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Fyn and PTP-PEST-mediated regulation of Wiskott-Aldrich syndrome protein (WASp) tyrosine phosphorylation is required for coupling T cell antigen receptor engagement to WASp effector function and T cell activation.

Badour K, Zhang J, Shi F, Leng Y, Collins M, Siminovitch KA - J. Exp. Med. (2004)

Bottom Line: By contrast, mutation of tyrosine residue Y291, identified here as the major site of TCR-induced WASp tyrosine phosphorylation, abrogated induction of WASp tyrosine phosphorylation and its effector activities, including nuclear factor of activated T cell transcriptional activity, actin polymerization, and immunological synapse formation.Although Fyn enhanced WASp-mediated Arp2/3 activation and was required for synapse formation, PTP-PEST combined with PSTPIP1 inhibited WASp-driven actin polymerization and synapse formation.These observations identify key roles for Fyn and PTP-PEST in regulating WASp and imply that inducible WASp tyrosine phosphorylation can occur independently of cdc42 binding, but unlike the cdc42 interaction, is absolutely required for WASp contributions to T cell activation.

View Article: PubMed Central - PubMed

Affiliation: Mount Sinai Hospital, 600 University Avenue, Room 656A, Toronto, Ontario M5G 1X5, Canada.

ABSTRACT
Involvement of the Wiskott-Aldrich syndrome protein (WASp) in promoting cell activation requires its release from autoinhibitory structural constraints and has been attributed to WASp association with activated cdc42. Here, however, we show that T cell development and T cell receptor (TCR)-induced proliferation and actin polymerization proceed normally in WASp-/- mice expressing a WASp transgene lacking the cdc42 binding domain. By contrast, mutation of tyrosine residue Y291, identified here as the major site of TCR-induced WASp tyrosine phosphorylation, abrogated induction of WASp tyrosine phosphorylation and its effector activities, including nuclear factor of activated T cell transcriptional activity, actin polymerization, and immunological synapse formation. TCR-induced WASp tyrosine phosphorylation was also disrupted in T cells lacking Fyn, a kinase shown here to bind, colocalize with, and phosphorylate WASp. By contrast, WASp was tyrosine dephosphorylated by protein tyrosine phosphatase (PTP)-PEST, a tyrosine phosphatase shown here to interact with WASp via proline, serine, threonine phosphatase interacting protein (PSTPIP)1 binding. Although Fyn enhanced WASp-mediated Arp2/3 activation and was required for synapse formation, PTP-PEST combined with PSTPIP1 inhibited WASp-driven actin polymerization and synapse formation. These observations identify key roles for Fyn and PTP-PEST in regulating WASp and imply that inducible WASp tyrosine phosphorylation can occur independently of cdc42 binding, but unlike the cdc42 interaction, is absolutely required for WASp contributions to T cell activation.

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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.
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fig4: 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.

Mentions: 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.


Fyn and PTP-PEST-mediated regulation of Wiskott-Aldrich syndrome protein (WASp) tyrosine phosphorylation is required for coupling T cell antigen receptor engagement to WASp effector function and T cell activation.

Badour K, Zhang J, Shi F, Leng Y, Collins M, Siminovitch KA - J. Exp. Med. (2004)

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.
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fig4: 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.
Mentions: 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.

Bottom Line: By contrast, mutation of tyrosine residue Y291, identified here as the major site of TCR-induced WASp tyrosine phosphorylation, abrogated induction of WASp tyrosine phosphorylation and its effector activities, including nuclear factor of activated T cell transcriptional activity, actin polymerization, and immunological synapse formation.Although Fyn enhanced WASp-mediated Arp2/3 activation and was required for synapse formation, PTP-PEST combined with PSTPIP1 inhibited WASp-driven actin polymerization and synapse formation.These observations identify key roles for Fyn and PTP-PEST in regulating WASp and imply that inducible WASp tyrosine phosphorylation can occur independently of cdc42 binding, but unlike the cdc42 interaction, is absolutely required for WASp contributions to T cell activation.

View Article: PubMed Central - PubMed

Affiliation: Mount Sinai Hospital, 600 University Avenue, Room 656A, Toronto, Ontario M5G 1X5, Canada.

ABSTRACT
Involvement of the Wiskott-Aldrich syndrome protein (WASp) in promoting cell activation requires its release from autoinhibitory structural constraints and has been attributed to WASp association with activated cdc42. Here, however, we show that T cell development and T cell receptor (TCR)-induced proliferation and actin polymerization proceed normally in WASp-/- mice expressing a WASp transgene lacking the cdc42 binding domain. By contrast, mutation of tyrosine residue Y291, identified here as the major site of TCR-induced WASp tyrosine phosphorylation, abrogated induction of WASp tyrosine phosphorylation and its effector activities, including nuclear factor of activated T cell transcriptional activity, actin polymerization, and immunological synapse formation. TCR-induced WASp tyrosine phosphorylation was also disrupted in T cells lacking Fyn, a kinase shown here to bind, colocalize with, and phosphorylate WASp. By contrast, WASp was tyrosine dephosphorylated by protein tyrosine phosphatase (PTP)-PEST, a tyrosine phosphatase shown here to interact with WASp via proline, serine, threonine phosphatase interacting protein (PSTPIP)1 binding. Although Fyn enhanced WASp-mediated Arp2/3 activation and was required for synapse formation, PTP-PEST combined with PSTPIP1 inhibited WASp-driven actin polymerization and synapse formation. These observations identify key roles for Fyn and PTP-PEST in regulating WASp and imply that inducible WASp tyrosine phosphorylation can occur independently of cdc42 binding, but unlike the cdc42 interaction, is absolutely required for WASp contributions to T cell activation.

Show MeSH
Related in: MedlinePlus