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Laboratoire d’Immunologie Cellulaire, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7627, Centre Hospitalier Pitié-Salpêtrière, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and, before that, on the phosphorylation of ZAP-70 and of the
linker molecule for activation of T cells, LAT. An anti-CD3-induced
increase in actin polymerization was observed in control cells but not
in cells transfected with a Rac mutant. In addition, latrunculin, which
binds to monomeric actin, simultaneously inhibited basal and
CD3-induced actin polymerization and Ca2+ signaling. These
findings suggest a link between the effects exerted by Rac mutants on
cortical actin polymerization and on TCR signaling. Rac cycling between
its GTP- and GDP-bound states is necessary for this signaling.
Alterations observed in early TCR-dependent signals suggest that Rac
contributes to the assembly of the TCR-associated multiprotein
transduction complex. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI-dependent activation of NF-AT in mast cells also
requires Rac (11). In T cells, the functional importance of Rac is an unsolved but important question. Indeed, in these cells, the main guanine nucleotide exchange factor (4) for Rac is Vav, a protooncogene product whose expression is largely limited to cells of hematopoietic origin and which has been shown to play a crucial role in lymphocyte development and stimulation. Thus, Vav-deficient mice display severe defects in T cell development and the number of their peripheral CD4+ and CD8+ T cells is reduced (12, 13, 14). In T lymphocytes from Vav-/- mice, the TCR-induced Ca2+ response is severely depressed, and the production of IL-2 and the CD3-induced proliferation are impaired. After stimulation with anti-CD3 mAbs, these lymphocytes also exhibit a defect in actin-cap formation and an impairment of actin polymerization (10, 15). Additionally, overexpression of Vav in Jurkat cells is sufficient to activate NF-AT, and it greatly potentiates the effect of TCR stimulation (16).
How could Vav deficiency affect Ca2+ signaling in T cells? What has been proposed so far is that in Vav-/- T cells, a weak production of inostitol 1,4,5-trisphosphate (IP3)4 could be the consequence of a low level of phosphatidylinositol-4,5-biphosphate (PIP2), itself due to a defective activity of phosphatidylinositol-4-phosphate 5-kinase (PIP5K) (15). However, this hypothesis was not based on the measurement of Vav-dependent variations in the level of PIP2. The reasoning was made by analogy with observations made in other cell types. Thus, in C3H cells, it has been convincingly shown that the activity of PIP5K is enhanced by activated Rho, an effect which takes place following integrin stimulation (17). Upon stimulation of phospholipase C (PLC), a larger amount of IP3 is indeed observed in C3H cells enriched in PIP2. In permeabilized platelets, activated Rac also induces a rapid increase in PIP2 (18). It has been suggested but not proven, that a similar effect might take place in B cells, following stimulation of Vav/Rac in the CD19 cascade (19). This hypothesis will be tested directly in the present work.
Are the consequences of deficiency or overexpression of Vav due to its function as a guanine nucleotide exchange factor for Rac? This answer is not yet clear. What is already known is that transgenic mice expressing constitutively active Rac-2 in the thymus have a reduced cellularity which could result from an enhanced apoptosis, related to an overstimulation of the cells (20). No transgenic mouse expressing the dominant-negative form of Rac has been described yet.
In this study, different elements of the response to TCR/CD3 stimulation (Ca2+ response, protein tyrosine phosphorylation, actin polymerization, PIP2 level, and IP3 production) have been examined in T cells transiently transfected with various mutants of Rac. We show that in T cells expressing the dominant inhibitory RacN17 and the constitutively active mutant RacV12 there is a comparable inhibition of the TCR-induced Ca2+ response with a parallel alteration of the pattern of tyrosine phosphorylations. This common inhibition of the Ca2+ response is accompanied by different effects on actin polymerization, IP3, and PIP2. Altogether, our data are indicative of a very early role of Rac, possibly in the assembly of the multimolecular transduction complex, which might explain at least part of the functional importance of Vav in TCR signaling.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experiments were performed with Jurkat cells expressing large T-Ag of SV40 (JTAg cells). Cells were maintained in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin.
