Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms.
RapGEF1
Ras-associated protein 1
Signal transduction
Src-homology 2 domain
Src-homology 3 domain
Tyrosine phosphorylation
Journal
Cell communication and signaling : CCS
ISSN: 1478-811X
Titre abrégé: Cell Commun Signal
Pays: England
ID NLM: 101170464
Informations de publication
Date de publication:
03 02 2023
03 02 2023
Historique:
received:
12
12
2022
accepted:
07
01
2023
entrez:
4
2
2023
pubmed:
5
2
2023
medline:
8
2
2023
Statut:
epublish
Résumé
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. 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. 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. 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.
Sections du résumé
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.
Identifiants
pubmed: 36737758
doi: 10.1186/s12964-023-01042-2
pii: 10.1186/s12964-023-01042-2
pmc: PMC9896810
doi:
Substances chimiques
CRK protein, human
0
Guanine Nucleotide Exchange Factors
0
Guanine Nucleotide-Releasing Factor 2
0
Nuclear Proteins
0
Nucleotides
0
Proto-Oncogene Proteins c-crk
0
Tyrosine
42HK56048U
TERF2IP protein, human
0
Types de publication
Video-Audio Media
Journal Article
Research Support, N.I.H., Intramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
30Subventions
Organisme : NIBIB NIH HHS
ID : Intramural Research program
Pays : United States
Informations de copyright
© 2023. U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.
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