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* Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121;
Departments of Immunology and Cell Biology, The Scripps Research Institute, La Jolla, CA 92037;
Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain; and
Institut de Génétique Moléculaire de Montpellier, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5535, Institut Fédératif de Recherche 24, Montpellier, France.
¶ Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 integrin ligation by fibronectin
induced Vav1 phosphorylation in peripheral blood lymphocytes and in two
different T cell lines. Vav1 overexpression led to massive T cell
spreading on 1 integrin ligands, and, conversely, two
dominant negative mutants blocked integrin-induced spreading. Vav1 and
1 integrin ligation synergistically activated Pak, but
not Rac, Cdc42, or c-Jun N-terminal kinase. In addition, Vav1
cooperated with constitutively active V12Rac mutant, but not with
V12Cdc42, to induce T cell spreading after integrin occupancy. More
importantly, a Vav1 mutant that lacked guanine exchange factor activity
still cooperated with V12Rac. In contrast, a point mutation in the SH2
domain of Vav1 abolished this synergistic effect. Therefore, our
results suggest a new regulatory effect of Vav1 in T cell spreading,
which is independent of its guanine exchange factor
activity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). Vav1-deficient mice
display a severe reduction in the number of T and B lymphocytes
(3, 4). Vav1 is essential for: the actin polymerization
that leads to TCR cap formation after TCR/CD3 ligation
(3, 4, 5), lipid raft clustering at the immune synapse
(6), TCR-induced T cell adhesion and integrin clustering
(7), and protein kinase C (PKC) 
membrane translocation
and its subsequent activation (8). Vav1 associates with an
array of cytoskeletal, cytoplasmic, and nuclear proteins. It is
believed that Vav1 controls cytoskeleton rearrangements because it
displays GEF activity toward Rac in vitro that can be stimulated by
tyrosine kinases and phosphoinositides (9, 10, 11). Vav1 also
causes Rac, Rho, and Cdc42 activation in vivo (12, 13). In
Jurkat T cells, Pak1 activation occurs downstream of Vav1 and is
required for activation of extracellular signal-regulated kinase 2
(ERK2) and NF-AT (14). Vav1 interacts with a ternary
complex composed by signal linker protein 76 (SLP-76)/Nck/Pak1,
facilitating the activation of Rac in the vicinity of its target Pak1
to induce maximal activation of this kinase (15, 16). In
contrast, SLP-76 is not required for Rac and Pak1 activation after TCR
stimulation (17). In addition, Vav couples TCR to serum
response factor-dependent transcription via a
Rac/Cdc42-Pak1-mitogen-activated protein 1-dependent pathway
(18).
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integrin cytoplasmic domains (22, 23).
Integrins induce tyrosine phosphorylation of Vav family proteins in
cells that express Syk. Additionally, Syk and Vav1 cooperatively
induced activation of c-Jun N-terminal kinase (JNK), ERK2, and Akt in
nonhemopoietic cells attached to fibrinogen (24, 25, 26), and
Vav is essential for integrin-induced activation of Pyk2
(7), a focal adhesion kinase that is inducible
phosphorylated downstream of the TCR and integrins (27)
and that is thought to be important in integrin-mediated pathways. In
NK cells, Vav1 and Rac control the activation of p38 mitogen-activated
protein kinase triggered by integrins (28). Rac, but not
other small Rho GTPases like Rho or Cdc42, regulates integrin-mediated
spreading and adhesion of T cells (29). However, it
remains unknown whether Vav cooperates with integrins to stimulate
mitogen-activated protein kinase activation, gene transcriptional
activation and cytoskeleton changes in cell lineages where Vav1 is
normally expressed, e.g., T lymphocytes. Here, we investigated the role
of Vav1 in T cell spreading after integrin occupancy. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The anti-CD3 mAb OKT3 and the anti-Myc mAb 9E10 were purified from culture supernatants of the corresponding hybridomas by protein A-Sepharose chromatography. The anti-hemagglutinin (HA; 12CA5) mAb was from Boehringer Mannheim (Indianapolis, IN). The anti-Vav1, anti-Rac, and the anti-phosphotyrosine mAb were obtained from Upstate Biotechnology (Lake Placid, NY). The anti-JNK mAb was from BD PharMingen (San Diego, CA). Polyclonal anti-Pak1 Ab (R626) was described previously (30). Fibronectin (FN) was prepared from human plasma as described (31), poly-L-lysine was from Sigma-Aldrich (St. Louis, MO), and rhodamine-phalloidin was from Molecular Probes (Eugene, OR).