Abs and reagents
CD3-specific UCHT1 myeloma cells were kindly given by Dr. P.
Beverley (Imperial Cancer Research, London, U.K.); Abs against bovine
PLC-1 (mixed mAbs), phosphotyrosine (mAb 4G10), and linker for
activation of T cells (LAT; rabbit polyclonal IgG) were obtained from
Upstate Biotechnology (Lake Placid, NY). Rabbit immune serum against
the C-terminal region of murine ZAP-70 was kindly provided by Dr. A.
Veillette (McGill University, Montreal, Canada).
Anti-PIP2 were obtained from PerSeptive
Biosystems (Framingham, MA). Fura 2 acetoxymethyl ester (fura 2-AM) was
obtained from Molecular Probes (Eugene, OR). Latrunculin was obtained
from Alexis Biochemicals (Paris, France). Protein A-Sepharose CL-4B,
and all other reagents were obtained from Sigma (St. Louis,
MO).
Cell transfections
JTAg cells were transiently transfected by electroporation (320V, 960 µF in culture medium plus 25 mM HEPES) with 30 µg Rac wild type (WT), RacV12, and RacN17 vector constructs, each bearing a myc tag, or with 30 µg of the empty vector (EV). Electroporated cells were purified on a Ficoll-Hypaque gradient 24 h after electroporation, and used at 24 h or 48 h. The transfection efficiency, checked by anti-myc immunofluorescence led to between 70 and 95% of positive cells. Rac constructs and anti-myc mAb 9E10 were kindly provided by Dr. D. Cantrell (Imperial Cancer Research).
Calcium measurements
T cells (2 x 106) were loaded with 1 µM fura 2-AM in culture medium. After 20-min incubation at 37°C, the cells were centrifuged and the pellet was resuspended in 2 ml of mammalian saline (140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES; pH 7.2). For experiments performed in the absence of external Ca2+, the cells were resuspended in 2 ml 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, plus 0.5 mM EGTA. Ca2+ measurements were performed at 37°C in a Perkin-Elmer LS-5B luminescence spectrometer (Bois d’Arcy, France). The cell suspension was excited alternatively at 340 and 380 nm and fluorescence was measured at 510 nm.
Inositol 1,4,5-trisphosphate (IP3) measurements
Transfected Jurkat cells (5.106 cells in 500 µl of RPMI 1640 plus HEPES) were stimulated with UCHT1 (1/500 ascite dilution) for 1 min at 37°C. Stimulation was stopped by the addition of 100 µl of ice-cold 100% trichloroacetic acid followed by a 15-min incubation on ice. The samples were centrifuged at 1000 x g for 10 min at 4°C. The supernatant was incubated for 15 min at room temperature. Trichloroacetic acid was extracted from samples by adding 1.2 ml of a solution 3:1 of 1,1,2-Trichloro-1,2,2-trifluoroethane-trioctylamine. The samples were vigorously shaken and low-speed centrifuged. IP3 present in the top layer was quantified in duplicate 100 µl samples by using a competitive [3H]IP3 binding assay (Du Pont de Nemours, Les Ulis, France) according to the manufacturer’s instructions.
Scanning electron microscopy
Transfected Jurkat cells were immobilized on polylysine-coated coverslips for 5 min and fixed with 2.5% glutaraldehyde for 30 min. After washing, the samples were dehydrated in five successive and graded ethanol baths (from 25 to 100%), dried by the critical-point method using liquid CO2, coated with gold by sputtering, and observed with a Scanning Electron Microscope (JSM.840.A, JEOL, Peabody, MA).
