From Onyx Pharmaceuticals, Richmond, California 94806
Received for publication, November 28, 2000, and in revised form, March 23, 2001
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ABSTRACT |
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Wiskott-Aldrich syndrome protein (WASP) plays a
key role in cytoskeletal rearrangement and transcriptional activation
in T-cells. Recent evidence links WASP and related proteins to actin
polymerization by the Arp2/3 complex. To study whether the role of WASP
in actin polymerization is coupled to T-cell receptor (TCR)-mediated
transcriptional activation, we made a series of WASP deletion mutants
and tested them for actin co-localization, actin polymerization, and
transcriptional activation of NFAT. A WASP mutant with a deletion in
the C-terminal region (WASP Wiskott-Aldrich syndrome protein
(WASP)1 was originally
isolated by positional cloning and identified as the gene product
responsible for the X-linked recessive disorder known as
Wiskott-Aldrich syndrome (1). Clinical features of Wiskott-Aldrich
syndrome point to a link between WASP function, the actin cytoskeleton,
and transcriptional activation (2-5). The cellular changes observed in
T-cells include an altered cytoskeleton, abnormal cell morphology,
decreased size and density of cell surface microvilli, and
transmembrane signaling defects (5). Similar cellular abnormalities are
observed in WASP WASP contains a number of domains known to interact with both the
cytoskeleton and various signaling complexes. These include a
GTPase-binding domain (GBD); a polyproline-rich region capable of
binding Src homology 3 (SH3) domains; two conserved domains, WH1 and
WH2, postulated to be involved in the regulation of the actin
cytoskeleton; and a conserved acidic carboxyl-terminal domain that
associates with the Arp2/3 complex (3, 4). The modular organization of
these domains is known to be shared by several proteins, suggesting
that a conserved family of related proteins exists (9-12). The WASP
protein family currently includes WASP, N-WASP, Scar/WAVE, and
Las17p/Bee1p (9-12).
The presence of a binding domain for activated Cdc42 suggests that WASP
may provide a link between Rho GTPases and the actin cytoskeleton. We
have previously shown that overexpression of WASP in different cell
types has a profound effect on Cdc42-regulated actin polymerization
(8). Cdc42 has been implicated in reorganization of the actin
cytoskeleton in many cell types and has also been shown to play a role
in polarization of the T-cell cytoskeleton during antigen presentation
(13). However, the precise mechanism by which WASP regulates actin
polymerization is not known.
Recent findings indicate that the carboxyl terminus of WASP, including
the WH2 domain and acidic residues (referred to as the WH2-C domain;
also known as the verprolin/cofilin/acidic domain), enhances nucleation
of actin filaments by the Arp2/3 complex (12, 14). Moreover,
introduction of the WH2-C domain into Swiss 3T3 fibroblast cells is
sufficient to block membrane ruffling and to disrupt Arp2/3
localization (12, 15). Although the WH2-C motif is required for actin
nucleation by Arp2/3, other WASP domains may also play a role in
regulating this activity. Structural and biochemical analyses suggest
that the WH2-C domain of WASP interacts with the Arp2/3 complex only
when both phosphatidylinositol 4,5-diphosphate (PIP2) and Cdc42 bind to WASP (16, 17).
The restriction of WASP expression to hematopoietic cells, as well as
studies of T-cells from Wiskott-Aldrich syndrome patients and
WASP-deficient mice, indicates that WASP function is vital for T-cells
(6, 7). Although WASP has been shown to play a role in reorganization
of the actin cytoskeleton and transcriptional activation, the specific
function of WASP in T-cells remains unknown. T-cells from
Vav To characterize the role of WASP in T-cell activation and to test the
hypothesis that actin polymerization and transcriptional activation are
functionally linked, we mapped the domains within WASP required for
each of these processes. Our results demonstrate that regulation of
actin polymerization by WASP is independent of its role in TCR-mediated
transcriptional activation. Furthermore, deletional analysis reveals a
distinct region of WASP, the WH1 domain, that is required for
NFAT-dependent IL-2 transcription.
