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INTRODUCTION |
The Rho GTPase proteins participate in cellular processes
such as cell cycle, movement and migration, metabolism, survival, proliferation, and differentiation (1-4). Rho GTPase proteins cycle
between the GDP-bound inactive and GTP-bound active forms. Extracellular signals can affect Rho GTPase activity through at least
three types of regulatory molecules: the GTPase-activating proteins
that stimulate conversion from the GTP-bound form to the GDP-bound
form; the GDP/GTP exchange factors
(GEFs),1 which facilitate the
shift from the GDP-bound form to the GTP-bound form; and the GDP
dissociation inhibitors (GDIs), which block GDP dissociation from Rho
GTPases, thus maintaining the inactive state (4). Despite accumulating
experimental evidence, many details of the regulation of GDP/GTP
exchange remain to be elucidated.
One of the well studied GEFs for Rho GTPases is Vav1, a hematopoietic
cell-specific signal transducer protein (5, 6). Vav1 contains several
characteristic structural motifs that enable its function as a signal
transducer protein. In fact, Vav1 and the other ubiquitously expressed
members of the Vav family of proteins (Vav2 and Vav3) are the only
known Rho GEFs that have SH2 domains, suggesting that their GEF
activity is regulated by tyrosine phosphorylation (7-10).
One of the best studied roles of Vav1 is as a signal transducer in
activated T cells. TCR stimulation with antigen or with cross-linking
antibodies initiates a complex signaling cascade. The earliest event in
this cascade is the activation of multiple cytoplasmic tyrosine
kinases, including the Src family tyrosine kinases Lck and Fyn. This
leads to the phosphorylation of the immunoreceptor tyrosine activation
motifs on the TCR (11). The phosphorylated tyrosines in these
immunoreceptor tyrosine activation motifs associate with the SH2
domains of ZAP-70, another cytoplasmic tyrosine kinase (12). Src
tyrosine kinases also phosphorylate and activate ZAP-70, leading to the
tyrosine phosphorylation of downstream signaling proteins, including
Vav1 (5, 6). TCR-induced tyrosine phosphorylation of Vav1 leads to
activation of its GEF function toward Rac, its preferred target GTPase
(9, 10). This signaling cascade results in the activation of the
nuclear factor of activated T cells (NFAT), which plays a critical role in the regulation of many genes including interleukin-2 (13). The
cardinal role of Vav1 in NFAT induction is inferred from numerous in vitro and in vivo experiments. Overexpression
of Vav1 was shown to induce NFAT stimulation in T cells (13). This
effect was enhanced upon stimulation of TCR (13). Furthermore,
Vav1-deficient cells fail to mobilize calcium and reorganize the
cytoskeleton, events that are related to Vav1 GEF activity and are
important for NFAT stimulation (14-18). The precise mechanism by which
Vav1 induces NFAT is not yet entirely resolved. It is clear that
Vav1's function as a GEF toward Rac is important for this process.
However, it has been suggested that Rac-independent Vav1 activities are also involved (5). In addition to its role in T cell activation, Vav1
is also involved in numerous other immune functions such as T-cell
development, differentiation, and cell cycle control (19).
Vav1 was first isolated as an oncogene (20). However, wild-type Vav1
transforms NIH3T3 fibroblasts only when it is overexpressed. It is
significant to note that converting wild-type Vav1 to an oncogene
involves mutations/deletions in its amino terminus (21, 22). Thus,
removal of 66 residues from the amino terminus, mimicking the mode of
activation of the originally isolated oncogene, is sufficient to induce
transformation by Vav1 in murine fibroblasts. A more potent
transforming form of Vav1 is obtained when an even larger (186 residues) region is removed (5). The amino terminus of Vav1 is not only
necessary for regulating its transforming activity but was also found
to be important for Vav1-mediated NFAT transcription. Overexpression of
wild-type Vav1 in Jurkat T cells leads to activation of NFAT; however,
the truncated oncogenic proteins, Vav1-66 and Vav1-186, do not cause
any changes in NFAT transcription, even when the TCR is stimulated (5,
13). The amino terminus region resembles a calponin homology region yet is unlikely to directly associate with F-actin, since two such regions
in tandem are needed for association with actin (23). Since this region
is devoid of any catalytic activity and is rich in 
-helical content,
it seemed plausible that it might participate in protein-protein
interactions. Indeed, we have recently demonstrated that the amino
terminus region of Vav1 interacts in vitro and in
vivo with another potential regulator of Rho GTPases, the
hematopoietic-specific GDI protein, Ly-GDI (24).
GDIs regulate Rho GTPase activity by inhibiting GDP dissociation,
promoting the inactive form (4, 25). The ubiquitously expressed Rho-GDI
also appears to function as a chaperone for the Rho GTPases, shuttling
them between the cytosol and the membrane. In addition, Rho-GDI blocks
both intrinsic and GAP-stimulated GTP hydrolysis (4, 25). Whether all
of these functions are also carried out by Ly-GDI in T cells is still
an open question. Stimulation of T lymphocytes and myelomonocytic cells
with phorbol esters leads to phosphorylation of Ly-GDI on
serine/threonine residues, raising the possibility that Ly-GDI is
involved in signaling pathways in these cells (26, 27). In addition,
Ly-GDI is constitutively phosphorylated on tyrosine residues in
neutrophils (28). It is not known if the function of Ly-GDI in these
cells is also regulated by extracellular signals.
The association between Vav1 and Ly-GDI is particularly intriguing
because, theoretically, these two proteins should have opposite effects
on the activity of small Rho GTPases. Since the function of Ly-GDI in T
cells is largely unknown, we examined its activity, localization,
response to TCR stimulation, and potential effect on the function of
Vav1 in T cells. Here, we show for the first time that the distribution
of Ly-GDI is altered in stimulated T cells and accumulates with Vav1 in
the membrane extensions in the periphery of the "immunological
synapse." Ly-GDI exhibits several characteristics of a protein
involved in signaling. First, Ly-GDI is phosphorylated on tyrosine
residues following TCR stimulation. Second, it associates with the SH2
region of an adapter protein, Shc. Third, Ly-GDI interacts with Vav1
when signaling is enabled in T cells. Ly-GDI can block calcium
mobilization in Jurkat cells; however, surprisingly, rather than
counteracting the effects of Vav1, Ly-GDI further enhances the
induction of NFAT when the TCR is stimulated in T cells overexpressing
Vav1. Thus, in T cells, these two regulators of Rho GTPases appear to
function cooperatively as signal transducers in the TCR pathway and may
be involved in the cytoskeletal reorganization required for formation
of the "immunological synapse."
