From the § INSERM Unité 343, IFR50, Hôpital
de l'Archet, 06202 Nice, Cédex 3, France, the
INSERM Unité 145, IFR50, Faculté de
Médecine, 06107 Nice, Cédex 2, France, and the
La
Jolla Institute for Allergy and Immunology,
San Diego, California 92121
Received for publication, November 22, 2000, and in revised form, March 16, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Vav1 and Vav2 are members of the Dbl
family of guanine nucleotide exchange factors for the Rho family
of small GTPases. Although the role of Vav1 during lymphocyte
development and activation is well characterized, the function of Vav2
is still unclear. In this study, we compared the signaling pathways
regulated by Vav1 and Vav2 following engagement of the T cell receptor
(TCR). We show that Vav2 is tyrosine-phosphorylated upon TCR
stimulation and by co-expressed Src and Syk family kinases. Using
glutathione S-transferase fusion proteins, we observed that
the Src homology 2 domain of Vav2 binds tyrosine-phosphorylated
proteins from TCR-stimulated Jurkat T cell lysates, including c-Cbl and
SLP-76. Like Vav1, Vav2 cooperated with TCR stimulation to increase
extracellular signal-regulated kinase activation and to promote
c-fos serum response element transcriptional activity.
Moreover, both proteins displayed a similar action in increasing the
expression of the early activation marker CD69 in Jurkat T cells.
However, in contrast to Vav1, Vav2 dramatically suppressed TCR signals
leading to nuclear factor of activated T cells
(NF-AT)-dependent transcription and induction of the
interleukin-2 promoter. Vav2 appears to act upstream of the phosphatase
calcineurin because a constitutively active form of calcineurin rescued
the effect of Vav2 by restoring TCR-induced NF-AT activation.
Interestingly, the Dbl homology and Src homology 2 domains of Vav2 were
necessary for its inhibitory effect on NF-AT activation and for
induction of serum response element transcriptional activity. Taken
together, our results indicate that Vav1 and Vav2 exert overlapping but
nonidentical functions in T cells. The negative regulatory pathway
elicited by Vav2 might play an important role in regulating lymphocyte
activation processes.
Antigen receptor engagement stimulates the activity of T cell
receptor (TCR)1-coupled
protein-tyrosine kinases (PTKs) of the Src and Syk families, which
induce the assembly of large signaling complexes composed of cellular
enzymes, adaptors, and other cytoplasmic transducers (1, 2). These
complexes initiate multiple signaling pathways coordinating the
activation of immediate-early genes (3) and nuclear factors that
control the transcription of several immunomodulatory genes, including
the interleukin-2 (IL-2) gene (4).
Optimal T cell activation requires the formation of a T cell synapse.
This process depends on reorganization of the actin cytoskeleton, which
is controlled by small GTPases of the Rho/Rac family (5). Upon
stimulation by extracellular signals, these proteins cycle from an
inactive, GDP-bound form to an active, GTP-bound enzyme that
translocates to the membrane and interacts with downstream effector
proteins. These proteins, in turn, regulate adhesion, motility, and
gene transcription in lymphocytes and other cell types (6, 7).
In lymphocytes, the activation-dependent exchange of GDP
for GTP is regulated by the guanosine nucleotide exchange factor (GEF)
Vav1. Vav1 contains several functional domains, including a Dbl
homology (DH) domain, a Pleckstrin homology domain, a cysteine-rich domain, one Src homology 2 (SH2) domain, and two Src homology 3 (SH3)
domains (8). Vav1 expression is mostly restricted to hematopoietic
cells, and different studies have shown that it is a critical link
between TCR-coupled PTKs of the Src and Syk families and the signaling
pathways regulated by Rho/Rac proteins (7-9). Analysis of
Vav1-deficient mice indicated that Vav1 is required for T cell
development and antigen receptor-mediated T or B lymphocytes activation
or apoptosis (10-12). Vav1 activity is also required for TCR
clustering and actin cytoskeleton reorganization (13, 14),
Ca2+ signaling, activation of mitogen-activated protein
kinase ERKs and transcription factors NF-AT and NF- Recent studies have identified other members of the Vav family, Vav2
and Vav3, which display a much broader tissue expression (9). Vav2
transcripts are nearly ubiquitously expressed in mouse, from embryonic
to adult stage (22). The enzymatic activities of all three isoforms are
subjected to a phosphorylation-dependent regulation (9).
Tyrosine phosphorylation of Vav proteins can be induced through the
stimulation of different receptors, including immune recognition
receptors, cytokine receptors, integrins, and PTK receptors such as the
epidermal growth factor and the platelet-derived growth factor
receptors (23-26). Despite the fact that each member of the Vav family
can induce cytoskeletal reorganization and transform rodent
fibroblasts, their catalytic specificity toward Rho/Rac proteins
appears to differ. Whereas Vav1 displays GEF activity for Rac1, Cdc42,
RhoA, and RhoG, Vav2 was shown to exhibit GEF activity for RhoA, RhoB,
and RhoG but not for Rac1 or Cdc42 (27). In this regard, the
morphological phenotypes induced by Vav1 and Vav2 expression in
fibroblasts are distinct (27). However, these findings were not
confirmed in another study (28). Together these studies suggest that
the Vav family might use overlapping but nonidentical signal
transduction pathways.
Although the physiological role of Vav1 during lymphocyte development
and activation is well established, the function of Vav2 is poorly
documented. In particular, it is not known whether Vav1 and Vav2 elicit
similar responses in lymphocytes. Here, we compared the involvement of
Vav1 and Vav2 in TCR signaling. We show that Vav2 shares with Vav1
several biological features, including tyrosine phosphorylation by
TCR-associated PTKs of the Src and Syk families, binding to
tyrosine-phosphorylated c-Cbl and SLP-76, and a positive effect on
activation of ERKs, c-fos serum response element (SRE), and
CD69 expression. However, in contrast to Vav1, Vav2 negatively
regulates TCR-induced NF-AT and IL-2 gene activation. We also
demonstrate that Vav2 functions upstream of calcineurin (Cn) and that
the intact DH and SH2 domains of Vav2 are required for activation of
c-fos SRE and inhibition of NF-AT induction. Therefore,
Vav1 and Vav2, two closely related members of the Vav family, are
functionally distinct in promoting gene activation in T cells.
Antibodies and Reagents--
The anti-CD3 monoclonal antibody
(mAb) OKT3 was purified from the corresponding hybridoma supernatant by
protein A-Sepharose chromatography. The anti-phosphotyrosine (Tyr(P))
and the anti-Myc mAbs were derived from the 4G10 and 9E10 hybridomas,
respectively. The anti-hemagglutinin mAb (12CA5) was from Roche
Molecular Biochemicals. The anti-SLP-76 mAb was provided by P. Findell
(Palo Alto, CA). The anti-human Vav2 mAb was a kind gift from J. Downward (London, UK). The anti-ERK1/2 polyclonal and anti-Cbl mAb were
from Santa Cruz Biotechnology, Inc. The anti-phospho-ERK
polyclonal antibody and anti-human Vav1 mAb were obtained from Upstate
Biotechnology, Inc. The phycoerythrin-conjugated anti-human CD69 mAb
was from PharMingen. Culture media and oligonucleotides were from Life Technologies, Inc. Chemicals were obtained from Sigma, and enzymes were
from New England Biolabs, Inc.
