Understanding the role of the G-actin-binding domain of Ena/VASP in actin assembly

Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, USA.
Journal of Structural Biology (Impact Factor: 3.23). 09/2006; 155(2):195-201. DOI: 10.1016/j.jsb.2006.01.012
Source: PubMed

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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    • "Interactions may also be significantly modified by numerous physiologically relevant factors such as buffer ionic strength, VASP and actin concentration, substrate confinement or molecular crowding (Breitsprecher et al. 2008). Because nucleation, tetramerization and bundling are all interrelated and accounted for by the EVH2 domain found in each of VASP's four flexible arms, VASP–actin interactions may be strongly influenced by the local cytoskeletal environment (Chereau and Dominguez 2006). Because VASP–actin interactions are complex, it is important to study them in simplified and controlled systems . "
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    ABSTRACT: Vasodilator-stimulated phosphoprotein (Ena/VASP) is an actin binding protein, important for actin dynamics in motile cells and developing organisms. Though VASP's main activity is the promotion of barbed end growth, it has an F-actin binding site and can form tetramers, and so could additionally play a role in actin crosslinking and bundling in the cell. To test this activity, we performed rheology of reconstituted actin networks in the presence of wild-type VASP or mutants lacking the ability to tetramerize or to bind G-actin and/or F-actin. We show that increasing amounts of wild-type VASP increase network stiffness up to a certain point, beyond which stiffness actually decreases with increasing VASP concentration. The maximum stiffness is 10-fold higher than for pure actin networks. Confocal microscopy shows that VASP forms clustered actin filament bundles, explaining the reduction in network elasticity at high VASP concentration. Removal of the tetramerization site results in significantly reduced bundling and bundle clustering, indicating that VASP's flexible tetrameric structure causes clustering. Removing either the F-actin or the G-actin binding site diminishes VASP's effect on elasticity, but does not eliminate it. Mutating the F-actin and G-actin binding site together, or mutating the F-actin binding site and saturating the G-actin binding site with monomeric actin, eliminates VASP's ability to increase network stiffness. We propose that, in the cell, VASP crosslinking confers only moderate increases in linear network elasticity, and unlike other crosslinkers, VASP's network stiffening activity may be tuned by the local concentration of monomeric actin.
    Biophysics of Structure and Mechanism 09/2012; 41(11):979-90. DOI:10.1007/s00249-012-0861-1 · 2.47 Impact Factor
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    • "The affinity of the GAB domain for actin monomers appears to be higher than for filaments so (similar to WH2 domains and profilin) incorporation of a bound monomer into the filament promotes dissociation of the GAB domain and frees it up to bind another monomer. The GAB domain is related to WH2 domains found in other actin regulatory proteins (Chereau and Dominguez, 2006; Ferron et al., 2007) and, consistent with our results, Co et al. (2007) found that WH2 domains from N-WASP can interact with barbed ends of actin filaments and recruit the molecule to sites with a high Because the filament elongation rate increases with VASP concentration, Breitsprecher et al. (2008) proposed that VASP binds barbed ends distributively, delivering a maximum of four actin monomers before either dissociating or remaining statically attached to the side of the filament. We find, however, that in the low ionic strength buffers used for these experiments, individual human VASP tetramers interact processively with growing filament ends and, in the presence of 2 µM actin, can deliver more than 60 monomers before dissociating. "
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    ABSTRACT: Ena/VASP proteins regulate the actin cytoskeleton during cell migration and morphogenesis and promote assembly of both filopodial and lamellipodial actin networks. To understand the molecular mechanisms underlying their cellular functions we used total internal reflection fluorescence microscopy to visualize VASP tetramers interacting with static and growing actin filaments in vitro. We observed multiple filament binding modes: (1) static side binding, (2) side binding with one-dimensional diffusion, and (3) processive barbed end tracking. Actin monomers antagonize side binding but promote high affinity (K(d) = 9 nM) barbed end attachment. In low ionic strength buffers, VASP tetramers are weakly processive (K(off) = 0.69 s(-1)) polymerases that deliver multiple actin monomers per barbed end-binding event and effectively antagonize filament capping. In higher ionic strength buffers, VASP requires profilin for effective polymerase and anti-capping activity. Based on our observations, we propose a mechanism that accounts for all three binding modes and provides a model for how VASP promotes actin filament assembly.
    The Journal of Cell Biology 11/2010; 191(3):571-84. DOI:10.1083/jcb.201003014 · 9.69 Impact Factor
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    • "It has been suggested that basic stretches within the EVH2 domain, that contains the S239 phosphorylation site, mediate VASP binding to F-actin via electrostatic forces (Huttelmaier and et al., 1999; Bachmann and et al., 1999). In addition, Chereau and Dominguez suggest that a subdomain of the EVH2 domain binds profilin-G-actin and mediates the incorporation of the actin monomer onto the barbed end of the growing actin filament (Chereau and Dominguez, 2006). Using an identical approach to ours (expression of a mutant VASP mimicking phosphorylation), Harbeck et al. (2000) were able to show that the introduction of negative charges by phosphorylation weakens the VASP-F-actin interaction resulting in less actin polymerization. "
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    ABSTRACT: Nitric oxide triggers cGMP-dependent kinase-mediated phosphorylation of the actin regulator vasodilator-stimulated phosphoprotein (VASP) at residue serine239. The function of this phosphorylation for smooth muscle cell (SMC) adhesion, spreading, matrix contraction, and invasion is not well understood. We reconstituted VASP deficient SMC with wild-type VASP (wt-VASP) or VASP mutants that mimic "locked" serine239 phosphorylation (S239D-VASP) or "blocked" serine239 phosphorylation (S239A-VASP). Collagen gel contraction was reduced in S239D-VASP compared to S239A-VASP and wt-VASP expressing cells and nitric oxide (NO) stimulation decreased gel contraction of wt-VASP reconstituted SMC. Invasion of collagen was enhanced in S239D-VASP and NO-stimulated wild-type SMCs compared to S239A-VASP expressing cells. Expression of S239D-VASP impaired SMC attachment to collagen, reduced the number of membrane protrusions, and caused cell rounding compared to expression of S239A-VASP. Treatment of wt-VASP reconstituted SMCs with NO exerted similar effects as expression of S239D-VASP. As unstimulated cells were spreading on collagen S239A-VASP and wt-VASP localized to actin fibers whereas S239D-VASP was enriched in the cytosol. NO interferes with SMC invasion and contraction of collagen matrices. This requires phosphorylation of VASP on serine239, which reduces VASP binding to actin fibers. These findings support the conclusion that VASP phosphorylation at serine239 regulates cytoskeleton remodeling.
    Journal of Cellular Physiology 01/2010; 222(1):230-7. DOI:10.1002/jcp.21942 · 3.87 Impact Factor
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