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EXPERIMENTAL PROCEDURES |
Antibodies and Immunofluorescence Reagents--
Antibodies were
obtained from the following sources: anti-Myc mAbs (clone 9E10; Upstate
Biotechnology, Inc., Lake Placid, NY); anti-CD28 mAbs (R&D
Systems). Anti-Vav polyclonal Abs were raised in rabbits against a
specific peptide of vav, residues 528-541 (21); anti-Vav
mAbs, anti-Shc Abs, and anti-phosphotyrosine mAb 4G10 (IgG1) were from
Upstate Biotechnology; anti-Vav1 mAb (Vav30, IgG1) was a gift of Dr. J. Griffin (Dana-Farber Cancer Institute, Boston, MA); anti-CD3 (OKT3,
IgG2a) mouse mAb was from the American Tissue Culture Collection
(Manassas, VA). Anti-CD3 (UCHT1, IgG1) was a gift of Dr. P. Beverley (Imperial Cancer Research Fund, London). Anti-Ly-GDI and
anti-Rho-GDI rabbit polyclonal Abs were from Upstate Biotechnology.
Anti-phospho-PLC
(Tyr783), and anti-PLC Abs were
purchased from Cell Signaling Technology (Beverly, MA).
Alexa488-coupled goat anti-mouse IgG Abs, Alexa633-coupled goat
anti-rabbit IgG Abs, Alexa546-coupled phalloidin, and Alexa488-coupled goat anti-fluorescein Abs were from Molecular Probes, Inc. (Eugene, OR). Rhodamine-coupled goat anti-rabbit IgG Ab was from Immunotech (Marseille, France). Fluorescein-coupled goat anti-mouse IgG1, Texas
Red-coupled goat anti-mouse Ig2a, and IgG2b were from Southern Biotechnology (Birmingham, AL).
Cell Lines--
The human leukemia T cell line Jurkat, clone
J77cl20, and the antigen-presenting cell (APC) Raji have been described
(29). Jurkat and Jurkat Tag were a gift of Dr. A. Weiss (University of
California, San Francisco). Cells were grown in RPMI medium containing
10% heat-inactivated fetal calf serum.
Expression Vectors--
pSecA containing Ly-GDI or Rho-GDI,
pEF-VavF (pEF115) that contains the wild-type Vav1, and pEF-oncoVav
(pEF67) that contains the oncogenic Vav1 mutant that lacks 66 residues
from its amino terminus and NFAT luciferase reporter construct
(NFAT-Luc) were gifts from Dr. Arthur Weiss (13). The glutathione
S-transferase bacterial expression vector encoding the SH2
region of Shc was a gift from Dr. N. Isakov (Ben-Gurion
University, Beer-Sheva, Israel).
Immunoprecipitation and Immunoblotting--
Cell lysis,
immunoprecipitation, and immunoblotting were performed as previously
described (24).
Immobilization of Bacterial Fusion Proteins on
Glutathione-Sepharose Beads--
Fusion proteins were purified from
transformed Escherichia coli bacteria and bound to
glutathione-Sepharose beads (Amersham Biosciences) as previously
described (24).
Activation of Jurkat T Cells--
Jurkat T cells at 2 × 107 cells/200 µl were activated with anti-CD3 mAb OKT3
(1:100; American Type Tissue Culture Collection) for 2 min at
37 °C.
Immunofluorescence Microscopy--
The procedure we followed was
similar to the one published by Roumier et al. (30).
Briefly, T cell activation with superantigen was performed by pulsing
APCs (Raji) for 15 min at 37 °C with 5 µg of
Staphylococcus enterotoxin E superantigen. T
cells were then incubated with the APCs at various periods as indicated
under "Results." Cells were then placed on
poly-L-lysine-coated coverslips, allowed to sediment for 5 min, and then centrifuged 1 min at 200 × g and fixed
for 30 min at room temperature in 4% paraformaldehyde. Intracellular
proteins were stained in 0.05% saponin, whereas immunofluorescence
staining of TCR was performed without permeabilization. Immunofluorescence and confocal microscopy analysis were performed as
previously described (30).
NFAT Activation--
Jurkat cells (Jurkat Tag) were transfected
as previously described (13). For the electroporations the following
amounts of plasmids were used: pEF-VavF, 8 µg; pEF-oncoVav, 8 µg;
Ly-GDI, 16 µg; Rho-GDI, 16 µg; NFAT-luc, 5 µg; and
-galactosidase, 1 µg. Cells were lysed after the indicated time
and treatment, and luciferase activity, in duplicate samples, was
measured and normalized to the -galactosidase values.
Subcellular Fractionation--
Cytosolic and particulate
(membrane) fractions were obtained as previously described (31).
Briefly, cells were resuspended in a buffer that contains 20 mM Tris/HCl, pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 10 mM -mercaptoethanol, 10 µg/ml
each of leupeptin and aprotinin. The cells were repeatedly aspirated
through a 1-ml syringe with a 26-gauge needle. Cell lysates were
centrifuged at 400 × g for 5 min to remove nuclear
pellets and then recentrifuged at 100,000 × g. Triton
X-100 was added to generate a final concentration of 1% in the
supernatants (cytosolic fractions) and the pellets (particulate,
membrane fraction) that were resuspended in the above mentioned buffer.
Calcium Mobilization--
Jurkat T cells (2 × 106/ml) treated as indicated under "Results" were
resuspended in RPMI and 2% fetal calf serum and dually loaded with
Fluo-3/AM (4 µg/ml; Molecular Probes) and Fura Red-AM (10 µg/ml;
Molecular Probes) for 45 min at 37 °C. Cells were then rinsed and
incubated for 20 min at room temperature. Each sample was left
untreated for 30 s, and then cells were stimulated by the addition
of anti-human CD3 and anti-mouse IgG. Calcium mobilization was
determined by the intensity ratio of Fluo-3/Fura Red fluorescence over
time as recorded by flow cytometry.
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RESULTS |
Following T Cell Activation, Vav1 and Ly-GDI Accumulate in
F-actin-rich Membrane Extensions That Protrude in the Contact Zone with
the APC--
Upon stimulation of the T cell, redistribution of
signaling molecules occurs leading to the formation of an organized
"immunological synapse" at the contact between a T cell and an APC
(32, 33). High resolution immunofluorescence imaging of T cell-APC
conjugates allows the visualization of two distinct regions within the
immunological synapse: the central zone and the peripheral zone. The
central zone (c-SMAC) contains the TCR, surface co-stimulatory
receptors such as CD28, and intracellular signaling molecules such as
protein kinase C
, whereas the peripheral zone (p-SMAC), is enriched
for the integrin LFA-1 and the actin-binding protein talin (34-36). It
is widely believed that reorganization of the immunological synapse is
required for TCR signaling (32, 33). F-actin plays a critical role in
this reorganization (32). The polymerization of F-actin is regulated by
Rac (37); therefore, it was of interest to establish the involvement of
the Rac regulators, Vav1 and the GDIs, in organization of the
immunological synapse.