Plasmids
The cDNAs encoding a c-Myc epitope-tagged Vav1 in the pEF
mammalian expression vector have been described (8, 32).
The Vav mutants VavGEF* (L213A), Vav
PH (PH deletion from 405 to 507
aa), and VavSH2* (R695L) were cloned in the same vector. HA-JNK1 was
cloned in pcDNA3. A constitutively active Rac1 mutant (Rac1V12) was
cloned in pEXV. The green-fluorescent protein (GFP) fusions of the Rac
and Cdc42 active mutants have been described (33).
Cell culture and transfection
SV40 T Ag-transfected human leukemic Jurkat T (Jurkat-TAg) cells and the mouse T cell hybridoma A1.1 were grown in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 1x MEM nonessential amino acid solution (Life Technologies), and 100 U/ml each penicillin G and streptomycin. Cells were starved for 24 h in 0.2% serum before stimulation and assays of cell spreading, Rac, Pak, or JNK. Jurkat cells in a logarithmic growth phase were transfected with the indicated amounts of plasmid DNAs by electroporation as described previously (8). In each experiment, cells in different groups were transfected with the same total amount of plasmid DNA by supplementing expression vector DNA with the proper amounts of the corresponding empty vector. Cell lysis and analysis were performed 48 h after transfection as described for each experiment.
Human peripheral blood lymphocytes cells were prepared from healthy volunteers by standard Ficoll-Hypaque centrifugation and cultured in the presence of an activating anti-CD3 mAb (OKT3; 1 µg/ml) plus recombinant human IL-2 (20 U/ml) for 6 days. Before restimulation, cells were deprived of anti-CD3 and IL-2 for 36 h.
FN coating and cell spreading
Bacterial plastic dishes (100 mm) or glass coverslips in 24-well plastic dishes were coated with 20 µg/ml human FN or 1 mg/ml poly-L-lysine at 4°C overnight. The dishes were washed three times with PBS, and the FN-coated ones were blocked with 10 mg/ml heat-denatured BSA. Cells were plated and allowed to spread on the substrates for different times and then fixed with 3% paraformaldehyde-PBS for 30 min, permeabilized in 0.2% Triton X-100 in PBS for 5 min, and blocked with 10% normal goat serum. F-actin was stained with rhodamine-phalloidin. Images were acquired using a Bio-Rad 1024 MRC laser scanning confocal imaging system. Spreading of GFP+ cells was quantified by scoring 0 points for round cells, 1 point for partially spread cells, and 2 points for fully spread cells. The index of cell spreading was calculated as (y + 2z)/(x + y + z) where x, y, and z are the number of cells that were scored as 0, 1, and 2, respectively.
Quantification of cell surface area
The area of the cell surface in contact with the FN-coated dishes was outlined and measured using the Inovision ISEE software (Inovision, Raleigh, NC) running on a UNIX workstation (Silicon Graphics, Mountain View, CA). At least 100 cells of each experimental condition were analyzed.
