Profilins are small proteins that form complexes with G‐actin and phosphoinositides and are therefore considered to link the microfilament system to signal transduction pathways. In addition, they bind to poly‐L‐proline, but the biological significance of this interaction is not yet known. The recent molecular cloning of the vasodilator‐stimulated phosphoprotein (VASP), an established in vivo substrate of cAMP‐ and cGMP‐dependent protein kinases, revealed the presence of a proline‐rich domain which prompted us to investigate a possible interaction with profilins. VASP is a microfilament and focal adhesion associated protein which is also concentrated in highly dynamic regions of the cell cortex. Here, we demonstrate that VASP is a natural proline‐rich profilin ligand. Human platelet VASP bound directly to purified profilins from human platelets, calf thymus and birch pollen. Moreover, VASP and a novel protein were specifically extracted from total cell lysates by profilin affinity chromatography and subsequently eluted either with poly‐L‐proline or a peptide corresponding to a proline‐rich VASP motif. Finally, the subcellular distributions of VASP and profilin suggest that both proteins also interact within living cells. Our data support the hypothesis that profilin and VASP act in concert to convey signal transduction to actin filament formation.

Although cGMP-dependent protein kinases (cGPKs) 1 have been recognized as important components of major signal transduction pathways (1-3), quantitative analysis of cGPK activation in intact cells has been very difficult (1-4). This is because of the relatively low expression of cGPK in most cell types compared with the relatively high expression of its closest functional homolog, the cAMP-dependent protein kinase (cAPK), and the scarcity of specific cGPK substrates. Unfortunately, the mediating role of cGPK for a given effect/function is often implied or excluded by the use of cGPK activators and/or inhibitors alone, which is clearly insufficient to establish or rule out functional roles of cGPKs (1-4). One of the few established cGPK substrates is the 46-kDa/50-kDa vasodilator-stimulated phosphoprotein (VASP), which was initially discovered and characterized as a substrate of both cAPK and cGPK in human platelets (5-8). VASP phosphorylation in response to cyclic nucleotide-regulating vasodilators (i.e. cAMP-elevating prostaglandins and cGMP-elevating nitric oxide donors) closely correlates with platelet inhibition and in particular with the inhibition of fibrinogen binding to the integrin ␣ IIb ␤ 3 of human platelets (9 -11). Molecular cloning of human, canine, and mouse VASP predicted highly homologous proteins and revealed a proline-rich protein that is organized into three structural segments of different sequence complexity (12,13). VASP is the founding member of a new family of proline-rich proteins, which includes Enabled (Ena), a dose-dependent suppressor of Drosophila Abl-and Disabled-dependent phenotypes, its mammalian homolog Mena, and the Ena-VASP-like protein Evl (14 -16). These proteins all share an overall domain organization consisting of highly homologous NH 2 -terminal and COOHterminal domains (Ena-VASP homology domains 1 and 2, EVH1 and EVH2), which are separated by a proline-rich central domain of low complexity (12-16). In platelets and many other cells including vascular smooth muscle cells, endothelial cells, and fibroblasts, VASP has been found to be associated with stress fibers, focal adhesions, cell-cell contacts, and highly dynamic membrane regions (16,17). VASP colocalizes with profilins and binds directly to their poly(L-proline) binding site (18), binds to and colocalizes with zyxin and vinculin (16,19), and also directly binds to Listeria monocytogenes surface protein ActA, which is essential for the actin polymerization-based intracellular motility of this pathogen (20). Functional evidence indicates that VASP is a crucial factor involved in the enhancement of spatially confined actin filament formation (16,20,21).Three distinct phosphorylation sites were biochemically identified in VASP (serine 157, serine 239, and threonine 278) which are used in vitro and in intact human platelets by both cAPK and cGPK and by the serine/threonine protein phosphatases 2A and 2B with overlapping selectivity (8,22). Phosphorylation of serine 157, the site preferred by the cAPK, leads to a marked...

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The haemolysin protein (HlyA) of Escherichia coli contains 11 tandemly repeated sequences consisting of 9 amino acids each between amino acids 739 and 849 of HlyA. We removed, by oligonucleotide-directed mutagenesis, different single repeats and combinations of several repeats. The resulting mutant proteins were perfectly stable in E. coli and were secreted with the same efficiency as the wild-type HlyA. HlyA proteins which had lost a single repeat only were still haemolytically active (in the presence of HlyC) but required elevated levels of Ca2+ for activity, as compared to the wild-type haemolysin. Removal of three or more repeats led to the complete loss of the haemolytic activity even in the presence of high Ca2+ concentrations. The mutant haemolysins were unable to compete with the wild-type haemolysin for binding to erythrocytes at low Ca2+ concentrations but could still generate ion-permeable channels in artificial lipid bilayer membranes formed of plant asolectin, even in the complete absence of Ca2+. These data indicate that the repeat domain of haemolysin is responsible for Ca2+-dependent binding of haemolysin to the erythrocyte membrane. A model for the possible functional role of Ca2+ in haemolysis is presented.