We used confocal microscopy to determine the localization of Vav1 and
Ly-GDI during the formation of the immunological synapse. This approach
allowed us to determine the specific subcellular localization of these
proteins in Jurkat T cells, information that could not be achieved by
biochemical approaches. Jurkat cells were incubated with APCs either
unpulsed (control) or prepulsed (activated) with S. enterotoxin E superantigen and the intracellular localization of Vav1 and Ly-GDI was determined with two-color immunofluorescence. As shown in Fig. 1,
an accumulation of Vav1 in membrane extensions in the contact area with
the APC was observed at 5 min (B), was maximal at 15 min
(C), and was mostly undetectable at 30 min (D).
Likewise, Ly-GDI accumulated in these membrane extensions overlapping
Vav1 accumulation in these areas and following the same kinetics (Fig.
1, B-D). In addition to the accumulation in membrane
extensions, a minute fraction of Vav1 was also observed in the central
zone of the junction, whereas Ly-GDI was mainly seen in the extensions
and not in the center. In contrast, in conjugates formed by T cells and
unpulsed APCs, no relocalization of Vav1 or Ly-GDI was observed at any
of the incubation times (Fig. 1A and data not shown).
Contrary to Ly-GDI, no accumulation of Rho-GDI in the contact area
seemed to occur (Fig. 2). Thus, the same
fluorescence density of Rho-GDI was observed in the membrane extensions
in contact with the APC and in other areas of the cell (Fig. 2,
B-D). These data indicate that Vav1 and Ly-GDI, but not Rho-GDI, translocate to membrane extensions in contact with the APC in
response to superantigen stimulation. Their accumulation was transient
and correlated with strong T cell shape changes in the contact zone
with the APC.
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Fig. 1.
Relocalization of Vav1 and Ly-GDI upon
activation of T Cells. Jurkat T cells (J77cl20) were incubated for
15 min at 37 °C with APCs (Raji) prepulsed with medium alone
(control; A) or with S. enterotoxin E
superantigen (activated) for different time intervals: 5 min
(B), 15 min (C), and 30 min (D). Cells
were fixed, and immunofluorescence staining was performed with
anti-Vav1 mouse mAbs and anti-Ly-GDI rabbit polyclonal Abs, followed by
Alexa488-coupled anti-mouse IgG and rhodamine-coupled
anti-rabbit IgG secondary Abs. The cells were then analyzed by confocal
microscopy. To this end, a Z-series of optical sections were acquired
at 0.5-µm steps. A central optical section per representative
conjugate is shown. The figure depicts the nonactivated T
cell-APC conjugate at 15 min (A). The results obtained for
the periods of 5 and 30 min were similar (data not shown). The position
of the T cell is indicated in the differential interference contrast
(DIC) image (T). An arrow in the DIC image shows
the extension of the T cell.
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Fig. 2.
Relocalization of Vav1 and Rho-GDI upon
activation of T cells. This experiment was identical to that
described in the legend to Fig. 1, except that anti-Rho-GDI Abs were
used in place of anti-Ly-GDI.
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Activation-induced changes in cell shape usually correlate with
modified actin dynamics in particular subcellular areas. Since Vav1 and
Ly-GDI are regulators of Rac GTPase and this in turn can control actin
dynamics (37), we analyzed whether subcellular accumulations of Vav1
and Ly-GDI coincided with actin polymerization in these areas. To this
end, three-color immunofluorescence and confocal microscopy analyzed
the presence of Vav1, Ly-GDI, and F-actin. As shown in Fig.
3B, the membrane extensions in
contact with the APC that displayed accumulation of Vav1 and Ly-GDI
were also enriched in F-actin. Therefore, a correlation seems to exist between accumulation of Vav1 and Ly-GDI and increased actin
polymerization in these subcellular areas. In contrast to activated
cells, nonactivated T cell-APC conjugates did not display accumulation
of F-actin, Vav1, and Ly-GDI in the contact area (Fig. 3A).
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Fig. 3.
Relocalization of Vav1 and Ly-GDI in
activated T cells occurs in membrane extensions rich in F-actin in the
periphery of TCR-CD3 clusters. Jurkat T cells (J77cl20) were
incubated for 15 min at 37 °C with APCs (Raji) prepulsed with medium
alone (control; A and C) or with S. enterotoxin E superantigen (activated; B and
D). Immunofluorescence staining was performed using the
following reagents. A and B, anti-Vav1 mouse mAbs
and anti-Ly-GDI polyclonal rabbit Abs, followed by Alexa488-coupled
anti-mouse IgG and Alexa633-coupled anti-rabbit secondary Abs. F-actin
was detected using Alexa546-coupled phalloidin. C and
D, anti-Vav1 mouse mAbs (IgG1), anti-CD3 mouse mAbs (IgG2a),
and anti-Ly-GDI polyclonal rabbit Abs, followed by fluorescein-coupled
anti-mouse IgG1, Texas Red-coupled anti-mouse IgG2a, and
Alexa633-coupled anti-rabbit IgG secondary Abs. Fluorescein labeling
was then amplified using Alexa488-coupled anti-fluorescein Abs.
Confocal analysis was carried out as described in the legend to Fig. 1.
The position of the T cell is indicated in the DIC image
(T). An arrow in the DIC image shows the
extension of the T cell.
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Comparison of staining for TCR-CD3, Vav1, and Ly-GDI revealed that Vav1
and Ly-GDI accumulations occurred at the periphery of the T cell-APC
contact site, whereas the TCR-CD3 clustered in the center. Some overlap
between TCR-CD3 and Vav1 staining was observed in the center of the
contact zone, although Vav1 was mostly present in the peripheral cell
extensions (Fig. 3D).
Altogether, these immunolocalization experiments indicate that Vav1 and
Ly-GDI accumulate in the contact zone between T cells and stimulatory
APCs within membrane extensions that transiently form and enlarge the
contact area between the cells. These extensions are also enriched in
F-actin, suggesting that a correlation exists between Vav1 and Ly-GDI
accumulation and increased actin dynamics in these cellular areas.
