PMC:1887720 / 19001-52876 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/1887720","sourcedb":"PMC","sourceid":"1887720","source_url":"https://www.ncbi.nlm.nih.gov/pmc/1887720","text":"Results and Discussion\n\nTCR-induced Proliferation and Actin Polymerization Proceed Normally in WAS−/−ΔGBD Mice.\nAlthough activated cdc42 can bind WASp and trigger WASp VCA domain–mediated Arp2/3 activity (2, 3), it is unclear whether this interaction is required and/or solely responsible for induction of WASp effector function after TCR stimulation. To address this issue, the relevance of the GBD–cdc42 interaction to WASp effects on T cell activation was studied using mice in which a CD2 promoter/enhancer-driven WASp cDNA lacking the GBD (WASpΔGBD; Fig. 1 A) was expressed on the C57BL/6 WAS−/− background. After confirmation that the WASpΔGBD species does not bind activated cdc42 (Fig. 1 B) and demonstration that the WASpΔGBD transgene is expressed in T lineage cells (Fig. 1 C), WAS−/−ΔGBD mice were characterized with respect to T cell phenotype and function. In contrast to WAS−/− mice, in which thymocyte and peripheral T cell numbers are markedly reduced and thymocyte maturation impaired (18), the WAS−/−ΔGBD mice showed no defects in either T cell numbers or development (not depicted). Analysis of TCR-induced proliferation also revealed proliferative responses to anti-CD3 antibody or anti-CD3 and anti-CD28 antibodies to be essentially comparable in WAS−/−ΔGBD and control thymocytes and peripheral T cells, and markedly reduced in WAS−/− cells (Fig. 1 D). Similarly, immunofluorescence analysis of FITC phalloidin–stained WAS−/−ΔGBD thymocytes revealed CD3/CD28-induced actin polymerization, which is abrogated in WAS−/− cells, to be essentially normal in WAS−/−ΔGBD cells (Fig. 1 E). Together, these observations indicate the coupling of TCR engagement to proliferation and actin polymerization to be unaffected by absence of the WASp GBD and thus imply that WASp effects on TCR-induced transcriptional activation and cytoskeletal arrangement can be evoked by mechanisms other than cdc42–GBD interaction.\nFigure 1. The GBD of WASp is not essential for WASp activity. (A) Scheme that shows the domain organization of wild-type and a GBD-deleted (WASpΔGBD) WASp cDNA used to derive WAS−/−ΔGBD mice. Positions of all tyrosine residues within WASp are indicated. (B) Lysates obtained from WASp or WASpΔGBD-expressing Cos-7 cells were incubated with GTPγS-loaded GST-cdc42 fusion protein bound to glutathione sepharose beads and the complexes were then resolved by SDS-PAGE and sequential immunoblotting analysis with anti-WASp and anti-cdc42 antibodies (top two panels). Immunoblotting analysis demonstrating WASp and WASpΔGBD expression in Cos-7 transfectants (Tfn) is shown in the bottom panel. (C) Immunoblotting analysis showing the WASp species detected in thymocytes and lymphocytes from wild-type (WT), WAS−/−, and WAS−/−ΔGBD mice using an anti-WASp antibody. (D) Lymphocytes and thymocytes isolated from 4–8-wk-old wild-type (WT), WAS−/−, and WAS−/−ΔGBD mice were cultured for 48 h in medium alone or with either 1 μg/ml anti-CD3 antibody or 0.2 μg/ml anti-CD3 plus anti-CD28 antibody. Antigen receptor–evoked proliferative responses were determined after an 18-h pulse with [3H]thymidine. Values represent the means (± SEM) of four independent experiments. (E) Thymocytes from wild-type (WT), WAS−/−, and WAS−/−ΔGBD mice were isolated and stimulated with anti-CD3 plus anti-CD28 antibodies for 30 min on ice followed by cross-linking with a secondary antibody. Cells were fixed with 5% paraformaldehyde and F-actin content was quantified by flow cytometric analysis of FITC phalloidin–stained resting (bold lines) and stimulated (dashed lines) cells. The results are representative of three independent experiments.\n\nPhosphorylation of WASp Tyrosine 291 Is Induced by TCR Engagement and Required for Induction of NFAT Translocation, Actin Polymerization, and Synapse Formation.