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INTRODUCTION |
Recognition of specific antigen by the T cell antigen receptor
(TCR)1 initiates biochemical
changes including protein tyrosine phosphorylation, calcium influx,
metabolism of phospholipids, and activation of the Ras/MAPK cascade (1,
2). Together with signaling events initiated by engagement of
co-receptors on the T cell, these biological second messengers lead to
cytokine production, proliferation, and differentiation, or conversely
programmed cell death (3-5). Activation of protein tyrosine kinases
(PTKs) of the Src and Syk families is the most membrane-proximal
biochemical event known following TCR engagement (6). Phosphorylation
of cellular proteins by these kinases is essential for all subsequent
TCR-initiated signaling pathways. PTK activation may result in
downstream signals through direct phosphorylation and modulation of the
catalytic activities of effector molecules (such as phospholipase
C-
1) or the formation of signaling complexes by the interaction of proteins newly phosphorylated on tyrosine residues with other Src
homology 2 (SH2) or phosphotyrosine-binding domain-containing proteins
within the cell. A widely studied example of this in T cells is the
recruitment of ZAP-70 to the newly phosphorylated tyrosines of immune
receptor activation motifs (ITAMs) on the TCR
and CD3 chains (7,
8).
Given the central importance of PTKs in T cell activation, a number of
laboratories have attempted to identify substrates of these PTKs in an
effort to gain insight into how biochemical signals are integrated.
Among the substrates identified are the hematopoietic cell-specific
molecules SH2 domain containing leukocyte phosphoprotein of 76 kDa
(SLP-76) and Vav, both of which play critical roles in the regulation
of TCR signals (9, 10). SLP-76 is an adapter protein which is comprised
of three motifs allowing for protein-protein interactions: an
amino-terminal acidic region containing tyrosine phosphorylation sites
(11), a middle proline-rich motif that binds to the SH3 domain of Grb2
family members (9, 12), and a carboxyl-terminal SH2 domain that associates with SLP-76-associated phosphoprotein of 130 kDa (SLAP-130 or FYB, for Fyn-binding protein) and another unidentified 62-kDa phosphoprotein (13-15). The importance of SLP-76 in T cell signaling has been demonstrated in experiments in the Jurkat human T cell leukemia line and in mice made deficient in SLP-76 expression through
homologous recombination. When SLP-76 is overexpressed in Jurkat cells,
there is a dramatic augmentation of TCR-mediated activation of the
full-length IL-2 promoter or a reporter construct driven by the nuclear
factor of activated T cells (NFAT) element from the IL-2 promoter (9,
16). Each of the three structural domains of SLP-76 are essential for
this enhanced promoter activity (17). Furthermore, TCR-mediated signals
are abrogated in a mutant variant of Jurkat which has lost expression
of SLP-76 (18). Studies of SLP-76-deficient mice indicate also that
this adapter protein is critical for signaling via the pre-TCR, as
these mice exhibit arrest of thymocyte development at the pro-T3,
CD3
CD4
CD8
stage (19,
20).
Vav is a guanine nucleotide exchange factor which acts on members of
the Rac/Rho family of small GTP-binding proteins (21, 22). Vav has a
pleckstrin homology, a Dbl homology, an SH2, and two Src homology 3 (SH3) domains (23, 24). The Dbl homology domain of Vav is responsible
for its nucleotide exchange activity while the pleckstrin homology
domain regulates interactions with inositol phospholipids (25). The SH2
and SH3 domains mediate the interaction of Vav with other signaling
molecules, including SLP-76 (26-29). Like SLP-76, Vav is critical for
effective TCR signaling. Its overexpression in Jurkat T cells also
augments the transcriptional activity of the IL-2 promoter following
TCR ligation (10). T cells from Vav-deficient mice do not respond effectively to TCR stimulation (30, 31). Interestingly, overexpression of SLP-76 and Vav together has a synergistic effect on activation of
the IL-2 promoter (29). However, previous studies have not addressed
whether the interaction between SLP-76 and Vav is required for their
synergy and if these two proteins function in the same signaling pathways.