Vav1 and Ly-GDI Associate in the Cytoplasm--
Activation of the
exchange of GDP for GTP on Rho family GTPases is accompanied by their
intracellular translocation from the cytoplasm to the plasma membrane.
The subcellular localization of the regulators of Rho GTPases is less
well established, although Vav1 was shown to be recruited to the plasma
membrane in response to activation of Fc receptors (38). The GDI
regulators, Ly-GDI and Rho-GDI, are considered to be predominantly
cytosolic (25). Our confocal microscopy results raised the possibility
that Vav1 and Ly-GDI proteins are present in the same cellular
microenvironment upon activation of Jurkat T cells (Figs. 1-3),
suggesting that these proteins might cooperate. To explore this
possibility, we first established whether Vav1 and Ly-GDI interact in
specific subcellular compartments in Jurkat T cells.
Vav1, Ly-GDI, and Rho-GDI were found predominantly in the
cytoplasm (Fig. 4A).
Immunoblotting with anti-CD28 mAbs and anti-actin mAbs verified the
purity of the particulate (membrane) and cytoplasmic fractions and
ruled out significant cross-contamination (Fig. 4A). We
demonstrated previously that Vav1 and Ly-GDI associate in T cells both
in vitro and in vivo (24). To further investigate the subcellular localization of their interaction, cytoplasmic and
particulate (membrane) fractions of Jurkat T cells were
immunoprecipitated with anti-Vav1 Abs (Fig. 4B) and
immunoblotted with either anti-Vav1 mAbs (Fig. 4B,
upper panel) or with anti-Ly-GDI Abs (Fig.
4B, lower panel).
Co-immunoprecipitation of Vav1 and Ly-GDI was observed in the
cytoplasmic fraction and was not apparent in the particulate fraction
(Fig. 4B). In contrast, Rho-GDI, previously shown to bind to
Vav1 in vitro (24), does not associate with Vav1 in Jurkat T
cells in vivo (Fig. 4C, lower
panel, lane 6 versus
lane 4). Longer exposures of the autoradiograms
did not reveal the presence of Rho-GDI in the immunoprecipitates of
Vav1. Thus, in vivo, Vav1 discriminates between the GDI
proteins and binds only to the hematopoietic-specific GDI, Ly-GDI.
These results imply that Vav1 and Ly-GDI might cooperate in T
cells.
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Fig. 4.
Ly-GDI, but not Rho-GDI, associates with Vav1
in the cytoplasm. A, lysates of Jurkat T cells were
separated into cytoplasmic and membrane (particulate) fractions as
detailed under "Experimental Procedures." Samples of both fractions
were separated on SDS-PAGE and immunoblotted (WB) with
antibodies raised against Vav1, Ly-GDI, Rho-GDI, CD28, and -actin as
indicated in the figure. B, cytoplasmic
(lane 1) and membrane (particulate;
lane 2) fractions of Jurkat T cells were
immunoprecipitated (IP) with anti-Vav1 Abs. Proteins were
separated on SDS-PAGE and immunoblotted with anti-Vav1 mAbs
(upper panel) and following stripping of the blot
with anti-Ly-GDI Abs (lower panel). C,
the cytoplasmic fraction of Jurkat T cells was immunoprecipitated with
the following Abs: anti-Rho-GDI Abs (lane 1),
anti-Vav1 (lanes 2 and 6), and
anti-Myc (lane 3). In addition, a sample of
anti-Vav1 Abs with lysis buffer only (lane 4) and
cell lysates without Abs were also used (lane 5).
The proteins were separated on SDS-PAGE and immunoblotted with
anti-Vav1 mAbs (upper panel). Following
stripping, these blots were reprobed with anti-Rho-GDI Abs
(lanes 1-5) and anti-Ly-GDI Abs (lane
6). D, control (lanes 1 and
2) and herbimycin A-treated (lanes 3 and 4) Jurkat cells that were transfected with Vav1 and
Ly-GDI were either stimulated with anti-CD3 Abs (lanes
2 and 4) or nonstimulated (lanes
1 and 3). Lysates of these cells were separated
on SDS-PAGE and immunoblotted with anti-Tyr(P) (pTyr) Abs
(left panel) or immunoprecipitated with anti-Vav1
Abs (right panel) and immunoblotted as indicated.
The figure depicts one representative experiment of four
performed. The level of Ly-GDI in these cell lysates was determined
(right panel, lower row).
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To determine whether the association between Vav1 and Ly-GDI is
regulated by T cell activation, Jurkat T cells transiently transfected
with Vav1 and Ly-GDI were stimulated with anti-CD3 mAbs (Fig.
4D). Cell lysates were immunoprecipitated with anti-Vav1 Abs
and immunoblotted with anti-Ly-GDI Abs. Co-immunoprecipitation of Vav1
and Ly-GDI was observed in control Jurkat T cells and increased
significantly following TCR stimulation with anti-CD3 mAbs (Fig.
4D, right panel, lane
2 versus lane 1). This
constitutive interaction might be the result of already
tyrosine-phosphorylated proteins such as Vav1 in a cell line such as Jurkat.
TCR stimulation induces tyrosine phosphorylation of Vav1 (39), and Vav1
activity is regulated by tyrosine phosphorylation (5). To explore the
role of tyrosine phosphorylation in the Ly-GDI/Vav1 association, cells
were treated with the protein-tyrosine kinase inhibitor herbimycin A
prior to the addition of anti-CD3 mAbs (Fig. 4D). No
significant co-immunoprecipitation of Ly-GDI and Vav1 was observed in
herbimycin A-treated Jurkat cells even following stimulation with
anti-CD3 (Fig. 4D, right panel,
lanes 3 and 4). Our results
demonstrate that tyrosine phosphorylation plays a role in the
interaction between Vav1 and Ly-GDI.
Ly-GDI as a Potential Signal Transducer Protein--
Both the fact
that tyrosine phosphorylation is important for the association between
Vav1 and Ly-GDI (Fig. 4) and the fact that Ly-GDI contains consensus
sequences for tyrosine phosphorylation prompted us to investigate
whether Ly-GDI is tyrosine-phosphorylated following engagement of the
TCR. To accomplish this, Jurkat T cells were stimulated with anti-CD3
mAbs, a treatment known to induce tyrosine phosphorylation of numerous
signaling molecules, including Vav1 (39). Total lysates and
anti-phosphotyrosine immunoprecipitates from these cells were
immunoblotted with anti-Ly-GDI Abs or anti-Rho-GDI Abs. The results
suggest that Ly-GDI is tyrosine-phosphorylated in response to anti-CD3
mAbs, whereas Rho-GDI is not (Fig.
