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The WAVE2 complex regulates T cell receptor signaling to integrins via Abl- and CrkL-C3G-mediated activation of Rap1.

Nolz JC, Nacusi LP, Segovis CM, Medeiros RB, Mitchell JS, Shimizu Y, Billadeau DD - J. Cell Biol. (2008)

Bottom Line: Furthermore, we show that WAVE2 regulates TCR-mediated activation of the integrin regulatory guanosine triphosphatase Rap1 via the recruitment and activation of the CrkL-C3G exchange complex.Moreover, we demonstrate that although Abl does not regulate the recruitment of CrkL-C3G into the membrane, it does affect the tyrosine phosphorylation of C3G, which is required for its guanine nucleotide exchange factor activity toward Rap1.These findings identify a previously unknown mechanism by which the WAVE2 complex regulates TCR signaling to Rap1 and integrin activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, College of Medicine, Mayo Clinic, Rochester, MN 55905, USA.

ABSTRACT
WAVE2 regulates T cell receptor (TCR)-stimulated actin cytoskeletal dynamics leading to both integrin clustering and affinity maturation. Although WAVE2 mediates integrin affinity maturation by recruiting vinculin and talin to the immunological synapse in an Arp2/3-dependent manner, the mechanism by which it regulates integrin clustering is unclear. We show that the Abl tyrosine kinase associates with the WAVE2 complex and TCR ligation induces WAVE2-dependent membrane recruitment of Abl. Furthermore, we show that WAVE2 regulates TCR-mediated activation of the integrin regulatory guanosine triphosphatase Rap1 via the recruitment and activation of the CrkL-C3G exchange complex. Moreover, we demonstrate that although Abl does not regulate the recruitment of CrkL-C3G into the membrane, it does affect the tyrosine phosphorylation of C3G, which is required for its guanine nucleotide exchange factor activity toward Rap1. This signaling node regulates not only TCR-stimulated integrin clustering but also affinity maturation. These findings identify a previously unknown mechanism by which the WAVE2 complex regulates TCR signaling to Rap1 and integrin activation.

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Affect of Abl suppression on CrkL–C3G membrane localization and activation. (A) Jurkat T cells transfected with control, WAVE2, or Abl EGFP suppression vectors were stimulated with IgG (column 1) or OKT3-coated beads (columns 2–4) and imaged for CrkL (aqua) and F-actin (red) recruitment. The percentage of EGFP+ T cell–bead conjugates showing localization of CrkL is indicated and was performed as described in Materials and methods. Representative images are shown. Bar, 5 μm. (B) Jurkat T cells were transfected with the indicated suppression vectors, stimulated by anti-CD3 cross-linking and cytosolic/membrane fractions, were prepared and immunoblotted with the indicated antibodies. Numbers below blots are arbitrary units based on densitometric analysis of the immunoblots. (C) Purified Abl and Fyn were incubated with the indicated GST fusion proteins in kinase buffer, and tyrosine phosphorylation was detected by anti-Tyr immunoblotting. Input levels of GST were detected by anti-GST immunoblotting. Numbers on the left are arbitrary units based on densitometric analysis of the immunoblots. (D) Jurkat T cells were transfected with the indicated suppression vectors, stimulated by anti-CD3 cross-linking, and C3G was precipitated as described in Fig. 5 E. Proteins were detected by immunoblotting with the indicated antibodies. Numbers below blots are arbitrary units based on densitometric analysis of the immunoblots.
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fig8: Affect of Abl suppression on CrkL–C3G membrane localization and activation. (A) Jurkat T cells transfected with control, WAVE2, or Abl EGFP suppression vectors were stimulated with IgG (column 1) or OKT3-coated beads (columns 2–4) and imaged for CrkL (aqua) and F-actin (red) recruitment. The percentage of EGFP+ T cell–bead conjugates showing localization of CrkL is indicated and was performed as described in Materials and methods. Representative images are shown. Bar, 5 μm. (B) Jurkat T cells were transfected with the indicated suppression vectors, stimulated by anti-CD3 cross-linking and cytosolic/membrane fractions, were prepared and immunoblotted with the indicated antibodies. Numbers below blots are arbitrary units based on densitometric analysis of the immunoblots. (C) Purified Abl and Fyn were incubated with the indicated GST fusion proteins in kinase buffer, and tyrosine phosphorylation was detected by anti-Tyr immunoblotting. Input levels of GST were detected by anti-GST immunoblotting. Numbers on the left are arbitrary units based on densitometric analysis of the immunoblots. (D) Jurkat T cells were transfected with the indicated suppression vectors, stimulated by anti-CD3 cross-linking, and C3G was precipitated as described in Fig. 5 E. Proteins were detected by immunoblotting with the indicated antibodies. Numbers below blots are arbitrary units based on densitometric analysis of the immunoblots.

Mentions: Because our data suggests that Abl regulates TCR-stimulated Rap1 activation and WAVE2 is involved in the recruitment of Abl into the membrane after TCR ligation, we reasoned that Abl might affect the membrane localization or activation of the CrkL–C3G complex. Consistent with the biochemical data shown in Fig. 6 C, we find that CrkL localization to the cell–bead contact site is affected in WAVE2-suppressed T cells (Fig. 8 A). In contrast, localization of CrkL to the cell–bead contact site, as well as the membrane recruitment of CrkL and C3G was only modestly affected in Abl-suppressed T cells (Fig. 8, A and B). Interestingly, suppression of Abl did not affect the recruitment of WAVE2 into the membrane fraction after TCR-stimulation (Fig. 8 B). Collectively, these data suggest that the recruitment of CrkL and C3G into the membrane is largely dependent on the WAVE2 complex.