Antibodies--
Monoclonal anti-CD3 antibody (UCHT1) was
purchased from Pharmingen; monoclonal anti-CD28 antibody
(CD28.2) was purchased from Immunotech; and monoclonal anti-FLAG
antibody (M2) was purchased from Sigma. Secondary antibodies conjugated
to horseradish peroxidase (used in Western blotting), fluorescein, and
Texas Red (used in three-color immunofluorescence microscopy) were from
Bio-Rad and Jackson ImmunoResearch Laboratories, Inc. Alexa
350-conjugated secondary antibodies and Texas Red- or FITC-conjugated
phalloidin were purchased from Molecular Probes, Inc. Monoclonal
anti-phospho-ERK antibody (clone E10) was from New England
Biolabs Inc., and monoclonal anti-Zap-70 antibody (clone 2F3.2) was
from Upstate Biotechnology, Inc.
Epitope-tagged Expression Constructs--
FLAG epitope-tagged
WASP-WT and WASP mutant constructs were made by subcloning various
cDNA fragments into the NotI site of the pEFmycHisA
mammalian expression vector (Invitrogen). WASP-WT, WASP Microinjections and Immunofluorescence
Microscopy--
Microinjections and immunofluorescence microscopy were
carried out as previously described (20). PAE cells grown in
Dulbecco's modified Eagle's medium/nutrient mixture F-12 containing
10% fetal bovine serum were plated on coverslips. Expression vectors
encoding FLAG-tagged WASP constructs diluted to a concentration of 50 ng/ml in injection buffer (5 mM potassium glutamate and 130 mM KCl) were microinjected into the nuclei of >100
subconfluent PAE cells. Injected cells were incubated for 4-6 h at
37 °C. Cells were washed once with PBS and fixed in 4% formaldehyde
in PBS. Cells were permeabilized with PBS containing 0.1% Triton X-100
and incubated in the presence of primary monoclonal anti-FLAG
antibodies for 60 min. Coverslips were washed with PBS containing 0.1%
Tween 20 and incubated for 30 min with secondary Alexa 350-conjugated anti-mouse antibody. Cells were incubated with FITC-conjugated phalloidin to visualize F-actin and with Texas Red-conjugated DNase I
to visualize G-actin. Fluorescence microscopy was carried out on a
Zeiss Axiophot 100 with appropriate filters for fluorescence detection.
Transfections and Reporter Gene Analysis--
Jurkat cells
(1 × 107) were cotransfected by electroporation (250 V, 960 microfarads) with FLAG-tagged WASP-WT, WASP mutants, or vector
control (pEF vector, 20 µg) and a plasmid containing the luciferase
reporter gene driven by the NFAT-responsive element (20 µg). At
24 h post-transfection, cells were treated with 500 ng/ml soluble
anti-CD3 antibody (UCHT1), anti-CD3 plus 500 ng/ml anti-CD28 (CD28.2)
antibodies, or 25 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 µM ionomycin for 5 h in the presence or absence of 1 µM latrunculin A or 10 µM cytochalasin D
(where indicated). Cell lysates were analyzed for luciferase activity.
Each graph represents the mean of at least three independent
experiments. Error bars represent the S.D. of these experiments. Equal
expression levels of full-length FLAG-tagged WASP and WASP mutants were
verified by immunoblotting lysates with anti-FLAG epitope tag antibody. To derive WASP Receptor Capping and Immunofluorescence
Photomicroscopy--
Jurkat T-cells were incubated at 37 °C for 30 min in the absence or presence of 10 µM cytochalasin D
and stimulated with 500 ng/ml anti-CD3 antibody (UCHT1). Cells were
cytospun onto poly-L-lysine-coated slides, fixed in
3% paraformaldehyde, and permeabilized in 0.1% Triton X-100. To
visualize the CD3 complex or polymerized actin, the slides were
incubated with anti-CD3 antibody or Texas Red-conjugated phalloidin
(Molecular Probes), respectively. The anti-CD3 antibody was
detected with FITC-conjugated anti-mouse immunoglobulin. Fluorescence photomicroscopy was carried out on a Zeiss Axiophot with appropriate filter sets for epifluorescence detection of FITC or Texas Red signals.