5A, lanes
1, 2, 5, and 6). Since
anti-Tyr(P) mAbs may immunoprecipitate Ly-GDI indirectly, we used a
more direct approach that also eliminated the cross-reactivity of light
chains of Abs (Fig. 5B). Jurkat T cells were transiently
transfected with a Myc-tagged pSec/Ly-GDI plasmid and then were either
left nonstimulated (lanes 1 and 2) or
stimulated with anti-CD3 mAbs (lane 3). Cells
were then lysed and immunoprecipitated with anti-Myc mAbs
(lanes 2 and 3) or nonimmune sera
(lane 1), separated on SDS-PAGE, and
immunoblotted with either anti-Ly-GDI Abs or anti-Tyr(P) mAbs (Fig.
5B). The results of this experiment verify that Ly-GDI
exhibits an increase in tyrosine phosphorylation in activated Jurkat
cells (lane 3 versus
lane 2). Using a computer program to screen the
deduced amino acids of the encoded Ly-GDI and Rho-GDI proteins for
consensus tyrosine phosphorylation sites, we located four tyrosines
that have a high probability of being phosphorylated in Ly-GDI
(Tyr24, Tyr125, Tyr153,
Tyr172) and three in Rho-GDI (Tyr128,
Tyr144, Tyr156). Nevertheless, a fundamental
difference exists between these two GDI proteins. Whereas Ly-GDI is
tyrosine-phosphorylated in activated T cells, Rho-GDI is not.
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Fig. 5.
Ly-GDI is tyrosine-phosphorylated (A
and B) and associates with the SH2 of Shc following
TCR stimulation in Jurkat T cells (C and
D). A, Jurkat T cells were activated
with anti-CD3 Abs for 2 min (+, lanes 2,
4, 6, and 8) or left nonactivated ( ,
lanes 1, 3, 5, and
7) and then lysed and either immunoprecipitated with
anti-Tyr(P) (pTyr) mAbs (lanes 1,
2, 5, and 6) or nonimmunoprecipitated
(lanes 3, 4, 7, and
8). Proteins were resolved on SDS-PAGE and immunoblotted
(WB) with either anti-Ly-GDI Abs (lanes
1-4) or anti-Rho-GDI Abs (lanes
5-8). B, lysates of Jurkat cells co-transfected
with Vav1 and Ly-GDI and either nonstimulated (lanes
1 and 2) or stimulated with anti-CD3 Abs
(lane 3) were immunoprecipitated with either
preimmune serum (p.i.; lane 1) or with
anti-Myc Abs (lanes 2 and 3). The
blots were hybridized with either anti-Ly-GDI or anti-Tyr(P) as
indicated. C, bacterially expressed glutathione
S-transferase (lane 1) and glutathione
S-transferase-SH2Shc (lanes 2-4) were
bound to glutathione beads. The bound proteins were then incubated with
lysates of Jurkat T cells that were either activated with anti-CD3 mAbs
for 2 min (lanes 1 and 3) or anti-CD3
and anti-CD28 mAbs incubated for 15 min (lane 4)
or left untreated (lanes 2). The bound proteins
were separated on SDS-PAGE and then immunoblotted with anti-Ly-GDI Abs.
D, Jurkat T cells were either nontreated (lane
1) or stimulated with anti-CD3 Abs (lane
2). Lysates of these cells were either immunoblotted with
anti-Tyr(P) (left panel) or immunoprecipitated
with anti-Shc Abs and immunoblotted as indicated (right
panel). The level of Ly-GDI in the cell lysates was
determined by immunoblotting as indicated (right panel). The
figure depicts one representative experiment of four
performed.
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Since Ly-GDI is phosphorylated on tyrosine residues in T cells, it is
conceivable that it will associate with SH2-containing proteins, a
characteristic feature of tyrosine-phosphorylated proteins. The
motif-based profile scanning approach developed by Yaffe et
al. (40) predicted that Ly-GDI would associate with the SH2 region
of Crk and Shc. The SH2 region of Shc was produced as glutathione
S-transferase fusion protein in a bacterial expression system, purified, bound to glutathione-Sepharose beads, and used in a
binding assay with Ly-GDI. Ly-GDI associated with the SH2 region of Shc
(Fig. 5C, lanes 2-4). Stimulation of
TCRs with anti-CD3 mAbs and with both anti-CD3 and anti-CD28 Abs
increased binding of Ly-GDI to Shc (Fig. 5C,
lanes 3 and 4 versus
lane 2). Ly-GDI also co-immunoprecipitates with
Shc in a TCR-induced fashion (Fig. 5D, right
panel, lane 2 versus
lane 1). Based on the fact that Ly-GDI is
tyrosine-phosphorylated upon TCR stimulation and it associates with the
SH2 region of the adapter protein Shc in a TCR-induced manner, we
conclude that Ly-GDI is a protein that participates in signaling
cascades in T cells.
Influence of GDI Proteins on NFAT Stimulation by
Vav1--
Wild-type Vav1 appears to be necessary for full activation
of the transcription factor NFAT following TCR stimulation (13). It has
been suggested that the ability of Vav1 to stimulate NFAT activation
depends on its ability to function as a GEF toward Rac as well as on
converging signals from the Ras pathway (5). We wished to analyze
whether Ly-GDI influences the NFAT induction by Vav1 overexpression in
T cells. The rationale for this experiment was based on the following.
First, Ly-GDI is considered to be a GDI for the Rho GTPase proteins,
including Rac; therefore, it might influence Rac-associated downstream
functions such as NFAT activity (4, 25). Second, Ly-GDI associates with
wild-type Vav1 but not with the Vav1 amino terminus oncogenic deletion
mutants (24). The amino terminus of Vav1 seems to control NFAT
stimulation by an as yet unknown mechanism. Third, Ly-GDI was found by
us to become phosphorylated on tyrosine residues following TCR
stimulation (Fig. 5), suggesting that it might play a role in T cell
signaling pathways.
To investigate the role of Ly-GDI in Vav1-induced NFAT activation,
Jurkat T cells were cotransfected with a luciferase reporter driven by
a promoter containing NFAT binding sites and several plasmids encoding
Vav1, Vav1 mutants, Ly-GDI, or Rho-GDI (outlined in Fig.
