GIT1 utilizes a focal adhesion targeting-homology domain to bind paxillin

Abstract

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 Cterminal domain of GIT1 responsible for paxillin binding. Combining structural and mutational analysis, we show that this region folds into an antiparallel 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. KeywordsGIT1 protein; paxillin; focal adhesion targeting domain; PIX protein; Arf GTPase-activating protein; guanine nucleotide exchange factor The GIT and PIX proteins tightly associate to form an oligomeric complex that functions as a recruitable scaffold for various signaling proteins [1]. The GIT proteins, GIT1 and GIT2 (also known as p95-APP1/2, p95-PKL, and Cat-2 proteins), are GTPase-activating proteins for the Arf family of small GTP-binding proteins [2][3][4]. The PIX proteins, α-PIX and β-PIX (also known as Cool-1/2, p85-SPR and ARHGEF6/7 proteins), are guanine nucleotide exchange 4To whom correspondence should be addressed: richard.premont@duke.edu.. 3 These authors contributed equally to this work.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptCell Signal. Author manuscript; available in PMC 2008 August 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript factors for the Rho family of small GTP-binding proteins [5]. In addition to functioning as regulators of small GTP-binding proteins, GIT/PIX complexes have been reported to ...

Differential Expression of the ARF GAP Genes GIT1 and GIT2 in Mouse Tissues

“…In the absence of Src family kinases phosphorylating these sites, GIT2 is unable to localize to focal adhesions, whereas GIT1 shows no defect in localization (Brown et al 2005). We recently showed that GIT1 bearing mutations preventing tyrosine phosphorylation of these conserved sites is still able to bind paxillin (Schmalzigaug et al 2007). In endothelial cells, Garcia and colleagues reported distinct patterns of GIT1 and GIT2 phosphorylation and recruitment to adhesions in response to cell activation (Shikata et al 2003b).…”

Expanding functions of GIT Arf GTPase-activating proteins, PIX Rho guanine nucleotide exchange factors and GIT–PIX complexes

“…The coiledcoil domain is responsible for dimerization of GIT proteins by forming a parallel coiled-coil Schlenker and Rittinger, 2009). The FAT domain is a four-helix bundle and the binding site for the focal adhesion adaptor protein paxillin in a manner that is similar to paxillin binding to the FAT domain of focal adhesion kinase (Schmalzigaug et al, 2007a;Zhang et al, 2008).…”

“…GITs are also prominent substrates for Src family kinases (Haendeler et al, 2000;Brown et al, 2005;Heckel et al, 2009). Src/FAK-mediated phosphorylation of GIT2 at amino acid residues Y286, Y392 and Y592 is required for binding to paxillin at focal adhesions (Brown et al, 2005) but the cognate residues in GIT1 are not necessary for paxillin association (Schmalzigaug et al, 2007a). However, both GIT1 and GIT2 phosphorylated at Y392 bind to the SH2-SH3 adaptor proteins Nck1 and Nck2 (Frese et al, 2006;Segura et al, 2007).…”

“…4). Therefore, in all our studies GIT proteins are tagged at the Cterminus, which does not affect their ArfGAP activity (Premont et al, 1998;Vitale et al, 2000) and does not interfere with their binding to paxillin or association with focal adhesions (Schmalzigaug et al, 2007a).…”

The Adaptor Protein and Arf GTPase-activating Protein Cat-1/Git-1 Is Required for Cellular Transformation

“…The QuikChange TM Site-Directed Mutagenesis kit (Stratagene) was used to generate point mutants of Cat-1 that are defective in binding Cool-1 (D294K/E295R) (24), paxillin (K663E/K758E) (25), or are defective as an Arf-GAP (R39A) (26). Likewise, dominant active forms of Arf1 (Q71L, T161A) (27,28) and Arf6 (D125N) (29), as well as dominant negative Arf6 (T27N) (30), were also generated.…”