In the experiments described in this report we investigated these
questions first by identifying the tyrosines of SLP-76 responsible for
the interaction with Vav both in vitro and in intact Jurkat cells. Using this information, we performed a series of co-transfection assays and determined that the intermolecular interaction between SLP-76 and Vav is not required for their functional synergy. This finding suggested the hypothesis that overexpression of SLP-76 versus Vav may impact distinct signaling pathways in T
cells. Experiments are presented demonstrating that these two molecules modulate distinct signaling cascades to link T cell surface receptors with IL-2 gene expression.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Antibodies--
The human Jurkat T cell line
was cultured in RPMI 1640 supplemented with 10% fetal calf serum,
penicillin (1,000 units/ml), streptomycin (1,000 units/ml), and
glutamine (20 mM). For stimulation through the TCR, ascites
of the clonotypic mAb C305 (32) were used. Anti-CD28 monoclonal
antibody 9.3A (ATCC, Manassas, VA) was used for stimulating cells
through CD28. Monoclonal antibody M2 recognizes the FLAG epitope
(International Biotechnologies, New Haven, CT). Monoclonal antibody
9E10 recognizes the Myc epitope. Phosphotyrosine-containing proteins
were detected by the 4G10 mAb (Upstate Biotechnology, Inc., Lake
Placid, NY).
cDNA Constructs and Fusion Proteins--
Wild-type SLP-76
and each of the mutants were cloned into modified pEF-BOS vector
containing a sequence encoding the FLAG epitope on the amino terminus
of the cDNAs, as described previously (11, 17). cDNAs encoding
Vav and ERK2 with a Myc epitope on the amino terminus were also cloned
into pEF-BOS vector (gifts of Dr. A. Weiss, University of California,
San Francisco, CA). The NFAT luciferase reporter construct (NFAT-luc)
was provided by Dr. G. Crabtree (Stanford University, Palo Alto, CA).
The full-length IL-2 promoter driven luciferase construct (pIL-2luc2kb)
was a gift of Dr. R. Abraham (Mayo Clinic, Rochester, MN). The AP-1
-Gal construct (pAP-1-lacZ) was a gift of Dr. D. Mueller (University of Minnesota, Minneapolis, MN). The CD28RE luciferase construct was
provided by Dr. K. L. McGuire (San Diego State University, San
Diego, CA). The GST Vav SH2 was provided by Dr. S. Katzav (Lady Davis
Institute, Jewish General Hospital, Montereal, Quebec, Canada). GST
fusion protein was induced and affinity purified as described (33).
Transfections--
For transient transfection, 107
cells were electroporated at 250 V, 960 microfarads using a Gene Pulser
(Bio-Rad) as described previously (17), with 20 µg of the NFAT, AP-1,
IL-2, or CD28RE reporter plasmids, or pEF-myc-ERK2, 10 µg of plasmids
encoding Vav or variant SLP-76 constructs in the synergy experiments,
and 40 µg of Vav or SLP-76 constructs elsewhere.
Reporter Assays--
Luciferase or
-Gal assays were performed
as described previously (17). Briefly, 24 h after transfection,
triplicates of 5 × 105 viable cells were stimulated
as indicated at 37 °C for 8 h, lysed, and subsequently assayed
for luciferase or
-Gal activity with a monolight luminometer
(Analytical Luminescence Laboratory, Ann Arbor, MI).
Immunoprecipitations, Protein Precipitations, and
Immunoblots--
24 h after transfection, cells were collected, washed
in phosphate-buffered saline, and left unstimulated, or stimulated with C305 ascites (1:1,000) for the indicated time, or with pervanadate for
2 min, or with PMA for 5 min. Lysates were prepared in Nonidet P-40
lysis buffer containing protease and phosphatase inhibitors, as
described previously (9), and precipitated with GST fusion proteins or
indicated antibodies at 4 °C for 2 h. Following precipitation, the protein complexes were washed extensively in Nonidet P-40 lysis
buffer, subjected to SDS-PAGE, and transferred to nitrocellulose for
immunoblot analysis with the indicated antibodies, followed by a
horseradish peroxidase-conjugated secondary Ab (Bio-Rad) and developed
by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech).
ERK2 in Vitro Kinase Activity Assays--
ERK2 kinase activity
assays were performed as described previously (17). Briefly, Jurkat
cells were transfected with pEF, pEF/SLP-76, pEF/Vav plus pEF/myc/ERK2.