Western blot analysis
Transfected Jurkat cells were washed in 10 mM HEPES RPMI 1640 and resuspended at 107 cells/ml in HEPES RPMI 1640. Aliquots (500 µl) of the cell suspension were then stimulated with UCHT1 (1/500 ascite dilution) for 3 min at 37°C. Reactions were stopped by rapid centrifugation and the pellet was resuspended for 45 min at 4°C in 100 µl of a lysis buffer containing 20 mM Tris (pH 7.5), 1 mM EDTA, 140 mM NaCl, and 1% Nonidet P-40 supplemented with protease and phosphatase inhibitors. Cell debris was removed by centrifugation at 10,000 x g and the detergent-soluble fraction was boiled in sample buffer (50 mM Tris (pH 6.8), 3% SDS, 10% glycerol, 5% 2-ME, and bromophenol blue). Solubilized proteins were then separated by SDS-PAGE. Gels were electrotransferred to a nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany) and the blots were probed with the anti-phosphotyrosine mAb 4G10. The blots were developed with HRP-conjugated goat anti-mouse IgG (Bio-Rad, Ivry/Seine, France) and an enhanced chemiluminescence detection system (Amersham, Paris, France) according to the manufacturers’ instructions.
In immunoprecipitation experiments, Nonidet P-40-soluble fractions of 107 cells, obtained as described above, were incubated for 2 h at 4°C with specific Abs as indicated. Immunoprecipitations were performed with 30 µl packed protein A-Sepharose CL-4B for 60 min at 4°C. The samples were washed three times with cold lysis buffer and the immune complexes were boiled in SDS sample buffer. The recovered proteins were separated by SDS-PAGE and the gels were subjected to Western blot analysis using anti-phosphotyrosine mAb.
Actin polymerization assay
Actin polymerization was tested as described before
(21). Transfected JTAg cells in glucose-containing
mammalian saline were stimulated with UCHT1 Ab (1/500) for 3 min at
37°C. To 400 µl of the cell suspension, 100 µl of a solution
containing 8 x 10-7 M tetramethylrhodamine
isothiocyanate (TRITC)-labeled phalloidin, 0.5 mg/ml
1-
-lysophosphatidylcholine, and 18% formaldehyde in PBS were added
at 37°C. The fixed cells were observed by fluorescence microscopy or
subjected to flow cytometry (FACScan, Becton Dickinson, San Jose, CA),
and the mean fluorescence intensity (MFI) of each sample was
determined. All data points are plotted relative to the MFI of
EV-transfected cells before CD3 stimulation.
PIP2 staining
Transfected Jurkat cells were washed and resuspended in 0.5% PBS-BSA (106 cells in 100 µl). The cells were fixed by adding 500 µl of a 4% paraformaldehyde PBS- BSA solution for 20 min at room temperature. The cells were washed with PBS-BSA and resuspended for 30 min in PBS containing 0.1% saponin and anti-PIP2 mAb or with a control isotype (IgG2b; CliniSciences, Paris, France). The cells were washed three times in PBS-BSA-saponin and stained with the PE-conjugated goat anti-mouse (Immunotech, Marseille, France). After two washes in PBS-saponin and three in PBS-BSA, cells were observed by fluorescence microscopy or subjected to flow cytometry (FACScan, Becton Dickinson) and the MFI of each sample was determined. According to the technical specifications, the cross-reactivity of this anti-PIP2 with 1-palmitoyl-oleoylphosphatidic acid is <5%, with phosphatidylinositol phosphate is <0.1%, and with phosphatidylinositol, phosphatidylcholine, phosphatidylserine, phosphatidyl ethanolamine, phosphatidyl glycerol, cardiolipin, cholesterol, and 1,2-diacylglycerol is <0.2%. Similar results have been published (22).
Statistics
Averages are expressed ± SD, and significance of differences was evaluated with the Student t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression of Rac in transfected cells was first checked by
anti-myc immunoblots (Fig. 1
A). Both Rac mutants and
RacWT were expressed at similar levels while in EV-transfected cells
the myc-tagged Rac protein was absent. Furthermore, the expression of
Rac constructions was analyzed by anti-myc intracellular staining
and quantification by flow cytometry to determine the transfection rate
and the expression level profile (Fig. 1B). The transfection
rate was high and the expression-level profile was quite similar with
the three Rac constructs.