DNA Constructs--
Plasmid construction, cloning, and DNA
sequencing were carried out according to standard protocols. The yeast
two-hybrid constructs LexA-Syk, LexA-Fyn, LexA-lamin, GAD-Vav1, and
GAD-raf were previously described (16, 29). The cDNA encoding
Myc-tagged Vav1 (16) was cloned into the pCDNA3 vector
(Invitrogen). The cDNA encoding Vav2 (amino acids 1-868) was
amplified by polymerase chain reaction (PCR) from a cDNA library of
mouse liver and cloned into pCDNA3-Myc vector (Invitrogen) or pACT2
vector (CLONTECH) yielding Vav2-Myc and GAD-Vav2,
respectively. PCRs were performed using the thermostable Pwo DNA
polymerase (Roche Molecular Biochemicals). Myc-ERK2 has been described
elsewhere (19). The NF-AT and IL-2 luciferase reporter plasmids have
been described (29). The c-fos SRE luciferase reporter
plasmid was a kind gift from R. Janknecht (Rochester, MN) and has been
described (30). Mammalian expression vectors encoding Syk, Lck, Fyn,
and ZAP-70 have been previously described (31, 32). A bacterial
expression plasmid of a GST fusion protein containing the SH3-SH2-SH3
domains of Vav2 was generated by PCR amplification of nucleotides
1797-2604 (encoding amino acid residues 599-868) from the
pCDNA3-Vav2-Myc vector, followed by in-frame insertion into the
pGEX-3X plasmid (Amersham Pharmacia Biotech). Mutants were created with
the Quick Change site-directed mutagenesis kit (Stratagene), and
mutations were verified by DNA sequence analysis.
Reverse Transcriptase-PCR Analysis--
Sets of primers were
designed for specific PCR amplification of human Vav1 or Vav2
fragments. These primers were used to screen a panel of cDNAs
generated using poly(A)+ RNA from different human tissues
(CLONTECH). PCRs were performed with
Superscript DNA polymerase (Life Technologies, Inc.), and GAPDH
amplification was used as an internal control. Products were analyzed
on a 1.5% agarose gel, stained with ethidium bromide, and photographed
using UV light.
Cell Culture, Transfection, and Yeast Manipulation--
Cell
lines were obtained from American Type Culture Collection. Cells were
grown in RPMI 1640 medium (Life Technologies, Inc.), supplemented with
10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 1× minimum Eagle's
medium nonessential amino acids solution (Life Technologies,
Inc.), and 100 units/ml each of penicillin G and streptomycin. Simian
virus 40 T antigen-transfected human leukemic Jurkat T cells
(Jurkat-TAg) were kindly provided by G. Crabtree (Stanford, CA).
Jurkat (clone JE6.1) and Jurkat-TAg cells were transfected with the
indicated plasmids by electroporation as described previously (16).
Growth and transformation of the yeast strain L40 and the
Immunoprecipitation and Immunoblotting--
Cells were
stimulated for 5 min with 5 µg/ml of anti-CD3 mAb, washed twice, and
lysed at 1 × 108 cells/ml in ice-cold lysis buffer
(1% Nonidet P-40 in 150 mM NaCl, 50 mM HEPES,
pH 7.4, 5 mM NaF, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, 1 mM phenylmethylsulfonyl fluoride) for 15 min.
Lysates were clarified by centrifugation at 15,000 × g
for 10 min at 4 °C, and protein concentration was determined using
the bicinchoninic acid protein assay (Pierce). Cleared lysates were
incubated for 3 h at 4 °C with the indicated antibodies and
protein G-Sepharose beads (Sigma). Pellets were then washed three times
with ice-cold lysis buffer containing 0.2% Nonidet P-40 and
resuspended in SDS sample buffer. Eluted immunoprecipitates or whole
cell lysates were separated by SDS-polyacrylamide gel electrophoresis
and analyzed by immunoblotting. Reactive proteins were visualized by ECL.
GST Pulldown Assays--
GST fusion proteins were expressed in
BL21 bacterial cells and produced as described (33). Jurkat cells
(1 × 108 cells) were lysed in 1 ml of ice-cold lysis
buffer for 20 min. After centrifugation, lysates were incubated with 5 µg of the indicated fusion proteins for 3 h at 4 °C, followed
by incubation with glutathione-Sepharose 4B beads (Amersham Pharmacia
Biotech) for 1 h. Bound proteins were washed four times with 1 ml
of lysis buffer and resuspended in SDS sample buffer. Samples were
resolved by SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting.
Reporter Assays--
For luciferase assays, transfected Jurkat
cells were left unstimulated or stimulated with anti-CD3 mAb for the
indicated times, as described in the legend to each figure. Cells were
washed twice in phosphate-buffered saline, pH 7.2, and lysed in 100 µl of reporter lysis buffer (Promega). Luciferase activity was
assayed by luminometry (Lumat, EG&G Berthold) using the Promega
luciferase assay system. Normalization of transfection efficiency was
done using a co-transfected
For ERK2 phosphorylation assays, cells were lysed in lysis buffer
containing 1% Triton X-100 in place of Nonidet P-40. After centrifugation, the supernatants were incubated overnight at 4 °C
with antibodies to Myc and protein G-Sepharose beads.
Immunoprecipitates were resolved on SDS-polyacrylamide gel
electrophoresis and transferred to nitrocellulose, and the membranes
were probed with antibodies to phospho-ERK according to the
manufacturer's instructions. After stripping, the membranes were
reprobed with antibodies to Myc to confirm equal levels of ERK2 in the immunoprecipitates.
CD69 Expression--
Jurkat-TAg cells were transfected with the
indicated plasmids together with the pEGFP-N1 reporter plasmid
(CLONTECH) and cultured for 24 h at 37 °C.
Cells were incubated with medium alone or with PMA (50 ng/ml) for the
final 18 h of culture and stained with a phycoerythrin-conjugated
anti-human CD69 mAb as described (19). CD69 expression was analyzed by
flow cytometry (FACScan, Becton Dickinson) after gating on
GFP-positive cells.
Expression Pattern of Vav2 in Human Cells and Tissues from Immune
Origin--
To address the role of Vav2 in the immune system, we first
examined its expression pattern by reverse transcriptase-PCR analysis on various immune tissues. As shown in Fig.
1A, the Vav2 message was
predominantly expressed in spleen, fetal liver, tonsil, and peripheral
blood leukocytes and to a lesser extend in thymus and bone marrow. As
previously reported, Vav1 message was detected in all the immune
tissues tested. We also examined the expression of Vav2 at the protein
level in different hematopoietic cell lines. Vav2 was well expressed in
several human T (Jurkat, CEM, HPBall) or B (88.66, Raji, SKW6.4) cell
lines but poorly expressed in the monocytic cell line U937 (Fig.
1B). The distinct expression pattern of Vav1 and Vav2 might
account for distinct functions of the two proteins in different cell
types.
