The GIT proteins, GIT1 and GIT2, are GTPase-activating proteins for the ADP-ribosylation factor family of small GTP-binding proteins, but also serve as adaptors to link signaling proteins to distinct cellular locations. One role for GIT proteins is to link the PIX family of Rho guanine nucleotide exchange factors and their binding partners, the p21-activated protein kinases, to remodeling focal adhesions by interacting with the focal adhesion adaptor protein paxillin. We here identified the C-terminal domain of GIT1 responsible for paxillin binding. Combining structural and mutational analyses, we show that this region folds into an anti-parallel four-helix domain highly reminiscent to the focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK). Our results suggest that the GIT1 FAT-homology (FAH) domain and FAT bind the paxillin LD4 motif quite similarly. Since only a small fraction of GIT1 is bound to paxillin under normal conditions, regulation of paxillin binding was explored. Although paxillin binding to the FAT domain of FAK is regulated by tyrosine phosphorylation within this domain, we find that tyrosine phosphorylation of the FAH domain GIT1 is not involved in regulating binding to paxillin. Instead, we find that mutations within the FAH domain may alter binding to paxillin that has been phosphorylated within the LD4 motif. Thus, despite apparent structural similarity in their FAT domains, GIT1 and FAK binding to paxillin is differentially regulated.
Bibliographical noteFunding Information:
We thank D. Svergun and M. Petoukhov from DESY beamline X33 (EMBL, Hamburg) for their assistance with data recording, and advice throughout data analysis, and Dr. Erin Whalen for helpful comments on the manuscript. This work was supported by the NIH grants GM59989 and DA016347 (RTP), the Agence National de la Recherche, the Agence Nationale de Recherches sur le SIDA et les Hépatites Virales, the Association pour la Recherche sur le Cancer and the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche (STA). The latter also provides support for MLG.
- Arf GTPase-activating protein
- Focal adhesion targeting domain
- GIT1 protein
- Guanine nucleotide exchange factor
- PIX protein
ASJC Scopus subject areas
- Cell Biology