Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms
Abstract Background C3G is a guanine nucleotide exchange factor (GEF) that activates Rap1 to promote cell adhesion. Resting C3G is autoinhibited and the GEF activity is released by stimuli that signal through tyrosine kinases. C3G is activated by tyrosine phosphorylation and interaction with Crk ada...
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| Format: | Article |
| Language: | English |
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BMC
2023-02-01
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| Series: | Cell Communication and Signaling |
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| Online Access: | https://doi.org/10.1186/s12964-023-01042-2 |
| _version_ | 1797557717769912320 |
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| author | Antonio Rodríguez-Blázquez Arturo Carabias Alba Morán-Vaquero Sergio de Cima Juan R. Luque-Ortega Carlos Alfonso Peter Schuck José Antonio Manso Sandra Macedo-Ribeiro Carmen Guerrero José M. de Pereda |
| author_facet | Antonio Rodríguez-Blázquez Arturo Carabias Alba Morán-Vaquero Sergio de Cima Juan R. Luque-Ortega Carlos Alfonso Peter Schuck José Antonio Manso Sandra Macedo-Ribeiro Carmen Guerrero José M. de Pereda |
| author_sort | Antonio Rodríguez-Blázquez |
| collection | DOAJ |
| description | Abstract Background C3G is a guanine nucleotide exchange factor (GEF) that activates Rap1 to promote cell adhesion. Resting C3G is autoinhibited and the GEF activity is released by stimuli that signal through tyrosine kinases. C3G is activated by tyrosine phosphorylation and interaction with Crk adaptor proteins, whose expression is elevated in multiple human cancers. However, the molecular details of C3G activation and the interplay between phosphorylation and Crk interaction are poorly understood. Methods We combined biochemical, biophysical, and cell biology approaches to elucidate the mechanisms of C3G activation. Binding of Crk adaptor proteins to four proline-rich motifs (P1 to P4) in C3G was characterized in vitro using isothermal titration calorimetry and sedimentation velocity, and in Jurkat and HEK293T cells by affinity pull-down assays. The nucleotide exchange activity of C3G over Rap1 was measured using nucleotide-dissociation kinetic assays. Jurkat cells were also used to analyze C3G translocation to the plasma membrane and the C3G-dependent activation of Rap1 upon ligation of T cell receptors. Results CrkL interacts through its SH3N domain with sites P1 and P2 of inactive C3G in vitro and in Jurkat and HEK293T cells, and these sites are necessary to recruit C3G to the plasma membrane. However, direct stimulation of the GEF activity requires binding of Crk proteins to the P3 and P4 sites. P3 is occluded in resting C3G and is essential for activation, while P4 contributes secondarily towards complete stimulation. Tyrosine phosphorylation of C3G alone causes marginal activation. Instead, phosphorylation primes C3G lowering the concentration of Crk proteins required for activation and increasing the maximum activity. Unexpectedly, optimal activation also requires the interaction of CrkL-SH2 domain with phosphorylated C3G. Conclusion Our study revealed that phosphorylation of C3G by Src and Crk-binding form a two-factor mechanism that ensures tight control of C3G activation. Additionally, the simultaneous SH2 and SH3N interaction of CrkL with C3G, required for the activation, reveals a novel adaptor-independent function of Crk proteins relevant to understanding their role in physiological signaling and their deregulation in diseases. Video abstract |
| first_indexed | 2024-03-10T17:20:18Z |
| format | Article |
| id | doaj.art-69835b309c8c4fcc8550fc5ffc4c0d4f |
| institution | Directory Open Access Journal |
| issn | 1478-811X |
| language | English |
| last_indexed | 2024-03-10T17:20:18Z |
| publishDate | 2023-02-01 |
| publisher | BMC |
| record_format | Article |
| series | Cell Communication and Signaling |