Immunoprecipitation and immunoblotting
Cells were lysed in 1 ml of lysis buffer (20 mM Tris-HCl (pH
7.5), 150 mM NaCl, 5 mM EDTA, 5 mM
Na4P2O7,
1 mM Na3VO4 plus 1%
Nonidet P-40, 20 mM
NaH2PO4, 3 mM
-glycerophosphate, 10 mM NaF, and 10 µg/ml quantities each of
aprotinin and leupeptin) for 10 min on ice. After centrifugation
(16,000 x g for 10 min at 4°C), the supernatants
were incubated with optimal concentrations of Abs for 30 min at 4°C,
followed by 30 µl of protein A/G plus agarose (Santa Cruz
Biotechnology, Santa Cruz, CA) for 30 min at 4°C. Samples were washed
four times in lysis buffer, and precipitates were dissolved in Laemmli
buffer and resolved by SDS-PAGE. Electrophoresed samples were processed
for Western blot analysis as previously described
(34).
Rac and Cdc42 GTPase assays
Cells were serum starved overnight 24 h after transfection. Jurkat cells were kept in suspension in medium with 0.2% BSA or plated on dishes coated with 20 µg/ml FN. Cells were then chilled on ice, washed with ice-cold PBS, and lysed in buffer containing 0.5% Nonidet P-40, 50 mM Tris (pH 7.4), 150 mM NaCl, 10% glycerol, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 20 µg of recombinant GST fused to the Rac/Cdc42-binding domain of Pak. Lysates were then incubated with glutathione-agarose beads (Pharmacia, Peapack, NJ) at 4°C for 30 min, washed with lysis buffer, and eluted with SDS sample buffer. Bound GTPase was analyzed by Western blotting using a monoclonal anti-Rac Ab or a polyclonal Ab to Cdc42. Whole cell lysates were also analyzed for the presence of each GTPase for normalization.
Pak kinase assays
Pak was precipitated from cell lysates with anti-Pak1 Abs and kinase activity determined using an in-gel kinase assay with myelin basic protein as substrate as previously described (30). Pak protein in the precipitates was determined by Western blotting with the same Ab. Autoradiographs were quantitated by scanning densitometry, and the specific activity (kinase activity normalized to Pak protein) was calculated.
JNK assays
Transfected Jurkat cells were washed twice in PBS, lysed for 10
min at 4°C in 1 ml of lysis buffer B (20 mM Tris-HCl (pH 7.5), 150 mM
NaCl, 5 mM EDTA, 1% Nonidet P-40, 5 mM
Na4P2O7, 1 mM
Na3VO4, 10 µg/ml
quantities each of aprotinin and leupeptin), and spun at 15,000 x
g for 10 min at 4°C; the supernatants were incubated
overnight with 2 µg of anti-HA mAb plus 25 µl of protein
G-Sepharose 4B beads (Pharmacia Biotech, Uppsala, Sweden) to
immunoprecipitate JNK. The kinase activities of washed
immunoprecipitates were assayed using 2 µg GST-c-Jun fusion proteins
as substrate, in 20 µl of JNK kinase buffer (25 mM HEPES (pH 7.5), 25
mM MgCl2, 50 µM cold ATP, 25 mM
-glycerophosphate, 1 mM DTT, and 0.1 mM
Na3VO4). Kinase reactions
were incubated for 20 min at 30°C with gentle shaking and were
stopped by addition of 20 µl of 2x Laemmli buffer. Proteins were
resolved by SDS, 13% PAGE, transferred to nitrocellulose, and
subjected to Western blotting. The nitrocellulose membranes were
routinely reprobed with anti-JNK Ab and quantified by NIH image
software. The level of phosphorylation was normalized to JNK protein
levels.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells plated on integrin ligands undergo cell polarization,
which involves profound changes in the structure and shape of their
cytoskeleton (33, 35). Vav1 is a major protein involved in
cytoskeleton reorganization in T cells (1). To analyze the
interaction between Vav1 and integrins in T lymphocytes, primary T
cells and two T cell lines were plated on FN-coated dishes. FN binds to
51 and
41 integrins in T
cells and induces downstream signals. We observed that Vav1 was
phosphorylated on tyrosine by integrin occupancy in all T cell types
tested: activated human PBLs, Jurkat T cells, and the mouse T cell
hybridoma A1.1 (Fig. 1). Jurkat T cells
showed a transient Vav1 phosphorylation with a peak 
10 min after
stimulation and return to near baseline in 1 h. Human PBLs showed
a peak after 30 min, and a plateau that remains for at least 1 h.
