FIG. 2.
Expression of LMP2A, Lyn, and Syk in LMP2A PY1PY2 and
Y112F LCLs. (A) The protein levels of LMP2A, Lyn, Syk, and tubulin in
BJAB cells (lanes 1) and in LMP2A (lanes 2),
LMP2A+ (lanes 3 and 4), and PY1PY2 (lanes 5 and 6) LCLs
were analyzed. (B) Levels of Lyn and tubulin expression in
LMP2A+ (lanes 1), PY1PY2 (lanes 2), and LMP2A Y112F (lanes
3 to 5) LCLs were analyzed. Triton X-100 lysates from the various cell
lines were separated on sodium dodecyl sulfate-7.5% polyacrylamide
gel electrophoresis gels, transferred to Immobilon membranes, and
immunoblotted with anti-LMP2A, anti-Lyn, anti-Syk, or anti-tubulin
antibody, followed by incubation with horseradish peroxidase-conjugated
secondary antibody and enhanced chemiluminescence detection. Tubulin
served as a protein loading control. The positions of LMP2A, Lyn (both
the 56- and 53-kDa forms), Syk, and tubulin are indicated by
arrows.
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In contrast, changes in Lyn protein levels were much more dramatic in
the PY1PY2 LCLs when compared to LMP2A
+ LCLs.
Compatible with previous results with LMP2A-expressing
BJAB cells,
LMP2A
+ LCLs demonstrated a reduction in Lyn
protein expression when
compared to LMP2A
LCLs
or BJAB cells (Fig.
2A). This decrease of Lyn expression
was not
observed in PY1PY2 LCLs; rather, the Lyn protein level
in the PY1PY2
LCLs was equal to that of LMP2A
LCLs or BJAB
cells (Fig.
2A). In contrast to the results obtained
with Lyn, Syk
protein levels were not changed in the LCLs with
or without the PY1PY2
mutation (Fig.
2A).
Next, the levels of Lyn protein in PY1PY2 LCLs were compared to Lyn
protein levels in Y112F LCLs (
12). Y112F LCLs contain
a
tyrosine-to-phenylalanine mutation at tyrosine 112 within the
LMP2A
amino-terminal domain. This mutation results in a loss of
binding of
Lyn to LMP2A, which results in an increase of Lyn protein
levels to
wild-type levels (
12). The protein level of Lyn was
similar in both the PY1PY2 LCLs and the Y112F LCLs, demonstrating
that
both the single tyrosine-to-phenylalanine mutation and the
double PY
mutations have similar effects on Lyn protein levels
(Fig.
2B).
In addition, the levels of other proteins known to be important in BCR
signal transduction were also tested. These included
BLNK, Btk, Shc,
PI3K p85, PLC
2, and Cas p130. However, as with
Syk and tubulin,
there was no change in the protein level of any
of these proteins in
the wild-type LCLs when compared to PY1PY2
LCLs (data not shown).
Therefore, the LMP2A effect on Lyn protein
level appears to be
specific. Single mutation of either PY motif
had little effect on the
protein levels of Lyn or LMP2A in LCLs
infected with the appropriate
mutant (data not shown). In summary,
mutation of both LMP2A PY motifs
results in a restoration of Lyn
protein levels to that seen in
LMP2A
LCLs and in EBV
BJAB cells and a possibly modest increase in LMP2A protein levels
when
compared to wild-type-infected
LCLs.
Phosphorylation in LMP2A PY1PY2 LCLs.
Induction of tyrosine
phosphorylation is one of the earliest known biochemical events
following BCR cross-linking in B lymphocytes. LMP2A functions to block
BCR-mediated tyrosine phosphorylation in LMP2A+
LCLs. To determine the effect of the PY motif mutations on tyrosine phosphorylation following BCR cross-linking, LCLs with mutations in the
LMP2A PY motifs were tested. The LCLs used in these experiments were
verified for surface immunoglobulin (Ig) expression by flow cytometry
(data not shown). The induction of tyrosine phosphorylation was
analyzed at 1, 5, and 20 min after BCR cross-linking. Cell lysates of
LMP2A PY motif mutant LCLs and control cells were immunoprecipitated with anti-phosphotyrosine antibody followed by an immunoblotting with
anti-phosphotyrosine monoclonal antibody. As previously reported, tyrosine phosphorylation was induced in the BJAB cells and the LMP2A LCL but not in the wild-type LCL (Fig.