24 h later, transfected cells were left unstimulated or stimulated
with anti-TCR mAb C305 or phorbol myristate acetate (PMA), lysed in
Triton X-100 lysis buffer, and precipitated with anti-Myc antibody for
2 h at 4 °C after stimulation, The anti-Myc immune complexes
were washed extensively and suspended in kinase buffer (20 mM Tris, pH 7.6, 13 mM MgCl2, and
1.5 mM EGTA) containing myelin basic protein (MBP, 1 mg/ml) and [
-32P]ATP (0.22 µCi/sample), incubated at room
temperature for 15 min, and subjected to SDS-PAGE. Each gel was stained
with Coomassie Blue to assure equal loading of the MBP substrate and
phosphorylation of MBP was visualized by autoradiography and
quantitated by densitometry using the NIH Image computer program.
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RESULTS |
Both Tyrosines 113 and 128 of SLP-76 Are Required for the
SLP-76/Vav Interaction--
The interaction between SLP-76 and Vav
depends on the SH2 domain of Vav and tyrosine phosphorylation of SLP-76
(28, 29). Using a phosphopeptide library, Songyang et al.
(34) found that the SH2 domain of Vav preferentially binds
phosphorylated tyrosines in a Y*XXP motif (where Y*
represents a phosphorylated tyrosine, X is any amino acid,
and P is a proline residue). There are three tyrosines within SLP-76
which fall into such a motif: tyrosines 113 (YESP), 128 (YESP), and 145 (YEPP). Several lines of evidence suggest that Tyr-113 and Tyr-128 are
good candidates for mediating the association between SLP-76 and Vav.
First, both Tyr-113 and Tyr-128 are substrates of the TCR-stimulated
PTKs (11). Second, a similar YESP motif found in both ZAP-70 and Syk is
known to be responsible for the interaction of these PTKs with Vav (35, 36). Finally, phosphorylated peptides corresponding to the two YESP
motifs of SLP-76 precipitate Vav from cell lysates and inhibit the
SLP-76/Vav interaction in vitro (28, 37).
To investigate further the importance of Tyr-113, Tyr-128, and Tyr-145
in mediating the interaction between full-length SLP-76 and Vav in
intact T cells, FLAG-tagged SLP-76 constructs encoding wild-type SLP-76
(WT) or point mutants with tyrosines 113, 128, or 145 individually
changed to phenylalanine (Y113F, Y128F, and Y145F) were transfected
into Jurkat T cells. Transfected cells were stimulated via their TCR,
lysed, and precipitated with anti-FLAG (Fig.
1A) or GST-Vav-SH2 fusion
protein (Fig. 1B). Each construct was expressed at similar
levels as revealed by immunoblot analysis for the FLAG epitope (Fig. 1,
A, top panel; B, left). As shown previously,
wild-type SLP-76 and each single tyrosine mutant is phosphorylated upon
TCR stimulation (Fig. 1A, bottom panel). Surprisingly, however, mutation of either Tyr-113 or Tyr-128 abrogates the binding of
SLP-76 to the SH2 domain of Vav (Fig. 1B). In contrast,
wild-type SLP-76 and the Y145F mutant still bind to the Vav-SH2 (Fig.
1B). To obtain optimal phosphorylation of SLP-76, we also
stimulated cells with pervanadate, a tyrosine phosphatase inhibitor and
found again that the Y113F and Y128F SLP-76 mutants fail to associate with the SH2 domain of Vav (data not shown).

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Fig. 1.