\nThe negligible overt T cell functional deficit observed in WAS−/− ΔGBD mice implies that WASp activation in T cells might be mediated through non-GBD–dependent mechanisms. Data showing WASp or its orthologue, N-WASp, to undergo inducible tyrosine phosphorylation in B cells, platelets, and neurons (14, 21, 22) raise the possibility that TCR-evoked WASp tyrosine phosphorylation is relevant to the regulation of WASp function in T cells. To determine whether WASp is inducibly tyrosine phosphorylated after T cell stimulation, WASp immunoprecipitates from resting and TCR-stimulated Jurkat cells were subjected to anti-pTyr immunoblotting analysis. This analysis revealed WASp to be tyrosine phosphorylated constitutively in these cells, but to undergo a marked increase in tyrosine phosphorylation after TCR engagement (Fig. 2 A). Moreover, both basal and TCR-evoked WASp tyrosine phosphorylation levels appeared equivalent in thymocytes from wild-type and WAS−/−ΔGBD mice, suggesting that in T cells, WASp can be inducibly tyrosine phosphorylated in the absence of its interaction with activated cdc42 (Fig. 2 B). To determine which of the seven tyrosine resides within WASp (Fig. 1 A) undergo TCR-induced phosphorylation in vivo, cDNAs in which each tyrosine residue was individually replaced with phenylalanine (Y→F) were expressed as GFP-tagged proteins in Jurkat cells and phosphorylation status of the mutant proteins was examined after cell stimulation. As revealed by anti-pTyr immunoblotting analysis of anti-GFP immunoprecipitates from the transfected cells, induction of WASp tyrosine phosphorylation was unaffected by mutation at six of the seven WASp tyrosine sites, but was abrogated in cells expressing WASpY291F (Fig. 2 C). These findings indicate that TCR engagement triggers the tyrosine phosphorylation of WASp and demonstrate that Y291, a residue known to be selectively targeted by Btk (14), represents the major and likely only tyrosine site on WASp targeted for phosphorylation after TCR stimulation.\nFigure 2. TCR-induced phosphorylation of WASp Y291 is required for induction of NFAT activity, actin polymerization, and immunological synapse formation. (A) Lysates prepared from resting or TCR-stimulated Jurkat T cells at the indicated times after stimulation were immunoprecipitated with anti-WASp antibody or control IgG subjected to SDS-PAGE and sequential immunoblotting analysis with anti-pTyr and anti-WASp antibodies. (B) Lysates prepared from wild-type (WT) or WAS−/−ΔGBD thymocytes at the indicated times after stimulation were immunoprecipitated with anti-WASp antibody or control IgG and the complexes were then subjected to SDS-PAGE and sequential immunoblotting analysis with anti-pTyr and anti-WASp antibodies. (C) Jurkat cells were transfected with pEGFP vector containing either the wild-type (WT) WASp cDNA or WASp cDNAs carrying each of the indicated Y→F substitutions. Lysates prepared from the TCR-stimulated cells were immunoprecipitated with anti-GFP antibody and the immunoprecipitated proteins were then subjected to SDS-PAGE and sequential immunoblotting analysis with anti-pTyr and anti-GFP antibodies. (D) Thymocytes from WAS−/− mice were cotransfected with pEGFP vectors containing WASp or the indicated WASp tyrosine phenylalanine (Y→F) mutant cDNAs and an NFAT luciferase reporter vector. At 4 h after transfection, cells were stimulated with anti-CD3 and anti-CD28 antibodies. After 8 h of incubation, cells were lysed and assayed by luminometry for luciferase expression. Values represent the means (± SEM) of three assays and the results are representative of four independent experiments. (E) Thymocytes from WAS−/− mice were transfected with pEGFP-WASp, WASpY291F, WASpY212F, or WASpY88F and the cells were either left unstimulated or stimulated with anti-CD3 and anti-CD28 antibodies for 30 min on ice followed by cross-linking with anti–hamster Ig secondary antibody. Cells were fixed with 5% paraformaldehyde and F-actin content was quantified by flow cytometric analysis of FITC phalloidin–stained cells. The results are representative of three independent experiments. (F) Lymphocytes from WAS−/−/OT-II mice were either untreated (a) or transfected with pEGFP-WASp (b), pEGFP-WASpY102F (c), or pEGFP-WASpY291F (d) and the cells were then incubated with OVA329–339–pulsed LB27.4 cells, fixed, and stained for actin and PKC-θ, and then visualized by immunofluorescent microscopy. The images on the far right of each panel represent merges of the other three