Targeting and activation of Rac1 are mediated by the exchange factor β-Pix

Klooster, Jean Paul ten; Jaffer, Zahara M.; Chernoff, Jonathan; Hordijk, Peter L.

doi:10.1083/jcb.200509096

Cited by 220 publications

(252 citation statements)

References 52 publications

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“…b-PIX (also known as ARHGEF7) is a RhoGEF involved in various small GTPase signaling pathways. 30 b-PIX contains a PDZ-binding motif at its C-terminal end. We recently demonstrated that EBP50 interacts with b-PIX through its first PDZ domain, and b-PIX associates with the podocalyxin-EBP50-ezrin complex.…”

Section: Resultsmentioning

confidence: 99%

Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis

Chen

Jou

2012

Cell Death Differ

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We demonstrated a protein kinase C (PKC)-dependent phosphorylation of canine ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50) at serine 347/348 by site-directed mutagenesis and a phospho-specific antibody. Cell fractionation and confocal imaging revealed the relocation of EBP50 from the plasma membrane to cytosol that accompanied this phosphorylation event. Increased phosphorylation at these serine residues led to the dissociation of EBP50 from ezrin and b-PIX, which are two upstream regulators of Rac1 activation. Cells overexpressing an EBP50 mutant, mimicking serine 347/348 phosphorylation, became refractory to hepatocyte growth factor-induced cell spreading and scattering, which is normally mediated by Rac1 activation. Detachment of cells from the substratum also elicited an increase in EBP50 phosphorylation, apparently due to counteracting activities of PKC and protein phosphastase 2A, which resulted in decreased Rac1 activation and induction of anoikis. Cells overexpressing an EBP50 mutant defective in serine 347/348 phosphorylation did not undergo apoptosis in suspension culture. These studies reveal a signaling cascade in which different phosphorylation states and subcellular localization of EBP50 regulate Rac1 function. Scaffold proteins have many modular domains that mediate protein-protein interactions involved in orchestrating different signaling cascades. As a result, scaffold proteins are thought to act as stable structural proteins that facilitate many cell signaling pathways. However, this traditional role of scaffold proteins has been challenged, 1 and the dynamic role of scaffold as both an activator and suppressor of cellular signaling remains poorly characterized. Furthermore, the factors that modulate such a bifunctional role of scaffold proteins remain largely unknown.Ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50) is a scaffold protein with two tandem PDZ domains and a C-terminal ERM-binding domain. 2,3 EBP50 binds proteins that contain a PDZ-binding motif including ion exchangers, 4,5 ion channels, 6 G protein-coupled receptors, 7-9 growth factor tyrosine kinase receptors 10,11 and PTEN. 10 EBP50 participates in the modulation of ion transport, organization of apical microvilli, cancer development, and the trafficking and stabilization of membrane proteins. 9,12-14 Understanding the mechanisms that regulate the function of EBP50 is, therefore, a critical problem.Ras family proteins are locked in a GDP-bound, inactivated state by binding GDI in the GTP-rich cellular environment. 15,16 Dissociation of small GTPases from GDI is required, but is not sufficient for their activation. GEFs drive the exchange of GTP for GDP, leading to the activation of small GTPases. 17 EBP50 can activate Rho-family small GTPases by recruiting ezrin, which is thought to cause the dissociation of GDI from the small GTPase, and b-PIX, a Rac1 GEF that contains a PDZ-binding motif. 18,19 EBP50 is a serine/threonine-phosphorylated protein. 3 The phosphorylation state of EBP50 regulates the acc...

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Section: Resultsmentioning

confidence: 99%

Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis

Chen

Jou

2012

Cell Death Differ

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“…localization of the Rac1 GEF, ␤-PIX. It was recently reported that the interaction with ␤-PIX was necessary and sufficient for Rac1 recruitment to membrane ruffles and to focal adhesions (ten Klooster et al, 2006). We therefore hypothesized that the depletion of ARF6 might allow the relocalization of ␤-PIX to the plasma membrane and the activation of Rac1.…”

Section: Discussionmentioning

confidence: 99%

“…The Rac1 Guanine-Nucleotide Exchange Factor ␤-PIX Is Relocalized to the Plasma Membrane in ARF6-depleted Cells It is generally accepted that relocalization of small GTPases is necessary for their activation, although the mechanisms that control targeting of Rho GTPases in general are poorly understood. It was recently demonstrated that Rac1 binds directly to ␤-PIX (p21-activated kinases [PAK]-interacting exchange factor) and that this interaction is necessary and sufficient for Rac1 recruitment to membrane ruffles, providing a model for the intracellular targeting and localized activation of Rac1 (ten Klooster et al, 2006). To begin to address why Rac1 is found basally activated in ARF6-depleted cells, we examined the localization of the Rac/Cdc42 GEF ␤-PIX.…”

Section: Remodelling Of the Actin Cytoskeleton Induced By Arf6 Depletmentioning

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

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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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“…In addition to their potential enzymatic activity on Rho and Arf GTPases, PIX and GIT proteins are essential to the recruitment of signaling complexes including Rac1 and PAK1. They also integrate signals from Src and FAK to regulate focal adhesion dynamics (Brown et al, 2005;ten Klooster et al, 2006). The multimolecular complex PIX-GIT-PAK may thus be important for regulating Rho GTPases and podosome organization in osteoclast.…”

Section: Exchange Factors and Osteoclast Biologymentioning

confidence: 99%

Rho GTPases in osteoclasts: Orchestrators of podosome arrangement

Ory

Brazier

Pawlak

et al. 2008

European Journal of Cell Biology

130

101

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Cells from the myeloid lineage, namely macrophages, dendritic cells and osteoclasts develop podosomes instead of stress fibers and focal adhesions to adhere and migrate.Podosomes share many components with focal adhesions but differ in their molecular organization, with a dense core of polymerized actin surrounded by scaffolding proteins, kinases and integrins. Podosomes are found either isolated both in macrophages and dendritic cells or arranged into superstructures in osteoclasts. When osteoclasts resorb bone, they form an F-actin rich sealing zone, which is a dense array of connected podosomes that firmly anchors osteoclasts to bone. It delineates a compartment in which protons and proteases are secreted to dissolve and degrade the mineralized matrix. Since Rho GTPases have been shown to control F-actin stress fibers and focal adhesions in mesenchymal cells, the question of whether they could also control podosome formation and arrangement in cells from the myeloid lineage, and particularly in osteoclasts, rapidly emerged. This article considers recent advances made in our understanding of podosome arrangements in osteoclasts and how Rho GTPases may control it.

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Targeting and activation of Rac1 are mediated by the exchange factor β-Pix

Cited by 220 publications

References 52 publications

Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis

Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis

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

Rho GTPases in osteoclasts: Orchestrators of podosome arrangement

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