Flow Cytometry Analysis--
Cells were resuspended in staining
buffer (PBS containing 1% fetal calf serum and 0.05%
NaN3). WASP Phospho-ERK Western Blotting--
Parent vector control and
WASP Actin Polymerization and Clustering in PAE Cells Require Discrete
WASP Domains--
Given the multifunctional domain structure of WASP
and its role in both signaling and cytoskeletal reorganization, WASP
likely represents a critical player that coordinates receptor-mediated signaling pathways with the actin cytoskeleton. Although WASP regulates
both cytoskeletal reorganization and transcriptional activation, it is
not clear whether TCR-mediated transcriptional activation by WASP is
controlled by a pathway that is independent of WASP-Arp2/3-directed
actin polymerization.
To determine whether the role of WASP in actin polymerization is
directly coupled to TCR-mediated transcription, we generated a series
of WASP deletion mutants and compared their functions in actin
polymerization and transcriptional activation assays. We previously
showed that overexpression of WASP-WT in PAE cells induces WASP
clustering that co-localizes with polymerized actin (8). To map the
region of WASP that is essential for WASP clustering and association
with F-actin (polymerized) or G-actin (monomeric), we microinjected
epitope-tagged WASP DNA constructs into PAE cells and immunostained for
WASP, G-actin, and F-actin (Figs. 1 and 2). WASP-WT-expressing cells had large extended cluster formations of
both F-actin and G-actin that co-localized with WASP (Fig. 2A). Similar results were
obtained for cells microinjected with the WASP
Cells expressing WASP
Cells microinjected with the WASP The WH1 Domain of WASP Is Required for Co-clustering with
Actin--
Since the WASP WASP
To rule out the possibility that WASP
To rule out the possibility that NFAT activation by WASP
The potentiation of NFAT activation by WASP The WH1 Domain of WASP
Numerous signaling proteins have been shown to interact both directly
and indirectly with the WH1 domain, including WIP and the SH3
domain-containing proteins Nck, Fyn, Vav, and Grb2 (3, 26). The
physiological relevance of these interactions remains unknown. The
WH1-related domain EVH1 is structurally similar to the pleckstrin
homology domain, containing a binding pocket for polyproline sequences
and basic residues (27-30). WH1 and EVH1 domains derived from
different proteins were found to bind proline-rich peptides with a
specific sequence motif (27, 30). For instance, WASP was shown to bind
the proline-rich peptide DFPPPPTDEEL derived from ActA (30). The
function of the proteins containing the WH1 domain implies that this
region acts to couple signaling pathways to actin polymerization. Our
results are consistent with such a hypothesis.
WASP
Several recent studies have linked cytoskeletal rearrangement to
signals originating from the TCR leading to downstream IL-2 transcription (3, 19). Our data suggest that the contribution of
WASP-Arp2/3-directed actin polymerization to the process of transcriptional activation of T-cells is marginal. To further address
whether the actin cytoskeleton plays a role in WASP
In summary, we have identified the WH1 domain of WASP as an important
domain required for TCR-mediated NFAT transcriptional activation in
T-cells. We demonstrated, by use of a dominant-active mutant for
actin polymerization (WASP-WH2-C), that this potentiation is
independent of WASP-Arp2/3-directed actin polymerization. Our data
suggest that the regulation of IL-2 transcription by WASP can be
uncoupled from its role in actin polymerization. The model in Fig.
7 describes a pivotal role for WASP in
integrating signals to activate both transcription and actin
polymerization. During TCR stimulation, activated Cdc42 binds to the
GBD of WASP to relieve intramolecular autoinhibitory interactions. The
active form of WASP can then deliver simultaneously at least two
signals through two different domains. The C terminus interacts with
the Arp2/3 complex to initiate actin polymerization, and the WH1 domain
binds to unknown protein(s), possibly WIP, to initiate transcriptional activation.