images within the panel. Data shown are representative of four independent experiments. To examine the biologic relevance of WASp tyrosine phosphorylation in T cells and specifically determine whether Y291 phosphorylation modulates WASp function, WAS−/− T cells were reconstituted with the WASp Y→F mutants and the cells were then assayed for NFAT transcriptional activity and actin polymerization. As revealed using an NFAT luciferase reporter, induction of NFAT activity after CD3 or CD3/CD28 stimulation was profoundly reduced in WASp Y291F relative to wild-type WASp-expressing cells, but unaffected in the context of other WASp tyrosine mutations (Fig. 2 D). Similarly, immunofluorescence analysis of phalloidin-labeled WAS−/− thymocytes expressing either wild-type or tyrosine-substituted WASp mutants, revealed induction of actin polymerization to be disrupted by either WASp deficiency or WASpY291F expression (Fig. 2 E), but to proceed normally in cells expressing wild-type WASp, WASpY88F, WASpY212F (Fig. 2 E), or any of the other WASp mutants (not depicted). Therefore, Y291 phosphorylation modulates and is, in fact, required for WASp contribution to the induction of transcriptional activation and actin polymerization after TCR engagement.\nThe critical role for WASp Y291 phosphorylation in linking TCR engagement to actin polymerization implies that WASp effects on actin-mediated processes associated with T cell activation are also dependent on its phosphorylation at this site. To address this possibility, the effects of Y291F mutation on WASp capacity to promote immunological synapse formation after TCR stimulation were investigated using T cells from WAS−/− mice expressing a TCR transgene (OT-II) that specifically recognizes an epitope of OVA. For those studies, WAS−/−/OT-II T cells transfected with wild-type or the tyrosine-mutated WASp cDNAs were stimulated with OVA-pulsed APCs and synapse formation was then evaluated using immunofluorescence microscopy to monitor accumulation of polymerized actin and PKC-θ at the T cell–APC interface of developing conjugates. As is consistent with the essential role identified for WASp in synapse development (5), actin and PKC-θ accumulation indicative of synapse formation at the T cell–APC contact site was absent in WAS−/−/OT-II cells, but was fully restored by expression of wild-type WASp (Fig. 2 F, a and b). Induction of synapse formation in these cells was also restored by expression of WASp Y102F (Fig. 2 F, c) and all other WASp tyrosine mutants (not depicted) with the exception of WASpY291F, the latter of which translocated to the synaptic region after cell stimulation, but failed to evoke the actin reorganization and PKC-θ localization within this region that demarcate synapse formation (Fig. 2 F, d). These data suggest that WASp translocation to the T cell–APC contact zone occurs independently of its tyrosine phosphorylation, whereas formation of the immunological synapse after TCR engagement requires not only WASp, but also phosphorylation of WASp at Y291. Together, these findings reveal phosphorylation at the Y291 site to be critical for activation of WASp effector function in T cells and thus imply that this modification enables the release of WASp from autoinhibitory structural constraints.\n\nTyrosine Phosphorylation of WASp Is Regulated by Fyn and PTP-PEST.\nPhosphorylation of WASp at Y291 by both Btk and Hck has been demonstrated in exogenous expression systems (14, 15), but the PTKs modulating Y291 phosphorylation in vivo in T cells have not been defined. Similarly, although WASp has been shown to bind GST fusion proteins containing the Itk, Lck, or Fyn SH3 domains and associate with Fyn in transformed monocytes (16–18), the relevance of these interactions to the in vivo induction of WASp tyrosine phosphorylation in T cells remains unknown. To address these issues, induction of WASp tyrosine phosphorylation was evaluated in T cells deficient for Itk, Lck, or Fyn. As shown in Fig. 3 A, immunoblotting analysis revealed no differences in the levels of WASp tyrosine phosphorylation elicited in T cells from Itk-deficient compared with wild-type mice. Similarly, comparisons between Lck-deficient JCam-1 cells and JCam-1 cells reconstituted for Lck