A1.1 cells showed a slower but progressive increase in Vav1
phosphorylation. We conclude that integrin occupancy leads to Vav1
phosphorylation in T cells.
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Next, we examined effects of FN and/or Vav1 on T cell spreading.
Transfected cells were recognized by cotransfection with GFP.
Jurkat T cells plated on FN showed very little spreading or
filopodia at 10 min after plating (Fig. 2A). At 30–60 min, cells
started to show mainly filopodia and short lamellipodia (see Fig. 2A, insets) Longer incubation did not increase
spreading (Fig. 2, A and B). By contrast,
Vav1-transfected cells showed clear lamellipodia formation after only
10 min on FN (Fig. 2A). Very large lamellipodia were found
after 30 min that corresponded to maximal spreading (Fig. 2B). We failed to observe cell spreading when Jurkat T cells
were plated on poly-L-lysine, even after 4 h
(data no shown), suggesting that integrin engagement is essential for T
cell spreading even when overexpressing Vav. Conversely, two dominant
negative Vav constructs, VavPH and VavGEF* (see scheme in Fig. 5
A and Refs. 6, 8) blocked integrin-induced T
cell spreading (Fig. 2A). Both mutants showed a 50%
reduction in spreading after 30 min of plating on FN (Fig. 2C). These mutants were expressed at slightly lower levels
than the wild type (Fig. 2C, bottom), but they
were still capable of blocking FN-induced T cell spreading. These
results suggest that Vav activity is essential for integrin-induced
spreading in hemopoietic T cells. We therefore decided to study in more
detail the mechanism of Vav1-induced T cell spreading. Because
Vav1-transfected cells replated on FN for 10 min showed maximal
spreading differences relative to mock-transfected cells, this time
point was used in the rest of the study.
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Cytoskeletal changes induced by Vav1 have been linked to its
ability to activate the small GTPase Rac (9), including
studies in Jurkat cells (13, 36). Vav1 overexpression led
to increased activity of Rac and Cdc42 (Fig. 3, A and B), two
known targets of Vav1 in vivo (12). We then studied the
effects of Vav-1 on two downstream targets of these GTPases, Pak and
JNK. Pak has been linked to cytoskeletal rearrangements and is
downstream of Vav1 (14) and was also activated by Vav1 in
Jurkat cells (Fig. 3C). JNK, which is activated by onco-Vav1
in nonhemopoietic cells and by Rac (9, 37), was also
activated by Vav1 in Jurkat cells (Fig. 3D). These data
suggested that Vav1 overexpression leads to activation of the JNK
pathway located downstream of its main targets Rac and Cdc42. In
contrast, 1 integrin engagement by FN failed
to activate Rac, Cdc42, or JNK and induced a partial activation of Pak.
In addition, 1 integrin engagement on cells
transfected with Vav1 induced a significant increase on Pak activity,
but not of Rac, Cdc42, or JNK activities. These data suggested that
Pak, but not other enzymes of this pathway, integrated signals derived
from Vav1 and integrins.
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T cell spreading is barely detectable in T cells plated for 10 min
on FN but is substantially increased by Vav overexpression (Figs. 2, 4A, and 5C). To determine whether these effects
of Vav1 on the cytoskeleton were dependent on Rac and/or Cdc42
activity, we used GFP chimeras of constitutively active mutants of both
GTPases. Expression of active Rac or Cdc42 induced T cell
spreading when plated for 10 min on FN (Fig. 4A). Coexpression of Vav1 and
constitutively active Rac induced a further increase in T cell
spreading (Fig. 4A), whereas Vav1 and V12Cdc42 showed almost
no cooperation. Western blot analysis showed that expression of active
Rac or Cdc42 did not alter Vav1 expression and vice versa (Fig. 4A, bottom). Direct measurement of cell area
showed that Vav1, V12Rac, and V12Cdc42 individually induced a 3-fold
increase in T cell area (Fig. 4B), whereas expression of
Vav1 and V12Rac together resulted in a nearly 7-fold increase. Similar
results were obtained when the cell spreading index was calculated as
in Fig. 2 (data not shown). Because V12Rac is maximally GTP loaded,
these results strongly suggest that Vav1 activates additional pathways
leading to T cell spreading apart from Rac activation.
