Identification and Relevance of the CD95-binding Domain in the N-terminal Region of Ezrin

Abstract

The CD95 (Fas/APO-1) linkage to the actin cytoskeleton through ezrin is an essential requirement for susceptibility to the CD95-mediated apoptosis in CD4؉ T cells. We have previously shown that moesin was not involved in the binding to CD95. Here we further support the specificity of the ezrin/CD95 binding, showing that radixin did not bind CD95. The ezrin region specifically and directly involved in the binding to CD95 was located in the middle lobe of the ezrin FERM domain, between amino acids 149 and 168. In this region, ezrin, radixin, and moesin show 60 -65% identity, as compared with the 86% identity in the whole FERM domain. Transfection of two different human cell lines with a green fluorescent protein-tagged ezrin mutated in the CD95-binding epitope, induced a marked inhibition of CD95-mediated apoptosis. In these cells, the mutated ezrin did not co-localize or co-immunoprecipitate with CD95. Further analysis showed that the mutated ezrin, while unable to bind CD95, was fully able to bind actin, thus preventing the actin linkage to CD95. Altogether, our results support the specificity of ezrin in the association to CD95 and the importance of the ezrin-to-CD95 linkage in CD95-mediated apoptosis. Moreover, this study suggests that a major role of ezrin is to connect CD95 to actin, thus allowing the CD95 polarization on the cells and the occurrence of the following multiple cascades of the CD95 pathway.A major requirement for the susceptibility to CD95 (APO-1/ Fas)-mediated apoptosis in CD4ϩ T cells is the CD95 polarization on cell uropods, as a result of CD95 linkage to the actin cytoskeleton through ezrin (1). Ezrin, radixin, and moesin (ERM) 1 are closely related proteins involved in cellular polarization and in various cellular functions (2-4). ERM are found in microvilli, filopodia, membrane ruffles, and cell-to-cell contact sites, where they co-localize and associate with F-actin (5-10). ERM are members of the erythrocyte protein 4.1 superfamily, characterized by a ϳ300-residue globular N-terminal domain highly conserved in the ERM family (FERM domain) (3,(11)(12)(13)(14). ERM interact directly with various membrane proteins, such as CD43, CD44, and intercellular adhesion molecule-1, -2, and 3, through their FERM amino-terminal domain (reviewed in Ref.3). These proteins can also indirectly bind to H ϩ /Na ϩ exchanger 3 via the cytoplasmic protein EBP50 (15, 16). ERM are present both in an inactive/closed and in an active/opened form (9, 14), and a direct consequence of ERM activation is their recruitment to the plasma membrane, allowing the ERM linkage to the membrane molecules (17, 18). The ERM/membrane protein interaction is stabilized at the plasma membrane by the ERM association to phosphatidylinositol 4, 5-bisphosphate (19, 20) and regulated by tyrosine phosphorylation in Tyr 145 and Tyr 353 (21-26). However, whereas the ERM domains involved in actin binding are known (27-30), the specific sites accounting for the binding to membrane proteins are much less defined. Particularly, the specific a...

Ezrin is a negative regulator of death receptor-induced apoptosis

“…Similar to previous reports (Parlato et al, 2000;Lozupone et al, 2004), recombinant ezrin bound directly to Fas in a glutathione S-transferase (GST) pull-down assay (Figure 1a). The association of ezrin with Fas during the course of Fas-mediated apoptosis was also monitored in H9 cells, a type I cell line known for prominent DISC formation (Scaffidi et al, 1998;Algeciras-Schimnich et al, 2002;Lee et al, 2006).…”

“…Fas is directly associated with ezrin (Parlato et al, 2000;Lozupone et al, 2004;He´bert et al, 2008), but the requirement for ezrin and actin filament in Fas-triggered cell death remains controversial. A study by Kondo et al (1997) showed that dephosphorylation of ERM proteins and translocation of ERM into cytoplasm is essential for Fas-induced apoptosis to proceed.…”

Pleiotropic function of ezrin in human metastatic melanomas

“…Experiments were performed in a human tumor/SCID mice xenograft model (15), inoculated with either untransfected, ez-146 or full length ezrin-GFP transfected cells (MM1-MM3 and HeLa).…”

“…10,[13][14][15][16][17][18] Notably, ezrin is primarily involved in both cell-to-cell adhesion and cell adhesion to the ECM through association with ICAMs, CD44, E-cadherin and b-catenin. 12,19 To this family belongs also the tumor suppressor merlin (NF-2, schwannomin), whose loss is related to the development of tumors 20,21 and metastasis formation.…”

Localization of Fas/CD95 into the Lipid Rafts on Down-Modulation of the Phosphatidylinositol 3-Kinase Signaling Pathway

“…The formation of this synapse has been extensively studied and cumulative data favor the involvement of the actin cytoskeleton and actin-linked adaptors belonging to the ezrin/ moesin/radixin family to the formation of this lipid raftenriched supramolecular complex (43). Ezrin has been reported as essential to induce the Fas-mediated apoptotic signal (44) and to interact directly to the regulatory subunit p85 of the PI3K (45). Furthermore, recent works by Stickney et al (46) showed that merlin, a protein related to the ezrin/moesin/radixin family, was able to link the actin cytoskeleton to the lipid rafts.…”