expression revealed induction of WASp tyrosine phosphorylation to be unaffected by Lck deficiency. By contrast, T cells from Fyn-deficient mice showed no detectable increase in tyrosine phosphorylation in response to TCR engagement. Moreover, TCR-elicited WASp tyrosine phosphorylation was markedly lower in Jurkat cells coexpressing wild-type and a catalytically inert Fyn (FynK296M) protein (23) compared with cells expressing only wild-type Fyn and was essentially abrogated by the sole overexpression of FynK296M (Fig. 3 B). These data indicate that Fyn, but not Itk or Lck, is required for WASp tyrosine phosphorylation and raise the possibility that WASp might be exclusively tyrosine phosphorylated by Fyn in T cells. Importantly, the coexpression in Jurkat cells of Fyn with either dominant negative (cdc42-N17) or activated (cdc42-V12) cdc42 mutant proteins had no effect on TCR-induced WASp tyrosine phosphorylation, implying that Fyn phosphorylates WASp independently of cdc42 activity (Fig. 3 C).\nFigure 3. Fyn binds and phosphorylates WASp after TCR engagement. (A) Lymphocytes from Itk−/−, Fyn−/−, and wild-type mice as well as JCam-1 (Lck-deficient) and Lck-reconstituted JCam-1 (JCam + Lck) cells were stimulated with anti-CD3/anti-CD28 antibodies and cell lysates were then prepared and immunoprecipitated with anti-WASp antibody. Complexes were subjected to SDS-PAGE and sequential immunoblotting analysis with anti-pTyr and anti-WASp antibodies. (B) Lysates were prepared from anti-CD3/anti-CD28–stimulated Jurkat cell transfectants expressing cDNAs for wild-type Fyn and FynK296M alone or in combination. After immunoprecipitation with anti-WASp antibody, complexes were subjected to immunoblotting analysis using anti-pTyr and then anti-WASp antibodies (top two panels). Fyn expression levels in the transfected cells lysates are shown in the bottom panel. (C) Lysates were prepared from unstimulated (U) or stimulated (S) Jurkat cells cotransfected with Fyn, WASp, and one of either the cdc42 wild-type (WT), the cdc42-V12, or N17 mutant cDNAs. Lysate proteins were immunoprecipitated with anti-WASp antibody and subjected to SDS-PAGE and sequential immunoblotting with anti-pTyr and anti-WASp antibodies (top two panels). Expression of Fyn and cdc42 in the lysates is shown in the bottom two panels. (D) Jurkat cells were stimulated for the indicated time periods with anti-CD3 and anti-CD28 antibodies, lysed, and the lysate proteins were immunoprecipitated with anti-WASp antibody. Complexes, lysate proteins, and IgG were subjected to SDS-PAGE and sequential immunoblotting analysis with anti-Fyn and anti-WASp antibodies. (E) Purified His-tagged Fyn fusion protein was incubated with GST-WASp, GST-WASpΔPro, or GST fusion proteins bound to glutathione sepharose beads. The complexes were washed and subjected to SDS-PAGE and sequential immunoblotting with anti-Fyn and anti-GST antibodies. (F) Jurkat cells were stimulated, lysed, and the lysate proteins were subjected to anti-Fyn antibody immunoprecipitation. Immunoprecipitates were then incubated in kinase buffer containing 10 μg [γ-32P] ATP and either GST, GST-WASp, GST-WASpΔPro, or GST-WASp Y291F fusion proteins bound to glutathione sepharose beads. Complexes were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and phosphorylation was analyzed by autoradiography. The position of phosphorylated GST-WASp is indicated. This result is representative of four independent assays. Because these findings suggest that WASp is a Fyn substrate, Fyn association with WASp was evaluated in Jurkat cells. As shown in Fig. 3 D, these proteins inducibly interact with one another after CD3/CD28 stimulation. The capacity of Fyn to bind directly to WASp was also examined by incubating polyhistidine-tagged Fyn (His Fyn) with glutathione sepharose–bound GST fusion proteins containing wild-type WASp or a WASp mutant lacking the proline-rich region (WASpΔPro). Anti-Fyn immunoblotting analysis of these complexes revealed interaction of His Fyn proteins with the GST-WASp fusion protein, but not with control GST or GST-WASpΔPro proteins (Fig. 3 E). Thus, the association of Fyn with WASp is direct and requires the WASp proline-rich region and its likely binding to the Fyn SH3 domain.