Tyrosines 113 and 128 of SLP-76 are required
for the association between SLP-76 and Vav. A and
B, Jurkat cells were transfected with FLAG-tagged WT SLP-76
and SLP-76 mutants Y113F, Y128F, and Y145F. 24 h after
transfection, the transfected cells were stimulated with anti-TCR mAb
C305, lysed, and precipitated by either anti-FLAG antibody
(A) or GST-Vav SH2 fusion protein (B). In
panel A, the anti-FLAG immune complexes were subjected to
SDS-PAGE and Western blotting with either anti-FLAG antibody
(top) or anti-phosphotyrosine antibody 4G10
(bottom). Lane 1, wild-type SLP-76; Lane
2, Y113F; Lane 3, Y128F; Lane 4, Y145F. In
panel B, cell lysates (Lanes 1-4) and GST-Vav
SH2 precipitates (Lanes 5-8) were subjected to SDS-PAGE and
immunoblot analysis with anti-FLAG. Lanes 1 and
5, WT SLP-76; Lanes 2 and 6, Y113F;
Lanes 3 and 7, Y128F; Lanes 4 and
8, Y145F. C, Jurkat cells were transfected with
Myc-tagged Vav and FLAG-tagged SLP-76 WT or mutant constructs. 24 h after transfection, cells were stimulated with pervanadate, lysed,
and precipitated with anti-Myc antibody. Cell lysates (Lanes
1-4) and anti-Myc immuno-complexes (Lanes 5-8) were
subjected to SDS-PAGE and blotted with anti-Myc antibody
(top) or anti-FLAG antibody (bottom), as
indicated. Lanes 1 and 5, cells transfected with
WT SLP-76; Lanes 2 and 6, Y113F; Lanes
3 and 7, Y128F; Lanes 4 and 8,
Y145F.
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These in vitro findings were followed by experiments in
intact Jurkat cells. For these studies, cells were co-transfected with
Myc-tagged Vav and FLAG-tagged SLP-76 constructs. Transfected Jurkat
cells were treated with pervanadate, lysed, and immunoprecipitated with
anti-Myc antibody. As shown, an equal amount of Vav is present in each
immune complex (Fig. 1C, top panel). However, both SLP-76 mutants Y113F and Y128F fail to co-immunoprecipitate with Myc-Vav, while wild-type SLP-76 and the Y145F mutant associate with Vav (Fig.
1C, bottom panel). Thus, in agreement with our in
vitro finding, both tyrosines 113 and 128 of SLP-76 are required
for the association of SLP-76 with the Vav SH2 in intact cells.
Synergy between Vav and SLP-76 Does Not Require Their
Interaction--
Although it is clear that SLP-76 and Vav synergize to
couple TCR ligation with cytokine gene expression (29), the mechanism for this cooperation is not clear. One possibility is that SLP-76, as
an adaptor molecule, recruits Vav to the proper subcellular location
where Vav executes its role as a guanine nucleotide exchange factor. If
this were true, the synergy between SLP-76 and Vav should require their
interaction. To test this possibility, we transfected cDNA encoding
Vav along with SLP-76 mutants Y113F and Y128F and assessed the effect
on IL-2-NFAT promoter activity. To optimize conditions for observing
synergy between SLP-76 and Vav, limiting amounts of cDNA encoding
SLP-76 and Vav were transfected into cells. As shown, overexpression of
SLP-76 or Vav alone at this level fails to augment IL-2-NFAT promoter
activity. However, co-transfection of Vav with wild-type SLP-76, or
either SLP-76/Y113F or SLP-76/Y128F augments IL-2-NFAT promoter
activity significantly (Fig. 2). In each
experiment, Western blot analysis demonstrated that all of the SLP-76
constructs were expressed at similar levels and that Vav was expressed
similarly in each transfection condition (not shown).

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Fig. 2.
The synergy between SLP-76 and Vav does not
require their interaction. A luciferase reporter construct driven
by three copies of the NFAT element of IL-2 promoter was transfected
into Jurkat cells alone with control vector or cDNA encoding SLP-76
(WT, Y113F, Y128F, and Y145F), Vav, or SLP-76 plus Vav. 24 h after
transfection, cells were left untreated (UN; ) or
stimulated with anti-TCR mAb C305 (TCR; ) or PMA plus
ionomycin at 37 °C for 8 h. Lysates were prepared and analyzed
for luciferase activity. Results are shown as the percentage of the PMA
plus ionomycin response (maximum) for each group. Expression of SLP-76
constructs and Vav constructs were documented by Western analysis.
SLP-76 expression level is not changed with co-overexpression of Vav
(data not shown). These data are representative of three independent
experiments.
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Overexpression of SLP-76, but Not Vav, Augments TCR-induced AP-1
Activity--
Our observation that the association between SLP-76 and
Vav is not required for their functional cooperation suggests the possibility that these two proteins modulate different TCR-induced signaling cascades. We and others have found that when overexpressed individually, both SLP-76 and Vav augment activity of the NFAT region
of the IL-2 promoter (9, 10). Work from several laboratories has shown
that this region of the IL-2 promoter includes two components; a
sequence which binds NFAT family members and a contiguous region which
binds AP-1 dimers (reviewed in Ref. 38). Thus, modulating signals
leading to either NFAT translocation and activity or AP-1 function
could increase responsiveness of an IL-2 promoter-derived NFAT reporter
(39-41).
