AlphaII-spectrin interacts with Tes and EVL, two actin-binding proteins located at cell contacts.
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 alpha- and beta-subunits are linked to actin microfilaments, forming a network that binds a multitude of proteins. The most prevalent alpha-spectrin subunit in non-erythroid cells, alphaII-spectrin, contains two particular spectrin repeats in its central region, alpha9 and alpha10, 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 alpha9-alpha10 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 alpha10 repeat of alphaII-spectrin whereas EVL interacts with the Src homology 3 domain located within the alpha9 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 alphaII-spectrin, Tes and EVL indicate new functions for spectrin in actin dynamics and focal adhesions.
Rotter
Björn
B
INSERM U409, Faculté de Médecine Bichat, Association Claude Bernard, Xavier Bichat, 16 rue Henri Huchard, BP416, 75870 Paris cedex 18, France.
Bournier
Odile
O
Nicolas
Gael
G
Dhermy
Didier
D
Lecomte
Marie-Christine
MC
D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion; D002460:Cell Line; D002462:Cell Membrane; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D018398:Homeodomain Proteins / Q000378:metabolism*; D060588:LIM Domain Proteins; D008840:Microfilament Proteins / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins; D013049:Spectrin / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; C092744:ENA-VASP proteins; D018398:Homeodomain Proteins; D060588:LIM Domain Proteins; D008840:Microfilament Proteins; D016601:RNA-Binding Proteins; C429688:TES protein, human; D025521:Tumor Suppressor Proteins; D013049:Spectrin