αII-Spectrin interacts with Tes and EVL, two actin-binding proteins located at cell contacts

Abstract

The spectrin-based membrane skeleton, a multi-protein scaffold attached to diverse cellular membranes, is presumed to be involved in the stabilization of membranes, the establishment of membrane domains as well as in vesicle trafficking and nuclear functions. Spectrin tetramers made of α- and β-subunits are linked to actin microfilaments, forming a network that binds a multitude of proteins. The most prevalent α-spectrin subunit in non-erythroid cells, αII-spectrin, contains two particular spectrin repeats in its central region, α9 and α10, which host an Src homology 3 domain, a tissue-specific spliced sequence of 20 residues, a calmodulin-binding site and major cleavage sites for caspases and calpains. Using yeast two-hybrid screening of kidney libraries, we identified two partners of the α9-α10 repeats: the potential tumour suppressor Tes, an actin-binding protein mainly located at focal adhesions; and EVL (Ena/vasodilator-stimulated phosphoprotein-like protein), another actin-binding protein, equally recruited at focal adhesions. Interactions between spectrin and overexpressed Tes and EVL were confirmed by co-immunoprecipitation. In vitro studies showed that the interaction between Tes and spectrin is mediated by a LIM (Lin-11, Isl-1 and Mec3) domain of Tes and by the α10 repeat of αII-spectrin whereas EVL interacts with the Src homology 3 domain located within the α9 repeat. Moreover, we describe an in vitro interaction between Tes and EVL, and a co-localization of these two proteins at focal adhesions. These interactions between αII-spectrin, Tes and EVL indicate new functions for spectrin in actin dynamics and focal adhesions.