We have shown that in the Jurkat T cell line, SLP-76 overexpression
augments TCR-induced activation of a
-galactosidase reporter driven
by 5 copies of an AP-1 response element (17). To test whether
overexpression of Vav has a similar impact on AP-1 function, we
transfected Jurkat cells with the AP-1 reporter construct along with
either SLP-76 or Vav and measured
-galactosidase activity in resting
and stimulated cells. As shown in Fig. 3,
TCR or PMA stimulation results in only a modest increase in AP-1
reporter activity in cells transfected with vector control. TCR
stimulation of cells transfected with SLP-76 cDNA results in marked
augmentation of reporter activity. As expected, SLP-76 overexpression
does not impact on PMA-stimulated AP-1 function as SLP-76 is not
tyrosine phosphorylated following PMA stimulation (Fig. 3 and data not shown). In contrast to results seen with SLP-76, overexpression of Vav
does not increase TCR stimulated AP-1 activity above that seen with the
vector transfected control cells. Thus it appears that although both
SLP-76 and Vav can modulate some TCR signals, those leading to AP-1
activation are affected by SLP-76 but not Vav.

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Fig. 3.
Overexpression of SLP-76, but not Vav,
augments AP-1 activity. An AP-1- -Gal reporter construct plus a
Rous sarcoma virus-luciferase construct were transfected into Jurkat
cells along with control vector, or cDNA encoding SLP-76 or Vav.
24 h after transfection, cells were left untreated (UN)
or treated with anti-TCR mAb C305 (TCR) or PMA at 37 °C for 8 h. Cells were then lysed and -Gal activity measured. The -Gal
counts were normalized to the Rous sarcoma virus-luciferase counts to
control for transfection efficiency. Expression of transfected SLP-76
or Vav was documented by Western analysis with anti-FLAG (for SLP-76)
or anti-Myc (for Vav) antibody (data not shown). This experiment is
representative of five independent experiments. , UN; , TCR; ,
PMA.
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SLP-76, but Not Vav, Leads to ERK Activation--
There is
considerable evidence to suggest that extracellular signal-regulated
kinase (ERK) family members stimulated by TCR ligation function
upstream of AP-1 activation (40, 42, 43). We have found previously that
overexpression of SLP-76 augments TCR induced activity of ERK2 (17). To
investigate if Vav has a similar ability to modulate TCR-stimulated ERK
function, a Myc-tagged ERK2 construct was co-transfected into Jurkat
cells along with cDNA encoding SLP-76 or Vav. Cells were left
unstimulated or stimulated via the TCR or PMA. Cellular lysates were
then subjected to immunoprecipitation with anti-Myc and in
vitro kinase assays were performed on each immune complex using
MBP as an artificial substrate. Phosphorylation of MBP was quantitated
by densitometry and normalized to PMA-induced MBP phosphorylation.
Whereas overexpression of SLP-76 consistently enhances TCR-induced
kinase activity of ERK2 to a modest degree, overexpression of Vav does
not affect ERK2 enzymatic activity (data not shown).
We reasoned that if SLP-76 affects AP-1 indirectly via augmentation of
ERK activity, overexpression of SLP-76 and ERK together should lead to
additive enhancement of AP-1 activity in TCR-, but not PMA-stimulated
cells. Furthermore, since Vav does not appear to modulate
TCR-stimulated ERK2, we reasoned that overexpression of Vav plus ERK2
would not result in AP-1 function above that seen with overexpression
of ERK2 alone. We tested these hypotheses in the experiments shown in
Fig. 4. For this study, the AP-1 reporter construct was transfected into Jurkat cells along with cDNAs for ERK2 alone or with either SLP-76 or Vav. As predicted, overexpression of ERK2, but not SLP-76, impacts PMA-stimulated AP-1 activity (Fig.
