Abstract
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 paralogues 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 (TD) 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.
Highlights
The Ena/VASP family comprises a group of highly conserved proteins involved in the regulation of cell morphology, adhesion and motility [1,2,3,4]
We found that the EVH2 domains of both Mena and VASP were readily detected to co-IP with FlagVASP, as expected
Our results demonstrate that Ena/VASP proteins can form mixed oligomers, the composition of these complexes is biased against certain paralogue combinations
Summary
The Ena/VASP family comprises a group of highly conserved proteins involved in the regulation of cell morphology, adhesion and motility [1,2,3,4]. The EVH1 domain mediates interactions between Ena/VASP members and proteins containing the highly conserved poly-proline core consensus motif ‘FPPPP’ (FP4) [12,13]. At the most C-terminal portion of the EVH2 domain, a highly conserved tetramerization domain (TD) mediates the formation of Ena/VASP tetramers [14,18,19,20]. Both full length VASP and the isolated EVH2 domain form stable tetramers as indicated by analytical centrifugation [14,19,21]. The TD of VASP has been crystalized and shown to form a right-handed, coiled-coil tetramer [34]
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