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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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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.
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fig5: 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.

Mentions: 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.


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)

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.
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Related In: Results  -  Collection

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fig5: 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.
Mentions: 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.

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