3). In contrast, the PY1PY2 LCLs showed
an apparent hyperphosphorylation of a protein with a molecular mass of
approximately 120 kDa and a modest increase in phosphorylation of 75- and 60-kDa proteins prior to BCR stimulation when compared to wild-type
LCLs (Fig. 3). This pattern remained unchanged following BCR
cross-linking (Fig. 3). This apparent hyperphosphorylation of the
120-kDa protein was particularly impressive in being the dominant
phosphoprotein present in any of the samples from the various cell
lines. Interestingly, the level of constitutive phosphorylation in PY1
or PY2 single-mutation LCLs was reduced when compared to the PY1PY2
LCLs, although there was some constitutive phosphorylation of the
120-kDa protein in PY1 LCLs (Fig. 3). In summary, despite the observed
differences in patterns of phosphorylation prior to BCR cross-linking,
both wild-type LCLs and the PY1, PY2, and PY1PY2 LCLs all exhibited a
block in BCR signal transduction, as measured by the induction of
tyrosine phosphorylation following BCR cross-linking (Fig. 3). In
addition, a 120-kDa protein was dramatically hyperphosphorylated in
PY1PY2 LCLs.
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FIG. 3.
Tyrosine phosphorylation following BCR cross-linking in
LMP2A PY motif mutant LCLs. BJAB cells and LCLs (107 cells)
were untreated ( ) or treated with anti-human surface Ig (sIg)
antibodies for the indicated times (1, 5, or 20 min), lysed in
Triton X-100 lysis buffer, and immunoprecipitated (IP) with
anti-phosphotyrosine antibody (PY20). Precipitated proteins were
separated on sodium dodecyl sulfate-7.5% polyacrylamide gel
electrophoresis gels and immunoblotted with horseradish
peroxidase-conjugated anti-phosphotyrosine antibody (RC20). The
hyperphosphorylated 120-, 75-, and 60-kDa proteins are indicated by
arrows. Molecular mass standards are indicated (in
kilodaltons).
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Tyrosine phosphorylation of LMP2A, Lyn, and Syk in LMP2A PY1PY2
LCLs.
To further characterize tyrosine-phosphorylated proteins in
the PY1PY2 LCLs, tyrosine phosphorylation of specific proteins was
examined in the PY1PY2 LCLs before and after BCR cross-linking. To
determine the level of LMP2A phosphorylation, cell lysates from PY1PY2
LCLs were immunoprecipitated with anti-LMP2A antibody followed by an
immunoblotting with anti-LMP2A antibody to verify similar amounts of
LMP2A in the immunoprecipitation or with anti-phosphotyrosine antibody
to determine the level of phosphorylation of LMP2A (Fig. 4A). Both wild-type and mutant LMP2A were
found at nearly the same level in the LMP2A immunoprecipitation (Fig.
4A). More interestingly, LMP2A in the PY1PY2 LCLs exhibited a much
greater level of phosphorylation when compared to LMP2A in wild-type
LCLs, indicating that the PY1PY2 mutation caused hyperphosphorylation
of LMP2A (Fig. 4A).
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FIG. 4.
Expression and tyrosine phosphorylation of LMP2A, Lyn,
and Syk after BCR cross-linking in LMP2A PY1PY2 LCLs. BJAB cells and
LCLs (107 cells) were untreated () or treated with
anti-human Ig antibodies for the indicated times (1 or 5 min), lysed in
Triton X-100 lysis buffer, and immunoprecipitated (IP) with anti-LMP2A
(A), anti-Lyn (B), or anti-Syk (C) antibody. Precipitated proteins were
separated on sodium dodecyl sulfate-7.5% polyacrylamide gel
electrophoresis gels and transferred to Immobilon membranes. To
determine the amount of each protein in the immunoprecipitation (top),
membranes were immunoblotted with anti-LMP2A (A), anti-Lyn (B), or
anti-Syk (C) antibody. To determine the level of tyrosine
phosphorylation (bottom), membranes were immunoblotted with horseradish
peroxidase-conjugated anti-phosphotyrosine antibody (RC20). Relevant
proteins are indicated with arrows, and molecular mass standards are
shown (in kilodaltons).
