Spectrin interacts with EVL (Enabled/vasodilator-stimulated phosphoprotein-like protein), a protein involved in actin polymerization

• Published in: Biology of the cell 2006-05, Vol.98 (5), p.279-293
• Main Authors: Bournier, Odile, Kroviarski, Yolande, Rotter, Björn, Nicolas, Gaël, Lecomte, Marie C., Dhermy, Didier
• Format: Article
• Language: English
• Subjects: Actins; Animals; Cell Adhesion Molecules - metabolism; Cercopithecus aethiops; COS Cells; Ena/vasodilator-stimulated phosphoprotein-like protein (EVL); Epithelial Cells; Gene Library; Immunoprecipitation; Kidney - metabolism; Mena; Microfilament Proteins - metabolism; Phosphoproteins - metabolism; Protein Interaction Mapping; Protein Isoforms - metabolism; Rats; spectrin; Spectrin - physiology; Src homology 3 (SH3) domain; src Homology Domains; Two-Hybrid System Techniques; vasodilator-stimulated phosphoprotein (VASP)
• ISSN: 0248-4900; 1768-322X
• DOI: 10.1042/BC20050024

Background information. The α‐ and β‐spectrin chains constitute the filaments of the spectrin‐based skeleton, which was first identified in erythrocytes. The discovery of analogous structures at plasma membranes of eukaryotic cells has led to investigations of the role of this spectrin skeleton in many cellular processes. The αII‐spectrin chain expressed in nucleated cells harbours in its central region several functional motifs, including an SH3 (Src homology 3) domain. Results. Using yeast two‐hybrid screening, we have identified EVL [Enabled/VASP (vasodilator‐stimulated phosphoprotein)‐like protein] as a new potential partner of the αII‐spectrin SH3 domain. In the present study, we investigated the interaction of the αII‐spectrin SH3 domain with EVL and compared this with other proteins related to EVL [Mena (mammalian Enabled) and VASP]. We confirmed the in vitro interaction between EVL and the αII‐spectrin SH3 domain by GST (glutathione S‐transferase) pull‐down assays, and showed that the co‐expression of EVL with the αII‐spectrin SH3 domain in COS‐7 cells resulted in the partial delocalization of the SH3 domain from cytoplasm to filopodia and lamellipodia, where it was co‐localized with EVL. In kidney epithelial and COS‐7 cells, we demonstrated the co‐immunoprecipitation of the αII‐spectrin chain with over‐expressed EVL. Immunofluorescence studies showed that the over‐expression of EVL in COS‐7 cells promoted the formation of filopodia and lamellipodia, and the expressed EVL was detected in filopodial tips and the leading edge of lamellipodia. In these cells over‐expressing EVL, the αII‐spectrin membrane labelling lagged behind EVL staining in lamellipodia and filopodia, with co‐localization of these two stains in the contact area. In kidney epithelial cell lines, focused co‐localization of spectrin with expressed EVL was observed in the membrane of the lateral domain, where the cell—cell contacts are reinforced. Conclusions. The possible link between the spectrin‐based skeleton and actin via the EVL protein suggests a new way of integrating the spectrin‐based skeleton in areas of dynamic actin reorganization.