@phdthesis{Offner2017,
  author    = {Offner, Kristin},
  title     = {SH3-mediated protein interactions of Mena and VASP},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-154481},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Regulation of actin cytoskeletal turnover is necessary to coordinate cell movement and cell adhesion. Proteins of the Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family are important mediators in cytoskeleton control, linking cyclic nucleotide signaling pathways to actin assembly. In mammals, the Ena/VASP family consists of mammalian Enabled (Mena), VASP, and Ena-VASP-like (EVL). The family members share a tripartite domain organization, consisting of an N-terminal Ena/VASP homology 1 (EVH1) domain, a central proline-rich region (PRR), and a C-terminal EVH2 domain. The EVH1 domain mediates binding to the focal adhesion proteins vinculin and zyxin, the PRR interacts with the actin-binding protein profilin and with Src homology 3 (SH3) domains, and the EVH2 domain mediates tetramerization and actin binding. Endothelial cells line vessel walls and form a semipermeable barrier between blood and the underlying tissue. Endothelial barrier function depends on the integrity of cell-cell junctions and defective sealing of cell-cell contacts results in vascular leakage and edema formation. In a previous study, we could identify a novel interaction of the PRR of VASP with αII-spectrin. VASP-targeting to endothelial cell-cell contacts by interaction with the αII-spectrin SH3 domain is sufficient to initiate perijunctional actin filament assembly, which in turn stabilizes cell-cell contacts and decreases endothelial permeability. Conversely, barrier function of VASP-deficient endothelial cells and microvessels of VASP- null mice is defective, demonstrating that αII-spectrin/VASP complexes regulate endothelial barrier function in vivo. The aim of the present study was to characterize the structural aspects of the binding of Ena/VASP proteins to αII-spectrin in more detail. These data are highly relevant to understand the cardiovascular function of VASP and its subcellular targeting. In the present study, the following points were experimentally addressed: 1. Comparison of the interaction between αII-spectrin and Mena, VASP, or EVL In contrast to the highly conserved EVH1/EVH2 domains, the PRR is the most divergent part within the Ena/VASP proteins and may differ in binding modes and mechanisms of regulation. More specifically, VASP contains a triple GP5 motif, whereas EVL and Mena contain one or more GP6 motifs or even longer proline stretches. In the present study, we used peptide scans and competitive αII-spectrin SH3 pull-down assays with the recombinant Mena, VASP, and VASP mutants to investigate the relative binding efficiency. Our results indicate that binding of the αII-spectrin SH3 domain to GP6 motifs is superior to GP5 motifs, giving a rationale for a stronger interaction of αII-spectrin with EVL and Mena than with VASP. 2. Interaction of SH3i with Ena/VASP proteins In the mammalian heart, an αII-spectrin splice variant exists (SH3i), which contains a 20 amino acid insertion C-terminal to the SH3 domain. We used GST-fusion proteins of αII-spectrin, comprising the SH3 domain with or without the alternatively spliced amino acids, to pull-down recombinant Mena, VASP or VASP mutants. The results demonstrate a substantially increased binding of the C-terminal extended SH3 domain as compared to the general αII-spectrin isoform without the 20 amino acid insertion. These findings were also confirmed in pull-down experiments with heart lysates and purified Mena from heart muscle. The increased binding was not due to an alternative, SH3-independent binding interface because a pointmutation of the SH3 domain (W1004R) in the alternatively spliced αII-spectrin isoform completely abrogated the interaction. To analyze the interaction of SH3i and Ena/VASP proteins in living cells, we expressed the extended SH3 domain as GFP fusion proteins in endothelial cells. Here, we observed an extensive co-localization with Mena and VASP at the leading edge of lamellipodia confirming the in vivo relevance of the interaction with potential impact on cell migration and angiogenesis. 