Fyn and PTP-PEST Modulate WASp Effects on Induction of Actin Polymerization and Immunological Synapse Formation.
In 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.
Figure 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.
The 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).
The 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.