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Next, Lyn phosphorylation was determined by the same procedure
described above. As already shown in Fig.
2, Lyn expression
was
remarkably decreased in the LMP2A
+ LCL, whereas
this decrease in Lyn protein levels was not observed
in the PY1PY2 LCL
(Fig.
4B). In the PY1PY2 LCLs, both the amount
of Lyn protein and the
degree of phosphorylation were similar
to what was observed in BJAB
cells or in LMP2A
LCLs for Lyn (Fig.
4B). Of
particularly interest was the appearance
of a band corresponding in
size to that of LMP2A. LMP2A has previously
been shown to bind tightly
to Lyn (
12). This band was identified
as being LMP2A by
stripping the blot and reprobing with LMP2A-specific
antibodies (data
not shown). The considerable increase of LMP2A
bound to Lyn is likely
due to the increased levels of Lyn in the
PY1PY2 LCLs and the increased
level of LMP2A phosphorylation which
may enhance Lyn binding to LMP2A.
Previous studies have indicated
that the interaction of Lyn with LMP2A
requires the phosphorylation
of LMP2A (
12).
Finally, the induction of Syk phosphorylation prior to and following
BCR cross-linking was determined. Syk was immunoprecipitated
from each
LCL, and the amount of Syk in each immunoprecipitate
was approximately
equal in each LCL (Fig.
4C). There was very
little induction of Syk
phosphorylation in the LMP2A
+ LCL following BCR
cross-linking when compared to the LMP2A
LCL
(Fig.
4C), but there was a modest increase in Syk constitutive
phosphorylation in the PY1PY2 LCLs when compared to wild-type
LCLs, and
this increase remained following BCR cross-linking (Fig.
4C). As
observed with the Lyn immunoprecipitations, LMP2A was
readily detected
in the Syk immunoprecipitates in the PY1PY2 LCLs
(Fig.
4C). This band
was verified as LMP2A by stripping the blot
and reprobing with
LMP2A-specific antibody (data not shown). As
with Lyn, this increased
binding of Syk to LMP2A is likely due
to the enhanced phosphorylation
of LMP2A in the PY1PY2 LCLs. The
binding of Syk to LMP2A has also been
shown to be dependent on
LMP2A phosphorylation (
11).
In order to identify the hyperphosphorylated 120-kDa protein in the
PY1PY2 LCLs, anti-phosphotyrosine immunoprecipitates from
PY1PY2 LCLs
were immunoblotted with antibodies against proteins
which are
phosphorylated following BCR stimulation and are known
to be
approximately 120 kDa in size. Antibodies directed against
RasGAP (120 kDa), Cbl (120 kDa), and Cas (130 kDa) were negative
in immunoblottings
using the anti-phosphotyrosine immunoprecipitates
(data not shown).
Thus, mutation of both LMP2A PY motifs results
in the
hyperphosphorylation of LMP2A and a modest increase in
Syk
phosphorylation and this phosphorylation does not change following
BCR
cross-linking. Thus, the 75-kDa protein identified in the
previous
section may be Syk, whereas the identities of the 60-
and 120-kDa
proteins await
determination.
BCR cross-linking does not induce BZLF1 expression in LMP2A PY1PY2
LCLs.
Previous studies have shown that lytic EBV replication can
be induced in LCLs with deletions of genes for LMP2A or LCLs which have
specific mutations which block the association of either Lyn or Syk
with LMP2A (11, 12). To determine if mutation of the LMP2A
PY motifs altered this LMP2A function, LCLs were either untreated or
treated for 48 h with goat anti-human Ig to cross-link the BCR and
then were analyzed for BZLF1 expression by immunoblotting with
BZLF1-specific monoclonal antibody (33). BZLF1 is the
immediate-early transactivator of EBV lytic replication and is
expressed upon switch from EBV latent infection to lytic replication
(3). The induction of BZLF1 expression was evident only in
the LMP2A LCL after BCR cross-linking but not
in wild-type LCLs as previously described (22) or in any
of the LMP2A PY motif mutants (Fig. 5).