Interaction of Vav2 with T Cell Signaling Molecules--
Jurkat T
cells are a widely used model for studying TCR signaling. Therefore, we
examined whether Vav2 undergoes tyrosine phosphorylation following TCR
stimulation. Jurkat E6.1 T cells were stimulated with an anti-CD3 mAb,
and after immunoprecipitation with Vav2-specific antibodies, the level
of tyrosine phosphorylation was assayed by immunoblotting with
antibodies to Tyr(P). Increased Vav2 tyrosine phosphorylation was
detectable after 30 s of stimulation, with peak levels detected
2-5 min following TCR ligation (Fig. 2A, lanes 1-5).
Tyrosine phosphorylation of Vav2 was also observed in
pervanadate-treated Jurkat E6.1 T cells (Fig. 2A, lane
6). We then compared the abilities of Vav1 and Vav2 to become
tyrosine-phosphorylated upon TCR stimulation. To do this, Jurkat-TAg
cells were transfected with the Myc-tagged forms of Vav1 or Vav2 and
stimulated with an anti-CD3 mAb. The level of tyrosine phosphorylation
was assayed by immunoblotting cell lysates and anti-Myc
immunoprecipitates with an anti-Tyr(P) mAb. Similar expression levels
of the transfected proteins were determined by immunoblotting with an
anti-Myc mAb (Fig. 2B). Both Vav1 and Vav2 were found
tyrosine-phosphorylated upon TCR stimulation (Fig. 2B,
upper panel, lanes 4 and 6). Of note,
basal tyrosine phosphorylation of Vav1 and Vav2 could be detected in
resting cells after prolonged exposure of the membrane (Fig.
2B, upper panel, lanes 3 and
5, and data not shown). To identify candidate PTKs involved
in the TCR-mediated phosphorylation of Vav2, we co-transfected
Jurkat-TAg cells with Vav2 and Lck, Fyn, Syk, or Zap-70. As shown in
Fig. 2C, Vav2 was prominently phosphorylated in intact T
cells by Lck, Fyn, and Syk and to a lesser extent by Zap-70. These
results indicate that, like Vav1, Vav2 is tyrosine-phosphorylated by T
cell nonreceptor PTKs.
Next, we used the yeast two-hybrid system to further examine the
physical interaction of Vav2 with Syk and Fyn. The interactions in
co-transformed L40 yeast cells were monitored by a
Finally, we assessed the nature of proteins interacting with the SH2
domain of Vav2 in lymphocytes. Two GST-Vav2 fusion proteins were
generated (Fig. 3A) and used
in GST pull-down assays. When GST-Vav2 599-868 was incubated with
lysates of resting Jurkat cells, it associated with two major
tyrosine-phosphorylated proteins of 120 and 62 kDa, respectively (Fig.
3B, lane 2). TCR stimulation did not
significantly increase the association of these two proteins with Vav2,
but it induced an additional interaction with a 75-kDa Tyr(P)-containing protein (Fig. 3B, lane 2 versus lane
5). In contrast, none of these proteins bound the SH2-mutated
GST-Vav2 protein or the GST alone (Fig. 3B, lanes
1, 3, 4, and 6). Probing the
membranes with a panel of antibodies to different candidate proteins
allowed us to identified pp120 and pp75 as c-Cbl and SLP-76,
respectively (Fig. 3B, two lower panels,
lane 5). Together, these results suggest that Vav2 may play
an important role as a component of the signaling complex assembled
during TCR-mediated cell activation.
Vav2 Promotes Activation of ERKs and SRE and Expression of CD69 in
T Cells--
Vav1 plays an important role in the immune system as an
inducer of gene transcription (8, 9), and a recent report indicated that Vav2 might play a similar role (26). Therefore, we wished to
compare the impact of Vav1 and Vav2 overexpression on several TCR-mediated downstream activation events, beginning with ERK activation. First, we examined the activation of endogenous ERK proteins by Vav2 in Jurkat E6.1 cells. Cells were transfected with the
Myc-tagged form of Vav2 or empty vector and stimulated with an anti-CD3
mAb for different times. Activation of endogenous ERK1 and 2 was
monitored by immunoblotting with a phospho-ERK-specific antibody. As
shown in Fig. 4A, expression
of Vav2 resulted in a significant increase of the activation of ERK1/2
following TCR stimulation (compare lanes 2-4 with
lanes 6-8). Next, we compared the ability of Vav1 and Vav2
to promote ERK2 activation. Jurkat-TAg cells were co-transfected with a
Myc-tagged ERK2 reporter, plus Vav1-Myc or Vav2-Myc. The transfected
cells were left unstimulated or stimulated with an anti-CD3 antibody.
Expression of MEK1 was used as a positive control for ERK2 activation.
Activation of ERK2 was monitored by immunoblotting with a
phospho-ERK-specific antibody. As shown in Fig. 4B,
expression of Vav1 and Vav2 induced no significant activation of ERK2
in unstimulated T cells (lanes 3 and 5). However,
either Vav1 or Vav2 further increased ERK2 activation following TCR
stimulation (lanes 4 and 6).
Next, we examined whether Vav2 overexpression results in stimulation of
c-fos SRE transcriptional activity. We transfected Jurkat-TAg cells with Myc-tagged Vav1 or Vav2 along with a luciferase reporter driven by SRE-binding sequences. Similarly both Vav1 and Vav2
induced a marked increase of either the basal or TCR-stimulated activities of SRE reporter plasmid (4- and 8-fold increase over basal
activity, respectively) (Fig.
5A). As a control, PMA plus ionomycin stimulation caused maximal SRE activation, which was not
affected by Vav1 or Vav2 expression (Fig. 5A). Proper
expression of the transfected Vav proteins was confirmed by immunoblot
analysis (Fig. 5A, inset).
Recently, we reported that Vav1 plays a role in the induction of the
early activation marker CD69 (19, 20). Therefore, we examined the
possibility that Vav2 could mediate a similar function in T cells.
Jurkat-TAg cells were transfected with Myc-tagged Vav1 or Vav2 together
with a pEF-GFP reporter plasmid and stimulated or not with PMA. CD69
expression of GFP-gated cells was then determined by FACScan analysis.
Interestingly, Vav2 overexpression led to a 4-fold increase of CD69
expression in the absence of stimulation (Fig. 5B). This
effect was similar to the one induced by Vav1. Moreover, Vav2, like
Vav1, further increased PMA stimulation to induce the surface
expression of CD69 (Fig. 5B). Taken together, our results
indicate that Vav2 shares with Vav1 the ability to activate pathways
that stimulate ERKs, SRE, and up-regulation of CD69 expression in T cells.
Vav2 Inhibits TCR-induced NF-AT and IL-2 Promoter
Activation--
Vav1 plays a crucial role in the signaling pathways
leading to NF-AT activation in T cells (13, 15, 16). To further explore
the function of Vav2 in T cell signaling, we transfected Jurkat-TAg
cells with Vav1 or Vav2 along with luciferase reporters driven by the
complete IL-2 promoter or its NF-AT-binding sequences. As reported
earlier (15, 16), overexpression of Vav1 increased the basal and
TCR-stimulated activity of both NF-AT and IL-2 promoter (Fig.
6, A and B).
However, in contrast to Vav1, Vav2 expression suppressed the
TCR-induced NF-AT and IL-2 promoter activation. Vav2 reduced the
TCR-induced activation of NF-AT and IL-2 promoter by 80 and 60%,
respectively. However, Vav2 had no significant effect on the basal
activities of these two reporters (Fig. 6, A and
B). Immunoblotting with anti-Myc mAb revealed similar
expression levels of the two Vav proteins (Fig. 6B,
inset). We also examined the effect of Vav2 on NF-AT
activation following TCR stimulation in the parental Jurkat clone E6.1.