6). Luciferase activity in cell extracts
was assessed following activation of the TCR by anti-CD3 mAbs. As
expected, in cells transfected with WT Vav1, luciferase activity is
significantly increased upon TCR activation (Fig. 6, lane
4 versus lane 3). Surprisingly, the Vav1-induced increase in luciferase activity was
further enhanced when Ly-GDI was co-introduced into Jurkat T cells
(lane 6 versus lane
4) despite the fact that Ly-GDI inhibits the luciferase
activity by 70% when transfected in the absence of Vav1
(lane 8 versus lane
4). No luciferase induction was observed when Ly-GDI was
co-transfected with the Vav1 amino terminus oncogenic deletion mutant
(OncVav1; lanes 9 and 10). Contrary to
the effect of Ly-GDI, Rho-GDI blocks the Vav1-induced increase in
luciferase activity (lane 14 versus
lane 4).
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Fig. 6.
Effect of Ly-GDI and Rho-GDI on the
stimulation of NFAT by wild-type Vav1 and oncogenic Vav. Jurkat
tag cells were electroporated with various plasmids: pSec vector
(lanes 1 and 2); pEFVav1 (pEF115;
lanes 3 and 4), pEFVav1 and Ly-GDI
(lanes 5 and 6), Ly-GDI
(lanes 7 and 8), oncogenic Vav
(OncVav1; pEF67) and Ly-GDI (lanes 9 and
10), Vav1 and Rho-GDI (lanes 11 and
12), and Rho-GDI (lanes 13 and
14). All of these plasmids were electroporated together with
-galactosidase and NFAT-luciferase reporter gene plasmids as
described under "Experimental Procedures." Eighteen hours following
electroporation, cells were activated with anti-CD3 and lysed 8 h
later. Luciferase activities were normalized to -galactosidase
activities to correct for transfection efficiency as described under
"Experimental Procedures." -Fold induction refers to the division
of values obtained in each lane by the ones obtained with vector alone
in nonactivated conditions. Histograms represent the mean ± S.E.
of duplicate values obtained in eight experiments performed. The paired
t test was used to evaluate the statistical significance in
luciferase activity between the following: (a) cells
transfected with vector alone (lane 2)
versus cells transfected with WT Vav1 (lane
4; p < 0.002); (b) cells
transfected with WT Vav1 (lane 4)
versus cells transfected with the combination of WT Vav1 and
Ly-GDI (lane 6; p < 0.025);
(c) cells transfected with Ly-GDI (lane
8), oncVav1 and Ly-GDI (lane 10), WT
Vav and Rho-GDI (lane 12), and Rho-GDI
versus cells transfected with WT Vav1 (lane
4; p < 0.002). The inset depicts
the levels of exogenous transfected proteins, as revealed by Western
blots. The levels of Ly-GDI and Rho-GDI from lysates of transfected
cells (lanes 6 and 12, respectively)
were assessed by immunoblotting with anti-Ly-GDI Abs and anti-Rho-GDI
Abs, respectively. The identity of the transfected proteins was
verified by immunoblotting with anti-Myc mAbs (data not shown). The
levels of Vav1 and the oncogenic Vav1 were determined by anti-Vav mAbs.
The exposure of the autoradiogram illustrates the level of the
transfected proteins (verified by anti-Myc mAbs).
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The fact that oncVav1 neither associates with Ly-GDI nor enhances NFAT
whereas Vav1 does both suggests that Ly-GDI association is required for
Vav1's ability to stimulate NFAT. The synergy of Vav1 and Ly-GDI in
the co-transfection experiments supports this notion.
Ly-GDI, Vav1, and Induction of PLC--
Vav1 was recently shown
to regulate the activation of PLC by phosphorylation and
GEF-dependent pathways, events that are critical for proper
calcium responses and various signal transduction pathways in
hematopoietic cells (41, 42). We next tested the possibility that
Ly-GDI contributes to this pathway and therefore influences
Vav1-induced NFAT stimulation. As shown in Fig.
7A, Jurkat T cells transfected
with Vav1 displayed increased tyrosine phosphorylation of PLC
compared with cells transfected with empty vector (Fig. 7A,
lane 4 versus lane
2). This increase was augmented when Ly-GDI was
co-transfected with Vav1 (Fig. 7A, lane
6 versus lane 4).
Interestingly, unlike WT Vav1, transfection with oncogenic Vav1
does not lead to an increase in phospho-PLC (lane
8 versus lane 4). When
Ly-GDI is co-transfected with oncogenic Vav1, levels of phospho-PLC
are not significantly different from levels in cells transfected with
oncogenic Vav1 alone (lane 10 versus
lane 6). In fact, the level of phospho-PLC
remained similar to that observed in cells transfected with vector only
(lanes 1 and 2). Interestingly, unlike
WT Vav1, oncogenic Vav1 does not lead to an increase in phospho-PLC
(lane 8 versus lane
4).
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Fig. 7.
Phosphorylation of PLC
(A) and calcium mobilization (B
and C) are influenced by Vav1 and Ly-GDI.
A, Jurkat cells were transfected with empty vector
(lanes 1 and 2), WT Vav1
(lanes 3 and 4), Vav1 and Ly-GDI
(lanes 6 and 7), oncogenic Vav
(Vavonc; lanes 7 and 8), and Vavonc
and Ly-GDI (lanes 9 and 10) and were
either nontreated (lanes 1, 3,
5, 7, and 9) or stimulated with
anti-CD3 (lanes 2, 4, 6,
8, and 10). Cell lysates were then
immunoprecipitated (IP) with anti-PLC and immunoblotted
(WB) with anti-phospho-PLC (Tyr783) Abs. The
levels of PLC in each sample were determined by immunoblotting with
anti-PLC. The figure depicts one representative
experiment of three performed. B, Jurkat T cells were
transfected with vector, Vav1, Vav1 and Ly-GDI, and Ly-GDI as
indicated. Cells were loaded with Fluo-3/AM and Fura Red as indicated
under "Experimental Procedures." Cells were stimulated by anti-CD3
Abs and anti-mouse IgG. Data shown are the ratios of Fluo-3 and Fura
Red fluorescence measured over time by a flow cytometry. One
representative experiment of three is depicted. C, a similar
experiment to the one described above was performed, but instead of WT
Vav1, oncogenic Vav1 was used as indicated. The figure
depicts one representative experiment of three performed.
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Several important conclusions can be drawn from these experiments.
First, WT Vav1 differs from oncogenic Vav1 in its ability to lead to
activation of PLC. Second, Ly-GDI can participate in PLC
activation only when it is expressed with WT Vav1, with which it
associates, but not when it is expressed with oncogenic Vav1, with
which it does not interact.