3. Binding affinity and influence of the Ena/VASP tetramerization domain We also determined the binding affinity of the general and the alternatively spliced αII-spectrin SH3 with Ena/VASP proteins by isothermal titration calorimetry (ITC) using a peptide from the PRR of Mena (collaboration with Dr. Stephan Feller, University of Oxford). Surprisingly, the binding affinity of the general SH3 domain was low (~900 μM) as compared to other SH3 domain- mediated interactions, which commonly display binding constants in the low micromolar range. Furthermore and in contrast to the pull-down assays, we could not detect an increased binding affinity of the C-terminally extended SH3 domain. This could be either explained by the existence of a third protein, which "bridges" the Mena/αII-spectrin complex in the pull-down assays, or, more likely, by the small size of the Mena peptide, which lacks major parts of the Mena protein, including the tetramerization domain. Indeed, it has been previously shown that the tetramerization of Ena is crucial for the interaction with the Abl- SH3 domain, although no SH3 binding sites are found in the tetramerization domain. To address this point experimentally, we used a VASP mutant that lacks the tetramerization domain in pull-down assays. Neither the general nor the alternatively spliced SH3 domain bound to the monomeric VASP, demonstrating the crucial (indirect) impact of Ena/VASP tetramerization on the interaction with αII-spectrin. In summary, we conclude that the αII-spectrin SH3 domain binds to the proline- rich region of all Ena/VASP proteins. However, binding to EVL and Mena, which both possess one or more GP6 motifs, is substantially more efficient than VASP, which only contains GP5 motifs. The C-terminally extended SH3 domain, which is present in the αII-spectrin splice variant SH3i, binds stronger to the Ena/VASP proteins than the general isoform and expression of the isolated domain is sufficient for co-localization with Ena/VASP in living endothelial cells. Finally, the tetramerization of the Ena/VASP proteins is indispensable for the interaction with either isoform of αII-spectrin.},
  language  = {en}
}
@phdthesis{Merkel2011,
  author    = {Merkel, Carla Jennifer},
  title     = {Characterisation of Mena Promoter Activity and Protein Expression in Wild-type and Gene-trapped Mice},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70414},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2011},
  abstract  = {Proteins of the Ena/VASP protein family are important regulators of actin and participate in cell-cell and cell-matrix adhesions. To date, the physiological importance of Ena/VASP proteins for integrity of the cardiovascular system has remained unclear. To study cardiovascular functions of Mena and VASP, we used an established VASP knockout mouse in combination with a novel gene-trap-based model to ablate Mena function. In the mutated Mena mouse, the endogenous Mena gene is disrupted by the insertion of a β-galactosidase construct and β-galactosidase expression is under the control of the endogenous Mena promoter. X-gal staining of mouse organs revealed Mena promoter activity in smooth muscle layers of vessels, intestines and bronchioles, but also in cells of the brain, in cardiomyocytes and in the respiratory epithelium of bronchioles. In wild-type mice, Western blotting revealed differing protein expression patterns of VASP and Mena. Mena expression was observed in almost every tissue, predominantly in heart, lung, stomach, large intestine, testis, brain and eye. Additionally, the neuronalspecific Mena isoform was expressed in brain, eye, and slightly in heart and stomach. VASP protein, in contrast, was predominantly detected in spleen and thrombocytes. In gene-trapped mice, Mena expression was largely reduced in heart, lung and stomach but only slightly decreased in brain and testis. Immunofluorescence microscopy revealed colocalisation of Mena and F-actin at intercalated discs of cardiomyocytes and strong colocalisation of Mena and α- smooth-muscle-actin in vessels and bronchioles. Functional analysis of Mena/VASP-mutated and wild-type mice using electrocardiography suggested that the depletion of either Mena or VASP does not interfere with normal heart function. However, in double-deficient mice, the resting heart rate was significantly increased, probably reflecting a mechanism to compensate defects in ventricle contraction and to maintain a normal cardiac output. In agreement, cardiac catheter investigations suggested dilated cardiomyopathy in doubledeficient mice. Thus, although Western blot analysis showed differing protein expression patterns of Mena and VASP, these findings suggest that Mena and VASP mutually compensate for each other. Concerning Mena, we propose an important role of the protein in vessel walls, cardiomyocytes and bronchioles.},
  subject      = {Spectrin},
  language  = {en}
}