C) that is defective in actin
polymerization potentiated NFAT transcription following TCR activation
by anti-CD3 and anti-CD3/CD28 antibodies, but not by phorbol
12-myristate 13-acetate/ionomycin. Furthermore, cotransfection of a
dominant-active mutant (WASP-WH2-C) for Arp2/3 polymerization did not
inhibit NFAT activation. Finally, by analyzing a series of WASP
double-domain deletion mutants, we determined that the WASP homology-1
domain is responsible for NFAT transcriptional activation. Our results
suggest that WASP activates transcription following TCR stimulation in
a manner that is independent of its role in Arp2/3-directed actin polymerization.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
/
mice, including impaired
antigen receptor-induced activation and cytoskeletal rearrangements (6,
7).
/
mice show many similarities to T-cells
from WASP
/
mice, including impaired
proliferative responses and defective TCR capping following TCR/CD3
engagement (18). These findings suggest that the proliferative defects
of WASP- and Vav-deficient T-cells result from inactivation of a common
signaling pathway. These studies also suggest that assembly of the
actin cytoskeleton, receptor clustering, and capping may have a direct
role in connecting signals originating at the TCR to downstream IL-2
transcription (18, 19).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
GBD (deletion
of amino acids 235-268), and WASP
C (deletion of amino acids
444-502) were prepared as described previously (7). WASP
WH1
(deletion of amino acids 1-146) was made by subcloning the
StuI-EcoRV fragment into a blunt XbaI
site of the same vector. WASP
PPr (deletion of amino acids 310-420)
was made by removing the KasI-BanI fragment
containing the proline-rich region. WASP
WH2 (deletion of amino acids
423-449) was a gift from Dan Kalman (University of California, San
Francisco). WASP
WH1
C, WASP
GBD
C, and WASP
PPr
C double-domain deletion mutants were all constructed by introducing a
C-terminal nucleotide deletion at position 1364 (polymerase chain
reaction mutagenesis) in each of the various single-domain deletion
mutant constructs, resulting in a frameshift and generation of a stop
codon at position 444. Production of the correct truncated protein in
transfected cells was verified by Western blotting with anti-FLAG
antibody (M2). pRK5-MycWASP-WH2-C was a gift from Dr. Laura Machesky.
C cell lines, Jurkat cells were transfected with WASP
and WASP
C plasmids by electroporation as in the transient transfection procedure. Briefly, Jurkat cells were transfected by
electroporation at 250 V and 960 microfarads in 0.4-cm cuvettes with
mammalian expression constructs (20 µg) all in the pEF vector. Cells
(1 × 107) were transfected with WASP-WT, the WASP
C
mutant, or the pEF vector control (20 µg). Electroporated cells were
transferred to a 10-cm dish containing 20 ml of prewarmed fresh RPMI
1640 medium. The cell suspension (100 µl) was transferred to a
flat-bottom 96-well plate and incubated overnight. Transfected cells
were then selected 18-24 h post-transfection with 1 mg/ml G418 for 2-3 weeks. The medium was changed every 3-4 days, and positive clones
(those occupying >50% of the wells) were expanded into 12-well plates
containing selection medium. Positive clones were then confirmed by
Western blot analysis of cell lysates using anti-FLAG antibody. Each
graph represents the average of at least three separate experiments.
The relative luciferase activity described in Figs. 3-5 is
expressed as fold increase in the NFAT luciferase activity of various
WASP mutants (stimulated over unstimulated) over vector control.
C and parent T-cell lines (2 × 106 cells/ml) were incubated for 30 min on ice with
anti-CD3 antibody (1 µg/ml) or isotype-matched control IgG antibody
and then washed and incubated for an additional 30 min on ice with
FITC-conjugated anti-mouse antibody (1 µg/ml). Cells were warmed to
37 °C and incubated for 60 min, followed by fixation for 15 min in
4% paraformaldehyde. Stained cells were analyzed using a
FACSCaliburTM with CellQuestTM software (Becton Dickinson).
