Tyrosine Phosphorylation of WASp Is Regulated by Fyn and PTP-PEST.
Phosphorylation 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).
Figure 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.
To 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.
The 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.
Figure 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.