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

Abstract

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...

A Molecular Switch for the Orientation of Epithelial Cell Polarization

“…Classical protein kinase C (PKC) phosphorylation of the FBM in either Podxl (S481) or NHERF1 (S339,340) abolishes interaction of either protein with Ezrin (Chen et al, 2012; Fukasawa et al, 2011; Garbett et al, 2010). Classical PKC(s) may therefore regulate the transient disassembly of the Podxl complex during polarity reorientation (Figures 3A and S3A).…”

“…To directly test this possibility, we first determined that PKCβ was a major regulator of Podxl complex phosphorylation using phosphorylation-specific antibodies to the NHERF1 FBM (pS339,340) (Chen et al, 2012). PKCβ, and to a lesser extent PKCα, depletion reduced PMA-induced phosphorylation of the FBM of endogenous NHERF1 (Figure 6F).…”

“…PKCβ, and to a lesser extent PKCα, depletion reduced PMA-induced phosphorylation of the FBM of endogenous NHERF1 (Figure 6F). Next, we expressed Podxl and NHERF1 PKC phosphorylation site mutants that control Ezrin association (Chen et al, 2012; Fukasawa et al, 2011). Expression of phosphorylation-deficient Podxl or NHERF1 strongly reduced SLF and induced front-rear polarization, which was potentiated by codepletion of endogenous Podxl/NHERF1.…”

“…The PP2A phosphatase complex binds and dephosphorylates NHERF1 (Boratkó et al, 2012; Chen et al, 2012). We examined the localization of the NHERF1-associating PP2A-B55α subunit (3×HA-PP2A-Bα).…”

SLC9A3R1 stimulates autophagy via BECN1 stabilization in breast cancer cells

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