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C). A similar inhibition was observed in a
Ca2+-free medium (not shown). This inhibition was
not due to differences in the expression of CD3 after transfection with
the different constructions of Rac (data not shown). One series of
experiments (anti-CD3-induced Ca2+ responses)
was performed in parallel with the Jurkat clone J.HM1, expressing the
muscarinic receptor type 1 (23), with very similar results
(not shown).
Stimulation of Jurkat T cells with an anti-CD3 mAb led to a rapid
and transient increase of IP3, peaking at 1 min.
Five minutes after the beginning of the stimulation, the level of
IP3 was almost back to the resting level (data
not shown). The IP3 responses at 1 min were
measured in the different Jurkat transfectants, and expressed relative
to the response observed in control condition (EV). As shown in Fig. 1D, a similar IP3 increase was
observed for cells transfected with EV or with RacWT, whereas
significantly smaller responses were observed in RacN17- or
RacV12-transfected cells (60 ± 23 and 46 ± 21% of EV;
n = 3 experiments). Thus, the inhibition of CD3-induced
Ca2+ response in Rac mutant-transfected cells is
directly associated with an inhibition of CD3-induced
IP3 production. Interestingly, although the
resting level of IP3 was similar in EV, RacN17-,
and RacWT-transfected cells (100%, 99 ± 2% (n =
3), and 97 ± 3% (n = 3) respectively), this
resting level was larger in RacV12-expressing cells (151 ± 45%;
n = 3).
The Rac-dependent inhibition of IP3 production is not due to a decreased level of PIP2
It has been suggested that the inhibition of the
Ca2+ responses associated to a defect in the
Rac/Vav pathway could be due to a decreased activity of PIP5K leading
to a low level of PIP2 (15, 18). We
have thus examined to what extent Rac mutants could affect the level of
PIP2. PIP2 can be stained
in permeabilized cells with an anti-PIP2 mAb
(Fig. 2A), and quantified by
flow cytometry. In stimulated cells, a small transient reduction in the
staining was observed at 30 s (not shown), similar to the one
reported in 32P-labeled platelets after FcRIIA
cross-linking (24). This PIP2
reduction is only transient presumably because hydrolysis and
resynthesis of PIP2 are tightly coupled. More
informative is the basal PIP2 level, which was
measured in Rac transfectants. Fig. 2B shows that this level
was not significantly different in cells transfected with an EV,
RacN17, or RacWT. It was higher in RacV12-transfected cells. These
results clearly demonstrate that the reduction of CD3-dependent
IP3 production cannot be explained by a default
in the resting PIP2 level. Nevertheless, the fact
that RacV12 transfectants present a higher basal level of
PIP2 than control cells, is consistent with the
higher IP3 level measured in the same cells, and
with the effect of activated Rac on PIP2 in
permeabilized platelets (25).
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There is a large set of data showing that Rac is involved in the
control of the cytoskeleton of various cell types including
lymphocytes. Therefore, we examined whether Rac-dependent alterations
of the cytoskeleton could be involved in the reduction of the
CD3-induced Ca2+ response. Overexpression of Rac
mutants resulted in alterations of the T cell shape, as shown in the
scanning electron micrographs of Fig. 3A. In RacWT-transfected
cells, the percentage of cells presenting a detectable membrane
ruffling was slightly increased compared with cells transfected with EV
(not shown). On the contrary, cells transfected with RacN17 were quite
round, with no sign of ruffling. RacV12-expressing cells had a striking
sea urchin-like appearance.
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B), most of the
T cell actin appears as a cortical ring, and additional F-actin is
present at the leading edge of membrane ruffles. Additionally, no clear
stress fibers are visible in lymphocytes, contrary to what is observed
in fibroblasts for instance.
The size of the F-actin pool was then quantified by flow cytometry to
see whether the increase in F-actin, which normally takes place upon
cell stimulation (26, 27), was affected by Rac mutants
(Fig. 3C). Overexpression of RacWT did not affect the basal
amount of F-actin, compared with EV. In contrast, RacN17 and RacV12
mutants exerted opposite effects on actin polymerization. In resting
RacN17-expressing cells, the average level of F-actin was significantly
lower than in EV (p < 0.02). On the contrary,
this pool was significantly increased in RacV12-transfected cells.