C stable cells (1 × 106/ml) were stimulated
with anti-CD3 antibody (1 µg/ml), pervanadate (0.02 mM),
or PMA/ionomycin. Cells were lysed in 1% Nonidet P-40 lysis buffer,
suspended in SDS sample buffer, separated on SDS-polyacrylamide gel,
and transferred to polyvinylidene difluoride membrane. The membranes
was blocked in 5% skim milk-containing PBS and then incubated with
anti-phospho-ERK antibody (New England Biolabs Inc.). After washing,
the membranes were incubated with a secondary mouse antibody linked to
horseradish peroxidase. Protein bands were subsequently detected with
the ECL chemiluminescence kit (Amersham Pharmacia Biotech).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
GBD or WASP
WH2
mutant (Fig. 2A). This is in contrast to a recent report
that described a role for the WH2 domain in actin polymerization and
co-clustering with WASP (21). It is possible that at higher levels of
WASP expression, the WH2 domain is not required for actin
polymerization.
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Fig. 1.
Wild-type WASP and deletion mutants.
Shown is a schematic representation of wild-type WASP and domain
deletion mutant structures used in this study. AR, acidic
region.
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Fig. 2.
Localization of WASP-WT and deletion mutants
with F-actin and G-actin in PAE cells. A, PAE cells
were microinjected with plasmids coding for FLAG-tagged WASP or WASP
deletion mutants, incubated for 4-6 h, fixed, and stained for
immunofluorescence. The fluorescence micrographs compare anti-FLAG
antibody (for expression), phalloidin (F-actin), and DNase I (G-actin).
B, PAE cells were microinjected with a series of plasmids
coding for FLAG-tagged WASP double-domain deletion mutants lacking the
WH1, GBD, or PPr domain in a WASP C background. Cells were treated as
described for A. C, PAE cells were co-injected
with the pEF vector control and green fluorescent protein
(GFP) plasmid constructs and treated as described for
A.
C had punctate staining throughout the
cytoplasm and co-localization of WASP
C with G-actin clusters (Fig.
2A). No F-actin clustering or polymerization was observed in
any of the cells expressing WASP
C. Recombinant WASP
C was also
inactive in in vitro pyrene-actin polymerization assays
(data not shown). These results suggest that the C-terminal 59 amino acids are essential for actin polymerization, whereas the remaining 443 amino acids retain the ability to co-localize with monomeric actin. Our
results, like those of Machesky et al. (14, 15), indicate
that the Arp2/3-interacting domain of WASP is critical for actin polymerization.
WH1 or WASP
PPr DNA construct had
a diffuse pattern of WASP staining (Fig. 2A). These two mutant proteins were detected throughout the cytoplasm and were not
co-localized with G-actin or F-actin clusters. Although our data
suggest that these regions are essential for WASP co-clustering and
co-localization with actin, the microinjection method may not fully
detect polymerized actin by a mutant that is defective in clustering.
Therefore, co-clustering of WASP with actin may be required for polymerization.
C mutant retains the ability to cluster
G-actin, it is likely that the region(s) responsible for clustering actin monomers resides in one or more of the remaining domains. To
further map the domain(s) on WASP required for G-actin co-localization and clustering, we constructed a series of WASP double-domain deletion
mutants lacking the WH1, GBD, or PPr domain in a WASP
C background.
As shown in Fig. 2B, WASP
WH1
C failed to cluster and
co-localize with G-actin. In contrast, WASP
PPr
C and
WASP
GBD
C were able to cluster or co-localize with G-actin like
the WASP
C mutant. These data indicate that the WH1 domain is
necessary for G-actin co-localization and co-clustering with WASP. Our
results also indicate that WASP clustering and G-actin clustering are linked to each other, in contrast to results reported by Kato et
al. (21). The discrepancies between our findings and those of Kato
et al. may be due either to different expression methods used (i.e. microinjection versus transient
transfection) or to cell type variations.
