Role of proteins of the Ena/VASP family in actin-based motility of Listeria monocytogenes

Intracellular propulsion of Listeria monocytogenes is the best understood f orm of motility dependent on actin polymerization. We have used in vitro mo tility assays of Listeria in platelet and brain extracts to elucidate the f unction of the focal adhesion proteins of the Ena (Drosophila Enabled)/VASP (vasodilator-stimulated phosphoprotein) family in actin-based motility. Im munodepletion of VASP from platelet extracts and of Evl (Ena/VASP-like prot ein) from brain extracts of Mena knockout (-/-) mice combined with add-back of recombinant (bacterial or eukaryotic) VASP and Evl show that VASP, Mena , and Evl play interchangeable roles and are required to transform actin po lymerization into active movement and propulsive force. The EVH1 (Ena/VASP homology 1) domain of VASP is in slow association-dissociation equilibrium high-affinity binding to the zyxin-homologous, proline-rich region of ActA. VASP also interacts with F-actin via its COOH-terminal EVH2 domain. Hence VASP/Ena/Evl link the bacterium to the actin tail, which is required for mo vement. The affinity of VASP for F-actin is controlled by phosphorylation o f serine 157 by cAMP-dependent protein kinase. Phospho-VASP binds with high affinity (0.5 x 10(8) M-1); dephospho-VASP binds 40-fold less tightly. We propose a molecular ratchet model for insertional polymerization of actin, within which frequent attachment-detachment of VASP to F-actin allows its s liding along the growing filament.