As shown in Fig. 6C, Vav2 reduced TCR-induced activation of
NF-AT by 80%, ruling out the possibility that the effect of Vav2 on
NF-AT was due to a cell type effect. As a control, PMA plus ionomycin
stimulation caused maximal NF-AT activation, which was not affected by
Vav2 overexpression, either in Jurkat-TAg (Fig. 6A) or in
Jurkat E6.1 cells (Fig. 6C, inset). Of note, Vav2
was also capable of blocking the stimulatory effect of Vav1 on NF-AT
activation (data not shown). These data provide evidence that Vav2
exerts a negative regulatory effect on TCR-induced NF-AT and IL-2
promoter activation.
Vav2 Functions Upstream of Cn to Inhibit TCR-induced NF-AT
Activation--
Cn is a calcium-activated serine/threonine phosphatase
that promotes the dephosphorylation of NF-AT and, thereby, its nuclear translocation and activation (4). Because NF-AT activation by Vav1 is
sensitive to cyclosporin A (15) and Vav1 modulates Ca2+
signaling pathways (17), Cn function has been implicated in the Vav1
signaling pathway leading to activation of the IL-2 gene. These
findings prompted us to examine whether the suppression of TCR-mediated
NF-AT activation by Vav2 can be rescued by co-expression of a
constitutively active Cn mutant (CA-Cn). As shown in Fig. 7A, expression of CA-Cn
increased the basal activity of NF-AT in Jurkat-TAg cells and further
enhanced anti-CD3 stimulation. Interestingly, the blocking effect of
Vav2 on TCR-induced NF-AT activation was not observed when CA-Cn was
co-expressed with Vav2. Of note, under these conditions, a 2-fold
increase in the basal activity of NF-AT was observed, suggesting the
existence of a functional interaction between the two proteins in T
cells. The absence of the Vav2 inhibitory effect was not due to
decreased protein expression as revealed by immunoblotting with the
relevant antibodies (Fig. 7B). These results indicate that
Vav2 functions upstream of Cn to inhibit TCR-induced NF-AT
activation.
Involvement of DH and SH2 Domains of Vav2 in Regulation of NF-AT
and SRE Activities--
The activation of distinct signaling pathways
by Vav1 requires different structural domains of the protein, including
the catalytic DH domain and the SH2 domain (8). Because these two domains are highly conserved in Vav2, we were interested in determining their potential involvement in Vav2-mediated transcriptional
activities. To inactivate the DH and the SH2 domains, we introduced
point mutations in the conserved leucine 212 and arginine 688 residues, respectively. The Myc-tagged mutated forms of Vav2 were transfected into Jurkat-TAg cells along with NF-AT- or SRE-luciferase reporters. Wild-type Vav2 reduced the TCR-induced activation of NF-AT by 60%
(Fig. 8A). In contrast,
overexpression of the SH2-mutated Vav2 (R688Q) had a minimal effect on
the TCR-induced NF-AT activation (15% reduction), whereas
overexpression of DH-mutated Vav2 (L212A) slightly increased NF-AT
activation (Fig. 8A). We also examined the effect of these
two mutations on the ability of Vav2 to increase SRE activity. As shown
before (Fig. 5A), Vav2 increased the basal and
TCR-stimulated activities of SRE. However, the ability of the L212A or
R688Q Vav2 mutants to stimulate the SRE reporter was severely impaired
(Fig. 8B). Immunoblotting with antibodies to Myc confirmed
the identical expression of the different proteins (Fig.
8C). These results indicate that activation of gene
transcription and suppression of TCR-induced NF-AT activation by Vav2
requires its intact DH and SH2 domains.
Recently, new members of the Vav family of Rho/Rac GEFs have been
identified, but little is known about their functions during T cell
activation. In this study, we investigated the role of Vav2 in
TCR-stimulated T cells. Tyrosine phosphorylation of Vav proteins is
thought to play a critical role in stimulating their catalytic
activation (7-9). Vav2 and other Vav proteins are
tyrosine-phosphorylated following stimulation of diverse membrane
receptors, including the epidermal and platelet-derived growth factor
receptors (24-26) and lymphocyte antigen receptors (23, 26).
Consistent with the latter studies, we observed that Vav2 was
tyrosine-phosphorylated following TCR stimulation and that this
phosphorylation can be mediated by co-expressed PTKs of the Src (Lck
and Fyn) and Syk (Zap-70 and Syk) families in Jurkat T cells. Moreover,
we showed that Vav2 interacts with Syk and Fyn via its SH2 domain.
Thus, similar mechanisms appear to be involved in the recruitment and activation of Vav1 and Vav2 induced by TCR engagement. Although additional studies are required to determine whether the interaction between Vav2 and members of the Src and Syk families is direct, it is
likely that TCR engagement stimulates the catalytic activity of Vav2
through direct phosphorylation by Src and/or the Syk PTKs. Therefore,
our findings connect Vav2 (in addition to Vav1) to TCR signaling pathways.
TCR engagement generates multiple signaling pathways that promote the
activation of immediate early genes (i.e. c-fos
and egr-1) and nuclear factors contributing to cytokine gene
expression (3, 4). The best characterized Vav family member, Vav1, plays a critical role in TCR signal transduction during lymphocyte development and activation (10-12). Analysis of
vav1 Our studies provide evidence that Vav1 and Vav2 exert overlapping but
nonidentical functions in T cells. Indeed, we showed that, similar to
Vav1, Vav2 cooperates with TCR stimulation to activate ERKs and
c-fos SRE and increases the expression of CD69. There was no
significant difference in the extent of c-fos SRE activation
in response to either Vav1 or Vav2. Transcriptional activation of
c-fos involves several control elements, one of which, the
SRE, is regulated by the activation of a Ras/MEK/ERK pathway (34).
Because the transcription factors that bind SRE or AP-1-binding sites
are major targets for ERK pathways, our studies suggest that Vav2 may
trigger in T cells an ERK pathway involved in the activation of
c-fos SRE and CD69 expression. Considering the broad tissue
expression of Vav2, it would be interesting to investigate whether Vav2
is also involved in the activation of ERK-dependent
pathways in cells of nonhematopoietic origins.
Our work also illustrates a major difference between Vav1 and Vav2 in
the way they regulate the transcription factor NF-AT and the IL-2 gene.
NF-AT proteins represent a large family of Ca2+/Cn-dependent nuclear factors, which are
involved in the regulation of several cytokine genes, including IL-2
(4). During the preparation of this manuscript, two studies reported
that Vav2 does not activate NF-AT in Jurkat T cells (26, 35). In
agreement with these reports, we found that Vav2 did not affect the
basal activity of NF-AT. However, our study revealed that Vav2 blocked
the activation of NF-AT induced by TCR stimulation. As a direct
consequence of this inhibition, the TCR-stimulated activity of the IL-2
promoter was also severely impaired in T cells overexpressing Vav2.
