Microsoft Word - bsr20150149

The members of the actin regulatory family of Ena/VASP proteins form stable tetramers. The vertebrate members of the Ena/VASP family, VASP, Mena, and EVL, have many overlapping properties and expression patterns, but functional and regulatory differences between paralogs have been observed. The formation of mixed oligomers may serve a regulatory role to refine Ena/VASP activity. While it has been assumed that family members can form mixed oligomers, this possibility has not been investigated systematically. Using cells expressing controlled combinations of VASP, Mena, and EVL, we evaluated the composition of Ena/VASP oligomers and found that VASP forms oligomers without apparent bias with itself, Mena, or EVL. However, Mena and EVL showed only weak hetero‐oligomerization, suggesting specificity in the association of Ena/VASP family members. Co‐ expression of VASP increased the ability of Mena and EVL to form mixed oligomers. Additionally, we found that the tetramerization domain at the C‐termini of Ena/VASP proteins conferred the observed selectivity. Finally, we demonstrate that replacement of the TD with a synthetic tetramerizing coiled‐coil sequence supports homo‐oligomerization and normal VASP subcellular localization Abbreviations used: Ena/VASP, Enabled/vasodilator‐stimulated phosphoprotein; EVH, Ena/VASP homology; F‐actin, filamentous actin; FAB, F‐actin binding; G‐actin, globular/monomeric actin; GAB, G‐actin binding; Lpd, lamellipodin; PRR, proline‐ rich regions; TD, tetramerization domain. Ac ce pt ed M an us cr ip t © 2015 The Author(s) Archiving permitted only in line with the archiving policy of Portland Press Limited. The final version of record will be available under the Creative Commons Attribution Licence 3.0 (http://creativecommons.org/licenses/by/3.0/). You are encouraged to use the final version of record.