After CD3 cross-linking, a modest but systematic and significant
(p < 0.05) increase of F-actin rapidly took
place in control cells (EV- and RacWT-transfected cells). Starting from
very different basal levels, neither RacN17 nor RacV12 transfectants
responded to CD3 stimulation by a significant increase in F-actin. In
RacV12 transfectants, the absence of further increase in actin
polymerization might be due to the fact that its level was already
close to its maximum. This maximum could not be precisely determined
with the usual PMA stimulation, because in Jurkat cells, PMA had only a
marginal effect on actin polymerization, contrary to what is observed
in other cell types (see e.g. Ref. 28).
Latrunculin simultaneously inhibits actin polymerization and CD3-induced Ca2+ signaling
We examined to which extent the effects of RacN17 could be
mimicked by latrunculin, a drug which prevents actin polymerization
(29). In Jurkat T cells, 0.5 µM latrunculin completely
disrupted the cortical actin cytoskeleton as can be seen by
fluorescence microscopy (Fig. 4A), and quantified by flow
cytometry (Fig. 4B). In latrunculin-treated cells, a marked
reduction of the CD3-induced Ca2+ response was
observed (Fig. 4C). This effect was dose-dependent and
maximal in the presence 0.5 µM latrunculin. The effect of latrunculin
was then tested on CD3-induced IP3 production. As
shown in Fig. 4D, a marked reduction of CD3-induced
IP3 production was observed in
latrunculin-treated cells, similar to the one observed in
RacN17-transfected cells. In latrunculin-treated cells, the level of
PIP2 was similar to the control one (data not
shown).
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Finally, we evaluated whether Rac mutants affected early tyrosine
phosphorylations triggered after TCR signaling. The global tyrosine
phosphorylation pattern was examined first in whole-cell lysates (Fig. 5A). We observed that the
increase in tyrosine phosphorylation induced after CD3 stimulation was
somewhat reduced in RacN17- and RacV12-transfected cells, as compared
with cells transfected with EV or with RacWT. More specifically, one
can see an impaired labeling of several bands migrating in the 21-kDa
range of the gel. As we and others have previously shown, these bands
presumably represent phosphorylated CD3 chains, the strongest one
corresponding to the fully tyrosine phosphorylated form of 
-chain,
p23 (30). An increased expression of p23 is
necessary to adequately recruit and trigger tyrosine phosphorylation of
the protein tyrosine kinase ZAP-70 after CD3 stimulation (31, 32). Therefore, we measured ZAP-70 phosphorylation in the
different transfectants after immunoprecipitation (Fig. 5B,
upper panel). The amount of phospho-ZAP-70 was clearly
reduced in RacN17- and RacV12-transfected cells.
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, A and
B, lower panel). LAT is a linker of fundamental
importance for T cell signaling. Once phosphorylated by ZAP-70, it
allows the recruitment of multiple signaling molecules, including
PLC-1 (33). We thus studied LAT in PLC-1
immunoprecipitates after CD3 stimulation (Fig. 5C). In
RacN17- and RacV12-transfected cells, one can see in the left panel
that the CD3-induced phosphorylation of PLC-1 was partially
inhibited in agreement with the Ca2+
measurements. The amount of PLC-1-associated phosphorylated LAT was
strongly reduced in parallel. Shown in the right panel are the
upper and lower parts of the same membrane reprobed with PLC-1 and
LAT Abs showing reduced amounts of LAT protein in PLC-1 precipitates
from RacN17- and RacV12-transfected cells. |
Discussion |
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In all the experiments of the present work, RacWT-transfected cells had characteristics very similar to those of cells transfected with EV. Therefore, the results observed with the two Rac mutants were not simply due to Rac overexpression, but to the fact that overexpressed Rac mutants did not allow the normal cycling of Rac between its GTP- and GDP-bound states. The only function which we found inhibited in a similar way by RacV12, RacN17, and RacWT was the CD3-induced nuclear translocation of NF-AT (data not shown). This effect was not considered as informative and included in the results, because it could simply have been due to Rac overexpression.