\nTo determine whether Fyn directly phosphorylates WASp, the effects of Fyn on the WASp phosphorylation state were assessed using an in vitro kinase assay. As shown in Fig. 3 F, Fyn immunoprecipitates from activated T cells induced the tyrosine phosphorylation of WASp, but neither WASpΔPro nor WASpY291F mutant proteins were phosphorylated in this assay. These findings suggest that Fyn-mediated WASp phosphorylation requires the physical association of these proteins and that it is Y291 and no other WASp tyrosine residue that is targeted by Fyn during T cell activation.\nThe relationship between tyrosine phosphorylation and WASp effector activity in T cells implies that PTP-mediated dephosphorylation of WASp is also important to WASp functions in T cell activation. Previous studies of WASp ligands in T cells have revealed that WASp inducibly associates with a cytosolic adaptor, PSTPIP1, also known to bind a hemopoietic PTP, PTP-PEST (5, 24). These observations raise the possibility that WASp might be juxtaposed to and thereby dephosphorylated by PST-PEST via its association with PSTPIP1, a paradigm observed in relation to PTP-PEST–mediated dephosphorylation of the c-Ab1 PTK (25). To determine whether these effectors associate with one another in T cells, PSTPIP1 immunoprecipitates from CD3/CD28-stimulated T cells were examined for the presence of WASp and PTP-PEST. As shown in Fig. 4 A, both WASp and PTP-PEST coimmunoprecipitated with PSTPIP1. PTP-PEST was also detected in WASp immunoprecipitates from stimulated T cells (Fig. 4 B), but recombinant WASp and PST-PEST did not associate in an in vitro binding assay, suggesting that their interaction is indirect and possibly mediated via PSTPIP1. To address this possibility and extend data on the structural basis for PSTPIP–PTP-PEST interaction (26), GST fusion proteins containing full-length PSTPIP1 or either of its major protein-binding domains (a coiled coil and an SH3 domain) were assessed for capacity to precipitate PTP-PEST from stimulated T cells. Results of this analysis revealed the interaction of PTP-PEST with GST fusion proteins containing full-length PSTPIP1 or the PSTPIP1 coiled coil region alone, but not with GST-PSTPIP1 SH3 domain fusion protein (Fig. 4 C). As PSTPIP has previously been shown to bind via its SH3 domain to WASp (5), these findings suggest that WASp and PST-PEST interact via their mutual binding to PSTPIP. Because these three proteins have each been implicated in actin cytoskeletal rearrangement (1, 23, 27), the functional relevance of a trimolecular WASp–PSTPIP1–PTP-PEST interaction was investigated using fluorescence microscopy to evaluate the localization of these proteins with respect to one another and to actin. As shown in Fig. 4 D, rhodamine phalloidin staining of Cos-7 cells expressing GFP-tagged WASp, PSTPIP1, or PTP-PEST revealed each of these proteins to be highly or entirely colocalized with actin structures, WASp being concentrated in actin-rich perinuclear aggregates, and PSTPIP1 and PTP-PEST localizing to actin fibrillary networks spanning the cytoplasm. Coexpression of PSTPIP1 and PTP-PEST revealed these protein to be colocalized and distributed similarly as when expressed alone (Fig. 4 D, d). When coexpressed with PSTPIP1 and PTP-PEST, WASp also showed a distribution pattern overlapping that of PSTPIP1/PTP-PEST (Fig. 4 D, e). Thus, as shown for PSTPIP1 (5), PSTPIP1/PTP-PEST association with WASp appears to evoke WASp relocalization such that these three effectors colocalize within the actin cytoskeleton, as is suggestive of a biological role for PSTPIP1-mediated linkage of WASp to PTP-PEST.