Moreover, our results imply that Vav2 might suppress NF-AT activation
by interfering with early TCR signals proximal to Cn activation. However, this role of Vav2 was observed when the protein was
transiently overexpressed in Jurkat cells. When we compared the
expression level of Vav1 and Vav2, we found that Vav1 and Vav2 proteins
could be simultaneously expressed in native leukocytes and in various immune cell lines, including Jurkat cells. Although the determination of the exact stoichiometry of the two molecules might require specific
experimentations such as Real-Time PCR analyses, our results
indicate that Vav2 was expressed in Jurkat cells at a lower level
relative to Vav1 expression. Thus, ascertaining Vav2 functions in T
cells will require further experimentations in native lymphocytes
and/or animal models. Nevertheless, our observations may partially
account for the loss of TCR-induced NF-AT and IL-2 gene activation in T
cells from vav1 A major question is how two highly homologous proteins such as Vav1 and
Vav2 can simultaneously exert antagonistic effects on
Cn/NF-AT-dependent pathways and similar effects on
ERK-dependent pathways? Cantrell and co-workers (36) have
shown that TCR-stimulated pathways leading to NF-AT and ERK2 activation
diverge early after Ras activation in T cells, at a level implicating
the GTPase Rac. Another recent study has shown that Vav1 and Vav2 have
distinct specificities toward Rac and Rho GTPases (27). Although these findings have not been confirmed in another study (28), such a
differential recruitment of Rho-regulated and Rac-regulated pathways by
individual Vav family members may provide one explanation for the
observed different biological responses in T cells. Our finding that
the GEF activity of Vav2 is required for NF-AT inhibition strongly
suggests that this is an active process and further supports the above
notion. The use of dominant-negative mutants of different small GTPases
of the Rho/Rac family should help to determine whether preferential
activation of Rho versus Rac GTPases by Vav2 underlies the
inhibition of NF-AT by Vav2.
Our results also indicate that the SH2 domain of Vav2 is required for
its function in T cells. Thus, an alternative mechanism for the
inhibition of NF-AT activity is that an SH2-dependent interaction of Vav2 with either stimulatory or inhibitory proteins modulates TCR signals to uncouple NF-AT activation from ERK activation, thereby blocking IL-2 gene activation but enhancing c-fos
SRE transcription and CD69 expression. This second mechanism is
supported by our observations that TCR stimulation increased the
association of Vav2 with proteins reminiscent of those interacting with
Vav1, including Syk, Fyn, c-Cbl, and SLP-76 (2, 8, 37). The association
of Vav2 with SLP-76 might play a critical role in Vav2-mediated
signaling pathways in T cells. Indeed, SLP-76 is a
hematopoietic-specific adapter protein that is tyrosine-phosphorylated by ZAP-70 following TCR engagement. Phosphorylated SLP-76 associates with a large number of signal transducers, including Vav1, LAT, Nck,
Grb2, and the Grb2-related protein Gads (38). SLP-76 cooperates with
Vav1 to induce NF-AT activity (39), and it is also involved in the
activation of ERK and AP-1 in T cells (40-42). These observations raise the possibility that association of Vav2 with SLP-76 may account
for the positive effect of Vav2 on a Ras-regulated pathway leading to
ERK and SRE activation. We consistently found that an SH2-inactive Vav2
mutant, which does not bind SLP-76, failed to activate SRE (and also
NF- Another important signal transducer in T cells, which we found to be
associated with the SH2 domain of Vav2 is c-Cbl. This finding might
provide a possible explanation for the negative regulation of NF-AT and
IL-2 transcription by Vav2. c-Cbl is a negative regulator of
PTK-mediated signaling pathways, and it inhibits Syk family
PTK-dependent signals in hematopoietic cells (46-50).
However, recent studies have shown that c-Cbl plays an important role
during T cell development but not during mature T cell activation in
the periphery (51, 52). Conversely, the Cbl-related protein Cbl-b
negatively regulates mature T cell activation (53, 54). In addition,
Cbl-b mutation in mouse T cells has revealed compensation of Vav1
function by other Vav proteins (18). Therefore, it would be interesting
to determine whether Vav2 also associates Cbl-b in mature T cells.
Finally, we cannot rule out the possibility that Vav2 interacts with
yet unidentified regulatory proteins that uncouple NF-AT activation
from ERK activation, thereby blocking IL-2 gene activation. In this
regard, it would be of interest to identify the phosphoprotein at 62 kDa that we found interacting with Vav2.
Taken together, our results show that, whereas Vav1 displays a positive
action on TCR-induced signaling pathways, Vav2 uncouples the activation
of a ERK/SRE/CD69 pathway from a NF-AT/IL-2 gene activation pathway. In
support of this notion, a recent study has shown that different Vav1
functions can be uncoupled in the Vav1×Cbl-b
double knockout mice, reflecting potential compensation by other Vav
proteins (18). Although the physiological relevance of the
Vav2-regulated signaling pathways are presently unknown, our results
raise the possibility that Vav2 negatively regulates major aspects of T
cell activation associated with IL-2 production. Alternatively, the
balance between expression levels of different Vav proteins could
determine T cell fates by fine tuning TCR-mediated gene activation.
Further studies are required to determine the relative expression and
role of each Vav isoform during T cell development and activation.
Finally, the identification of proteins that specifically interact with
different Vav proteins should help to understand the functions of the
Vav family in immune cell signaling pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B, up-regulation
of CD69, and IL-2 or IL-4 production (13-21).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase filter assay were performed as previously described
(16).
-galactosidase expression vector.
Luciferase activity was determined in triplicate and expressed as fold
increase relative to the basal activity seen in unstimulated
mock-transfected cells.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (48K):
[in a new window]
Fig. 1.
A, reverse transcriptase-PCR analysis of
Vav1 and Vav2 mRNA expression. cDNAs prepared from various
human tissues CLONTECH) were used as
templates for PCR. 35 cycles of PCR with primers specific for Vav1 or
Vav2 were used. The predicted sizes for Vav1 and Vav2 fragments are 568 and 840 base pairs (bp), respectively. The products were subjected to
agarose gel electrophoresis in presence of ethidium bromide and were
subsequently photographed using ultraviolet light. The expression of
the housekeeping GAPDH gene is shown. B, expression of Vav2
protein in lymphoid cell lines. 75 µg of lysates prepared from the
indicated cell lines was subjected to SDS-polyacrylamide gel
electrophoresis and was immunoblotted for Vav2 protein (top
panel). After being stripped, the membrane was then blotted for
Vav1 (middle panel), and actin (bottom panel) to
control for loading.
View larger version (27K):
[in a new window]
Fig. 2.
A, tyrosine phosphorylation of
endogenous Vav2 in Jurkat cells. Jurkat E6.1 cells were stimulated with
5 µg/ml of anti-CD3 mAb or 100 µM of pervanadate
(PV) for the indicated times. Lysates were subjected to
immunoprecipitation (IP) using polyclonal Vav2-specific
antibodies, and tyrosine phosphorylation (top panel) and
total Vav2 protein levels (bottom panel) were determined by
immunoblotting. B, TCR-induced tyrosine phosphorylation of
Vav1 and Vav2 in Jurkat-TAg cells. Cells were transfected with the
empty pcDNA3 vector (lanes 1 and 2) or with
the same vector expressing Myc-tagged Vav1 (lanes 3 and
4) or Myc-tagged Vav2 (lanes 5 and 6).
