The Experts below are selected from a list of 717 Experts worldwide ranked by ideXlab platform
Linda K Nicholson - One of the best experts on this subject based on the ideXlab platform.
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a noncanonical binding site in the EVH1 Domain of vasodilator stimulated phosphoprotein regulates its interactions with the proline rich region of zyxin
Biochemistry, 2017Co-Authors: Lucila Andrea Acevedo, Alexander I Greenwood, Linda K NicholsonAbstract:Vasodilator-stimulated phosphoprotein (VASP) is a processive actin polymerase with roles in the control of cell shape and cell migration. Through interaction with the cytoskeletal adaptor protein Zyxin, VASP can localize to damaged stress fibers where it serves to repair and reinforce these structures. VASP localization is mediated by its N-terminal Ena/VASP homology (EVH1) Domain, which binds to the (W/F)PxφP motif (most commonly occurring as FPPPP) found in cytoskeletal proteins such as vinculin, lamellipodin, and Zyxin. Sequentially close clusters of four or five of these motifs frequently occur, as in the proline rich region of Zyxin with four such motifs. This suggests that tetrameric VASP might bind very tightly to Zyxin through avidity, with all four EVH1 Domains binding to a single Zyxin molecule. Here, quantitative nuclear magnetic resonance titration analysis reveals a dominant bivalent 1:1 (Zyxin:EVH1) interaction between the Zyxin proline rich region and the VASP EVH1 Domain that utilizes the ...
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A Noncanonical Binding Site in the EVH1 Domain of Vasodilator-Stimulated Phosphoprotein Regulates Its Interactions with the Proline Rich Region of Zyxin
2017Co-Authors: Lucila Andrea Acevedo, Alexander I Greenwood, Linda K NicholsonAbstract:Vasodilator-stimulated phosphoprotein (VASP) is a processive actin polymerase with roles in the control of cell shape and cell migration. Through interaction with the cytoskeletal adaptor protein Zyxin, VASP can localize to damaged stress fibers where it serves to repair and reinforce these structures. VASP localization is mediated by its N-terminal Ena/VASP homology (EVH1) Domain, which binds to the (W/F)PxφP motif (most commonly occurring as FPPPP) found in cytoskeletal proteins such as vinculin, lamellipodin, and Zyxin. Sequentially close clusters of four or five of these motifs frequently occur, as in the proline rich region of Zyxin with four such motifs. This suggests that tetrameric VASP might bind very tightly to Zyxin through avidity, with all four EVH1 Domains binding to a single Zyxin molecule. Here, quantitative nuclear magnetic resonance titration analysis reveals a dominant bivalent 1:1 (Zyxin:EVH1) interaction between the Zyxin proline rich region and the VASP EVH1 Domain that utilizes the EVH1 canonical binding site and a novel secondary binding site on the opposite face of the EVH1 Domain. We further show that binding to the secondary binding site is specifically inhibited by mutation of VASP EVH1 Domain residue Y39 to E, which mimics Abl-induced phosphorylation of Y39. On the basis of these findings, we propose a model in which phosphorylation of Y39 acts as a stoichiometry switch that governs binding partner selection by the constitutive VASP tetramer. These results have broader implications for other multivalent VASP EVH1 Domain binding partners and for furthering our understanding of the role of Y39 phosphorylation in regulating VASP localization and cellular function
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Novel Bivalent Interaction between VASP-EVH1 And Zyxin is Critical for Binding Orientation
Biophysical Journal, 2016Co-Authors: Lucila Andrea Acevedo, Alexander I Greenwood, Eric B. Gibbs, Scott A. Showalter, Linda K NicholsonAbstract:The interaction of EVH1 Domain of the actin polymerization regulatory protein VASP with the cytoskeletal protein Zyxin shows a novel secondary binding site by NMR spectroscopy. Previous studies had shown the importance of the Y39 phosphorylation in EVH1 for co-localization of VASP and Zyxin in the cell. We found a novel secondary binding site that is located in the structural vicinity of Y39. Phosphomimetic mutation Y39E eliminates binding to this secondary interaction site. By analyzing NMR populations, we determined that the secondary binding site provides an orientation preference for the four-motif Zyxin ligand. We further ran double/triple resonance NMR experiments, including carbon-detected, to determine the resonance assignments and structure of a chimera that includes EVH1 and motifs two-four of Zyxin. This structure shows EVH1 bound simultaneously and specifically to two of the four proline-rich binding motifs in Zyxin. This works reveals a secondary binding site in EVH1 that could represent a previously unrecognized coordination mechanism for partners with multiple proline-rich binding motifs.