4A). In contrast, overexpression of either SLP-76 or ERK2 augments TCR-induced AP-1 activity (Fig. 4A). Consistent
with our hypothesis, overexpression of the two molecules together leads to further augmentation of AP-1 activity in TCR-stimulated Jurkat cells. However, when we perform the same AP-1 reporter analysis in
lysates from cells transfected with Vav, we find no augmentation of
promoter activity above that seen with vector control transfectants. Additionally, transfection with Vav plus ERK2 provides no greater AP-1
response than transfection with ERK2 alone (Fig. 4B). For each experiment, Western blot analysis was performed and showed expression of Myc-tagged ERK2 and Vav, or FLAG-tagged SLP-76 constructs (data not shown).

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Fig. 4.
Overexpression of SLP-76, but not Vav,
cooperates with ERK2 to augment TCR-induced AP-1 activity. An
AP-1- -Gal reporter construct plus an Rous sarcoma virus-luciferase
construct were transfected into Jurkat cells along with control vector
or cDNA encoding SLP-76, ERK2, SLP-76 plus ERK2 (A); or
Vav, ERK2, Vav plus ERK2 (B). 24 h later, cells were
left untreated (UN) or treated with anti-TCR mAb C305
(TCR) or PMA at 37 °C for 8 h. Cells were then lysed
and -Gal activity was measured. The -Gal counts were normalized
to the Rous sarcoma virus-luciferase counts to control for transfection
efficiency. Western blot analysis was performed to verify the
expression of transfected ERK2, SLP-76, and Vav (data not shown). The
results shown in both panels A and B are
representative of three independent experiments. , UN; , TCR;
, PMA.
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Vav, but Not SLP-76, Augments CD28-induced IL-2 Promoter
Activity--
Our experiments assessing the effect of SLP-76 or Vav on
ERK and AP-1 indicate that SLP-76 modulates TCR-induced signaling pathways which are not affected by Vav. We wondered, therefore, if the
converse is true as well. In T cells, signals initiated from the TCR
cooperate with those arising from co-receptors, such as CD28, to
optimize IL-2 gene transcription (44, 45). Work from a number of
investigators has shown that the TCR and CD28 stimulate transactivation
of different regions within the IL-2 promoter (46-48). A CD28-response
element within the IL-2 promoter, CD28RE, has been defined which
contains binding sites for Rel family members in conjunction with other
transcription factors (48-50). In combination with TCR ligation or
mitogenic stimulators such as PMA or phytohemagglutinin, engagement of
CD28 induces these transcription factors to bind to and transactivate
the CD28RE (48, 49).
To investigate the potential roles of SLP-76 and Vav in CD28-stimulated
IL-2 transcription, we performed reporter assays using either a
full-length IL-2 promoter construct (which includes the CD28RE) or a
reporter construct driven by four copies of the CD28RE. The full-length
IL-2 promoter construct was co-transfected into Jurkat T cells with
cDNAs encoding either SLP-76 or Vav (Fig. 5A). Transfected cells were
then left untreated or treated with TCR plus PMA or CD28 plus PMA. As
shown, although overexpression of either SLP-76 or Vav augments IL-2
promoter activity induced by stimulation via the TCR plus PMA,
overexpression of Vav but not SLP-76 augments the CD28 plus PMA signal.
PMA by itself is not able to induce IL-2 promoter activity and
overexpression of either Vav or SLP-76 does not affect its function
(data not shown). Thus, it appears that Vav, but not SLP-76, can
mediate the signaling pathways linking CD28 to IL-2 gene expression.
Expression of transfected Vav or SLP-76 constructs were verified by
Western blot (data not shown).

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Fig. 5.
Vav, but not SLP-76, regulates CD28-induced
IL-2 transcription. A, a luciferase reporter gene
driven by the full-length IL-2 promoter was transfected into Jurkat
cells along with control vector, SLP-76, or Vav. 24 h after
transfection, the transfected cells were left untreated (UN)
or treated with anti-TCR mAb C305 plus PMA (TCR + PMA), or anti-CD28
mAb A9.3 plus PMA (CD28 + PMA), or PMA plus ionomycin at 37 °C for
8 h. Cells were then lysed and luciferase activity analyzed.
