A role for Wiskott-Aldrich syndrome protein in T-cell receptor-mediated transcriptional activation independent of actin polymerization.

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 (WASPDeltaC) 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.

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)(3)(4)(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 Ϫ/Ϫ mice, including impaired antigen receptor-induced activation and cytoskeletal rearrangements (6,7).
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 4,5diphosphate (PIP 2 ) 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 Ϫ/Ϫ mice show many similarities to T-cells from WASP Ϫ/Ϫ 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).
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.

EXPERIMENTAL PROCEDURES
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 350conjugated 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⌬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.
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 Ͼ100 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 Redconjugated 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 ϫ 10 7 ) were cotransfected by electroporation (250 V, 960 microfarads) with FLAG-tagged WASP-WT, WASP mutants, or vector control (pEF vector, 20 g) and a plasmid containing the luciferase reporter gene driven by the NFAT-responsive element (20 g). 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 M ionomycin for 5 h in the presence or absence of 1 M latrunculin A or 10 M 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⌬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 g) all in the pEF vector. Cells (1 ϫ 10 7 ) were transfected with WASP-WT, the WASP⌬C mutant, or the pEF vector control (20 g). Electroporated cells were transferred to a 10-cm dish containing 20 ml of prewarmed fresh RPMI 1640 medium. The cell suspension (100 l) 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 Ͼ50% 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.
Receptor Capping and Immunofluorescence Photomicroscopy-Jurkat T-cells were incubated at 37°C for 30 min in the absence or presence of 10 M 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% NaN 3 ). WASP⌬C and parent T-cell lines (2 ϫ 10 6 cells/ml) were incubated for 30 min on ice with anti-CD3 antibody (1 g/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 g/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 FACSCalibur TM with CellQuest TM software (Becton Dickinson).
Phospho-ERK Western Blotting-Parent vector control and WASP⌬C stable cells (1 ϫ 10 6 /ml) were stimulated with anti-CD3 antibody (1 g/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 milkcontaining 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).

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.

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 polymeri-

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. zation is directly coupled to TCR-mediated transcription, we generated a series of WASP deletion mutants and compared their functions in actin polymerization and transcriptional ac-tivation assays. We previously showed that overexpression of WASP-WT in PAE cells induces WASP clustering that colocalizes 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⌬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.
Cells expressing WASP⌬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.
Cells microinjected with the WASP⌬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.
The WH1 Domain of WASP Is Required for Co-clustering with Actin-Since the WASP⌬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. WASP⌬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 WASPdeficient mice that suggest the defect is downstream of early TCR activation in that all defined signaling pathways appear to be intact (5)(6)(7). T-cells from WASP Ϫ/Ϫ and Vav Ϫ/Ϫ mice exhibit normal tyrosine phosphorylation of Zap-70, TCR-, and other major substrates following TCR engagement (7,18). Furthermore, IB␣ phosphorylation and the c-Jun NH 2 -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.
To rule out the possibility that WASP⌬C acts in a dominant- Cells were plated on poly-L-lysine-coated slides. TCR and actin were visualized with anti-CD3 antibody (1 g/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 g/ml), followed by incubation with FITCconjugated anti-mouse antibody (1 g/ml). Cells were then fixed, and CD3 expression was analyzed by flow cytometry. The graph is a representative experiment of four independent measurements. 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 Ϫ/Ϫ 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 dominantnegative way to block actin polymerization and receptor capping during T-cell activation.
To rule out the possibility that NFAT activation by WASP⌬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).
The potentiation of NFAT activation by WASP⌬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)(23)(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)(23)(24). Binding of Cdc42⅐GTP therefore relieves this interaction, opening the molecule to allow multiple protein complexes to form.
The WH1 Domain of WASP⌬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 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.

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 M) (C) or cytochalasin D (Cyt.D; 10 M) (D), and lysates were analyzed for luciferase activity. 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 doubledomain 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.
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)(28)(29)(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⌬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.
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⌬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 ϳ50% 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.
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.