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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: 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.

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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WAVE2 is required for phosphorylation of CrkL and C3G. (A) Jurkat T cells were stimulated with OKT3-bound beads, and the localization of WAVE2 (green) or CrkL (green) and F-actin (red) was analyzed by confocal microscopy. Representative images are shown. Bar, 5 μm. (B) Jurkat T cells were transfected with the indicated vectors and stimulated with anti-CD3. Whole cell extracts were prepared and immunoblotted for pCrkL (Y207), total CrkL, and WAVE2. (C) Jurkat T cells were transfected with the indicated suppression/reexpression vectors, stimulated with anti-CD3, and whole cell extracts were immunoblotted as in B. (D) Jurkat T cells were transfected with the indicated expression vectors, stimulated with anti-CD3, and lysates immunoblotted with the indicated antibodies. (E) Jurkat T cells were transfected with control vector, CrkL-suppression vector, or suppression/reexpression vectors, which reexpressed either wild-type CrkL or point mutants disrupting the function of the SH2, SH3-N, or SH3-C domains. Rap1 activation was assessed as previously described. (F) Cells were transfected and stimulated as in B, and C3G was purified using a GST-CrkL SH3-N fusion protein and subsequently blotted using the indicated antibodies. (G) Purified human CD4+ T cells were transfected with the indicated siRNAs, stimulated with anti-CD3, and Rap1 activation and phosphorylation of CrkL was analyzed by immunoblot. Numbers below top blots are arbitrary units based on densitometric analysis of the immunoblots. Black lines indicate that intervening lanes have been spliced out.
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fig5: WAVE2 is required for phosphorylation of CrkL and C3G. (A) Jurkat T cells were stimulated with OKT3-bound beads, and the localization of WAVE2 (green) or CrkL (green) and F-actin (red) was analyzed by confocal microscopy. Representative images are shown. Bar, 5 μm. (B) Jurkat T cells were transfected with the indicated vectors and stimulated with anti-CD3. Whole cell extracts were prepared and immunoblotted for pCrkL (Y207), total CrkL, and WAVE2. (C) Jurkat T cells were transfected with the indicated suppression/reexpression vectors, stimulated with anti-CD3, and whole cell extracts were immunoblotted as in B. (D) Jurkat T cells were transfected with the indicated expression vectors, stimulated with anti-CD3, and lysates immunoblotted with the indicated antibodies. (E) Jurkat T cells were transfected with control vector, CrkL-suppression vector, or suppression/reexpression vectors, which reexpressed either wild-type CrkL or point mutants disrupting the function of the SH2, SH3-N, or SH3-C domains. Rap1 activation was assessed as previously described. (F) Cells were transfected and stimulated as in B, and C3G was purified using a GST-CrkL SH3-N fusion protein and subsequently blotted using the indicated antibodies. (G) Purified human CD4+ T cells were transfected with the indicated siRNAs, stimulated with anti-CD3, and Rap1 activation and phosphorylation of CrkL was analyzed by immunoblot. Numbers below top blots are arbitrary units based on densitometric analysis of the immunoblots. Black lines indicate that intervening lanes have been spliced out.

Mentions: Because CrkL and C3G are both required for TCR-mediated Rap1 activation and WAVE2-suppressed T cells are impaired in Rap1 activation, we next determined whether CrkL is recruited to the IS. For this experiment, we used latex beads coated with control mouse IgG or anti-CD3 because the high level of expression of CrkL in the Nalm 6 B cell line impaired visualization of CrkL recruitment to the IS. We did not observe any colocalization of F-actin with either WAVE2 or CrkL when Jurkat T cells were stimulated with mouse IgG-coated beads (unpublished data). However, we observed recruitment of F-actin and WAVE2, as well as F-actin and CrkL to the cell–bead contact site when stimulating with anti-CD3–coated beads (Fig. 5 A).


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)

WAVE2 is required for phosphorylation of CrkL and C3G. (A) Jurkat T cells were stimulated with OKT3-bound beads, and the localization of WAVE2 (green) or CrkL (green) and F-actin (red) was analyzed by confocal microscopy. Representative images are shown. Bar, 5 μm. (B) Jurkat T cells were transfected with the indicated vectors and stimulated with anti-CD3. Whole cell extracts were prepared and immunoblotted for pCrkL (Y207), total CrkL, and WAVE2. (C) Jurkat T cells were transfected with the indicated suppression/reexpression vectors, stimulated with anti-CD3, and whole cell extracts were immunoblotted as in B. (D) Jurkat T cells were transfected with the indicated expression vectors, stimulated with anti-CD3, and lysates immunoblotted with the indicated antibodies. (E) Jurkat T cells were transfected with control vector, CrkL-suppression vector, or suppression/reexpression vectors, which reexpressed either wild-type CrkL or point mutants disrupting the function of the SH2, SH3-N, or SH3-C domains. Rap1 activation was assessed as previously described. (F) Cells were transfected and stimulated as in B, and C3G was purified using a GST-CrkL SH3-N fusion protein and subsequently blotted using the indicated antibodies. (G) Purified human CD4+ T cells were transfected with the indicated siRNAs, stimulated with anti-CD3, and Rap1 activation and phosphorylation of CrkL was analyzed by immunoblot. Numbers below top blots are arbitrary units based on densitometric analysis of the immunoblots. Black lines indicate that intervening lanes have been spliced out.
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Related In: Results  -  Collection

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fig5: WAVE2 is required for phosphorylation of CrkL and C3G. (A) Jurkat T cells were stimulated with OKT3-bound beads, and the localization of WAVE2 (green) or CrkL (green) and F-actin (red) was analyzed by confocal microscopy. Representative images are shown. Bar, 5 μm. (B) Jurkat T cells were transfected with the indicated vectors and stimulated with anti-CD3. Whole cell extracts were prepared and immunoblotted for pCrkL (Y207), total CrkL, and WAVE2. (C) Jurkat T cells were transfected with the indicated suppression/reexpression vectors, stimulated with anti-CD3, and whole cell extracts were immunoblotted as in B. (D) Jurkat T cells were transfected with the indicated expression vectors, stimulated with anti-CD3, and lysates immunoblotted with the indicated antibodies. (E) Jurkat T cells were transfected with control vector, CrkL-suppression vector, or suppression/reexpression vectors, which reexpressed either wild-type CrkL or point mutants disrupting the function of the SH2, SH3-N, or SH3-C domains. Rap1 activation was assessed as previously described. (F) Cells were transfected and stimulated as in B, and C3G was purified using a GST-CrkL SH3-N fusion protein and subsequently blotted using the indicated antibodies. (G) Purified human CD4+ T cells were transfected with the indicated siRNAs, stimulated with anti-CD3, and Rap1 activation and phosphorylation of CrkL was analyzed by immunoblot. Numbers below top blots are arbitrary units based on densitometric analysis of the immunoblots. Black lines indicate that intervening lanes have been spliced out.
Mentions: Because CrkL and C3G are both required for TCR-mediated Rap1 activation and WAVE2-suppressed T cells are impaired in Rap1 activation, we next determined whether CrkL is recruited to the IS. For this experiment, we used latex beads coated with control mouse IgG or anti-CD3 because the high level of expression of CrkL in the Nalm 6 B cell line impaired visualization of CrkL recruitment to the IS. We did not observe any colocalization of F-actin with either WAVE2 or CrkL when Jurkat T cells were stimulated with mouse IgG-coated beads (unpublished data). However, we observed recruitment of F-actin and WAVE2, as well as F-actin and CrkL to the cell–bead contact site when stimulating with anti-CD3–coated beads (Fig. 5 A).

Bottom Line: 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.

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