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A). The L213A mutation
(VavGEF*) does not display GEF activity toward Rac and/or Cdc42.
Deletion of residues 405–507 (VavPH) eliminates the region
responsible for the correct localization of Vav1. A point mutation
(R695L) in the SH2 domain, which is responsible for the interaction
with adaptor proteins such as SLP-76, makes the VavSH2* mutant
defective in some of the Vav1 adaptor functions. All these mutants
were expressed in Jurkat T cells at levels similar to those of
wild-type Vav1 (Fig. 5B).
To study the effect of these mutants on spreading, we plated Jurkat T
cells on FN-coated plates for 10 min, at which time nontransfected
cells showed minimal spreading (Fig. 5C). VavSH2* induced an
increase in spreading that was only slightly smaller than that of
wild-type Vav1 (Fig. 5, C and D), whereas PH
or GEF* Vav1 mutants did not show any effect. Mock-transfected cells
plated on FN for only 10 min exhibit a very low level of spreading, so
that no inhibition (as described at later time points; see Fig. 2)
could be observed. In summary, GEF activity toward Rac and/or Cdc42 is
required for Vav-mediated T cell spreading.
To identify the domain(s) of Vav1 involved in cooperation with
GTP-loaded Rac, we coexpressed these mutants with V12Rac and then
plated cells on FN for 10 min (Fig. 5, C and D).
Coexpression of VavGEF* with V12Rac still cooperated to induce T cell
spreading, further demonstrating that Vav1 can regulate the actin
cytoskeleton independently of its GEF activity (as suggested in Fig. 4). Strikingly, VavSH2* failed to cooperate with V12Rac, strongly
suggesting that the SH2 domain of Vav1 mediates an additional,
GEF-independent pathway. Coexpression of VavPH had no effect. This
mutant entirely localizes to the insoluble compartment
(38), suggesting that its function is impaired by
mislocalization. In fact, VavPH behaves as the most efficient
dominant negative Vav1 mutant (Fig. 2; Refs. 6, 8, 32 and
38).
Altogether, these results place Rac both downstream and parallel to Vav1 in the integrin-mediated T cell spreading pathway. Vav1 could activate two independent pathways leading to T cell spreading after integrin occupancy: 1) Vav1 activates Rac through its GEF domain; 2) Vav1 facilitates Rac function through its SH2 adaptor domain, likely by bringing Rac targets to its vicinity (15, 16).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data place Vav1 downstream of integrins in the pathway leading to T
cell spreading, as has been shown in other cellular systems (15, 16, 24). Thus, inhibition of Vav1 activity by two dominant
negative constructs, VavGEF* and VavPH, blocks T cell spreading
induced by FN. Conversely, Vav1 greatly increases T cell spreading
after integrin occupancy. However, our attempts to show defective
spreading on FN of vav1-/- mouse T
lymphocytes were unsuccessful, suggesting that other members of the Vav
family could compensate in vivo. In fact, Martinez-Gakidis et al.
(39) have recently shown that Vav1/Vav3 double knockout
macrophages were unable to migrate on FN, whereas macrophages deficient
in either one of the genes exhibited no significant alterations. This
contrasts with the fact that VavPH and VavGEF* block T cell spreading
(Fig. 2). The two more likely explanations are that these
mutants also block the activity of other members of the Vav family or
that in vav1-/- T cells there is a
mechanism of compensation for Vav function that is not present in
normal T cells.