Cells were left unstimulated ( ) or stimulated (+) for 5 min with an
anti-CD3 mAb. Lysates were subjected to immunoprecipitation
(IP) using an anti-Myc mAb. The immunoprecipitates were
analyzed by immunoblotting with an anti-Tyr(P) mAb (top
panel). The levels of Vav proteins in the immunoprecipitates were
measured by reprobing the membrane with an anti-Myc mAb (bottom
panel). The mobilities of Vav proteins are indicated by
arrows. C, expression of members of the Src and
Syk tyrosine kinases families induced tyrosine phosphorylation of Vav2.
Jurkat-TAg cells were transfected with empty vectors (lane
1) or a plasmid expressing Myc-tagged Vav2 alone (lanes
2) or in combination with plasmids encoding Lck, Fyn, Zap70, or
Syk (lanes 3-6). Cells were lysed, and proteins were
immunoprecipitated using an anti-Myc mAb. Immunoprecipitated proteins
were immunoblotted with antibodies to Tyr(P) (top panel) or
to Myc (bottom panel). The mobility of Vav2 protein is
indicated by arrows. D, interaction of Vav2 with
Syk and Fyn in the yeast two-hybrid system. Syk or Fyn cDNAs were
fused to the DNA-binding domain of LexA, whereas the indicated Vav2
constructs were fused to the Gal4 activation domain (GAD).
Yeast (L40) was co-transformed with the indicated plasmids
combinations, and interactions were assayed using a
-galactosidase
filter test. The panel summarizes three independent experiments.
, no
interaction; +++, strong interaction. Co-expression of GAD-Vav1 with
LexA-Syk or LexA-Fyn is shown as a positive control. Co-expression of
GAD constructs with LexA-lamin is shown as a negative control. Vav2
599-868 consists of the two SH3 domains and the SH2 domain of Vav2.
R688Q variant has a mutation in a conserved arginine residue within the
SH2 domain.
-galactosidase filter assay (16). GAD-Vav2 and GAD-Vav1 similarly interacted with
LexA-Syk and LexA-Fyn (Fig. 2D). As expected, a construct containing the C-terminal SH3-SH2-SH3 domain of Vav2 (Vav2 599-868) still interacted with Syk. The SH2 domain of Vav2 was required because
an SH2-inactivating point mutation (R688Q) abolished the interaction
between Vav2 and Syk (Fig. 2D). As a negative control, no
interactions between GAD-Vav2 constructs and a LexA DNA-binding domain
fused to lamin were detected.
View larger version (42K):
[in a new window]
Fig. 3.
Association of the SH2 domain of Vav2 with
Cbl and SLP-76. A, schematic representation of the Vav2
constructs used in this study. Vav2 599-868 consists of the two SH3
and the SH2 domain of Vav2. R688Q variant has a mutation in a conserved
arginine residue within the SH2 domain. B, lysates from
unstimulated ( ) or TCR-stimulated (+) Jurkat-TAg cells were mixed
with 5 µg of GST fusion proteins that were immobilized on glutathione
beads. Bound proteins were detected by immunoblotting with antibodies
to Tyr(P) (top panel), c-Cbl (middle panel), or
SLP-76 (bottom panel). For reference, whole cell lysate
aliquots were immunoblotted with the same antibodies (lanes
7 and 8). The positions of major phosphoproteins
(pp) are indicated by arrows. The data shown are
representative of three independent experiments.
View larger version (43K):
[in a new window]
Fig. 4.
A, effect of Vav2 on endogenous ERK1 and
2 activation. Jurkat E6.1 cells were transfected with empty vector
(lanes 1-4) or Myc-tagged Vav2 (lanes 5-8).
Cells were stimulated for the indicated times with an anti-CD3 mAb.
Lysates were immunoblotted using a phospho-ERK-specific antibody
(top panel). After being stripped, the membrane was then
blotted for ERK1/2 (middle panel) or Myc (bottom
panel). B, effect of Vav1 and Vav2 on ERK2 activation.
Jurkat-TAg cells were transfected with Myc-tagged ERK2 and either empty
vector (lanes 1 and 2), Myc-tagged Vav1
(lanes 3 and 4), Myc-tagged Vav2 (lanes
5 and 6), or MEK1 (lane 7). Cells were left
unstimulated ( ) or stimulated (+) for 5 min with an anti-CD3 mAb.
Lysates were subjected to immunoprecipitation (IP) using
antibody to Myc. ERK2 activation and expression were assayed as
described above (top and middle panels). Lysates
were blotted with antibody to Myc to analyze Myc-tagged Vav1 and Vav2
expression (bottom panel). The mobilities of Vav proteins
and ERK2 are indicated by arrows. The data shown are
representative of three independent experiments.
View larger version (29K):
[in a new window]
Fig. 5.
Effect of Vav2 on c-fos
SRE-dependent transcriptional activity and CD69
up-regulation. A, Jurkat-TAg cells were transfected
with the empty pcDNA3 vector or with the same vector expressing
Myc-tagged Vav1 or Myc-tagged Vav2 in the presence of a SRE luciferase
reporter plasmid and cultured for 24 h. Cells were left
unstimulated (open bars) or stimulated with an anti-CD3 mAb
(gray bars) or with a combination of PMA (50 ng/ml) plus
ionomycin (1 µg/ml) (dark bars) for the final 6 h of
culture and lysed for the luciferase assay. The bars
represent the means ± S.D. of triplicate samples. The data shown
are representative of three independent experiments. The
inset shows expression levels of Myc-tagged Vav proteins in
transfected cells. B, Jurkat-TAg cells were transfected with
the indicated plasmids together with a GFP expression plasmid and
cultured for 24 h. Cells were left unstimulated (open
bars) or stimulated (closed bars) with PMA (50 ng/ml)
for the final 18 h of culture. CD69 expression was determined by
FACScan analysis of GFP-gated cells. The inset shows
expression levels of Myc-tagged Vav proteins in transfected cells.
MFI, mean of fluorescence intensity.
View larger version (31K):
[in a new window]
Fig. 6.
Effect of Vav2 on TCR-induced NF-AT and IL-2
promoter activation. Jurkat-TAg cells were transfected with the
empty pcDNA3 vector or with the same vector expressing Myc-tagged
Vav1 or Myc-tagged Vav2 in the presence of NF-AT reporter construct
(A) or IL-2 promoter reporter construct (B) and
cultured for 24 h. Cells were left unstimulated (open
bars) or stimulated with an anti-CD3 mAb (gray bars) or
with a combination of PMA (50 ng/ml) plus ionomycin (1 µg/ml)
(dark bars) for the final 6 h (A) or 18 h (B) of culture and lysed for the luciferase assay. The
bars represent the means ± S.D. of triplicate samples.
The data shown are representative of five independent experiments. The
inset in B shows representative expression levels
of Myc-tagged Vav proteins. C, Jurkat E6.1 cells were
transfected with Myc-tagged Vav2 as described in A. Cells
were stimulated with an anti-CD3 mAb or with a combination of PMA (50 ng/ml) plus ionomycin (1 µg/ml) (inset) for the final
6 h of culture. Luciferase assays were performed as described
above.
View larger version (24K):
[in a new window]
Fig. 7.
