Understanding the role of the G-actin-binding domain of Ena/VASP in actin assembly
ABSTRACT The Ena/VASP and WASP family of proteins play distinct roles in actin cytoskeleton remodeling. Ena/VASP is linked to actin filament elongation, whereas WASP plays a role in filament nucleation and branching mediated by Arp2/3 complex. The molecular mechanisms controlling both processes are only emerging. Both Ena/VASP and WASP are multidomain proteins. They both present poly-Pro regions, which mediate the binding of profilin-actin, followed by G-actin-binding (GAB) domains of the WASP-homology 2 (WH2) type. However, the WH2 of Ena/VASP is somewhat different from that of WASP, and has been poorly characterized. Here we demonstrate that this WH2 binds profilin-actin with higher affinity than actin alone. The results are consistent with a model whereby allosteric modulation of affinity drives the transition of profilin-actin from the poly-Pro region to the WH2 and then to the barbed end of the filament during elongation. Therefore, the function of the WH2 in Ena/VASP appears to be to "process" profilin-actin for its incorporation at the barbed end of the growing filament. Conformational changes in the newly incorporated actin subunit, resulting either from nucleotide hydrolysis or from the G- to F-actin transition, may serve as a "sensor" for the processive stepping of Ena/VASP. Conserved domain architecture suggests that WASP may work similarly.
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ABSTRACT: The WAVE complex is the main activator of the Arp2/3 complex for actin filament nucleation and assembly in the lamellipodia of moving cells. Another important player in lamellipodial protrusion is Ena/VASP proteins, which enhance actin filament elongation. Here we examine the molecular coordination between the nucleating activity of the Arp2/3 complex and the elongating activity of Ena/VASP proteins for the formation of actin networks. Using an in vitro bead motility assay, we show that WAVE directly binds VASP, resulting in an increase in Arp2/3 complex-based actin assembly. We show that this interaction is important in vivo as well for the formation of lamellipodia during the ventral enclosure event of Caenorhabditis elegans embryogenesis. Ena/VASP's ability to bind F-actin and profilin-complexed G-actin are important for its effect, while Ena/VASP tetramerization is not necessary. Our data are consistent with the idea that binding of Ena/VASP to WAVE potentiates Arp2/3 complex activity and lamellipodial actin assembly.Molecular Biology of the Cell 10/2014; 26(1). DOI:10.1091/mbc.E14-07-1200 · 4.55 Impact Factor