Ezrin Regulates E-Cadherin-dependent Adherens Junction Assembly through Rac1 Activation

Abstract

Ezrin, a membrane cytoskeleton linker, is involved in cellular functions, including epithelial cell morphogenesis and adhesion. A mutant form of ezrin, ezrin T567D, maintains the protein in an open conformation, which when expressed in Madin-Darby canine kidney cells causes extensive formation of lamellipodia and altered cell-cell contacts at low cell density. Furthermore, these cells do not form tubules when grown in a collagen type I matrix. While measuring the activity of Rho family GTPases, we found that Rac1, but not RhoA or Cdc 42, is activated in ezrin T567D-expressing cells, compared with cells expressing wild-type ezrin. Together with Rac1 activation, we observed an accumulation of E-cadherin in intracellular compartments and a concomitant decrease in the level of E-cadherin present at the plasma membrane. This effect could be reversed with a dominant negative form of Rac1, N17Rac1. We show that after a calcium switch, the delivery of E-cadherin from an internalized pool to the plasma membrane is greatly delayed in ezrin T567D-producing cells. In confluent cells, ezrin T567D production decreases the rate of E-cadherin internalization. Our results identify a new role for ezrin in cell adhesion through the activation of the GTPase Rac1 and the trafficking of E-cadherin to the plasma membrane. INTRODUCTIONThe ezrin, radixin, and moesin (ERM) proteins act as linkers between the plasma membrane and the actin cytoskeleton. They have been implicated in the organization of specific domains of the plasma membrane, such as the apical domain of epithelial cells, the immunological synapse, or the formation of uropods Crepaldi et al., 1997;Serrador et al., 1997;Allenspach et al., 2001;Delon et al., 2001;Roumier et al., 2001). Their functions are regulated by changes in their conformation. ERM proteins exist in the cytoplasm as dormant monomers in which the F-actin cytoskeleton and the plasma membrane binding sites are masked. This closed conformation is due to an intramolecular N-to C-ERM association domain (ERMAD) interaction Gary and Bretscher, 1995). Thus, abrogation of the N/C-ERMAD interaction is required to "open up" the molecules and to expose their cryptic binding sites . Two factors have been implicated in the activation of ERM proteins. The binding to phosphatidylinositol 4,5-biphosphate is required for their interaction with actin in vitro and with membrane proteins in vivo (Nakamura et al., 1999;Barret et al., 2000). In addition, phosphorylation of a conserved threonine residue in the C-ERMAD, T567, inhibits the N/C-ERMAD interaction in vitro (Matsui et al., 1998;Nakamura et al., 1999) and in vivo, converts inactive oligomers to active monomers . Expression of an ezrin mutant, ezrin T567D, which mimics its phosphorylation, induces lamellipodia formation in nonconfluent cells and tufts of microvilli in confluent cells. Moreover, production of ezrin T567D perturbs the organization of epithelial cell monolayers (Yonemura et al., 1999;Gautreau et al., 2000).A number of studies have implicated ERM proteins in...

Merlin regulates transmembrane receptor accumulation and signaling at the plasma membrane in primary mouse Schwann cells and in human schwannomas

“…Merlin regulates the delivery of growth factor receptors to the plasma membrane in SCs Several reports have proposed a role for merlin in protein transport (Maitra et al, 2006;Curto et al, 2007) and the involvement of the ERM proteins in vesicular trafficking is well established (Pujuguet et al, 2003;Tamma et al, 2005;Stanasila et al, 2006). We compared growth factor internalization in Nf2 þ / þ and Nf2 À/À SC cultures using a surface protein biotin pulsechase approach and found no differences (data not shown).…”

“…Interestingly, in a schwannoma cell line, merlin regulates membrane levels of PDGFR-b by inducing its degradation (Fraenzer et al, 2003), revealing an additional degree of complexity in merlin regulation of growth factor steady state levels. ERM proteins also are implicated in vesicular trafficking (Pujuguet et al, 2003;Deretic et al, 2004;Stanasila et al, 2006). Thus, merlin and ERM proteins regulate protein transport to and from the plasma membrane.…”

Interaction of Ezrin with the Novel Guanine Nucleotide Exchange Factor PLEKHG6 Promotes RhoG-dependent Apical Cytoskeleton Rearrangements in Epithelial Cells

“…An association of ERM proteins with the exchange factor Dbl in vitro as well as in vivo has been reported (Takahashi et al, 1998;Lee et al, 2004;Vanni et al, 2004). In epithelial cells, an active form of ezrin has been shown to activate the small GTPase Rac1 with a concomitant disassembly of adherens junctions (Pujuguet et al, 2003). A genetic analysis in Drosophila, however, has suggested that Drosophila moesin negatively regulates the Rho1 pathway (Speck et al, 2003).…”

Phosphorylated ezrin is located in the nucleus of the osteosarcoma cell

“…11,12 Phosphorylation of ezrin promotes morphogenesis of apical microvilli and regulates E-cadherindependent adherent junction assembly. [13][14][15] Ezrin directly interacts with signaling enzymes such as phosphoinositide 3-kinase (PI3-kinase), and the phosphorylated tyrosine 353 (Tyr-353) residue of ezrin is regulated through the PI3-kinase/Akt pathway. 11 Ezrin is also preferentially degraded and resynthesized after oxidative stress 16 and the phosphorylation at threonine 567 (Thr-567) depends on the p38 MAP-kinase activity.…”