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isomerase catalyzed binding of interleukin 1 receptor associated kinase 1 to the EVH1 Domain of vasodilator stimulated phosphoprotein
Biochemistry, 2014Co-Authors: Alexander I Greenwood, Jeahoo Kwon, Linda K NicholsonAbstract:Interleukin-1 receptor-associated kinase 1 (IRAK1) is a crucial signaling kinase in the immune system, involved in Toll-like receptor signaling. Vasodilator-stimulated phosphoprotein (VASP) is a central player in cell migration that regulates actin polymerization and connects signaling events to cytoskeletal remodeling. A VASP–IRAK1 interaction is thought to be important in controlling macrophage migration in response to protein kinase C-e activation. We show that the monomeric VASP EVH1 Domain directly binds to the 168WPPPP172 motif in the IRAK1 undefined Domain (IRAK1-UD) with moderate affinity (KDApp = 203 ± 3 μM). We further show that this motif adopts distinct cis and trans isomers for the Trp168–Pro169 peptide bond with nearly equal populations, and that binding to the VASP EVH1 Domain is specific for the trans isomer, coupling binding to isomerization. Nuclear magnetic resonance line shape analysis and tryptophan fluorescence experiments reveal the complete kinetics and thermodynamics of the bindin...
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Isomerase-Catalyzed Binding of Interleukin‑1 Receptor-Associated Kinase 1 to the EVH1 Domain of Vasodilator-Stimulated Phosphoprotein
2014Co-Authors: Alexander I. Greenwood, Jeahoo Kwon, Linda K NicholsonAbstract:Interleukin-1 receptor-associated kinase 1 (IRAK1) is a crucial signaling kinase in the immune system, involved in Toll-like receptor signaling. Vasodilator-stimulated phosphoprotein (VASP) is a central player in cell migration that regulates actin polymerization and connects signaling events to cytoskeletal remodeling. A VASP–IRAK1 interaction is thought to be important in controlling macrophage migration in response to protein kinase C-ε activation. We show that the monomeric VASP EVH1 Domain directly binds to the 168WPPPP172 motif in the IRAK1 undefined Domain (IRAK1-UD) with moderate affinity (KDApp = 203 ± 3 μM). We further show that this motif adopts distinct cis and trans isomers for the Trp168–Pro169 peptide bond with nearly equal populations, and that binding to the VASP EVH1 Domain is specific for the trans isomer, coupling binding to isomerization. Nuclear magnetic resonance line shape analysis and tryptophan fluorescence experiments reveal the complete kinetics and thermodynamics of the binding reaction, showing diffusion-limited binding to the trans isomer followed by slow, isomerization-dependent binding. We further demonstrate that the peptidyl-prolyl isomerase cyclophilin A (CypA) catalyzes isomerization of the Trp168–Pro169 peptide bond and accelerates binding of the IRAK1-UD to the VASP EVH1 Domain. We propose that binding of IRAK1 to tetrameric VASP is regulated by avidity through the assembly of IRAK1 onto receptor-anchored signaling complexes and that an isomerase such as CypA may modulate IRAK1 signaling in vivo. These studies demonstrate a direct interaction between IRAK1 and VASP and suggest a potential mechanism for how this interaction might be regulated by both assembly of IRAK1 onto an activated signaling complex and PPIase enzymes
Kathleen G Morgan - One of the best experts on this subject based on the ideXlab platform.
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Actin polymerization in differentiated vascular smooth muscle cells requires vasodilator-stimulated phosphoprotein
American journal of physiology. Cell physiology, 2009Co-Authors: Hak Rim Kim, Francois Ferron, Malgorzata Boczkowska, Philip Graceffa, Cynthia Gallant, Roberto Dominguez, Kathleen G MorganAbstract:Our group has previously shown that vasoconstrictors increase net actin polymerization in differentiated vascular smooth muscle cells (dVSMC) and that increased actin polymerization is linked to contractility of vascular tissue (Kim et al., Am J Physiol Cell Physiol 295: C768-778, 2008). However, the underlying mechanisms are largely unknown. Here, we evaluated the possible functions of the Ena/vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongation factors in dVSMC. Inhibition of actin filament elongation by cytochalasin D decreases contractility without changing myosin light-chain phosphorylation levels, suggesting that actin filament elongation is necessary for dVSM contraction. VASP is the only Ena/VASP protein highly expressed in aorta tissues, and VASP knockdown decreased smooth muscle contractility. VASP partially colocalizes with alpha-actinin and vinculin in dVSMC. Profilin, known to associate with G actin and VASP, also colocalizes with alpha-actinin and vinculin, potentially identifying the dense bodies and the adhesion plaques as hot spots of actin polymerization. The EVH1 Domain of Ena/VASP is known to target these proteins to their sites of action. Introduction of an expressed EVH1 Domain as a dominant negative inhibits stimulus-induced increases in actin polymerization. VASP phosphorylation, known to inhibit actin polymerization, is decreased during phenylephrine stimulation in dVSMC. We also directly visualized, for the first time, rhodamine-labeled actin incorporation in dVSMC and identified hot spots of actin polymerization in the cell cortex that colocalize with VASP. These results indicate a role for VASP in actin filament assembly, specifically at the cell cortex, that modulates contractility in dVSMC.