C Enhances TCR-mediated Transcriptional
Activation--
Recent studies with WASP-deficient T-cells suggest
that WASP links T-cell receptor engagement to cytoskeletal
reorganization, receptor clustering, and cap assembly (6, 7). Deletion
of the WASP gene has been shown to impair T-cell proliferation,
cytokine production, and IL-2 transcription (6, 7). Although numerous studies have suggested a role for WASP in actin reorganization and
transcriptional activation, it is not clear whether these two functions
are directly linked by WASP. To investigate the role of WASP in
TCR-mediated transcriptional activation, Jurkat T-cells were
cotransfected with either WASP or WASP deletion mutants and a reporter
gene driven by the NFAT-responsive element. Transfected Jurkat cells
stimulated with soluble anti-CD3 antibody revealed a selective
8-10-fold enhancement of activation of NFAT-dependent transcription by the C-terminally truncated WASP
C mutant (Fig. 3, A and B), but
not by any other mutants (Fig. 3B). Similar results were
seen when cells were stimulated with antibodies cross-linking both CD3
and CD28 (Fig. 3C). No statistically significant differences between the various WASP mutants were observed in cells stimulated with
soluble stimuli such as PMA and ionomycin (Fig. 3D).
Enhancement of NFAT activity was also observed in stable Jurkat cell
lines expressing the WASP
C mutant (Fig. 3, F and
G). Moreover, ERK phosphorylation was significantly
increased in the WASP
C cell line over vector control following
stimulation with either anti-CD3 antibody or pervanadate, but not with
PMA/ionomycin (Fig. 3H). ERK phosphorylation in the WASP-WT
cell line was comparable to that in the vector control cell line (data
not shown). These results indicate that enhancement of transcriptional
activation by WASP
C is dependent on proximal signals coming from the
TCR, but independent of CD28 co-stimulation or downstream signaling
events initiated by PMA and ionomycin. These data are consistent with
previous reports describing T-cells from Wiskott-Aldrich syndrome
patients and WASP-deficient mice that suggest the defect is downstream of early TCR activation in that all defined signaling pathways appear
to be intact (5-7). T-cells from WASP
/
and
Vav
/
mice exhibit normal tyrosine
phosphorylation of Zap-70, TCR-
, and other major substrates
following TCR engagement (7, 18). Furthermore, I
B
phosphorylation
and the c-Jun NH2-terminal kinase/stress-activated protein
kinase, p38 kinase, and mitogen-activated protein kinase pathways also
appear intact in these cells (7, 18). These data suggest that WASP may
function as a cytoskeletal scaffold, integrating one or more of these
pathways and thereby overcoming an activation threshold by assembling
receptors and signaling molecules at the cap.
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Fig. 3.
Activation of NFAT by the
WASP C mutant. Jurkat cells were
cotransfected with WASP or WASP deletion mutants and a plasmid
containing the luciferase reporter gene driven by the NFAT-responsive
element. A, after 24 h, cells were treated with soluble
anti-CD3 antibody, and the luciferase activity of WASP
C and vector
control was compared with that in unstimulated cells. B-D,
cells transfected with the indicated WASP constructs were treated with
anti-CD3 antibody, anti-CD3/CD28 antibodies, or PMA/ionomycin
(P/I), respectively, for 5 h. Lysates were then
analyzed for luciferase activity. The luciferase activity was
normalized and is expressed as relative activity over vector control.
E, shown are the results from Western blot analysis of
WASP-WT and deletion mutants using anti-FLAG antibody. F,
shown is a comparison of NFAT activation in stable Jurkat T-cell lines
expressing WASP-WT, WASP
C, or plasmid control. G, shown
are the results from Western blot analysis of cell lysates from stable
Jurkat T-cell lines expressing WASP-WT, WASP
C, or vector control
using anti-FLAG antibody. H, WASP
C or vector control
Jurkat cell lines were stimulated with the indicated stimulus, and cell
lysates were prepared and immunoblotted with anti-phospho-ERK antibody.
The membranes were reprobed with anti-Zap-70 antibody, indicating that
equal amounts of proteins were loaded in each lane (lower
panels). Io, ionomycin.
C acts in a dominant-negative
way to block TCR- and WASP-mediated actin polymerization, we examined
the induction of TCR capping and internalization in the WASP
C stable
cell line. It was previously shown that
WASP
/
cells are defective in actin-mediated
TCR capping and internalization (7). Normal ligand-induced capping was
observed in our WASP
C cell line as visualized by immunofluorescence
using anti-CD3 antibody (Fig.