Vav1 overexpression combined with 1 integrin
ligation leads to substantial changes in the cytoskeleton (Figs. 1 and 2), whereas changes in Cdc42 and Rac activity were much more moderate
(Fig. 3). These modest changes could suggest that these GTPases are
constitutively active in these cells. However, the induction of cell
spreading by Vav-1, V12 Rac, and V12 Cdc42 (Figs. 2, 4, and 5) and the
absence of constitutive JNK activation in these cells (see, e.g., Refs.
8, 40 and 41) rule out this simplistic
explanation. A more likely explanation for this result is that T cells
can have more than one pool of Rac/Cdc42. Integrin engagement can
induce tyrosine phosphorylation of Vav1 that would activate a specific
Rac/Cdc42 pool, leading to a modest increase in total Rac/Cdc42
activity. However, this fraction could be critical for cytoskeletal
reorganization. Consistent with this idea, different Rac-GEFs are able
to selectively induce certain downstream targets but not others
(42). It is also possible that certain Rac/Cdc42 effectors
(i.e., Pak) might be sensitive to slight increases in the level of
GTPase activation. In fact, T cell spreading after combined Vav1 and
integrin stimulation correlated better with activity levels of Pak than
with those of Rac or Cdc42 (Fig. 3). This suggests that Pak could be
integrating signals coming from several domains of Vav1. These domains
would include those that directly activate Rac/Cdc42, plus an adapter
function that may facilitate Pak activation by the active GTPases.
Both V12Rac and V12Cdc42 were able to induce T cell spreading, although
the latter was slightly less efficient (Fig. 4). The effect of V12Cdc42
was not detected in a previous study where T cells were incubated on FN
for periods of time up to 16 h after overexpression
(29). Our assay was probably more sensitive, because we
observed cells at longer time points after transfection. Strikingly,
Vav1 cooperates with V12Rac but not with V12Cdc42 to induce T cell
spreading (Fig. 4). Because these mutants are constitutively active,
Vav1 cannot further increase their GTP loading. Therefore, synergy
between Vav1 and maximally GTP-loaded V12Rac suggests that Vav1
activates pathways distinct from GTPase activation. Some Vav1
functions have already been reported to be independent of its GEF
activity, i.e., NF-AT activation (43). Furthermore, our
results show, for the first time, that Vav1 can cooperate with Rac to
induce cytoskeletal rearrangements in a GEF activity-independent
manner. Moreover, VavSH2*, which induces cytoskeletal changes in
resting or FN-activated cells, fails to cooperate with V12Rac, strongly
suggesting that the SH2 domain of Vav1 is required for its cooperation
with V12Rac. Thus, the SH2 domain is likely mediating activation of a
pathway different from Vav1 enzymatic activity. The SH2 domain of Vav1
is necessary for JNK activation (44), and for its
translocation to the membrane and subsequent filopodia formation
(45). This domain binds SLP-76, which facilitates Pak
activation downstream of integrins (15, 16), although not
downstream of the TCR (17). An attractive possibility is
that the SH2 domain of Vav1 would bring the complex SLP-76/Nck/Pak to
the vicinity of Rac (15), facilitating Pak activation by
Rac. Consistent with this idea, our results show a synergistic effect
of Vav1 and FN on Pak activation (Fig. 3C). Additionally, T
cell spreading correlates better with Pak than with Rac or Cdc42
activity levels (Fig. 3). Consequently, Pak could be integrating
signals coming from the Vav1 SH2 and GEF domains. However, our results
show that GEF activity is absolutely essential for Vav-mediated T cell
spreading, whereas the SH2 domain is not absolutely required (Figs. 2 and 5). By using V12Rac, we have unmasked this second function that is
probably related to the adaptor function of Vav1. In summary, we favor
a model in which 1) the GEF activity of Vav is first required for
T cell spreading and 2) the SH2 domain recruits the final
components.
