Our biochemical studies provide insight into the mechanism for how Ena/VASP proteins enhance actin polymerization at filament barbed ends. We propose that Ena/VASP proteins bind actin filaments at or near barbed ends in a manner that permits association of actin and profilin-actin with barbed ends but prevents association of barbed end capping proteins. Three observations support this hypothesis. First, VASP anti-capping activity depended on the concentrations of both CP and VASP; second, VASP decreased the amount of G-actin in steady state solutions of actin and CP; and third, VASP increased the rate of depolymerization of pre-formed actin filaments in the presence of CP. VASP also protected barbed ends from other barbed end capping proteins, suggesting that VASP generally restricts access of barbed end binding proteins to filament ends. VASP also enhanced the rate of filament elongation from SAS, suggesting that VASP anti-capping activity may result, in part, from changes in the rates of actin subunit addition or dissociation at barbed ends. The relative contributions of VASP’s effects on filament elongation and direct competition with CP for barbed ends to anti-capping activity remain to be determined.
The EVH2 domain of VASP or Mena was sufficient for anti-capping activity, and three structural motifs within EVH2 were essential: the G-actin binding motif, the F-actin binding motif, and the tetramerization motif. These structural features suggest a model for how Ena/VASP proteins associate with F-actin in which the GAB motif within EVH2 interacts with the terminal actin subunits at barbed ends. The basic GAB motif may interact with an electronegative patch on the surface of subdomain 1 of the terminal actin subunits, overlapping the region occupied by the actin-capping domain of gelsolin domain 1 and by barbed end binding toxins (48
). However, unlike capping proteins that inhibit actin assembly, Ena/VASP proteins promote actin assembly, and may do so via interactions of GAB with terminal actin subunits similar to those of the central actin binding motifs of thymosin β4 (51
) and ciboulot (52
). Additional interactions via the F-actin binding motif within EVH2 may stabilize the association at barbed ends. The requirement for VASP tetramers for anti-capping activity suggests that Ena/VASP proteins may interact with each protofilament in order to stably dock at barbed ends.
Profilin further enhanced VASP anti-capping activity via a mechanism that required direct interactions of VASP and profilin-actin. Profilin-actin and VASP could stimulate anti-capping via several possible mechanisms. Binding of profilin-actin to the central proline-rich region could stabilize the interactions of VASP at barbed ends, thereby making VASP more effective in competing with CP. Alternatively, binding of profilin-actin to VASP could increase the local concentration of actin at barbed ends (35
). However, since subunit addition at barbed ends is considered to be diffusion-limited in dilute solutions (20
), locally increased profilin-actin concentrations may not result solely in increased rates of actin assembly. Notably, the FH1-FH2 fragment of some formin proteins, together with profilin, increase the rate for elongation at barbed ends greater than 10-fold (54
). VASP may similarly exploit profilin-actin to increase actin polymerization rates.
Some formin family proteins also compete with barbed end capping proteins, in part, by associating processively with barbed ends as the filaments grow (21
). Our data do not support a processive association of VASP at barbed ends during anti-capping because VASP remains bound to filaments and bundles them. Phosphorylation or interactions with other binding partners may regulate the interactions of Ena/VASP proteins with actin filaments in vivo
to allow persistent association of VASP with elongating filament ends, but this possibility remains to be investigated. Nonetheless, the ability of profilin-actin to enhance VASP anti-capping activity is similar to that of profilin and some formin proteins to allow rapid elongation at barbed ends, even in the presence of capping proteins.
The structural elements of VASP required for anti-capping activity are similar to those required for Mena to restore normal motility to Ena/VASP-deficient fibroblasts in vivo
. Both the G-actin and F-actin binding motifs were essential for normal cell motility (14
) and for anti-capping activity in vitro
. In contrast, profilin-actin was less critical for Ena/VASP function during whole cell motility, since EVH2 or mutant proteins lacking the proline-rich region restored normal cell motility (14
). In cells, regulation of full-length VASP by profilin may be important under conditions where maximal anti-capping activity is required, such as during extension of filopodia. Some of the features required for anti-capping (i.e., FAB and the C-terminal tetramerization motif) were not required for Ena/VASP proteins to stimulate intracellular Listeria
), suggesting that anti-capping activity is not essential for Ena/VASP proteins to support Listeria
VASP binds actin filaments and bundles them, which may have implications for anti-capping activity. VASP was most effective in protecting barbed ends from CP when actin filament mass was low (i.e., at the start of seeded polymerization reactions). Under conditions where filament mass was high, such as in experiments with pre-formed filaments, VASP was less effective in competing with CP, presumably because association of VASP with the sides of filaments would decrease the amount of VASP available to compete with CP for binding filament ends.
Ena/VASP proteins are often situated with subcellular structures having bundled actin filament, such as filopodia and focal adhesions, but the proteins are generally restricted to regions near the ends of filament bundles, rather than along their length. Scaffolding molecules that bind Ena/VASP proteins via interactions with EVH1-motifs also concentrate at the tips of filopodia and lamellipodia and could restrict Ena/VASP proteins to growing filament barbed ends (58
). The loosely packed filament bundles formed by Ena/VASP proteins at physiologic ionic conditions are similar to those observed in nascent filopodia (59
) and may provide access to other filament bundling proteins such as fascin as filopodia mature.
VASP anti-capping and filament bundling activities in vitro
are inhibited upon phosphorylation by PKA. Phosphorylation at S235, adjacent to the G-actin binding motif, was primarily responsible for loss of anti-capping activity. In contrast, phosphorylation of Ena/VASP proteins by PKA at the phosphorylation site conserved in all vertebrate Ena/VASP proteins (S153 in VASP) did not alter anti-capping activity in vitro
. Therefore, phosphorylation at the conserved site likely regulates other functions during cell migration or filopodia formation (8
). Interestingly, in platelets, S235 in VASP is the preferred site for phosphorylation by protein kinase G (44
), suggesting that distinct Ena/VASP activities may result via different cyclic nucleotide-mediated signaling pathways. Spatially regulated phosphorylation at sites within the EVH2 domain could also influence VASP anti-capping and bundling activities in vivo
Actin filament barbed ends play a central role in determining the form and function of cellular structures dependent on actin filaments. The ability of Ena/VASP proteins and profilin-actin to modulate filament elongation at barbed ends and protect barbed ends from capping highlights them as key regulators of actin-based processes that determine cellular form and function. We have focused here on the ability of Ena/VASP proteins and profilin to modulate actin dynamics at barbed ends by preventing barbed end capping. Ena/VASP proteins also interact with several other proteins that influence cellular signaling pathways. Specific recruitment of signaling factors to barbed ends via Ena/VASP proteins may be a direct conduit for transducing extracellular cues toward a dynamic actin cytoskeleton.