Matthias Reinhard

A novel proline‐rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP family

The ActA protein of the intracellular pathogen Listeria monocytogenes induces a dramatic reorganization of the actin‐based cytoskeleton. Two profilin binding proteins, VASP and Mena, are the only cellular proteins known so far to bind directly to ActA. This interaction is mediated by a conserved module, the EVH1 domain. We identify E/DFPPPPXD/E, a motif repeated 4‐fold within the primary sequence of ActA, as the core of the consensus ligand for EVH1 domains. This motif is also present and functional in at least two cellular proteins, zyxin and vinculin, which are in this respect major eukaryotic analogs of ActA. The functional importance of the novel protein–protein interaction was examined in the Listeria system. Removal of EVH1 binding sites on ActA reduces bacterial motility and strongly attenuates Listeria virulence. Taken together we demonstrate that ActA–EVH1 binding is a paradigm for a novel class of eukaryotic protein–protein interactions involving a proline‐rich ligand that is clearly different from those described for SH3 and WW/WWP domains. This class of interactions appears to be of general importance for processes dependent on rapid actin remodeling.

A focal adhesion factor directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-based cytoskeleton of mammalian cells.

The surface‐bound ActA polypeptide of the intracellular bacterial pathogen Listeria monocytogenes is the sole listerial factor needed for recruitment of host actin filaments by intracellularly motile bacteria. Here we report that following Listeria infection the host vasodilator‐stimulated phosphoprotein (VASP), a microfilament‐ and focal adhesion‐associated substrate of both the cAMP‐ and cGMP‐dependent protein kinases, accumulates on the surface of intracytoplasmic bacteria prior to the detection of F‐actin ‘clouds’. VASP remains associated with the surface of highly motile bacteria, where it is polarly located, juxtaposed between one extremity of the bacterial surface and the front of the actin comet tail. Since actin filament polymerization occurs only at the very front of the tail, VASP exhibits properties of a host protein required to promote actin polymerization. Purified VASP binds directly to the ActA polypeptide in vitro. A ligand‐overlay blot using purified radiolabelled VASP enabled us to identify the ActA homologue of the related intracellular motile pathogen, Listeria ivanovii, as a protein with a molecular mass of approximately 150 kDa. VASP also associates with actin filaments recruited by another intracellularly motile bacterial pathogen, Shigella flexneri. Hence, by the simple expedient of expressing surface‐bound attractor molecules, bacterial pathogens effectively harness cytoskeletal components to achieve intracellular movement.

The EVH2 Domain of the Vasodilator-stimulated Phosphoprotein Mediates Tetramerization, F-actin Binding, and Actin Bundle Formation

Vasodilator-stimulated phosphoprotein (VASP) is a member of the Ena/VASP family of proteins that are implicated in regulation of the actin cytoskeleton. All family members share a tripartite structural organization, comprising an N-terminal Ena/VASP homology (EVH) 1 domain, a more divergent proline-rich central part, and a common C-terminal EVH2 region of about 160–190 amino acids. Using chemical cross-linking, sucrose gradient sedimentation, and gel filtration analyses of different truncated VASP constructs, we demonstrate that the VASP EVH2 region is both necessary and sufficient for tetramerization. Moreover, co-sedimentation and fluorescent phalloidin staining showed that the EVH2 region binds and bundles F-actin in vitro and localizes to stress fibers in transfected cells. Analysis of the functional contribution of highly conserved blocks within this region indicated that residues 259–276 of human VASP are essential for the interaction with F-actin, whereas residues 343–380 are required for tetramerization, probably via coiled-coil formation. Interactions with F-actin are enhanced by VASP tetramerization. The results demonstrate that the C-terminal EVH2 segment is not only conserved in sequence but also forms a distinct functional entity. The data suggest that the EVH2 segment represents a novel oligomerization and F-actin binding domain.

Actin-based motility: stop and go with Ena/VASP proteins

Proteins of the Ena/VASP (Enabled/vasodilator-stimulated phosphoprotein) family are involved in Abl and/or cyclic nucleotide-dependent protein kinase signaling pathways. These proteins are also crucial factors in regulating actin dynamics and associated processes such as cell-cell adhesion, platelet function and actin-based motility of both cytopathogenic Listeria and their eukaryotic host cells. Although biochemical mechanisms have emerged depicting Ena/VASP proteins as enhancers of actin filament formation, increasing evidence also suggests that these proteins have inhibitory functions in integrin regulation, cell motility and axon guidance.

VASP interaction with vinculin: a recurring theme of interactions with proline-rich motifs

VASP (vasodilator‐stimulated phosphoprotein), a protein associated with microfilaments at cellular contact sites, has been identified as a ligand for profilin and zyxin, two proteins also involved in microfilament dynamics and organization at these regions. Here, we report that VASP also directly binds to vinculin, another component of adherens junctions. Competition experiments with a vinculin‐derived peptide showed that a proline‐rich motif, located in the hinge region that connects vinculins head and tail domains, is involved in VASP binding. The same motif is present in zyxin but the interactions of VASP with vinculin and zyxin differ in detail. Hence, this motif may be recognized by VASP in different ways when presented in distinct cellular sites.