Vav2 functions upstream of Cn to inhibit
TCR-induced NF-AT activation. Jurkat-TAg cells were transfected
with empty vectors, Myc-tagged Vav2 alone, hemagglutinin-tagged CA-Cn
alone, or together in the presence of NF-AT reporter construct. Cells
were left unstimulated (open bars) or stimulated
(closed bars) with an anti-CD3 mAb for the final 6 h of
culture and lysed for the luciferase assay (A). The
bars represent the means ± S.D. of triplicate samples.
The data shown are representative of three independent experiments.
B, samples of the same lysates were analyzed for Vav2 and
CA-Cn proteins expression by immunoblotting with antibody to Myc
(top panel) or to hemagglutinin (bottom panel).
The mobilities of Vav2 and CA-Cn are indicated by
arrows.
View larger version (24K):
[in a new window]
Fig. 8.
Involvement of DH and SH2 domains of Vav2 in
regulation of NF-AT and SRE activities. Jurkat-TAg cells were
transfected with the empty pcDNAvector or with the same vector
expressing Myc-tagged wild-type form of Vav2 (wt) or
Myc-tagged mutant forms of Vav2 (L212A or R688Q) in the presence of a
NF-AT reporter construct (A) or a SRE luciferase reporter
construct (B) and cultured for 24 h. Cells were left
unstimulated (open bars) or stimulated (closed
bars) with an anti-CD3 mAb for the final 6 h and lysed for
the luciferase assay. The bars represent the means ± S.D. of triplicate samples. The data shown are representative of three
independent experiments. C, samples of the same lysates were
analyzed for expression of the different transfected Vav2 constructs by
immunoblotting with an anti-Myc mAb. L212A and R688Q variants have a
mutation in a conserved residue within the DH domain or the SH2 domain,
respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
mice indicated that Vav1 activity is
required for TCR capping and cytoskeleton reorganization, maximal
Ca2+ signaling, ERK activation, and IL-2 gene induction
(13, 14, 17). However, it is unclear whether other Vav family members can compensate for the absence of Vav1 and stimulate at least a subset
of the Vav1-dependent pathways (13, 14, 17). A very recent
study suggested that other Vav proteins may substitute for Vav1
function in pathways involved in TCR capping, actin cytoskeleton reorganization, and lipid raft clustering (18). However, the identity
of the relevant Vav protein was not determined in this study.
/
mice (13) and the inability
of other Vav proteins to rescue NF-AT/IL-2 gene activation in the
Vav1×Cbl-b double knockout mice (18). Taken
together, our results further support the notion that Vav1 and Vav2 are
functionally distinct in promoting gene activation.
B; data not shown), suggesting that binding to SLP-76 may be
critical for Vav2 function in T cells. Moreover, the expression of
CD69, a known target of the Ras pathway in T cells (43-45), was also
up-regulated by Vav2. Because a Ras-dependent pathway is
required for CD69 up-regulation by Vav1 in T cells (19), Vav2 may also
promote an increase of CD69 expression by recruiting the Ras pathway
through a direct association with SLP-76.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank G. Crabtree, M. Karin, J. Downward, R. Janknecht, A. Vojtek, and P. Findell for reagents. The technical assistance of Valérie Brun is gratefully acknowledged.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the INSERM, by Association pour la Recherche sur le Cancer Grants 5395 (to S. T.-D.) and 9989 (to M. D.), by the Fondation pour la Recherche Médicale, and by National Institutes of Health Grant GM50819 (to A. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipients of doctoral fellowships from the Ministère de l'Enseignement Supérieur et de la Recherche.
** To whom correspondence should be addressed. E-mail: deckert@unice.fr.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M010588200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TCR, T cell receptor; PTK, protein-tyrosine kinase; GEF, guanine nucleotide exchange factor; DH, Dbl homology; SH2, Src homology 2; SH3, Src homology 3; GST, glutathione S-transferase; ERK, extracellular signal-regulated kinase; Cn, calcineurin; CA-Cn, constitutively active Cn; SRE, serum response element; NF-AT, nuclear factor of activated T cells; IL-2, interleukin-2; PMA, phorbol 12-myristate 13-acetate; mAb, monoclonal antibody; PCR, polymerase chain reaction.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Weiss, A., and Littman, D. R. (1994) Cell 76, 263-274[Medline] [Order article via Infotrieve] |
2. | van Leeuwen, J. E., and Samelson, L. E. (1999) Curr. Opin. Immunol. 11, 242-248[CrossRef][Medline] [Order article via Infotrieve] |
3. | Treisman, R. (1994) Curr. Opin. Genet. Dev. 4, 96-101[Medline] [Order article via Infotrieve] |
4. | Crabtree, G. R. (1999) Cell 96, 611-614[Medline] [Order article via Infotrieve] |
5. | Penninger, J. M., and Crabtree, G. R. (1999) Cell 96, 9-12[Medline] [Order article via Infotrieve] |
6. | Nobes, C. D., and Hall, A. (1995) Cell 81, 53-62[Medline] [Order article via Infotrieve] |
7. | Altman, A., and Deckert, M. (1999) Adv. Immunol. 72, 1-101[Medline] [Order article via Infotrieve] |
8. | Collins, T., Deckert, M., and Altman, A. (1997) Immunol. Today 18, 221-225[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Bustelo, X. R.
(2000)
Mol. Cell. Biol.
20,
1461-1477 |
10. | Fischer, K. D., Zmuldzinas, A., Gardner, S., Barbacid, M., Bernstein, A., and Guidos, C. (1995) Nature 374, 474-477[CrossRef][Medline] [Order article via Infotrieve] |
11. | Tarakhovsky, A., Turner, M., Schaal, S., Mee, P. J., Duddy, L. P., Rajewsky, K., and Tybulewicz, L. J. (1995) Nature 374, 467-470[CrossRef][Medline] [Order article via Infotrieve] |
12. | Zhang, R., Alt, F. W., Davidson, L., Orkin, S. H., and Swat, W. (1995) Nature 374, 470-473[CrossRef][Medline] [Order article via Infotrieve] |
13. | Fischer, K. D., Kong, Y. Y., Nishina, H., Tedford, K., Marengere, L. E., Kozieradzki, I., Sasaki, T., Starr, M., Chan, G., Gardener, S., Nghiem, M. P., Bouchard, D., Barbacid, M., Bernstein, A., and Penninger, J. M. (1998) Curr. Biol. 8, 554-562[Medline] [Order article via Infotrieve] |
14. | Holsinger, L. J., Graef, I. A., Swat, W., Chi, T., Bautista, D. M., Davidson, L., Lewis, R. S., Alt, F. W., and Crabtree, G. R. (1998) Curr. Biol. 8, 563-572[Medline] [Order article via Infotrieve] |
15. | Wu, J., Katzav, S., and Weiss, A. (1995) Mol. Cell. Biol. 15, 4337-4346[Abstract] |
16. | Deckert, M., Tartare-Deckert, S., Couture, C., Mustelin, T., and Altman, A. (1996) Immunity 5, 591-604[Medline] [Order article via Infotrieve] |
17. |
Costello, P. S.,
Walters, A. E.,
Mee, P. J.,
Turner, M.,
Reynolds, L. F.,
Prisco, A.,
Sarner, N.,
Zamoyska, R.,
and Tybulewicz, V. L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
3035-3040 |
18. | Krawczyk, C., Bachmaier, K., Sasaki, T., Jones, R. G., Snapper, S. B., Bouchard, D., Kozieradzki, I., Ohashi, P. S., Alt, F. W., and Penninger, J. M. (2000) Immunity 13, 463-470[Medline] [Order article via Infotrieve] |
19. | Villalba, M., Hernandez, J., Deckert, M., Tanaka, Y., and Altman, A. (2000) Eur. J. Immunol. 30, 1587-1596[CrossRef][Medline] [Order article via Infotrieve] |
20. | Villalba, M., Coudronniere, N., Deckert, M., Teixeiro, E., Mas, P., and Altman, A. (2000) Immunity 12, 151-160[Medline] [Order article via Infotrieve] |
21. |
Hehner, S. P.,
Li-Weber, M.,
Giaisi, M.,
Droge, W.,
Krammer, P. H.,
and Schmitz, M. L.