Comparing the effects of Vav and Rac
Qualitatively, the effects of Rac mutants in Jurkat T cells are
comparable to those observed in Vav-/- mice:
inhibition of CD3-dependent Ca2+ signaling and of
actin polymerization (10, 13, 15). This is consistent with
the fact that Vav is a major guanine nucleotide exchange factor for
Rac. In addition, overexpression of Vav (16), like
overexpression of Rac, had no significant effect on CD3-dependent
Ca2+ signaling or activation of tyrosine kinases.
However, despite the fact that phosphorylation of CD3 was inhibited
by Rac mutants (this study) but not by Vav deficiency
(15), the Ca2+ signaling defect due
to Vav deficiency in primary T cells (10) appeared more
severe than that induced by RacN17 in Jurkat cells. Thus, one cannot
exclude the possibility that, in addition to Rac, Vav has other
downstream molecular partners which can affect
Ca2+ signaling.
Are the effects of Rac mutants mediated by PIP5K?
It has been suggested that in Vav-/- T
cells, the inhibition of the TCR/CD3-induced Ca2+
response could be due to a Rac-dependent decrease of the
PIP2 level. In the present work, we have used
anti-PIP2 mAbs to measure directly the
influence of Rac on the PIP2 level in T cells.
Our results show that RacN17 causes a small but nonsignificant
decrease, and RacV12 causes an increase of PIP2
in resting cells. Even though activated Rac may augment the
PIP2 level, the CD3-dependent increase in
IP3 and Ca2+ was inhibited
to the same extent in both Rac mutants. This inhibition could not be a
reflection of the level of PIP2, but was more
likely explained by a decreased activation of PLC-1. Additionally,
it has been proposed that an increase of PIP2 can
result in uncapping actin barbed ends, creation of new nucleation sites
for actin monomer addition close to the membrane, and formation of
lamellipodia (18). One can hypothesize that such a
phenomenon may directly link the increase in basal PIP2 and
the intense membrane ruffling induced by RacV12.
Why do both RacN17 and RacV12 inhibit TCR signaling?
Both RacN17 and RacV12 inhibit TCR signaling, and both of them
prevent the normal cycling of Rac between its GTP- and GDP-bound
states. However, the initial consequences of this cycling inhibition
are likely to be distinct for the two mutants. The subcellular
localization of the two mutants is quite different; strictly
cytoplasmic for RacN17 and preferentially at the membrane for RacV12 as
well as RacWT (data not shown). RacN17 cells present no sign of
activation; these cells are entirely devoid of membrane ruffles, and
their F-actin level and global tyrosine-phosphorylation level are lower
than in the control cells. Upon stimulation, several responses are
inhibited, including tyrosine phosphorylation of ZAP-70, LAT, and
PLC-1; production of IP3;
Ca2+ release; and actin polymerization. On the
contrary, in the absence of CD3 stimulation, RacV12 cells seem already
partially activated; they present a marked membrane ruffling and their
F-actin, PIP2, and IP3
levels are high. It is possible that this sustained basal activation
leads to the desensitization of some pathways. Such a mixed
activated/desensitized state of RacV12 cells might explain why, upon
stimulation, early signaling events are also inhibited.
Effects of inhibitors of actin polymerization
The functional importance of actin polymerization on signaling is
frequently addressed by testing the effects of cytochalasin D.
