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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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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.
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fig2: 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.

Mentions: The 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.


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)

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.
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Related In: Results  -  Collection

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fig2: 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.
Mentions: The 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.

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