| spelling | doaj.art-69835b309c8c4fcc8550fc5ffc4c0d4f2023-11-20T10:22:07ZengBMCCell Communication and Signaling1478-811X2023-02-0121112210.1186/s12964-023-01042-2Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanismsAntonio Rodríguez-Blázquez0Arturo Carabias1Alba Morán-Vaquero2Sergio de Cima3Juan R. Luque-Ortega4Carlos Alfonso5Peter Schuck6José Antonio Manso7Sandra Macedo-Ribeiro8Carmen Guerrero9José M. de Pereda10Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de SalamancaCentro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de SalamancaCentro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de SalamancaCentro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de SalamancaCentro de Investigaciones Biológicas Margarita Salas, CSICCentro de Investigaciones Biológicas Margarita Salas, CSICLaboratory of Dynamics of Macromolecular Assembly, National Institute of Biomedical Imaging and Bioengineering, National Institutes of HealthIBMC-Instituto de Biologia Molecular e Celular, Universidade do PortoIBMC-Instituto de Biologia Molecular e Celular, Universidade do PortoCentro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de SalamancaCentro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de SalamancaAbstract Background C3G is a guanine nucleotide exchange factor (GEF) that activates Rap1 to promote cell adhesion. Resting C3G is autoinhibited and the GEF activity is released by stimuli that signal through tyrosine kinases. C3G is activated by tyrosine phosphorylation and interaction with Crk adaptor proteins, whose expression is elevated in multiple human cancers. However, the molecular details of C3G activation and the interplay between phosphorylation and Crk interaction are poorly understood. Methods We combined biochemical, biophysical, and cell biology approaches to elucidate the mechanisms of C3G activation. Binding of Crk adaptor proteins to four proline-rich motifs (P1 to P4) in C3G was characterized in vitro using isothermal titration calorimetry and sedimentation velocity, and in Jurkat and HEK293T cells by affinity pull-down assays. The nucleotide exchange activity of C3G over Rap1 was measured using nucleotide-dissociation kinetic assays. Jurkat cells were also used to analyze C3G translocation to the plasma membrane and the C3G-dependent activation of Rap1 upon ligation of T cell receptors. Results CrkL interacts through its SH3N domain with sites P1 and P2 of inactive C3G in vitro and in Jurkat and HEK293T cells, and these sites are necessary to recruit C3G to the plasma membrane. However, direct stimulation of the GEF activity requires binding of Crk proteins to the P3 and P4 sites. P3 is occluded in resting C3G and is essential for activation, while P4 contributes secondarily towards complete stimulation. Tyrosine phosphorylation of C3G alone causes marginal activation. Instead, phosphorylation primes C3G lowering the concentration of Crk proteins required for activation and increasing the maximum activity. Unexpectedly, optimal activation also requires the interaction of CrkL-SH2 domain with phosphorylated C3G. Conclusion Our study revealed that phosphorylation of C3G by Src and Crk-binding form a two-factor mechanism that ensures tight control of C3G activation. Additionally, the simultaneous SH2 and SH3N interaction of CrkL with C3G, required for the activation, reveals a novel adaptor-independent function of Crk proteins relevant to understanding their role in physiological signaling and their deregulation in diseases. Video abstracthttps://doi.org/10.1186/s12964-023-01042-2Ras-associated protein 1RapGEF1Signal transductionTyrosine phosphorylationSrc-homology 2 domainSrc-homology 3 domain |
| spellingShingle | Antonio Rodríguez-Blázquez Arturo Carabias Alba Morán-Vaquero Sergio de Cima Juan R. Luque-Ortega Carlos Alfonso Peter Schuck José Antonio Manso Sandra Macedo-Ribeiro Carmen Guerrero José M. de Pereda Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms Cell Communication and Signaling Ras-associated protein 1 RapGEF1 Signal transduction Tyrosine phosphorylation Src-homology 2 domain Src-homology 3 domain |
| title | Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms |
| title_full | Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms |
| title_fullStr | Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms |
| title_full_unstemmed | Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms |
| title_short | Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms |
| title_sort | crk proteins activate the rap1 guanine nucleotide exchange factor c3g by segregated adaptor dependent and independent mechanisms |
| topic | Ras-associated protein 1 RapGEF1 Signal transduction Tyrosine phosphorylation Src-homology 2 domain Src-homology 3 domain |
| url | https://doi.org/10.1186/s12964-023-01042-2 |
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