Ena/VASP Proteins Enhance Actin Polymerization in the Presence of Barbed End Capping Proteins

Melanie Barzik,Tatyana I Kotova,Henry N Higgs,Larnele Hazelwood,Dorit Hanein,Frank B Gertler,Dorothy A Schafer

doi:10.1074/jbc.m503957200

Abstract

Ena/VASP proteins influence the organization of actin filament networks within lamellipodia and filopodia of migrating cells and in actin comet tails. The molecular mechanisms by which Ena/VASP proteins control actin dynamics are unknown. We investigated how Ena/VASP proteins regulate actin polymerization at actin filament barbed ends in vitro in the presence and absence of barbed end capping proteins. Recombinant His-tagged VASP increased the rate of actin polymerization in the presence of the barbed end cappers, heterodimeric capping protein (CP), CapG, and gelsolin-actin complex. Profilin enhanced the ability of VASP to protect barbed ends from capping by CP, and this required interactions of profilin with G-actin and VASP. The VASP EVH2 domain was sufficient to protect barbed ends from capping, and the F-actin and G-actin binding motifs within EVH2 were required. Phosphorylation by protein kinase A at sites within the VASP EVH2 domain regulated anti-capping and F-actin bundling by VASP. We propose that Ena/VASP proteins associate at or near actin filament barbed ends, promote actin assembly, and restrict the access of barbed end capping proteins.

Highlights

Ena/VASP proteins influence the organization of actin filament networks within lamellipodia and filopodia of migrating cells and in actin comet tails
We propose that Ena/VASP proteins associate at or near actin filament barbed ends, promote actin assembly, and restrict the access of barbed end capping proteins
Biochemical experiments in which recombinant VASP enhanced actin polymerization in the presence of heterodimeric capping protein (CP)1 supported the hypothesis that Ena/VASP proteins promote the formation of long, unbranched actin filaments, in part, by protecting actin filament barbed ends from capping [9]

Summary

EXPERIMENTAL PROCEDURES

Recombinant Protein Expression and Purification—All VASP proteins were recombinant, His6-tagged and purified from Escherichia coli. Plasmids to express N-terminal His6-tagged murine VASP and mutant VASP proteins were constructed from PCR fragments cloned into pQE80L (Qiagen). The wild-type and mutant VASP proteins used in this study are shown in schematic form in supplementary materials Fig. 1. VASP proteins were expressed in E. coli strain BL21 (DE3) CodonPlus and purified by chromatography on TALON resin (BD Biosciences) and Superdex-200 in MKEI-200 buffer (20 mM imidazole, pH 7.0, 200 mM KCl, 1 mM EGTA, 2 mM MgCl2, and 1 mM DTT). VASP proteins were stored on ice and used within 2 weeks of purification. Recombinant murine CP (␣1␤2) was purified as described [15]. Plasmids for expression of human profilin I, profilin I-Y6D, and profilin I-R88E were constructed by D.

VASP and Profilin Enhance Actin Assembly

RESULTS

VASP protein

DISCUSSION