\nFigure 4. WASp inducibly associates and colocalizes with PSTPIP1 and PTP-PEST. (A) Jurkat T cells were stimulated for the indicated times with anti-CD3 and anti-CD28 antibodies and lysates were then prepared and immunoprecipitated with anti-PSTPIP1 antibody. The immune complexes were subjected to SDS-PAGE and sequentially immunoblotted with anti-PST-PEST, anti-WASp, and anti-PSTPIP1 antibodies. (B) Jurkat T cells were stimulated with anti-CD3 and anti-CD28 antibodies and lysates were then prepared and immunoprecipitated with anti-WASp antibody. Complexes were resolved by SDS-PAGE followed by sequential immunoblotting with anti–PTP-PEST and anti-WASp antibodies. (C) Lysates prepared from Jurkat T cells were incubated with GST, GST-PSTPIP1, full-length (FL), GST-PSTPIPCOIL, or GST-PSTPIPSH3 fusion proteins bound to glutathione- sepharose beads. Complexes were resolved by SDS-PAGE and immunoblotted using an anti–PTP-PEST antibody. (D) Cos-7 cells were transiently transfected with pEGFP-PSTPIP1 (a), pEGFP-PTP-PEST (b), pEGFP-WASp (c), pEGFP-PSTPIP1 and pcΔNA3-PTP-PEST (d), or pEGFP-PSTPIP1, DSRED-WASp, and pcDNA3-PTP-PEST (e). Cells were fixed, stained with rhodamine phalloidin for actin (a–c) or with anti–PTP-PEST antibody (d and e) and Cy5 anti–rabbit Ig (e), and then analyzed by confocal immunofluorescent microscopy. The images shown are representative of three independent experiments. (E) pcDNA3 constructs for expression of wild-type or catalytically inactive (C231S) PTP-PEST were cotransfected with pEGFP-WASp (WASp-GFP) into Jurkat cells. The cells were either left unstimulated or stimulated with anti-CD3 and anti-CD28 antibodies, lysed, and the lysate proteins were immunoprecipitated with anti-GFP antibodies. The complexes were subjected to SDS-PAGE and immunoblotted sequentially with anti–p-Tyr and anti-GFP antibodies. In view of its capacity to associate and colocalize with PTP-PEST, the potential for WASp to serve as a PTP-PEST substrate in T cells was examined by coexpressing WASp with PTP-PEST or a catalytically inactive form of PTP-PEST (PTP-PEST C231S) in Jurkat cells and examining these cells with respect to inducible WASp tyrosine phosphorylation status. As shown in Fig. 4 E, TCR-induced WASp tyrosine phosphorylation in these cells is essentially abrogated by PTP-PEST overexpression, but is enhanced by expression of catalytically inactive and likely dominant negative–acting PTP-PEST C231S. Thus, PTP-PEST–mediated tyrosine dephosphorylation may counteract Fyn-mediated phosphorylation of WASp after T cell stimulation and, by extension, WASp contributions to T cell activation might be modulated by the relative balance of these respective enzyme activities.\n\nFyn and PTP-PEST Modulate WASp Effects on Induction of Actin Polymerization and Immunological Synapse Formation.\nIn view of the effects of Fyn and PTP-PEST on WASp tyrosine phosphorylation and relevance of Y291 phosphorylation to WASp function, the possibility that Fyn and/or PTP-PEST modulate the ability of WASp to activate the Arp2/3 complex was directly tested using a pyrene fluorescence in vitro assay of actin polymerization. As shown in Fig. 5 A, WASp alone stimulated some polymerization of the pyrene-labeled actin, but the effect of WASp on actin polymerization was dramatically increased by the addition of Fyn. Because WASp effector activity can also be stimulated by cdc42 (1, 3), the efficacies of Fyn and cdc42 in triggering either WASp or WASpΔGBD-mediated actin polymerization were also compared using a GST fusion protein containing constitutively active cdc42 (cdc42-V12). Although GST–cdc42-V12 substantively enhanced WASp-mediated actin polymerization, when added at a concentration generating maximum Arp2/3 activation, its effects on actin polymerization were no greater than those induced by Fyn and were not increased by the addition of GST-Fyn (Fig. 5 A). Similarly, the addition of either GST-Fyn or GST–cdc42-V12 alone or in combination at concentrations yielding lower levels of WASp-Arp2/3 activation revealed the effects of cdc42-V12 and Fyn together to be less than additive (Fig. 5 B). Thus, although Fyn and cdc42 both stimulate WASp-mediated actin polymerization, they do not act synergistically to activate WASp. By contrast, the addition of PTP-PEST and PSTPIP1 had a dramatic effect on Fyn's capacity to stimulate WASp activity with the level of WASp-Arp2/3 activity evoked by the combination of Fyn with PTP-PEST and PSTPIP1 being comparable to that triggered by WASp alone and being even further diminished by the combination of PTP-PEST and PSTPIP1 (Fig. 5 A). Thus, as is consistent with a key role for tyrosine phosphorylation in regulating WASp function, these data reveal WASp's effects on actin polymerization to be modulated by both Fyn and PTP-PEST/PSTPIP1. The data also reveal Fyn to be as efficacious as cdc42 in stimulating WASp-Arp2/3 activity and modulating this activity independently of the WASp GBD.