Cytochalasins bind to the barbed end of actin microfilaments, thereby
preventing their elongation (34). With 1 µM cytochalasin
D in rat basophilic leukemia cells, stress fibers are disrupted but
cortical actin is still present (11). The functional
relevance of data obtained in the presence of high concentrations of
cytochalasin D is questionable, because 10 µM cytochalasin D causes
the formation of aggregates of F-actin in T cells (data not shown). In
addition, in NIH3T3 cells, 10 µM cytochalasin D triggers an
IP3 increase larger than that elicited by
platelet-derived growth factor (35). Thus, we have chosen
to use latrunculin, which prevents actin polymerization by binding to
monomeric actin (29), and at 0.5 µM causes the clear
disappearance of cortical actin in T cells without the multiple
aggregates seen with 10 µM cytochalasin D. This is the first report
that Ca2+ signaling can be inhibited by
latrunculin, which strongly suggests that in T cells,
Ca2+ signaling requires the integrity of cortical
actin. Surprisingly, in rat basophilic leukemia cells, a different
phenomenon was observed; disruption of the actin cytoskeleton appeared
to enhance signaling through FcRI, as if association of FcRI with
actin microfilaments was involved in turning off the FcRI signal
(36).
TCR signaling and T cell actin cytoskeleton
There is a growing evidence that actin cytoskeleton can be involved in TCR signaling (see Ref. 37 for a review). However, two levels should be clearly distinguished. The first one concerns the importance of the cytoskeleton during the APC-T cell interaction, which involves costimulation/adhesion molecules in addition to the TCR. The second one concerns TCR signaling per se, which can be probed when stimulating T cells with anti-CD3 mAbs. In the first case, the integrity of the actin cytoskeleton is necessary for the formation of the immunological synapse and for the maintenance of its integrity (38, 39). In addition to the TCR, LFA-1 and CD28 also contribute to the T cell cytoskeletal reorganization (40, 41, 42). At the immunological synapse formed between cloned D10 T cells and CH12 cells as APCs, the "supramolecular cluster" includes a central TCR aggregate surrounded by an LFA-1 ring associated with talin, an actin-binding protein (43). Finally, during sustained T cell-B cell interactions, reorganizations of the microtubule network play an important role in the polarized secretion of cytokines by T cells (44), a phenomenon which involves the GTPase Cdc42 (45).
Concerning the importance of the actin cytoskeleton for
anti-CD3-induced responses, the evidences are more scarce. In
resting T cells, a fraction of CD3 is associated with actin
(46), and this fraction increases following CD3
stimulation (46, 47). Anti-CD3-induced
Ca2+ responses are not affected by 1 µM
cytochalasin D in murine primary T cells (38) or in Jurkat
T cells (data not shown). In addition, early CD3-dependent tyrosine
phosphorylations are not affected by 5 µM cytochalasin D
(10), even though an inhibition was observed on
NF-AT-dependent transcription, and on the formation of TCR/CD3 patches
induced by cross-linking Abs. The data obtained in the present work
with latrunculin add further evidence to the importance of an intact
cortical cytoskeleton for TCR signaling.
Rac and TCR signaling
In conclusion, we have shown that in T cells, Rac mutants affect
very early TCR signaling events, such as phosphorylation of key
downstream molecules like ZAP-70, LAT, and PLC-1 involved in the
Ca2+ signaling cascade. Our data are best
explained by the hypothesis that Rac normally contributes to the
assembly of a multiprotein transduction complex associated to the TCR.
This complex would also include Vav, SLP-76, Nck, and p21-activated
kinase (33, 48, 49, 50). It could be preformed to a large
extent before stimulation, as it has been proposed for B cell receptor
signaling (51). To fulfill this function, Rac needs to
cycle between its GTP and GDP-bound forms.
Rac exerts simultaneous effects on TCR/CD3 signaling and on the polymerization state of the actin cytoskeleton. In addition, disrupting the cortical actin cytoskeleton with latrunculin results in alterations of TCR/CD3 signaling which offer several similarities with those observed in RacN17 cells. Thus, it is likely that at least part of the effects of Rac mutants on TCR signaling are exerted via the Rac-induced alterations of the actin cytoskeleton.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Molecular Pharmacology, Stanford University Medical Center, Center for Clinical Sciences Research, 269 Campus Drive, Stanford, CA 94305-5174
3 Address correspondence and reprint requests to Dr. Alain Trautmann, Centre National de la Recherche Scientifique, UMR 7627, Center Hospitalier Pitié-Salpêtrière/CERVI, 83 Bd de l’Hôpital, 75013, Paris, France.