The vasodilator‐stimulated phosphoprotein (VASP), a substrate for cAMP‐ and cGMP‐dependent protein kinases in vitro and in intact cells, is associated with actin filaments, focal adhesions and dynamic membrane regions. VASP, cloned here from human HL‐60 and canine MDCK cells, is organized into three distinct domains. A central proline‐rich domain contains a GPPPPP motif as a single copy and as a 3‐fold tandem repeat, as well as three conserved phosphorylation sites for cyclic nucleotide‐dependent protein kinases. A C‐terminal domain contains a repetitive mixed‐charge cluster which is predicted to form an alpha‐helix. The hydrodynamic properties of purified human VASP together with the calculated molecular mass of cloned VASP suggest that the native protein is a homotetramer with an elongated structure. VASP over‐expressed in transiently transfected BHK21 cells was predominantly detected at stress fibres, at focal adhesions and in F‐actin‐containing cell surface protrusions, whereas truncated VASP lacking the C‐terminal domain was no longer concentrated at focal adhesions. These data indicate that the C‐terminal domain is required for anchoring VASP at focal adhesion sites, whereas the central domain is suggested to mediate VASP interaction with profilin. Our results provide evidence for the structural basis by which VASP, both a target of the cAMP and cGMP signal transduction pathways and a component of the actin‐based cytoskeleton, including the cytoskeleton‐membrane interface, may be able to exchange signals between these networks.

Drosophila enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domains are 115 residue proteinp rotein interaction modules which provide essential links for their host proteins to various signal transduction pathways. Many EVH1-containing proteins are associated closely with actinbased structures and are involved in re-organization of the actin cytoskeleton. EVH1 domains are also present in proteins enriched in neuronal tissue, thus implicating them as potential mediators of synaptic plasticity, linking them to memory formation and learning. Like Src homology 3, WW and GYF domains and profilin, EVH1 domains recognize and bind specific proline-rich sequences (PRSs). The binding is of low affinity, but tightly regulated by the high specificity encoded into residues in the protein:peptide interface. In general, a small (3^6 residue) core' PRS in the target protein binds a`recognition pocket' on the domain surface. Further affinity-and specificity-increasing interactions are then formed between additional domain epitopes and peptide`core-flanking' residues. The three-dimensional structures of EVH1:peptide complexes now reveal, in great detail, some of the most important features of these interactions and allow us to better understand the origins of specificity, ligand orientation and sequence degeneracy of target peptides, in low affinity signalling complexes. ß

The Ena-VASP family of proteins act as molecular adaptors linking the cytoskeletal system to signal transduction pathways. Their N-terminal EVH1 domains use groups of exposed aromatic residues to speci®cally recognize`FPPPP' motifs found in the mammalian zyxin and vinculin proteins, and ActA protein of the intracellular bacterium Listeria monocytogenes. Here, evidence is provided that the af®nities of these EVH1±peptide interactions are strongly dependent on the recognition of residues¯anking the core FPPPP motifs. Determination of the VASP EVH1 domain solution structure, together with peptide library screening, measurement of individual K d s bȳ uorescence titration, and NMR chemical shift mapping, revealed a second af®nity-determining epitope present in all four ActA EVH1-binding motifs. The epitope was shown to interact with a complementary hydrophobic site on the EVH1 surface and to increase strongly the af®nity of ActA for EVH1 domains. We propose that this epitope, which is absent in the sequences of the native EVH1-interaction partners zyxin and vinculin, may provide the pathogen with an advantage when competing for the recruitment of the host VASP and Mena proteins in the infected cell.

Focal contact assembly involves interaction between VASP and vinculin, which is enhanced by PIP2-induced vinculin activation and oligomerization. Given that vinculin and VASP both bind to F-actin, vinculin-VASP complexes might bundle the distal ends of actin filaments in focal contacts. We propose that PIP2-dependent signalling modulates microfilament organization at cellular adhesion sites by regulating vinculin-VASP complexes.