This indicates that the LMP2A PY motifs are not required for blocking
the induction of EBV lytic replication through BCR-mediated signal
transduction.
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FIG. 5.
Induction of BZLF1 expression in LMP2A PY motif mutant
LCLs. LCLs (4 × 106 cells) were untreated () or
treated (+) with anti-human surface Ig (sIg) antibody for 48 h.
Triton X-100 lysates from LCLs were separated on sodium dodecyl
sulfate-7.5% polyacrylamide gel electrophoresis gels and
immunoblotted with anti-BZLF1 antibody, followed by incubation with
horseradish peroxidase-conjugated secondary antibody and enhanced
chemiluminescence detection. Molecular mass standards are indicated (in
kilodaltons).
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Comparative growth of LMP2A PY1PY2 LCLs.
Although routine
passage of the PY1PY2 LCLs did not indicate any growth phenotype in
tissue culture, the growth of PY1PY2 LCLs was investigated at various
cell concentrations and in media supplemented with 10, 1, 0.1, or 0%
fetal bovine serum. PY1PY2 LCLs, LMP2A LCLs,
and the wild-type LCLs were plated, and growth was assayed 10 days
later by microscopic examination and monitoring of medium pH (Table
1). As previously reported, there was no
difference in growth between wild-type and
LMP2A LCLs (20, 21, 28). The
growth characteristics for the PY1PY2 LCLs were very similar to those
of the control LCLs (Table 1). All LCLs were unable to grow or grew
poorly in wells seeded with 2.5 × 104 cells
in 0.1% or lower serum or seeded with 5 × 103 cells in 1% or lower serum. Two PY1PY2 LCLs
seeded at 105 cells grew somewhat better than the
control LCLs in 0% serum, but the other PY1PY2 LCLs behaved similarly
to the control LCLs. Therefore, there was no apparent difference in
growth between the PY1PY2 LCLs and control LCLs.
LMP1 stability in LMP2A PY1PY2 LCLs.
Previous studies
indicated that LMP1 and LMP2A colocalize in EBV-infected LCLs grown in
tissue culture (18). Recently, it was reported that LMP1
is specifically ubiquinated and that this ubiquination is important for
LMP1 degradation (2). Although previous studies indicated
that there was no difference in the level of LMP1 protein in
LMP2A LCLs or in wild-type
LMP2A+ LCLs, indicating no apparent effect of
LMP2A on LMP1 protein levels (20, 21), it was of interest
to confirm that LMP2A did not direct the ubiquitination of LMP1 by
association with E3 ubiquitin ligases. LMP1 levels were therefore
investigated by immunoblotting in PY1PY2 LCLs and a variety of LCLs
containing tyrosine-to-phenylalanine point mutations in LMP2A which had
previously been described (10, 11, 29). Figure
6 shows similar LMP1 expression in all
LCLs. The Y112F LMP2A mutation blocked LMP2A phosphorylation and the
binding of Lyn to LMP2A. Both Y74F and Y85F blocked the binding of Syk
to LMP2A, but this did not affect overall LMP2A phoshorylation. All
three of the mutations resulted in a nonfunctional LMP2A in regard to
the LMP2A function of blocking B-cell signal transduction. The other
LMP2A tyrosines as of yet have no identified function
(29). In summary, these results confirm the previous
experimental results (20, 21) demonstrating that
expression of LMP2A does not affect the protein level of LMP1. In
addition, they indicate that LMP2A phosphorylation or interaction with
Nedd4 family E3s, Syk, or Lyn does not have any role in LMP1 protein
levels.
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FIG. 6.