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actin polymerization in differentiated vascular smooth muscle cells requires vasodilator stimulated phosphoprotein vasp
Biophysical Journal, 2009Co-Authors: Francois Ferron, Malgorzata Boczkowska, Philip Graceffa, Cynthia Gallant, Paul C Leavis, Roberto Dominguez, Kathleen G MorganAbstract:Our group has shown that alpha agonists and phorbol esters increase net actin polymerization in differentiated vascular smooth muscle cells (dVSMC) and that actin polymerization is linked to contractility. However, the underlying mechanisms are still largely unknown. Inhibition of actin filament elongation by cytochalasin-D treatment decreases contractility without changing the level of myosin light chain phosphorylation in this tissue, suggesting that actin filament elongation processes are necessary for smooth muscle contraction. The enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins is associated with actin filament elongation in non-muscle systems. In this study, we evaluated the possible functions of Ena/VASP in dVSMC. Among Ena/VASP proteins, only VASP is highly expressed in ferret aorta. High resolution 3-D deconvolved fluorescent images of immunostained freshly dissociated aorta cells show that VASP partially colocalizes with both alpha-actinin and vinculin, markers of dense bodies and dense plaques in dVSMC. Profilin, which is known to associate with monomeric G-actin and VASP to facilitate actin filament elongation also colocalizes with both alpha-actinin and vinculin, potentially identifying both the dense bodies and the dense plaques as hot spots of actin polymerization. Differential centrifugation and imaging data indicate that VASP may undergo subtle conformational or/and positional changes in response to stimuli. The EVH1 Domain of VASP is known to be responsible for targeting VASP to its sites of action. Introduction of an expressed EVH1 Domain of Ena/VASP, made as a chimeric protein with the TAT transduction tag, acted as a decoy to inhibit stimulus-induced increases in actin polymerization. In contrast, introduction of the EVH1 mutant F78S, which does not bind target poly-Pro sequences, had no effect. Thus, VASP may be involved in actin filament assembly at dense bodies and dense plaques in dVSMC. Support: NIH P01 HL86655.
Hiroshi Kitani - One of the best experts on this subject based on the ideXlab platform.
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Identification of Fyn as the binding partner for the WASP N-terminal Domain in T cells
International immunology, 2011Co-Authors: Mitsuru Sato, Ryoko Sawahata, Takato Takenouchi, Hiroshi KitaniAbstract:Wiskott-Aldrich syndrome protein (WASP) plays important roles in TCR signaling. In transgenic (Tg) mice, over-expression of the WASP N-terminal region (exons 1-5) including the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) homology 1 (EVH1) Domain and anti-WASP-EVH1 single-chain variable fragment (scFv) intracellular expressed antibodies (intrabodies) impairs IL-2 production in activated T cells. However, it largely remains unknown that how this Domain transduces TCR signaling. Here, we demonstrate for the first time that the WASP N-terminal Domain specifically associates with the Fyn SH3 Domain; the interaction was uncovered by screening a λgt11 cDNA expression library obtained from the mouse T-cell line KKF. The interaction between Fyn and WASP was inhibited by over-expression of the WASP N-terminal Domain and anti-WASP-EVH1 scFv intrabodies in gene-transfected NIH3T3 cells and T cells derived from these Tg mice. WASP-interacting protein binding to the EVH1 Domain of WASP was also inhibited in these Tg mice T cells. Furthermore, tyrosine phosphorylation of WASP and nuclear translocation of nuclear factor of activated T cells following TCR stimulation was severely inhibited by over-expression of the WASP N-terminal Domain. These observations strongly suggest that the WASP N-terminal Domain plays a pivotal role in the TCR signaling cascade by binding to Fyn.