(2000)
J. Immunol.
164,
3829-3836 |
22. | Schuebel, K. E., Bustelo, X. R., Nielsen, D. A., Song, B. J., Barbacid, M., Goldman, D., and Lee, I. J. (1996) Oncogene 13, 363-371[Medline] [Order article via Infotrieve] |
23. |
Movilla, N.,
and Bustelo, X. R.
(1999)
Mol. Cell. Biol.
19,
7870-7885 |
24. |
Pandey, A.,
Podtelejnikov, A. V.,
Blagoev, B.,
Bustelo, X. R.,
Mann, M.,
and Lodish, H. F.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
179-184 |
25. |
Liu, B. P.,
and Burridge, K.
(2000)
Mol. Cell. Biol.
20,
7160-7169 |
26. |
Moores, S. L.,
Selfors, L. M.,
Fredericks, J.,
Breit, T.,
Fujikawa, K.,
Alt, F. W.,
Brugge, J. S.,
and Swat, W.
(2000)
Mol. Cell. Biol.
20,
6364-6373 |
27. |
Schuebel, K. E.,
Movilla, N.,
Rosa, J. L.,
and Bustelo, X. R.
(1998)
EMBO J.
17,
6608-6621 |
28. |
Abe, K.,
Rossman, K. L.,
Liu, B.,
Ritola, K. D.,
Chiang, D.,
Campbell, S. L.,
Burridge, K.,
and Der, C. J.
(2000)
J. Biol. Chem.
275,
10141-10149 |
29. | Deckert, M., Tartare-Deckert, S., Hernandez, J., Rottapel, R., and Altman, A. (1998) Immunity 9, 595-605[Medline] [Order article via Infotrieve] |
30. | Janknecht, R., Ernst, W. H., Pingoud, V., and Nordheim, A. (1993) EMBO J. 12, 5097-5104[Abstract] |
31. | Williams, S., Couture, C., Gilman, J., Jascur, T., Deckert, M., Altman, A., and Mustelin, T. (1997) Eur. J. Biochem. 245, 84-90[Abstract] |
32. |
Deckert, M.,
Elly, C.,
Altman, A.,
and Liu, Y.-C.
(1998)
J. Biol. Chem.
273,
8867-8873 |
33. |
Sawka-Verhelle, D.,
Baron, V.,
Mothe, I.,
Filloux, C.,
White, M. F.,
and Van Obberghen, E.
(1997)
J. Biol. Chem.
272,
16414-16420 |
34. | Treisman, R. (1995) EMBO J. 14, 4905-4913[Medline] [Order article via Infotrieve] |
35. | Billadeau, D. D, Mackie, S. M., Schoon, R. A., and Leibson, P. J. J. Exp. Med. 192, 381-391 |
36. | Genot, E., Cleverley, S., Henning, S., and Cantrell, D. (1996) EMBO J. 15, 3923-3933[Abstract] |
37. | Pivniouk, V. I., and Geha, R. S. (2000) Curr. Opin. Immunol. 12, 173-178[CrossRef][Medline] [Order article via Infotrieve] |
38. | Myung, P. S., Boerthe, N. J., and Koretsky, G. A. (2000) Curr. Opin. Immunol. 12, 256-266[CrossRef][Medline] [Order article via Infotrieve] |
39. | Wu, J., Motto, D. G., Koretzky, G. A., and Weiss, A. (1996) Immunity 4, 593-602[Medline] [Order article via Infotrieve] |
40. | Musci, M. A., Motto, D. G., Ross, S. E., Fang, N., and Koretzky, G. A. (1997) J. Immunol. 159, 1639-1647[Abstract] |
41. |
Fang, N.,
and Koretzky, G. A.
(1999)
J. Biol. Chem.
274,
16206-16212 |
42. |
Kaminuna, O.,
Deckert, M.,
Elly, C.,
Liu, Y.-C.,
and Altman, A.
(2001)
Mol. Cell. Biol.
21,
3126-3136 |
43. | D'Ambrosio, D., Cantrell, D. A., Frati, L., Santoni, A., and Testi, R. (1994) Eur. J. Immunol. 24, 616-620[Medline] [Order article via Infotrieve] |
44. | Taylor Fishwick, D. A., and Siegel, J. N. (1995) Eur. J. Immunol. 25, 3215-3221[Medline] [Order article via Infotrieve] |
45. | Castellanos, M. C., Munoz, C., Montoya, M. C., Lara-Pezzi, E., Lopez-Cabrera, M., and de Landazuri, M. O. (1997) J. Immunol. 159, 5463-5473[Abstract] |
46. | Rudd, C. E., and Schneider, H. (2000) Curr. Biol. 10, 344-347 |
47. |
Ota, Y.,
and Samelson, L. E.
(1997)
Science
276,
418-420 |
48. |
Miyake, S.,
Lupher, M. L. J.,
Druker, B.,
and Band, H.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
7927-7932 |
49. |
Lupher, M. L. J.,
Rao, N.,
Lill, N. L.,
Andoniou, C. E.,
Miyake, S.,
Clark, E. A.,
Druker, B.,
and Band, H.
(1998)
J. Biol. Chem.
273,
35273-35281 |
50. | Liu, Y.-C., and Altman, A. (1998) Cell. Signal. 10, 377-385[CrossRef][Medline] [Order article via Infotrieve] |
51. | Marengere, L. E., Mirtsos, C., Kozieradzki, I., Veillette, A., Mak, T. W., and Penninger, J. M. (1997) J. Immunol. 159, 70-76[Abstract] |
52. |
Naramura, M.,
Kole, H. K.,
Hu, R.-J.,
and Gu, H.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
15547-15552 |
53. | Bachmaier, K., Krawczyk, C., Kozieradzki, I., Kong, Y. Y., Sasaki, T., Oliveira-dos-Santos, A., Mariathasan, S., Bouchard, D., Wakeham, A., Itie, A., Lee, J., Ohashi, P. S., Sarosi, I., Nishina, H., Lipkowitz, S., and Penninger, J. M. (2000) Nature 403, 211-216[CrossRef][Medline] [Order article via Infotrieve] |
54. | Chiang, Y. J., Kole, H. K., Brown, K., Naramura, M., Fukuhara, S., Hu, R. J., Jang, I. K., Gutkind, J. S., Shevach, E., and Gu, H. (2000) Nature 403, 216-220[CrossRef][Medline] [Order article via Infotrieve] |