Ly-GDI and Calcium Mobilization--
NFAT activation is dependent
on calcium mobilization following TCR stimulation (43). Several
proteins including Vav1 and Cdc42/Rac1 were shown to play a critical
role in receptor-stimulated Ca2+ mobilization; therefore,
it seemed reasonable that Ly-GDI, a regulator of Rho GTPases, might
also be involved and thus affect NFAT stimulation. Changes in calcium
mobilization of TCR-stimulated cells transfected with vector alone
(red line), Vav1 (blue
line), Vav1 and Ly-GDI (green line),
and Ly-GDI (black line) was determined by the
ratio of fluorescence intensities of Fluo-3 and Fura Red over time. As
shown in Fig. 7B, Jurkat T cells transfected with Ly-GDI
exhibited significant reduced calcium mobilization compared with cells
transfected with the empty vector. In contrast, transfection with Vav1
led to an increase in calcium mobilization that was larger, more rapid,
and more sustained than that observed in cells transfected with vector
only. Cells transfected with both Vav1 and Ly-GDI exhibit a pattern
similar to that obtained in cells transfected with Vav1 alone, although
some increase in calcium mobilization can be noted. Thus, transfection
of Jurkat cells with Vav1 overrides the inhibition in calcium
mobilization exerted by Ly-GDI. Unlike WT Vav1, oncogenic Vav1 appears
to exert an inhibitory effect on calcium mobilization; Jurkat T cells
transfected with oncogenic Vav1 display reduced calcium mobilization
similar to that observed in cells transfected with Ly-GDI (Fig.
7C). There is no combinatorial effect of transfecting Jurkat
cells with both Ly-GDI and oncogenic Vav (Fig. 7C).
Our results demonstrate that Ly-GDI and WT Vav1 have opposite effects
on calcium mobilization; Ly-GDI is inhibitory, and Vav1 is stimulatory.
Furthermore, WT Vav1 not only reverses the Ly-GDI-generated inhibition,
but its positive effect is not hindered by the presence of Ly-GDI.
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DISCUSSION |
The regulation of Rho GTPase activity is integral to many basic
cellular functions and can be controlled by extracellular signals
(1-4). Regulatory proteins for small Rho GTPases include GEFs and
GDIs, which have opposite effects on GTPase activity. Vav1 exhibits a
regulated GEF activity in T cells following engagement of the TCR (5).
Intriguingly, we found recently that a potential negative regulator of
Rho GTPases, Ly-GDI, associates with Vav1 in vivo and
in vitro (24). The function and regulation of the hematopoietic-specific Ly-GDI in stimulated T cells have not been well described.
Here, we show that the distribution of both Vav1 and Ly-GDI is altered
in stimulated T cells, with both proteins accumulating in the membrane
extensions of the "immunological synapse" (32-36). The formation
of the immunological synapse at the contact between a T cell and an APC
was recently suggested to play a cardinal role in signaling in
activated T cells. Thus, determining the subcellular location of all
known signaling proteins during engagement of the TCR appears to be
critical to understanding the TCR-induced signaling cascade. The
relocalization of Ly-GDI and Vav1 following immunological synapse
formation was dependent upon activation of the APCs. The distribution
of these two Rho GTPase regulators displayed a partial overlap with one
distinct difference. Vav1 accumulated mainly in the extensions of the T
cells at the contact area with the APC; however, a minute amount was
also present in the central contact zone. Ly-GDI also concentrated at
the T cell extensions but was excluded from the central zone. We did
not observe any preferential accumulation of Rho-GDI in the extensions or the central zone. This was consistent with our biochemical results
showing no association of Rho-GDI with Vav1 with or without TCR
stimulation, suggesting that the Vav1-Ly-GDI association is very
specific. It is noteworthy that in T cells exhibiting synapses that
look more mature, with strong and compact TCR accumulations, smaller
protrusions can be found (data not shown). In these cases, the
accumulation of Vav1 and Ly-GDI was weaker, suggesting that Vav1 and
Ly-GDI relocalization precede TCR clustering. Furthermore, we showed
that the relocalization of Vav1 and Ly-GDI coincided with a strong
accumulation of F-actin in the same subcellular areas. This suggests a
link between the accumulation of Vav1 and Ly-GDI and increased actin
dynamics in those cellular regions. Experiments with T cells from mice
deficient for Vav1 also indicated that Vav1, Rac, and actin
cytoskeleton participate in the lipid raft formation that is needed for
the immunological synapse (44). Furthermore, using a green fluorescent
protein-tagged binding domain of WASP, that binds the active GTP-bound
Cdc42, a Rho family GTPase, it was demonstrated that Cdc42 localizes
along the T cell/APC contact site in an antigen-dependent
manner (45), thus exhibiting the role of GTPases in the organization of
the immunological synapse. Taken together, our results support the
common notion that Vav1 and possibly Ly-GDI are involved in
cytoskeletal rearrangement that occur during the formation of the
immunological synapse.
The relocalization of Ly-GDI during the organization of the
immunological synapse suggested to us that it might be regulated by
engagement of the TCR. Our results illustrate for the first time that
induction of the TCR leads to increased tyrosine phosphorylation of
Ly-GDI but not of Rho-GDI (Fig. 5). In addition, TCR stimulation leads
to association of Ly-GDI, but not Rho-GDI, with the SH2 domain of the
adapter protein Shc (Fig. 5). Moreover, the association of Ly-GDI with
Vav1 depends on tyrosine phosphorylation following TCR activation (Fig.
4). Consequently, our results suggest that Ly-GDI is specifically
activated by TCR engagement and participates in transmitting
extracellular signals in T cells.
Stimulation of T cells results in activation of NFAT, which is involved
in the production of interleukin-2 and in relaying signals that
coordinate immune responses (46-48). Stimulation of TCR leads to
calcium mobilization and activation of the phosphatase calcineurin (49,
50). Calcineurin dephosphorylates NFAT, enabling it to translocate to
the nucleus, where it binds specific DNA sequences and enhances
transcription by coordinating signals from other pathways such as the
Ras/mitogen-activated protein kinase (51, 52). Vav1 can stimulate the
transcription of the interleukin-2 gene proximal promoter via the
activation of NFAT by leading to the generation of robust calcium
fluxes in stimulated lymphocytes possibly through
Rac-dependent mechanisms (13, 15, 16, 53). However, the
Vav1-induced NFAT stimulation is probably more complex and might
involve Rac-independent mechanisms as well (54, 55). For instance, it
was recently demonstrated that Vav1 regulates PLC1 phosphorylation
through the Tec family kinase Itk and by promoting assembly of a
signaling complex containing PLC1 and the adapter molecule SLP-76
(41, 42). The fact that amino terminus-truncated oncogenic Vav1
proteins are unable to stimulate NFAT-dependent
transcription despite having intact GEF activity implies that GEF
activity is not sufficient for Vav1's role in NFAT stimulation (5,
13). It appears that the amino terminus of Vav1 is highly relevant for
its ability to induce NFAT activity. Based on its structure, it has
been suggested that the amino terminus of Vav1 serves as an
autoinhibitor of the exchange domain (DH domain) and that
phosphorylation or truncation of a Tyr174 Lck kinase
recognition site within the N terminus could therefore result in
stimulation of GEF activity (56). However, this result does not fully
explain the complex and diverse results obtained with amino terminus
Vav1 mutants. Specifically, if this hypothesis was correct, the Vav1
Y174F mutant should exhibit a phosphorylation-independent GEF activity.