An α-Actinin Binding Site of Zyxin Is Essential for Subcellular Zyxin Localization and α-Actinin Recruitment

The LIM domain protein zyxin is a component of adherens type junctions, stress fibers, and highly dynamic membrane areas and appears to be involved in microfilament organization. Chicken zyxin and its human counterpart display less than 60% sequence identity, raising concern about their functional identity. Here, we demonstrate that human zyxin, like the avian protein, specifically interacts with α-actinin. Furthermore, we map the interaction site to a motif of approximately 22 amino acids, present in the N-terminal domain of human zyxin. This motif is both necessary and sufficient for α-actinin binding, whereas a downstream region, which is related in sequence, appears to be dispensable. A synthetic peptide comprising human zyxin residues 21–42 specifically binds to α-actinin in solid phase binding assays. In contrast to full-length zyxin, constructs lacking this motif do not interact with α-actinin in blot overlays and fail to recruit α-actinin in living cells. When zyxin lacking the α-actinin binding site is expressed as a fusion protein with green fluorescent protein, association of the recombinant protein with stress fibers is abolished, and targeting to focal adhesions is grossly impaired. Our results suggest a crucial role for the α-actinin-zyxin interaction in subcellular zyxin localization and microfilament organization.

Role of Cyclic Nucleotide-Dependent Protein Kinases and Their Common Substrate VASP in the Regulation of Human Platelets

The activation of human platelets is inhibited by two intracellular pathways regulated by either cGMP- or cAMP-elevating agents. There is considerable evidence that the inhibitory effects of cGMP and cAMP are mediated by the cGMP-PK and cAMP-PK, respectively, in human platelets. The cGI-PDE is an additional target for cGMP, and the cGMP-mediated elevation of cAMP levels contributes to the well known synergism between cAMP- and cGMP-elevating platelet inhibitors. Stimulation of both cAMP-PK and cGMP-PK prevents the agonist-induced activation of MLCK and PKC and inhibits the agonist-induced calcium mobilization from intracellular stores without any major effect on the ADP-regulated cation channel. These studies suggest that the inhibition of an early event of platelet activation, e.g. activation of PLC, is an effect common to both cGMP-PK and cAMP-PK stimulation. A common substrate of both cGMP-PK and cAMP-PK, the 46/50 kDa protein VASP, has been recently identified as a novel microfilament- and focal contact-associated protein whose phosphorylation correlates very well with platelet inhibition. Future investigations will have to identify the precise molecular mechanism of cyclic nucleotide inhibition of Ca2+ discharge from intracellular stores and whether cGMP-PK- and cAMP-PK-mediated VASP phosphorylation is an important component of this effect of cyclic nucleotides in human platelets.

The lipoma preferred partner LPP interacts with α-actinin

The lipoma preferred partner LPP is a member of the zyxin family of proteins. In this paper, we demonstrate that the structural similarities observed between zyxin and LPP also extend to their interaction capabilities. Similar to zyxin, LPP was found to bind to α-actinin in vitro. This interaction was confirmed in yeast and mammalian cells. Studies utilizing the three-hybrid system further indicated that zyxin and LPP compete for the same binding site in α-actinin. This site was mapped to the central rod ofα -actinin, which contains spectrin-like repeats 2 and 3. In the case of LPP, a conserved motif present at the N-terminus was shown to be responsible for the interaction. Constructs lacking this motif did not bind toα -actinin in the yeast two-hybrid system and were not able to recruitα -actinin to an ectopic site in mammalian cells. Quantitative data obtained with the two-hybrid and the three-hybrid system suggest that LPP has a lower affinity for α-actinin than zyxin. It is likely that this difference leads to slightly different roles played by LPP and zyxin during the assembly and disassembly of focal adhesions.

VASP-dependent regulation of actin cytoskeleton rigidity, cell adhesion, and detachment

Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are established regulators of actin-based motility, platelet aggregation, and growth cone guidance. However, the molecular mechanisms involved essentially remain elusive. Here we report on a novel mechanism of VASP action, namely the regulation of tensile strength, contractility, and rigidity of the actin cytoskeleton. Compared to wild-type cells fibroblasts derived from VASP-deficient mice have thicker and more stable actin stress fibres. Furthermore focal adhesions are enlarged, myosin light chain phosphorylation is increased, and the rigidity of the filament-supported plasma membrane is elevated about three- to fourfold, as is evident from atomic force microscopy. Moreover, fibronectin-coated beads adhere stronger to the surface of VASP-deficient cells. The resistance of these beads to mechanical displacement by laser tweezers is dramatically increased in an F-actin-dependent mode. Cytoskeletal stabilization coincides with slower cell adhesion and detachment, while overall adhesion is increased. Interestingly, many of these effects observed in VASP (−/−) cells are recapitulated in VASP-overexpressing cells, hinting towards a balanced stoichiometry necessary for appropriate VASP function. Taken together, our results suggest that VASP regulates surface protrusion formation and cell adhesion through modulation of the mechanical properties of the actin cytoskeleton.