4A). Furthermore, TCR-mediated
actin recruitment and polymerization at the cap were not
affected in the WASP
C cell line. In contrast, treating cells
with the actin inhibitor cytochalasin D completely blocked receptor
capping and actin polymerization. These data suggest that WASP
C does
not act in a dominant-negative way to block actin polymerization and
receptor capping during T-cell activation.
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Fig. 4.
A, WASP C does not block receptor
capping and internalization. WASP
C and control cell lines were
stimulated with soluble anti-CD3 antibody in the absence or presence of
10 µM cytochalasin D. Cells were plated on
poly-L-lysine-coated slides. TCR and actin were visualized
with anti-CD3 antibody (1 µg/ml) and with Texas Red-conjugated
phalloidin, respectively, as described under "Experimental
Procedures." B, WASP
C and parent Jurkat T-cell lines
were incubated with anti-CD3 antibody (1 µg/ml), followed by
incubation with FITC-conjugated anti-mouse antibody (1 µg/ml). Cells
were then fixed, and CD3 expression was analyzed by flow cytometry. The
graph is a representative experiment of four independent
measurements.
C may be a
consequence of aberrant TCR internalization, we tested the rate of
receptor endocytosis in WASP
C and control cell lines. Comparable TCR
internalization was detected in stable Jurkat T-cell lines expressing
WASP-WT, WASP
C, or vector control (Fig. 4B).
C may result from
stimulation of a novel signal transduction pathway (19). It is possible
that this mutant interacts with an unidentified protein(s) that
normally associates with activated endogenous WASP. In this model, WASP
may exist in one of two activation states (17, 22-24). It was recently
proposed that in the inactive state, the acidic C terminus of WASP
interacts with the basic region immediately upstream of the GBD
(22-24). Binding of Cdc42·GTP therefore relieves this interaction,
opening the molecule to allow multiple protein complexes to form.
C Is Required for TCR-mediated
Potentiation of NFAT--
It has recently been suggested that WASP
normally exists in an autoinhibited state similar to that observed in
the structurally related Cdc42 effector, PAK1 (23). In both cases, it
has been shown that the N-terminal half of the molecule interacts with the C terminus, thereby sequestering protein activity. We hypothesized that the NFAT potentiation observed with the C-terminally truncated WASP
C mutant might be due to a loss of this autoinhibition. To determine which of the remaining regions of WASP is responsible for
enhanced transcriptional activation, a series of WASP double-domain deletion mutants were constructed in a WASP
C background (Fig. 1).
Jurkat T-cells were then cotransfected with WASP
C or WASP
C double-domain deletion mutants and a reporter gene driven by the NFAT-responsive element. Cells transfected with WASP
GBD
C or WASP
PPr
C and stimulated with anti-CD3 antibody exhibited enhanced activation of the NFAT reporter by 8- and 7-fold, respectively (Fig.
5A). Cells transfected with
the WASP
WH1
C mutant and stimulated with anti-CD3 antibody did not
enhance NFAT activation, suggesting that the WH1 domain is required for
activation (Fig. 5A). In agreement with these results, a
recent study showed that WASP-interacting protein (WIP) and Vav
synergize to enhance NFAT-dependent transcription (25). WIP
was previously shown to interact with the WH1 domain of WASP, and
WIP-dependent NFAT activation requires the WASP-interacting region on WIP. (25, 26). Although our result clearly suggests that the
WH1 domain is required for transcriptional activation, we were unable
to effect NFAT activation with the WH1 domain alone (data not shown).
These results indicate that the WH1 domain is required but not
sufficient for transcriptional activation. In addition, it is possible
that the dominant-negative or dominant-positive effects could
not be achieved due to misfolding or mislocalization of the expressed
WH1 protein.
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Fig. 5.