Expression of LMP1 in LMP2A PY motif and tyrosine motif
mutant LCLs. The protein levels of LMP1 was analyzed in various LMP2A
phenylalanine-to-tyrosine-mutation LCLs. Triton X-100 lysates from
PY1PY2 (lanes 1 and 2), Y23F (lanes 3 and 4), Y23F/Y31F (lane 5), Y31F
(lane 6), Y60F (lanes 7 and 8), Y64F (lanes 9 and 10), Y74F (lanes 11 and 12), Y85F (lanes 13 and 14), Y101F (lanes 15 and 16), and Y112F
(lanes 17 and 18) LCLs were separated on sodium dodecyl sulfate-7.5%
polyacrylamide gel electrophoresis gels and immunoblotted with
anti-LMP1 antibody. The position of LMP1 is indicated by an arrow, and
molecular mass standards are indicated (in kilodaltons).
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Ligand-induced receptor activation requires membrane recruitment of
signaling proteins stimulated by tyrosine phosphorylation
of docking
proteins (for review, see reference
27). Most docking
proteins contain a membrane-targeting region, multiple tyrosine
phosphorylation sites that function as binding sites for SH2 domains
of
a variety of signaling proteins, and specific domains such
as PTB
domains that are responsible for complex formation with
cell surface
receptors (
27). In this regard, LMP2A resembles
a
constitutively active receptor bound to its relevant docking
protein.
The 12 LMP2A transmembrane domains mediate constitutive
aggregation of
LMP2A while the eight tyrosines, which are phosphorylated
independently
of ligand binding, and the two PY motifs of LMP2A
provide binding sites
for a variety of proteins involved in cell
signaling (
11,
12,
14), much like an activated receptor.
Relevant to this study is
the binding of the SH2 domain-containing
Lyn and the WW
domain-containing Nedd4 family E3s to LMP2A, which
results in the
specific degradation of Lyn. This degradation is
dependent on Lyn
binding to LMP2A since the phenylalanine-to-tyrosine
mutation at amino
acid 112 of LMP2A, which does not bind Lyn,
has Lyn protein levels
similar to those of the PY1PY2 LCLs and
LCLs with a null mutation in
LMP2A. Interestingly, we observed
no difference in the level of the Syk
PTK which also binds LMP2A
via an SH2-phoshotyrosine interaction
(
14). There was also no
change in protein levels for BLNK,
Btk, PI3K p85, PLC
2, and
Cas p130, indicating that the LMP2A effect
on Lyn was relatively
specific. Recently, in another study, it was
reported that transiently
transfected Lyn and Syk are specifically
ubiquitinated in LMP2A-expressing
HEK 293 cells (
32).
Although the observed Lyn ubiquitination
is compatible with our
results, the Syk ubiquitination, as noted
by these investigators, did
not result in lower levels of Syk
similar to our results
(
14). The significance of this difference
is not known but
will require future investigation of the Nedd4
family E3s association
with
LMP2A.
From our previous studies (
14), in which we identified the
association of LMP2A with AIP4 and other ubiquitin ligases of
the Nedd4
family, and from the results presented in the present
study, we
hypothesize that rather than being a static complex,
the LMP2A complex
is dynamic. Our refined model hypothesizes that
once LMP2A is
synthesized, it becomes phosphorylated and activates
Lyn, Syk, and
potentially other SH2 domain-containing proteins.
This LMP2A complex is
able to deliver a B-cell survival and developmental
signal, as observed
in LMP2A transgenic animals, and block BCR
signal transduction, as
observed in wild-type LCLs grown in culture.
Binding of LMP2A with AIP4
and other Nedd4 ubiquitin ligases would
then be important for the
specific internalization and degradation
of these LMP2A complexes in a
ubiquitin-dependent fashion (
14).
The internalization and
degradation does not occur or is much
reduced in the PY1PY2 LCLs, which
results in an increase in Lyn
protein levels since it is not targeted
for degradation by the
Nedd4 family E3s. This results in the
hyperphosphophorylation
of the unknown 120-kDa protein and LMP2A. The
identification of
the 120-kDa protein is currently being investigated
since it will
likely have an important role in LMP2A function and,
quite possibly,
normal BCR signal
transduction.
| |
ACKNOWLEDGMENTS |
We thank members of the Longnecker and Spear laboratories for
providing invaluable advice and help.
M.I. is a special fellow of the Leukemia and Lymphoma Society. R.L. is
a Stohlman Scholar of the Leukemia and Lymphoma Society and 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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0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.12.5711-5718.2001
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