The GIT/PIX complex: an oligomeric assembly of GIT family ARF GTPase-activating proteins and PIX family Rac1/Cdc42 guanine nucleotide exchange factors

Premont, Richard T.; Perry, Stephen J.; Schmalzigaug, Robert; Roseman, J. Tyler; Xing, Yanghui; Claing, Audrey

doi:10.1016/j.cellsig.2004.02.002

Cited by 71 publications

(77 citation statements)

References 31 publications

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“…Interestingly, no defect in GIT1 localization (Ն88%) was observed ( Figure 7C). Thus, despite the reported ability of PKL/GIT (and PIX) to form heterooligomers (Kim et al, 2003;Paris et al, 2003;Premont et al, 2004), focal adhesion localized GIT1 cannot substitute for the specific requirement for PKL tyrosine phosphorylation in its focal adhesion localization providing additional evidence for the structural, regulatory and functional divergence of PKL/GIT2 and GIT1.…”

Section: Loss Of Src or Fak But Not Nck Results In A Profound Inhibitmentioning

confidence: 99%

Src and FAK Kinases Cooperate to Phosphorylate Paxillin Kinase Linker, Stimulate Its Focal Adhesion Localization, and Regulate Cell Spreading and Protrusiveness

Brown

Cary

Jamieson

et al. 2005

MBoC

166

179

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The ArfGAP paxillin kinase linker (PKL)/G protein-coupled receptor kinase-interacting protein (GIT)2 has been implicated in regulating cell spreading and motility through its transient recruitment of the p21-activated kinase (PAK) to focal adhesions. The Nck-PAK-PIX-PKL protein complex is recruited to focal adhesions by paxillin upon integrin engagement and Rac activation. In this report, we identify tyrosine-phosphorylated PKL as a protein that associates with the SH3-SH2 adaptor Nck, in a Src-dependent manner, after cell adhesion to fibronectin. Both cell adhesion and Rac activation stimulated PKL tyrosine phosphorylation. PKL is phosphorylated on tyrosine residues 286/392/592 by Src and/or FAK and these sites are required for PKL localization to focal adhesions and for paxillin binding. The absence of either FAK or Src-family kinases prevents PKL phosphorylation and suppresses localization of PKL but not GIT1 to focal adhesions after Rac activation. Expression of an activated FAK mutant in the absence of Src-family kinases partially restores PKL localization, suggesting that Src activation of FAK is required for PKL phosphorylation and localization. Overexpression of the nonphosphorylated GFP-PKL Triple YF mutant stimulates cell spreading and protrusiveness, similar to overexpression of a paxillin mutant that does not bind PKL, suggesting that failure to recruit PKL to focal adhesions interferes with normal cell spreading and motility. INTRODUCTIONCell attachment, spreading, and motility are complex processes requiring the integration of diverse signaling networks and structural assemblies (Jockusch et al., 1995;Sastry and Burridge, 2000;Juliano, 2002). One of the earliest steps in transducing extracellular cues through integrins to the cytoskeleton is the activation of the tyrosine kinases Src and FAK (Schwartz et al., 1995;Giancotti and Tarone, 2003). FAK is an integrin-binding nonreceptor tyrosine kinase that upon integrin ligation is activated to autophosphorylate Y397 and bind to the Src SH2 domain (Schaller et al., 1994;Calalb et al., 1995). Src then phosphorylates FAK on multiple residues that increase FAK kinase activity and several downstream binding partners for Src/FAK are targeted for phosphorylation, including the focal adhesion proteins p130Cas and paxillin (reviewed in Brown and Turner, 2004;Playford and Schaller, 2004;Mitra et al., 2005). Phosphorylated paxillin binding to the SH2/SH3 adaptor protein Crk is implicated in Rac activation and stimulation of cell motility (Petit et al., 2000;Lamorte et al., 2003;Valles et al., 2004), whereas binding of paxillin to the p120RasGAP SH2 domain may displace and allow for the activation of p190RhoGAP and subsequent decrease in RhoA activity (Iwasaki et al., 2002).The Cdc42, Rac1, and RhoA members of the Ras superfamily of p21 GTPases are central intermediaries in coordinating the defined temporal-spatial progression of signals emanating from initial integrin ligation, through acquisition of a polarized state, to initiation and maintenance of directed ce...

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Section: Loss Of Src or Fak But Not Nck Results In A Profound Inhibitmentioning

confidence: 99%

Src and FAK Kinases Cooperate to Phosphorylate Paxillin Kinase Linker, Stimulate Its Focal Adhesion Localization, and Regulate Cell Spreading and Protrusiveness

Brown

Cary

Jamieson

et al. 2005

MBoC

166

179

View full text Add to dashboard Cite

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“…In cells, it is likely that the signaling complex necessary to promote migration involves other signaling partners/regulators/effectors of both small GTPases. For example, we and others have shown that GIT proteins, characterized as ARF GAPs, and ␤-PIX proteins, a Rac GEF are tightly associated (Bagrodia et al, 1999;Premont et al, 2004). One would therefore expect these proteins to associate with ARF6 and Rac1 to promote signal transduction in a cellular context.…”

Section: Discussionmentioning

confidence: 99%

Endogenous ARF6 Interacts with Rac1 upon Angiotensin II Stimulation to Regulate Membrane Ruffling and Cell Migration

Cotton

Boulay

Houndolo

et al. 2007

MBoC

Self Cite

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ARF6 and Rac1 are small GTPases known to regulate remodelling of the actin cytoskeleton. Here, we demonstrate that these monomeric G proteins are sequentially activated when HEK 293 cells expressing the angiotensin type 1 receptor (AT 1 R) are stimulated with angiotensin II (Ang II). After receptor activation, ARF6 and Rac1 transiently form a complex. Their association is, at least in part, direct and dependent on the nature of the nucleotide bound to both small G proteins. ARF6-GTP preferentially interacts with Rac1-GDP. AT 1 R expressing HEK293 cells ruffle, form membrane protrusions, and migrate in response to agonist treatment. ARF6, but not ARF1, depletion using small interfering RNAs recapitulates the ruffling and migratory phenotype observed after Ang II treatment. These results suggest that ARF6 depletion or Ang II treatment are functionally equivalent and point to a role for endogenous ARF6 as an inhibitor of Rac1 activity. Taken together, our findings reveal a novel function of endogenously expressed ARF6 and demonstrate that by interacting with Rac1, this small GTPase is a central regulator of the signaling pathways leading to actin remodeling.

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“…GIT1 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].…”

Section: Introductionmentioning

confidence: 99%

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

Schmalzigaug

Garron

Roseman

et al. 2007

Cellular Signalling

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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 ...

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The GIT/PIX complex: an oligomeric assembly of GIT family ARF GTPase-activating proteins and PIX family Rac1/Cdc42 guanine nucleotide exchange factors

Cited by 71 publications

References 31 publications

Src and FAK Kinases Cooperate to Phosphorylate Paxillin Kinase Linker, Stimulate Its Focal Adhesion Localization, and Regulate Cell Spreading and Protrusiveness

Src and FAK Kinases Cooperate to Phosphorylate Paxillin Kinase Linker, Stimulate Its Focal Adhesion Localization, and Regulate Cell Spreading and Protrusiveness

Endogenous ARF6 Interacts with Rac1 upon Angiotensin II Stimulation to Regulate Membrane Ruffling and Cell Migration

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

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