4 Abbreviations used in this paper: IP3, inositol 1,4,5-trisphosphate; EV, empty vector; LAT, linker for activation of T cells; MFI, mean fluorescence intensity; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP5K, phosphatidylinositol-4-phosphate 5-kinase; PLC, phospholipase C; RacWT, Rac wild type; TRITC, tetramethylrhodamine isothiocyanate.
Received for publication July 22, 1999. Accepted for publication June 23, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 is an early key event in FcRIIA-mediated activation of human platelets. J. Biol. Chem. 273:24314.-chain and displays a unique activation-dependent phosphorylation pattern. J. Biol. Chem. 271:20705.-chain associates with the actin cytoskeleton upon activation of mature T lymphocytes. Immunity 3:623.[Medline]
1 in an SLP-76-deficient T cell. Science 281:413.This article has been cited by other articles:
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  C. M. Johnson, G. R. Chichili, and W. Rodgers Compartmentalization of Phosphatidylinositol 4,5-Bisphosphate Signaling Evidenced Using Targeted Phosphatases J. Biol. Chem., October 31, 2008; 283(44): 29920 - 29928. [Abstract] [Full Text] [PDF]
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 M. Humar, N. Andriopoulos, S. E. Pischke, T. Loop, R. Schmidt, A. Hoetzel, M. Roesslein, H. L. Pahl, K. K. Geiger, and B. H. J. Pannen Inhibition of Activator Protein 1 by Barbiturates Is Mediated by Differential Effects on Mitogen-Activated Protein Kinases and the Small G Proteins Ras and Rac-1 J. Pharmacol. Exp. Ther., December 1, 2004; 311(3): 1232 - 1240. [Abstract] [Full Text] [PDF]
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 E. J. Park, K.-A. Ji, S.-B. Jeon, W.-H. Choi, I.-o. Han, H.-J. You, J.-H. Kim, I. Jou, and E.-H. Joe Rac1 Contributes to Maximal Activation of STAT1 and STAT3 in IFN-{gamma}-Stimulated Rat Astrocytes J. Immunol., November 1, 2004; 173(9): 5697 - 5703. [Abstract] [Full Text] [PDF]
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S. Jordan and W. Rodgers T Cell Glycolipid-Enriched Membrane Domains Are Constitutively Assembled as Membrane Patches That Translocate to Immune Synapses J. Immunol., July 1, 2003; 171(1): 78 - 87. [Abstract] [Full Text] [PDF]
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 T. A Lyubchenko, G. A Wurth, and A. Zweifach The actin cytoskeleton and cytotoxic T lymphocytes: evidence for multiple roles that could affect granule exocytosis-dependent target cell killing J. Physiol., March 15, 2003; 547(3): 835 - 847. [Abstract] [Full Text] [PDF]
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 H. Nishihara, M. Maeda, A. Oda, M. Tsuda, H. Sawa, K. Nagashima, and S. Tanaka DOCK2 associates with CrkL and regulates Rac1 in human leukemia cell lines Blood, December 1, 2002; 100(12): 3968 - 3974. [Abstract] [Full Text] [PDF]
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M. M. Morgan, C. M. Labno, G. A. Van Seventer, M. F. Denny, D. B. Straus, and J. K. Burkhardt Superantigen-Induced T Cell:B Cell Conjugation Is Mediated by LFA-1 and Requires Signaling Through Lck, But Not ZAP-70 J. Immunol., November 15, 2001; 167(10): 5708 - 5718. [Abstract] [Full Text] [PDF]
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 H. Yu, D. Leitenberg, B. Li, and R. A. Flavell Deficiency of Small Gtpase Rac2 Affects T Cell Activation J. Exp. Med., October 1, 2001; 194(7): 915 - 926. [Abstract] [Full Text] [PDF]
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