Results are shown as the percentage of the PMA plus ionomycin response
(maximum) for each group. Expression of transfected SLP-76 and Vav was
documented by Western blot analysis (not shown). These data are
representative of three independent experiments. B, a
luciferase reporter gene driven by CD28RE/AP-1 was transfected into
Jurkat cells along with control vector, SLP-76, or Vav. 24 h
later, transfected cells were left untreated (UN) or treated
with anti-TCR mAb C305 (TCR), or anti-CD28 mAb 9.3A plus PMA (CD28 + PMA) at 37 °C for 8 h. Cell lysates were prepared and
luciferase activity was measured. Results were calculated as extent of
luciferase activity induced by TCR or CD28 treatment compared with
unstimulated cells ("Fold Induction"). Expression of transfected
SLP-76 and Vav was documented by Western blot analysis (data not
shown). The results are representative of three independent
experiments. , UN; , TCR + PMA (A) or TCR alone
(B); , CD28 + PMA.
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To test more directly whether Vav can regulate CD28RE activation
following CD28 ligation, we performed experiments with the CD28RE
reporter (Fig. 5B). Jurkat cells were transfected with this
reporter along with a vector control, SLP-76, or Vav. Cells were left
untreated or were stimulated via their TCR or with anti-CD28 plus PMA.
Consistent with previous findings of others (48, 50), TCR stimulation
or PMA by itself fails to induce CD28RE activity in vector control
transfectants (Fig. 5B). Additionally, we find that
overexpression of neither SLP-76 nor Vav modifies the ability of the
TCR or PMA to induce this reporter. Interestingly, however, transfection of Vav, but not SLP-76, augments CD28/PMA-induced CD28RE
activation, again supporting the notion that Vav and SLP-76 differ in
their ability to impact on CD28-stimulated signaling pathways.
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DISCUSSION |
Numerous signaling cascades function together to link antigen
recognition to gene expression in T cells. Recently, a number of
studies have demonstrated the importance of adaptor molecules in
regulating this network (reviewed in Refs. 51-54). Having no intrinsic
enzymatic activity, adaptor molecules recruit other signaling proteins
to form multimeric complexes through intermolecular associations.
SLP-76 is one such adaptor protein which has been shown to bind Gads
(12), Vav (28, 29, 37), SLAP-130 (FYB) (14, 15), and other as yet
unidentified molecules. Although these protein-protein interactions
have been documented, their physiological importance remains unclear.
Because SLP-76 and Vav function synergistically in their ability to
augment TCR signaling leading to activation of the IL-2 gene (29), we
initiated a series of experiments to map the tyrosines of SLP-76
important for the SLP-76/Vav interaction and to ask about the
functional consequences of their binding. These experiments have led to
a number of surprising findings.
We and others have shown previously that the association between SLP-76
and Vav is mediated by the Vav SH2 domain binding to
tyrosine-phosphorylated residues within the SLP-76 amino terminus (28,
29, 37). In experiments presented in this study we found that although
the binding is mediated by a single Vav SH2 domain, two SLP-76
tyrosines, Tyr-113 and Tyr-128, both appear essential for this
association. This is particularly surprising since both Tyr-113 and
Tyr-128 are found in identical sequence motifs (DYESP), either of which
would be predicted to bind the Vav SH2. Although we are not yet sure
why both SLP-76 tyrosines are required for Vav binding, several models
come to mind. One possibility is that tyrosine phosphorylation of
SLP-76 is sequential, and that phosphorylation of either tyrosine 113 or 128 is required for induced or maintained phosphorylation of the
second residue. Thus, although only one tyrosine may be involved in
direct binding to Vav, the second tyrosine is required for the
phosphorylation of the Vav-binding tyrosine. However, while mutation of
both tyrosines 113 and 128 abolishes TCR-induced tyrosine
phosphorylation of SLP-76, mutants with either Tyr-113 or Tyr-128
mutated individually are still phosphorylated following TCR or
pervanadate stimulation, making this explanation unlikely. A second
possibility is that one phosphorylated tyrosine is responsible for
directing the interaction with the Vav SH2 domain, while the second
tyrosine is required for stabilizing this interaction, perhaps through
maintaining a structural microenvironment or via an interaction with a
third molecule. In this regard, SLP-76 was recently shown to
simultaneously bind to the SH2 domains of Vav and the adapter protein,
Nck, to form a trimolecular complex (55). Thus, it is possible that the
SLP-76/Nck interaction is important for stabilizing the association between SLP-76 and Vav. Crystal or solution nuclear magnetic resonance determination of the structures of SLP-76, Vav, and Nck, when bound
together, will be necessary to test this hypothesis.