\nFigure 5. Fyn and PTP-PEST modulate WASp effects on induction of actin polymerization and synapse formation. (A) Polymerization of 2.8 μM pyrene-labeled actin monomer was assayed in the presence of 20 nM Arp2/3 complex, 100 nM GST-WASp or GST-WASpΔGBD, and GST fusion proteins containing 50–500 nM either Fyn, PTP-PEST, PSTPIP, or cdc42-V12. Polymerization was monitored by the increase in prenyl-actin fluorescence. B. The pyrene actin assay was used to compare the WASp-Arp2/3–actin polymerizing activities of cdc42 at low (15 nM), medium (250 nM), or high (500 nM) concentration and Fyn at low (10 nM), medium (100 nM), or high (200 nM) concentration alone or in combination. (C) Fyn effects on synapse formation were evaluated by incubating lymphocytes from OT-II (a and b) and Fyn−/−/OT-II (c) mice with unpulsed (a) or OVA peptide–pulsed (b and c) LB27.4 cells followed by cell fixation, staining for WASp, Fyn, and actin, and visualization by immunofluorescent microscopy. Images on the far left of each panel represent a merge of the other three images within each panel. A computer-generated three-dimensional reconstruction of the synaptic region formed between wild-type T cells and APCs (d) shows the localization of Fyn in the central area of the synapse and the distribution of WASp in both the central and peripheral synaptic region. Synapses were quantified (e) by counting the numbers of T cell–APC conjugates showing clustered actin at the conjugation site. Values shown are the percent of conjugates with synapse formation and represent means (± SEM) of three independent experiments. (D) PTP-PEST effects on synapse formation were assessed using WAS−/−/OT-II lymphocytes transfected with pDSRED-WASp (a), pcDNA3-WASp and pDSRED-PSTPIP1 (b), or pcDNA3-WASp, pEGFP-PSTPIP1, and pDSRED-PTP-PEST (c). Cells were incubated with OVA peptide–pulsed LB27.4 B cells, fixed, and stained for actin and/or PKC-θ, and visualized by immunofluorescent microscopy. The image on the far right of each panel is a merged image of all other images in the panel. Synapses were quantified by counting the number of T cell–B cell conjugates showing clustered actin at the synaptic site. Values shown are the percent of conjugates with synapse formation and represent the means (± SEM) of three independent experiments. To directly establish the relevance of Fyn and PTP-PEST to WASp functions in T cells, the capacity of these effectors to modulate WASp effects on immunological synapse formation was also investigated using the OT-II transgenic mice as well as mice expressing the OT-II transgene on the Fyn−/− background. As shown in Fig. 5 C, stimulation of T cells from OT-II TCR mice with OVA peptide–pulsed APCs induced translocation of both WASp and Fyn to the T cell–APC interface where they colocalized with a region of intense actin accumulation demarcating the developing synapse. Three-dimensional reconstruction and 90°C rotation of the interface area confirmed published data indicating Fyn to be concentrated centrally within the synapse (28) and revealed WASp to be distributed between both the central and peripheral synaptic region. By contrast, synapse formation, as revealed by actin accumulation at the T cell–APC interface, was essentially not detectable in conjugates formed between Fyn−/−/OT-II cells and antigen-pulsed APCs (Fig. 5 C, c and e). Synapse formation was also found to be impaired by the coexpression of PTP-PEST with WASp in WAS−/−/OT-II cells with percentages of synapses formed being at least 50% lower than observed between APCs and WASp-expressing cells (Fig. 5 D, d). As is consistent with the role of PSTPIP1 in coupling PTP-PEST to WASp, the coincident expression of WASp, PSTPIP1, and PTP-PEST in these cells had an even more deleterious effect on synapse formation and, as with Fyn deficiency, abrogated actin accumulation at the T cell–APC interface (Fig. 5 D, c and d). These results are in agreement with published data indicating that Fyn and PSTPIP1 translocate to the T cell–APC contact region after TCR stimulation (5, 28) and suggest that Fyn and PSTPIP/PTP-PEST colocalization with WASp within this region influences WASp capacity to evoke the actin polymerization and cytoskeletal change required for synapse formation. Thus, these data provide further evidence that Fyn and PTP-PEST effects on the tyrosine phosphorylation of WASp are critical to the induction of WASp effector activities required for T cell activation.