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...

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.The mammalian vasodilator-stimulated phosphoprotein (VASP) 1 (1) and Drosophila Enabled (Ena) (2) are the founding members of the Ena/VASP family of proteins. While Ena is a substrate of the Abelson tyrosine kinase (Abl) and is also genetically linked to the Abl signaling pathway (2), VASP is a substrate of both cGMP-and cAMP-dependent protein kinases (Ref. 3; for a review see Ref. 4). Three common cGMP-dependent protein kinase/cAMP-dependent protein kinase phosphorylation sites have been biochemically identified in human VASP (Ser-157, Ser-239, and Thr-278) (5, 6), two of which are also conserved in Mena (mammalian Enabled) and one in Evl (Ena-VASP-like), two other family members (7). VASP phosphorylation in response to cyclic nucleotide regulating vasodilators (i.e. cAMP-elevating prostaglandins and cGMP-elevating NO donors) closely correlates with platelet inhibition and in particular with the inhibition of fibrinogen binding to the human platelet integrin ␣ IIb ␤ 3 (3,8). In agreement with these earlier studies, recent analysis of platelets from VASP-deficient mice support the concept that VASP is an important component in modulating agonist-induced integrin ␣ IIb ␤ 3 and P-selectin activation (9, 10). Similar to Ena (11) and its mammalian homologue Mena (7), VASP is an actin filament-associated protein that is predominantly localized at stress fibers, cell-matrix and cell-cell adherens junctions, and highly dynamic membrane areas (12, 13). There is a large body of evidence that both VASP and Mena are involved in the facilitation of spatially confined actin filament formation (for a review see Refs. 14 and 15).All Ena/VASP family members share a tripartite structural ...

Drosophila Enabled (Ena) was initially identified as a dominant genetic suppressor of mutations in the Abelson tyrosine kinase and, more recently, as a member of the Ena/human vasodilator-stimulated phosphoprotein (VASP) family of proteins. We have used genetic, biochemical, and cell biological approaches to demonstrate the functional relationship between Ena and human VASP. In addition, we have defined the roles of Ena domains identified as essential for its activity in vivo. We have demonstrated that VASP rescues the embryonic lethality associated with loss of Ena function in Drosophila and have shown that Ena, like VASP, is associated with actin filaments and focal adhesions when expressed in cultured cells. To define sequences that are central to Ena function, we have characterized the molecular lesions present in two lethal ena mutant alleles that affected the Ena/VASP homology domain 1 (EVH1) and EVH2. A missense mutation that resulted in an amino acid substitution in the EVH1 domain eliminated in vitro binding of Ena to the cytoskeletal protein zyxin, a previously reported binding partner of VASP. A nonsense mutation that resulted in a C-terminally truncated Ena protein lacking the EVH2 domain failed to form multimeric complexes and exhibited reduced binding to zyxin and the Abelson Src homology 3 domain. Our analysis demonstrates that Ena and VASP are functionally homologous and defines the conserved EVH1 and EVH2 domains as central to the physiological activity of Ena.

BackgroundThe recent concept of secretome-based tissue regeneration has profoundly altered the field of regenerative medicine and offers promising novel therapeutic options. In contrast to medicinal products with a single active substance, cell-derived secretomes comprise pleiotropic bioactive ingredients, representing a major obstacle for reproducible drug product efficacy and warranting patient safety. Good manufacturing practice (GMP)-compliant production guarantees high batch-to-batch consistency and reproducible efficacy of biological medicinal products, but different batches of cellular secretomes produced under GMP have not been compared yet, and suitable quality control parameters have not been established. To this end, we analyzed diverse biological and functional parameters of different batches produced under GMP of the secretome obtained from γ-irradiated peripheral blood mononuclear cells with proven tissue regenerative properties in infarcted myocardium, stroke, spinal cord injury, and skin wounds.MethodsWe quantified key secretome ingredients, including cytokines, lipids, and extracellular vesicles, and functionally assessed potency in tube formation assay, ex vivo aortic ring sprouting assay, and cell-based protein and reporter gene assays. Furthermore, we determined secretome stability in different batches after 6 months of storage at various ambient temperatures.ResultsWe observed that inter-batch differences in the bioactive components and secretome properties were small despite considerable differences in protein concentrations and potencies between individual donor secretomes. Stability tests showed that the analytical and functional properties of the secretomes remained stable when lyophilisates were stored at temperatures up to + 5 °C for 6 months.ConclusionsWe are the first to demonstrate the consistent production of cell-derived, yet cell-free secretome as a biological medicinal product. The results from this study provide the basis for selecting appropriate quality control parameters for GMP-compliant production of therapeutic cell secretomes and pave the way for future clinical trials employing secretomes in tissue regenerative medicine.

Drosophila Enabled (Ena) was first identified as a genetic suppressor of mutations in the Abelson tyrosine kinase and subsequently was shown to be a member of the Ena͞vasodilator-stimulated phosphoprotein family of proteins. All members of this family have a conserved domain organization, bind the focal adhesion protein zyxin, and localize to focal adhesions and stress fibers. Members of this family are thought to be involved in the regulation of cytoskeleton dynamics. The Ena protein sequence has multiple poly-(L-proline) residues with similarity to both profilin and src homology 3 binding sites. Here, we show that Ena can bind directly to the Drosophila homolog of profilin, chickadee. Furthermore, Ena and profilin were colocalized in spreading cultured cells. We report that the proline-rich region of Ena is responsible for this interaction as well as for mediating binding to the src homology 3 domain of the Abelson tyrosine kinase. These data support the hypothesis that Ena provides a regulated link between signal transduction and cytoskeleton assembly in the developing Drosophila embryo.