On the surface, a second finding from our studies, that the synergy
between SLP-76 and Vav does not appear to require their association,
seems surprising. One potential explanation could be that the SLP-76
tyrosine mutants dimerize with endogenous, wild-type SLP-76 and thus
interact with Vav indirectly. However, multiple experimental approaches
in our laboratory have failed to demonstrate evidence for a
SLP-76/SLP-76 interaction,2
making this explanation unlikely. Moreover, our result is consistent with several other observations. First, when SLP-76 mutants Y113F and
Y128F are overexpressed at high levels, they augment IL-2-NFAT promoter
activity to a level similar to that of wild-type SLP-76 (11),
suggesting that in this assay, SLP-76 can function independently of its
ability to bind Vav. Second, SLP-76 and Vav synergize in augmenting
basal IL-2-NFAT promoter activity in Jurkat cells, although under these
conditions, SLP-76 is not phosphorylated and does not bind Vav (29).
Third, it was reported that in the T cell hybridoma DC27.10, where
there is no detectable association between SLP-76 and Vav (even
following TCR stimulation), TCR ligation still induces IL-2 production
(37). Finally, we have found recently that mice made deficient in
SLP-76 expression by homologous recombination have a much more profound
defect in T cell development than do mice which lack expression of Vav,
suggesting that SLP-76 and Vav do not function entirely in the same
pathway (19, 20, 30, 31, 56). Collectively, these observations,
together with the experiments presented in our current study, suggest
that the Vav/SLP-76 interaction is not essential for their functional synergy, at least for inducing IL-2 gene transcription. This conclusion led us to ask if SLP-76 and Vav may work together by modulating different signaling pathways in T cells.
There are several examples of how different pathways may enhance
signals leading to IL-2 gene expression in T cells. One involves the
cooperation between signals initiated from the TCR and those stimulated
by engagement of CD28 (47). Another is the interdependence of signals
leading to translocation of NFAT from the cytoplasm to the nucleus with
other signals leading to AP-1 activation resulting in stimulating
promoter elements within many cytokine genes (38). Our studies suggest
that the synergy between SLP-76 and Vav may reflect the cooperation of
different, but overlapping signals initiated from the TCR and CD28 as
Vav, but not SLP-76 impacts on the ability of CD28 to augment IL-2 gene
promoter activity. This finding is consistent with the observation that
while Vav is one of the major substrates for CD28-induced PTKs (57), we find that SLP-76 is phosphorylated to only a very limited extent, if at
all following CD28 ligation (data not shown). Our data suggest also
that SLP-76 and Vav function in different TCR-stimulated pathways as
SLP-76, but not Vav couples the TCR with AP-1. This is consistent with
the recent observation that activation of ERK following TCR ligation is
diminished markedly in SLP-76-deficient Jurkat cells (18) while
stimulation of this second messenger is retained in Vav-deficient T
cells (56, 58).
Further insight into the potential mechanisms by which SLP-76 exerts
its effects on TCR-mediated signaling have come from studies of the
SLP-76 defective Jurkat mutant. In this cell line, TCR engagement
results in activation of PTKs, but fails to couple effectively with the
phosphatidylinositol as well as Ras signaling pathways (18). Thus, it
appears that SLP-76, via its adaptor function, is critical for allowing
TCR-stimulated PTK activation to result in optimal phosphorylation of
phospholipase C
1 in addition to signals leading to ERK activation.
It remains to be determined, however, exactly which protein-protein
interactions mediated by SLP-76 are critical for these more downstream
signals to occur.
Although the experiments in this study indicate that for TCR-stimulated
IL-2 gene regulation, the interaction between SLP-76 and Vav does not
appear to play a critical role, it is possible, and perhaps likely,
that other T cell activation events are influenced by SLP-76 and Vav
and require their interaction. For example, it has been reported that
the interaction between SLP-76 and Vav may be required for TCR-induced
PAK activation as well as the capping process (55). Since several
studies have shown that Vav plays a critical role in formation of the
cap following TCR stimulation (56, 58, 59), it will be important to
study capping function in SLP-76-deficient T cells. We are currently working to reconstitute our SLP-76-deficient mice with wild-type and
mutant variants of SLP-76 to begin to address this and other related questions.