However, Vav1 Y174F retains phosphorylation-dependent exchange activity which can stimulate NFAT activity once the TCR is
activated (57, 58). Thus, this explanation of the role of the amino
terminus of Vav1 in NFAT induction is unsatisfactory and might imply
that other explanations for the role of the amino terminus of Vav1 for
NFAT induction must exist.
Ly-GDI associates with the amino terminus region of Vav1 (24). Could
Ly-GDI be part of the missing link between Vav1 and NFAT activation?
Ly-GDI does increase the activation of NFAT in T cells when
co-expressed with Vav1, yet it does not activate NFAT when co-expressed
with the oncogenic Vav1, which does not bind Ly-GDI (Fig. 6) (24).
Thus, the ability of Vav1 to induce NFAT seems to be correlated with
its ability to associate with Ly-GDI. When co-expressed with Vav1,
Ly-GDI also enhances Vav1's effects on PLC phosphorylation and
possibly calcium mobilization; however, it does not alter the
regulation of these phenomena by oncogenic Vav1. Again, this strongly
supports the hypothesis that Vav1 and Ly-GDI must physically associate
to exert their cooperative functions.
How could the association of Ly-GDI with the amino terminus of Vav1
promote NFAT activation? Ly-GDI seems to inhibit NFAT stimulation (Fig.
6), which correlates with its capacity to block calcium mobilization
(Fig. 7). This activity most probably stems from its ability to inhibit
members of the Rho GTPases. Indeed, it was recently shown that a strong
structural similarity exists between Ly-GDI and Rho-GDI (59-61).
Furthermore, several in vitro studies suggest that Ly-GDI is
as efficient as Rho-GDI in inhibiting GDP to GTP exchange on Rho GTPase
proteins (62). Yet, conflicting studies claim that Ly-GDI is less
efficient than Rho-GDI in its activity as a GTPase inhibitor (26, 63).
Nonetheless, apparently the inhibitory activity of Ly-GDI toward Rho
GTPases is sufficient to block calcium mobilization as well as NFAT
stimulation. Moreover, it is possible that Ly-GDI inhibits an unknown
hematopoietic cell-specific GTPase more potently than it inhibits known
Rho GTPases.
Cdc42 and Rac were shown to act upstream of the calcium influx pathway.
Cells expressing the dominant active mutants of Cdc42 and Rac exhibit
elevated levels of antigen-stimulated inositol 1,4,5-trisphosphate
production, leading to calcium mobilization upon activation (64, 65).
When Vav1 and Ly-GDI are concomitantly overexpressed in Jurkat T cells,
both calcium mobilization and NFAT are stimulated, thus indicating that
Vav1 overrides the inhibition exerted by Ly-GDI. It is conceivable that
the interaction between these proteins extracts Ly-GDI away from its
target GTPase and enables Vav1 to function more efficiently as a GEF.
Indeed Rho-GDI, which does not associate with Vav1, reduced NFAT
activation regardless of the expression of Vav1 (Fig. 6). This result
indicates that the ability of Rho-GDI to function as an inhibitor for
GTPases is not hampered in the presence of Vav1. Whereas its ability to remove Ly-GDI from the target GTPase can explain why WT Vav1, but not
OncVav1, can overcome inhibition by Ly-GDI and not Rho-GDI, it does not
explain the observed synergy of Vav1 and Ly-GDI on NFAT activity. One
plausible scenario is that the association of Vav1 and Ly-GDI also
influences other signaling pathways. The observed increase in PLC
tyrosine phosphorylation in cells transfected with Vav1 and Ly-GDI as
compared with cells transfected with Vav1 alone (Fig. 7A)
may be one example of this.
Additional signaling cascades might also be influenced by Ly-GDI. We
demonstrate that Ly-GDI associates with the SH2 domain of the adapter
protein Shc. Shc is tyrosine-phosphorylated by ZAP-70 upon TCR
engagement, and it then forms complexes with Grb2 as well as with other
proteins (66). Moreover, Shc was shown to be involved in activation of
c-Rel and mitogen-activated protein kinase, and it is required for
TCR-induced interleukin-2 production (67). Vav1 also binds multiple
signaling proteins through its SH2, SH3, and proline-rich regions,
including adapter molecules such as Grb2, Crk, and Shc (68, 69) and
signaling proteins such as ZAP-70 (70) and SLP-76 (71, 72). The
functional significance of the interaction of Vav1 with the adapter
proteins is unknown as yet. However, the association of Vav1 with
signaling molecules such as ZAP-70 and SLP-76 influences NFAT activity
(73, 74). The fact that Ly-GDI and Vav1 associate with adapter proteins that form multicomplexes as well as with signaling proteins and of
course with each other suggests that they might influence various signaling pathways in T cells, including stimulation of
phosphatidylinositol 3-kinase and/or the Ras/mitogen-activated protein
kinase pathway.
Ly-GDI and Rho-GDI are very similar in structure (59-61).
Here we show that major differences exist between Rho-GDI and Ly-GDI in
their subcellular distribution, association with other intracellular proteins, post-translational modification, and mode of action in T
cells. In all of our experiments in T cells in this study, Rho-GDI and
Ly-GDI exhibited opposite results. Most interesting is the effect of
Ly-GDI on Vav1-induced NFAT stimulation. Our results suggest that
Ly-GDI and Vav1 complex and regulate intracellular signaling molecules
and pathways, including PLC and calcium mobilization, leading to an
increase in NFAT activation. Since overexpression of Ly-GDI alone
inhibits NFAT activation, our results suggest that the relative
titration of these two molecules and/or the regulation of their
association by tyrosine phosphorylation may serve as a sensitive
bidirectional regulatory mechanism for NFAT activation.
In summary, these studies reveal a novel molecular mechanism involved
in the response of T cells to TCR engagement and might represent a more
complex level of control of GTPases. More studies are necessary to
fully comprehend the interaction of these two regulatory proteins.
,
TOP
ABSTRACT
INTRODUCTION