The WAVE2 complex regulates T cell receptor signaling to integrins via Abl- and CrkL-C3G-mediated activation of Rap1.

Nolz JC, Nacusi LP, Segovis CM, Medeiros RB, Mitchell JS, Shimizu Y, Billadeau DD - J. Cell Biol. (2008)

Affect of Abl suppression on CrkL–C3G membrane localization and activation. (A) Jurkat T cells transfected with control, WAVE2, or Abl EGFP suppression vectors were stimulated with IgG (column 1) or OKT3-coated beads (columns 2–4) and imaged for CrkL (aqua) and F-actin (red) recruitment. The percentage of EGFP+ T cell–bead conjugates showing localization of CrkL is indicated and was performed as described in Materials and methods. Representative images are shown. Bar, 5 μm. (B) Jurkat T cells were transfected with the indicated suppression vectors, stimulated by anti-CD3 cross-linking and cytosolic/membrane fractions, were prepared and immunoblotted with the indicated antibodies. Numbers below blots are arbitrary units based on densitometric analysis of the immunoblots. (C) Purified Abl and Fyn were incubated with the indicated GST fusion proteins in kinase buffer, and tyrosine phosphorylation was detected by anti-Tyr immunoblotting. Input levels of GST were detected by anti-GST immunoblotting. Numbers on the left are arbitrary units based on densitometric analysis of the immunoblots. (D) Jurkat T cells were transfected with the indicated suppression vectors, stimulated by anti-CD3 cross-linking, and C3G was precipitated as described in Fig. 5 E. Proteins were detected by immunoblotting with the indicated antibodies. Numbers below blots are arbitrary units based on densitometric analysis of the immunoblots.
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fig8: Affect of Abl suppression on CrkL–C3G membrane localization and activation. (A) Jurkat T cells transfected with control, WAVE2, or Abl EGFP suppression vectors were stimulated with IgG (column 1) or OKT3-coated beads (columns 2–4) and imaged for CrkL (aqua) and F-actin (red) recruitment. The percentage of EGFP+ T cell–bead conjugates showing localization of CrkL is indicated and was performed as described in Materials and methods. Representative images are shown. Bar, 5 μm. (B) Jurkat T cells were transfected with the indicated suppression vectors, stimulated by anti-CD3 cross-linking and cytosolic/membrane fractions, were prepared and immunoblotted with the indicated antibodies. Numbers below blots are arbitrary units based on densitometric analysis of the immunoblots. (C) Purified Abl and Fyn were incubated with the indicated GST fusion proteins in kinase buffer, and tyrosine phosphorylation was detected by anti-Tyr immunoblotting. Input levels of GST were detected by anti-GST immunoblotting. Numbers on the left are arbitrary units based on densitometric analysis of the immunoblots. (D) Jurkat T cells were transfected with the indicated suppression vectors, stimulated by anti-CD3 cross-linking, and C3G was precipitated as described in Fig. 5 E. Proteins were detected by immunoblotting with the indicated antibodies. Numbers below blots are arbitrary units based on densitometric analysis of the immunoblots.
Mentions: Because our data suggests that Abl regulates TCR-stimulated Rap1 activation and WAVE2 is involved in the recruitment of Abl into the membrane after TCR ligation, we reasoned that Abl might affect the membrane localization or activation of the CrkL–C3G complex. Consistent with the biochemical data shown in Fig. 6 C, we find that CrkL localization to the cell–bead contact site is affected in WAVE2-suppressed T cells (Fig. 8 A). In contrast, localization of CrkL to the cell–bead contact site, as well as the membrane recruitment of CrkL and C3G was only modestly affected in Abl-suppressed T cells (Fig. 8, A and B). Interestingly, suppression of Abl did not affect the recruitment of WAVE2 into the membrane fraction after TCR-stimulation (Fig. 8 B). Collectively, these data suggest that the recruitment of CrkL and C3G into the membrane is largely dependent on the WAVE2 complex.

Bottom Line: Furthermore, we show that WAVE2 regulates TCR-mediated activation of the integrin regulatory guanosine triphosphatase Rap1 via the recruitment and activation of the CrkL-C3G exchange complex.Moreover, we demonstrate that although Abl does not regulate the recruitment of CrkL-C3G into the membrane, it does affect the tyrosine phosphorylation of C3G, which is required for its guanine nucleotide exchange factor activity toward Rap1.These findings identify a previously unknown mechanism by which the WAVE2 complex regulates TCR signaling to Rap1 and integrin activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, College of Medicine, Mayo Clinic, Rochester, MN 55905, USA.

ABSTRACT
WAVE2 regulates T cell receptor (TCR)-stimulated actin cytoskeletal dynamics leading to both integrin clustering and affinity maturation. Although WAVE2 mediates integrin affinity maturation by recruiting vinculin and talin to the immunological synapse in an Arp2/3-dependent manner, the mechanism by which it regulates integrin clustering is unclear. We show that the Abl tyrosine kinase associates with the WAVE2 complex and TCR ligation induces WAVE2-dependent membrane recruitment of Abl. Furthermore, we show that WAVE2 regulates TCR-mediated activation of the integrin regulatory guanosine triphosphatase Rap1 via the recruitment and activation of the CrkL-C3G exchange complex. Moreover, we demonstrate that although Abl does not regulate the recruitment of CrkL-C3G into the membrane, it does affect the tyrosine phosphorylation of C3G, which is required for its guanine nucleotide exchange factor activity toward Rap1. This signaling node regulates not only TCR-stimulated integrin clustering but also affinity maturation. These findings identify a previously unknown mechanism by which the WAVE2 complex regulates TCR signaling to Rap1 and integrin activation.

Show MeSH
Related in: MedlinePlus