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

• Published in: The Journal of biological chemistry 2004-03, Vol.279 (10), p.9199-9207
• Main Authors: Lozupone, Francesco, Lugini, Luana, Matarrese, Paola, Luciani, Francesca, Federici, Cristina, Iessi, Elisabetta, Margutti, Paola, Stassi, Giorgio, Malorni, Walter, Fais, Stefano
• Format: Article
• Language: English
• Subjects: Actins - metabolism; Apoptosis - physiology; Binding Sites; Cytoskeletal Proteins; fas Receptor - genetics; fas Receptor - metabolism; HeLa Cells; Humans; Mutation; Phosphoproteins - analysis; Phosphoproteins - metabolism; Protein Binding; Protein Structure, Tertiary; Signal Transduction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M305561200

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.