Another possibility is that the SH2 domain could be targeting a Rac-specific effector, such as WAVE/Scar (46). This would account for the lack of synergy between Vav1 and V12Cdc42 in T cell spreading. In contrast, more than one mechanism could explain the lack of cooperation between V12Rac and VavSH2*. For example, phosphorylation of VavSH2* is deficient after integrin and anti-CD3 stimulation (M. A. del Pozo and M. Villalba, unpublished results). Therefore, it is possible that other adaptor proteins with SH2 domains, which bind tyrosine-phosphorylated residues of wild-type Vav1, do not recognize this mutant, thus impairing Vav1 cooperation with V12Rac.
Intriguingly, Vav1 also activates two intracellular pathways involved in cytoskeletal reorganization after TCR engagement (7). One of them regulates TCR capping via Wiscott-Aldrich syndrome protein (WASP), and the other regulates integrin clustering via a yet unidentified signaling component. We also describe herein a dual effect of Vav1 (GEF dependent and independent) in integrin-mediated T cell spreading. It is feasible that the WASP-dependent pathway could account for the GEF-dependent effects of Vav1, whereas the WASP-independent pathway could be similar to the GEF-independent pathway unmasked in the present work. Clearly, further analysis is required to test this possibility.
We propose that Vav1 influences Rac downstream signaling specificity. There are several examples in which other GEFs were found to influence which effector proteins become activated when a Rho GTPase is GTP loaded. Thus, Tiam1 and Dbl stimulate Pak but not JNK activity mediated by Rac and Cdc42, whereas FGD1 and GFKG1 show the opposite effect (42). Their large size, subcellular targeting motifs, and interaction with other proteins might explain these effects. GEFs may form complexes with GTPase downstream targets influencing the biological outcome, as we propose here for Vav1 and Rac effectors. Sos1 acts both upstream and downstream of Ras (47, 48). PIX transduces signals from Cdc42 to Rac, forming part of a complex that also involves Pak (49, 50), and Dbl is activated by the Cdc42 effector ACK1 (51).
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Acknowledgments |
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Footnotes |
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2 This is publication number 475 from the La Jolla Institute for Allergy and Immunology and publication number 15180-VB from The Scripps Research Institute.
3 Address correspondence and reprint requests to Dr. Miguel A. del Pozo, Departments of Immunology and Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, E-mail address: mdelpozo{at}scripps.edu; Dr. Amnon Altman, Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, E-mail address: amnon{at}liai.org; or Dr. Martin Villalba, Institut de Génétique Moléculaire de Montpellier, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5535, Institut Fédératif de Recherche 24, 1919 Route de Mende, 34293 Montpellier, France, E-mail address: villalba{at}igm.cnrs-mop.fr
4 Abbreviations used in this paper: GEF, guanine exchange factor; PH, pleckstrin homology domain; PKC, protein kinase C; FN, fibronectin; GFP, green-fluorescent protein; WASP, Wiscott-Aldrich syndrome protein; JNK, c-Jun N-terminal kinase; ERK2, extracellular signal-regulated kinase 2; SLP-76, signal linker protein 76; HA, hemagglutinin; Jurkat-TAg, SV40 T Ag-transfected human leukemic Jurkat T cells.