The WH1 domain is required for NFAT
activation by WASP C. A, Jurkat
cells were cotransfected with WASP
C or WASP double-domain deletion
mutants and a plasmid containing the luciferase reporter gene driven by
the NFAT-responsive element. Cells were treated as described in the
legend to Fig. 3. B, shown are the results from Western blot
analysis of WASP
C and WASP double-domain deletion mutants using
anti-FLAG antibody.
C Potentiates Transcriptional Activation Independent of
WASP-Arp2/3-directed Actin Polymerization--
WASP
C did not
polymerize actin, but did activate NFAT. It is possible that endogenous
levels of WASP effected polymerization and subsequent transcriptional
activation. To determine whether activation of NFAT by WASP
C is
linked to WASP-directed actin polymerization, Jurkat T-cells were
cotransfected with WASP
C and WASP-WH2-C, a WASP mutant shown to be
dominant-active for WASP-Arp2/3-directed polymerization (Fig.
6, A and B) (31). Coexpression of WASP
C with WASP-WH2-C had little or no effect on NFAT activation (Fig. 6A). This suggests that the ability
of WASP to regulate IL-2 transcription can be uncoupled from its role
in actin polymerization.
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Fig. 6.
WASP C potentiation
of NFAT is independent of Arp2/3-mediated actin polymerization.
A and B, Jurkat cells were cotransfected with
WASP
C, WASP-WH2-C, or WASP
C and WASP-WH2-C and with NFAT
plasmids, followed by the treatment described in the legend to Fig. 3.
C and D, activation of NFAT by WASP
C is
sensitive to latrunculin A or cytochalasin D. Jurkat cells were
transfected with WASP or WASP
C and NFAT plasmids as described in the
legend to Fig. 3. After 24 h, cells were treated with soluble
anti-CD3 antibody, anti-CD3/CD28 antibodies, or PMA/ionomycin
(Io) for 5 h in the presence or absence of latrunculin
A (Lat A; 1 µM) (C) or cytochalasin
D (Cyt.D; 10 µM) (D), and lysates
were analyzed for luciferase activity.
C activation of
transcription, Jurkat T-cells were cotransfected with WASP
C and the
reporter gene driven by the NFAT-responsive element, followed by
treatment with an actin inhibitor (latrunculin A or cytochalasin D).
Transcriptional activation of NFAT by WASP
C following anti-CD3 or
anti-CD3/CD28 antibody stimulation was reduced by ~50% with both
agents (Fig. 6, C and D). In addition, no
statistically significant changes were observed in latrunculin A- or
cytochalasin D-treated cells stimulated with PMA/ionomycin (Fig.
6, C and D). This confirms that TCR-mediated
transcriptional activation of NFAT by WASP
C requires an intact actin cytoskeleton.
View larger version (18K):
[in a new window]
Fig. 7.
WASP integrates TCR-mediated transcriptional
activation with actin polymerization. See "Results and
Discussion" for details.
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ACKNOWLEDGEMENTS |
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We thank Drs. J. Crabtree, A. Weiss, L. Machesky, and D. Kalman for plasmids; Drs. J. Chernoff, D. Drubin, G. Servant, and M. Welch for critical review of the manuscript; and Dr. P. Paz for many helpful discussions.
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FOOTNOTES |
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* This work was supported by Grant RO1 GM56478-01A1 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Onyx Pharmaceuticals,
3031 Research Dr., Richmond, CA 94806. Tel.: 510-262-8757; Fax:
510-222-9758; E-mail: aabo@onyx-pharm.com.
Published, JBC Papers in Press, March 30, 2001, DOI 10.1074/jbc.M010729200
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ABBREVIATIONS |
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The abbreviations used are: WASP, Wiskott-Aldrich syndrome protein; GBD, GTPase-binding domain; SH, Src homology; WH, WASP homology; Arp, actin-related protein; TCR, T-cell receptor; IL-2, interleukin-2; NFAT, nuclear factor of activated T-cells; FITC, fluorescein isothiocyanate; ERK, extracellular signal-regulated kinase; WT, wild-type; PPr, polyproline-rich; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; WIP, WASP-interacting protein; PAE, porcine aortic endothelial.
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