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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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The Abl tyrosine kinase interacts with and is localized to the membrane by the WAVE2 complex. (A) Purified human T cells were stimulated with anti-CD3. WAVE2 was immunoprecipitated and bound proteins were immunoblotted as indicated. (B) Control or WAVE2-suppressed Jurkat T cells (green) were allowed to form conjugates with antigen-pulsed Nalm 6 B cells (blue) and then stained for Abl (red). The percentage of T–B cell conjugates showing Abl accumulation at the IS were quantified as described in the Materials and methods. Error bars represent SD. Bar, 5 μm. (C) Jurkat T cells were transfected with either control or WAVE2 suppression vector and stimulated with anti-CD3. Subsequently, cytosolic and membrane fractions were collected as described in Materials and methods and immunoblotted with the indicated antibodies. Numbers below blots are arbitrary units based on densitometric analysis of the immunoblots.
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fig6: The Abl tyrosine kinase interacts with and is localized to the membrane by the WAVE2 complex. (A) Purified human T cells were stimulated with anti-CD3. WAVE2 was immunoprecipitated and bound proteins were immunoblotted as indicated. (B) Control or WAVE2-suppressed Jurkat T cells (green) were allowed to form conjugates with antigen-pulsed Nalm 6 B cells (blue) and then stained for Abl (red). The percentage of T–B cell conjugates showing Abl accumulation at the IS were quantified as described in the Materials and methods. Error bars represent SD. Bar, 5 μm. (C) Jurkat T cells were transfected with either control or WAVE2 suppression vector and stimulated with anti-CD3. Subsequently, cytosolic and membrane fractions were collected as described in Materials and methods and immunoblotted with the indicated antibodies. Numbers below blots are arbitrary units based on densitometric analysis of the immunoblots.

Mentions: It has been proposed that phosphorylation of CrkL on Y207 inhibits its localization through an autoinhibitory mechanism, where the SH2 domain of the protein binds with high affinity to a phosphorylated tyrosine between the amino- and carboxy-terminal SH3 domains (Feller et al., 1994; Rosen et al., 1995), thereby preventing the association with other binding partners and decreasing CrkL-mediated signaling. In fact, expression of an Y207F CrkL point mutant resulted in enhanced basal adhesion to fibronectin as compared with control and wild-type CrkL-expressing Jurkat T cells (Fig. S2, B and C). We therefore next determined which domains of CrkL were required for its phosphorylation and regulation of Rap1 after TCR ligation. Although wild-type CrkL underwent tyrosine phosphorylation, mutation of the critical residue within the SH2 domain (R39A) prevented TCR-stimulated phosphorylation of CrkL, whereas mutation of either SH3 domain had no effect (Fig. 5 D). In addition, we found that the CrkL SH2 domain and N-terminal SH3 domain (which binds to C3G) are involved in regulating Rap1 activation downstream of the TCR (Fig. 5 E). Collectively, these results suggest that the WAVE2 complex is involved in the recruitment of CrkL (via its SH2 domain) for optimal TCR-stimulated Rap1 activation (Fig. 6 C).


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)

The Abl tyrosine kinase interacts with and is localized to the membrane by the WAVE2 complex. (A) Purified human T cells were stimulated with anti-CD3. WAVE2 was immunoprecipitated and bound proteins were immunoblotted as indicated. (B) Control or WAVE2-suppressed Jurkat T cells (green) were allowed to form conjugates with antigen-pulsed Nalm 6 B cells (blue) and then stained for Abl (red). The percentage of T–B cell conjugates showing Abl accumulation at the IS were quantified as described in the Materials and methods. Error bars represent SD. Bar, 5 μm. (C) Jurkat T cells were transfected with either control or WAVE2 suppression vector and stimulated with anti-CD3. Subsequently, cytosolic and membrane fractions were collected as described in Materials and methods and immunoblotted with the indicated antibodies. Numbers below blots are arbitrary units based on densitometric analysis of the immunoblots.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2542481&req=5

fig6: The Abl tyrosine kinase interacts with and is localized to the membrane by the WAVE2 complex. (A) Purified human T cells were stimulated with anti-CD3. WAVE2 was immunoprecipitated and bound proteins were immunoblotted as indicated. (B) Control or WAVE2-suppressed Jurkat T cells (green) were allowed to form conjugates with antigen-pulsed Nalm 6 B cells (blue) and then stained for Abl (red). The percentage of T–B cell conjugates showing Abl accumulation at the IS were quantified as described in the Materials and methods. Error bars represent SD. Bar, 5 μm. (C) Jurkat T cells were transfected with either control or WAVE2 suppression vector and stimulated with anti-CD3. Subsequently, cytosolic and membrane fractions were collected as described in Materials and methods and immunoblotted with the indicated antibodies. Numbers below blots are arbitrary units based on densitometric analysis of the immunoblots.
Mentions: It has been proposed that phosphorylation of CrkL on Y207 inhibits its localization through an autoinhibitory mechanism, where the SH2 domain of the protein binds with high affinity to a phosphorylated tyrosine between the amino- and carboxy-terminal SH3 domains (Feller et al., 1994; Rosen et al., 1995), thereby preventing the association with other binding partners and decreasing CrkL-mediated signaling. In fact, expression of an Y207F CrkL point mutant resulted in enhanced basal adhesion to fibronectin as compared with control and wild-type CrkL-expressing Jurkat T cells (Fig. S2, B and C). We therefore next determined which domains of CrkL were required for its phosphorylation and regulation of Rap1 after TCR ligation. Although wild-type CrkL underwent tyrosine phosphorylation, mutation of the critical residue within the SH2 domain (R39A) prevented TCR-stimulated phosphorylation of CrkL, whereas mutation of either SH3 domain had no effect (Fig. 5 D). In addition, we found that the CrkL SH2 domain and N-terminal SH3 domain (which binds to C3G) are involved in regulating Rap1 activation downstream of the TCR (Fig. 5 E). Collectively, these results suggest that the WAVE2 complex is involved in the recruitment of CrkL (via its SH2 domain) for optimal TCR-stimulated Rap1 activation (Fig. 6 C).

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