Received for publication June 11, 2002. Accepted for publication October 21, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IIb3 to the actin cytoskeleton. J. Biol. Chem. 276:5916. cytoplasmic domains. Curr. Biol. 11:1799.[Medline]
1 integrin-induced interleukin-8 production in human natural killer cells. Immunity 12:7.[Medline]
is a necessary component, and cooperates with calcineurin, to induce FasL expression during activation-induced T cell death. J. Immunol. 163:5813. in T cells is mediated by a nonconventional, PI3-K- and Vav-dependent pathway, but does not absolutely require phospholipase C. J. Cell Biol. 157:253. to activate JNK and IL-2 promoter in T lymphocytes. EMBO J. 17:3101.[Medline]
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C. Papaharalambus, W. Sajjad, A. Syed, C. Zhang, M. O. Bergo, R. W. Alexander, and M. Ahmad Tumor Necrosis Factor {alpha} Stimulation of Rac1 Activity: ROLE OF ISOPRENYLCYSTEINE CARBOXYLMETHYLTRANSFERASE J. Biol. Chem., May 13, 2005; 280(19): 18790 - 18796. [Abstract] [Full Text] [PDF]
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 M. Vicente-Manzanares, A. Cruz-Adalia, N. B. Martin-Cofreces, J. R. Cabrero, M. Dosil, B. Alvarado-Sanchez, X. R. Bustelo, and F. Sanchez-Madrid Control of lymphocyte shape and the chemotactic response by the GTP exchange factor Vav Blood, April 15, 2005; 105(8): 3026 - 3034. [Abstract] [Full Text] [PDF]
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G. Rosenberger, A. Gal, and K. Kutsche {alpha}PIX Associates with Calpain 4, the Small Subunit of Calpain, and Has a Dual Role in Integrin-mediated Cell Spreading J. Biol. Chem., February 25, 2005; 280(8): 6879 - 6889. [Abstract] [Full Text] [PDF]
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T. Kawakatsu, H. Ogita, T. Fukuhara, T. Fukuyama, Y. Minami, K. Shimizu, and Y. Takai Vav2 as a Rac-GDP/GTP Exchange Factor Responsible for the Nectin-induced, c-Src- and Cdc42-mediated Activation of Rac J. Biol. Chem., February 11, 2005; 280(6): 4940 - 4947. [Abstract] [Full Text] [PDF]
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 R. A. Clemens, S. A. Newbrough, E. Y. Chung, S. Gheith, A. L. Singer, G. A. Koretzky, and E. J. Peterson PRAM-1 Is Required for Optimal Integrin-Dependent Neutrophil Function Mol. Cell. Biol., December 15, 2004; 24(24): 10923 - 10932. [Abstract] [Full Text] [PDF]
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 W. T. Arthur, L. A. Quilliam, and J. A. Cooper Rap1 promotes cell spreading by localizing Rac guanine nucleotide exchange factors J. Cell Biol., October 11, 2004; 167(1): 111 - 122. [Abstract] [Full Text] [PDF]
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M. A. M. Gakidis, X. Cullere, T. Olson, J. L. Wilsbacher, B. Zhang, S. L. Moores, K. Ley, W. Swat, T. Mayadas, and J. S. Brugge Vav GEFs are required for {beta}2 integrin-dependent functions of neutrophils J. Cell Biol., July 19, 2004; 166(2): 273 - 282. [Abstract] [Full Text] [PDF]
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A. W. Zimmerman, J. M. D. T. Nelissen, S. E. van Emst-de Vries, P. H. G. M. Willems, F. de Lange, J. G. Collard, F. N. van Leeuwen, and C. G. Figdor Cytoskeletal restraints regulate homotypic ALCAM-mediated adhesion through PKC{alpha} independently of Rho-like GTPases J. Cell Sci., June 1, 2004; 117(13): 2841 - 2852. [Abstract] [Full Text] [PDF]
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L. Sanchez-Martin, N. Sanchez-Sanchez, M. D. Gutierrez-Lopez, A. I. Rojo, M. Vicente-Manzanares, M. J. Perez-Alvarez, P. Sanchez-Mateos, X. R. Bustelo, A. Cuadrado, F. Sanchez-Madrid, et al. Signaling through the Leukocyte Integrin LFA-1 in T Cells Induces a Transient Activation of Rac-1 That Is Regulated by Vav and PI3K/Akt-1 J. Biol. Chem., April 16, 2004; 279(16): 16194 - 16205. [Abstract] [Full Text] [PDF]
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N. Hogg, M. Laschinger, K. Giles, and A. McDowall T-cell integrins: more than just sticking points J. Cell Sci., December 1, 2003; 116(23): 4695 - 4705. [Abstract] [Full Text] [PDF]
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