\nThe data reported here identify phosphorylation at Y291 as a major mechanism for induction of WASp effector activity after TCR engagement. A requirement for WASp “activation” in T cells is consistent with cumulative data revealing WASp to be constitutively autoinhibited (1–3) and identifying induction of actin polymerization as a critical facet of the T cell response to antigen stimulation (29). The current data identifying Fyn as the PTK responsible for WASp phosphorylation in T cells and implicating PTP-PEST in WASp dephosphorylation in these cells are also in agreement with previous data indicating an essential role for Fyn in induction of cytoskeletal reorganization after TCR engagement (30) and demonstrating that PTP-PEST acts as a negative regulator of lymphocyte activation (31). Taken together with the observed capacity of Fyn to promote and PTP-PEST to inhibit WASp stimulatory effects on Arp2/3 polymerizing function, these findings suggest that tyrosine phosphorylation alone can mediate induction of WASp activity after TCR engagement. By contrast, based on the results of in vitro binding assays, it has recently been suggested that tyrosine phosphorylation effects on WASp activity require its binding to cdc42 (32). Although the current data do not preclude cdc42 binding as a mechanism for activating WASp, the normal behavior of the WASpΔGBD T cells studied here together with the catastrophic effects of Y291 mutation on WASp function and T cell activation indicate that the in vivo induction of WASp effector activity absolutely requires WASp tyrosine phosphorylation and can occur independently of cdc42 binding to WASp. These data do not exclude the possibility that the ΔGBD mutation alters WASp structure so as to disrupt normal autoinhibitory constraints on WASp activity. However, no evidence for “constitutive” T cell activation was observed in the WAS−/− ΔGBD T cells and the data suggest that WASpΔGBD is constitutively inactive and requires an activation signal for function. Further, although Lyn/Btk-mediated phosphorylation of WASp has been reported to require cdc42 (33), the current data revealing Fyn-mediated tyrosine phosphorylation to be unaffected by constitutively active or dominant negative forms of cdc42 and the GBD deletion to have no effect on Fyn-driven WASp-Arp2/3 actin polymerization in vitro, provide compelling evidence to support previous data indicating that cdc42 binding is not essential for triggering WASp activity (13, 15).\nThe association of Y291 phosphorylation with induction of WASp effector activity raises the possibility that WASp activation also involves its interactions with SH2 domain–containing signaling effectors. Such effectors may include WASp-binding adaptors, such as Nck and Grb2, and kinases, such as Fyn, whose interactions with WASp are mediated via SH3 domain binding to the WASp polyproline region, but may also involve SH2 domain binding to the WASp phosphorylated Y291 residue. Phosphorylation at the Y291 site may also enable currently undefined WASp interactions with SH2 domain–containing effectors and thereby broaden the repertoire of WASp binding so as to facilitate WASp activation as well as its participation in TCR-evoked signaling cascades. Similarly, although Fyn appears to represent the major mediator of WASp tyrosine phosphorylation in T cells, the current data do not exclude the possibility that WASp ligands include tyrosine phosphatases, which like PTP-PEST, target WASp for dephosphorylation and functional deactivation. Although these issues require further investigation, the data presented here indicate the modulation of WASp tyrosine phosphorylation by Fyn and PTP-PEST to be critical to the induction of WASp effector activity in T cells and thus identify WASp tyrosine phosphorylation as essential to its contributions to the coupling of TCR engagement to T cell activation. 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