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Impaired LPS-induced signaling in microglia overexpressing the Wiskott-Aldrich syndrome protein N-terminal Domain.
International immunology, 2007Co-Authors: Mitsuru Sato, Kazumasa Ogihara, Ryoko Sawahata, Kenji Sekikawa, Hiroshi KitaniAbstract:Wiskott–Aldrich syndrome protein (WASP) plays important roles in TCR signaling, but its roles in signal transduction in innate immune cells have not been well characterized. As microglia are the primary immune effector cells in the brain, WASP may possibly have important roles in microglial activation, such as production of inflammatory and anti-inflammatory cytokines and neurotoxic factors. Here, we established a microglial cell line from WASP dominant-negative transgenic (Tg) mice overexpressing the N-terminal enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) Domain. WASP Tg microglia were impaired in production of inflammatory cytokines such as tumor necrosis factor-a, IL-6 and IL-1b upon LPS stimulation, whereas anti-inflammatory IL-10 production was significantly enhanced. Also, LPS-induced phosphorylation of nuclear factor kB was reduced in WASP Tg microglia. Furthermore, WASP Tg microglia exhibited less cytotoxicity against co-cultured neurons after stimulation by LPS and IFN-g, with a concordant decrease in nitric oxide production. These results strongly suggest that WASP may have pivotal roles through the EVH1 Domain in the LPS signaling cascade, either directly or indirectly, and modulates inflammatory immune responses in microglia.
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Intrabodies against the EVH1 Domain of Wiskott-Aldrich syndrome protein inhibit T cell receptor signaling in transgenic mice T cells.
The FEBS journal, 2005Co-Authors: Mitsuru Sato, Ryo Iwaya, Kazumasa Ogihara, Ryoko Sawahata, Hiroshi Kitani, Joe Chiba, Yoshikazu Kurosawa, Kenji SekikawaAbstract:Intracellularly expressed antibodies (intrabodies) have been used to inhibit the function of various kinds of protein inside cells. However, problems with stability and functional expression of intrabodies in the cytosol remain unsolved. In this study, we show that single-chain variable fragment (scFv) intrabodies constructed with a heavy chain variable (V(H)) leader signal sequence at the N-terminus were translocated from the endoplasmic reticulum into the cytosol of T lymphocytes and inhibited the function of the target molecule, Wiskott-Aldrich syndrome protein (WASP). WASP resides in the cytosol as a multifunctional adaptor molecule and mediates actin polymerization and interleukin (IL)-2 synthesis in the T-cell receptor (TCR) signaling pathway. It has been suggested that an EVH1 Domain in the N-terminal region of WASP may participate in IL-2 synthesis. In transgenic mice expressing anti-EVH1 scFvs derived from hybridoma cells producing WASP-EVH1 mAbs, a large number of scFvs in the cytosol and binding between anti-EVH1 scFvs and native WASP in T cells were detected by immunoprecipitation analysis. Furthermore, impairment of the proliferative response and IL-2 production induced by TCR stimulation which did not affect TCR capping was demonstrated in the scFv transgenic T cells. We previously described the same T-cell defects in WASP transgenic mice overexpressing the EVH1 Domain. These results indicate that the EVH1 intrabodies inhibit only the EVH1 Domain function that regulates IL-2 synthesis signaling without affecting the overall Domain structure of WASP. The novel procedure presented here is a valuable tool for in vivo functional analysis of cytosolic proteins.
Mitsuru Sato - One of the best experts on this subject based on the ideXlab platform.
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Identification of Fyn as the binding partner for the WASP N-terminal Domain in T cells
International immunology, 2011Co-Authors: Mitsuru Sato, Ryoko Sawahata, Takato Takenouchi, Hiroshi KitaniAbstract:Wiskott-Aldrich syndrome protein (WASP) plays important roles in TCR signaling. In transgenic (Tg) mice, over-expression of the WASP N-terminal region (exons 1-5) including the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) homology 1 (EVH1) Domain and anti-WASP-EVH1 single-chain variable fragment (scFv) intracellular expressed antibodies (intrabodies) impairs IL-2 production in activated T cells. However, it largely remains unknown that how this Domain transduces TCR signaling. Here, we demonstrate for the first time that the WASP N-terminal Domain specifically associates with the Fyn SH3 Domain; the interaction was uncovered by screening a λgt11 cDNA expression library obtained from the mouse T-cell line KKF. The interaction between Fyn and WASP was inhibited by over-expression of the WASP N-terminal Domain and anti-WASP-EVH1 scFv intrabodies in gene-transfected NIH3T3 cells and T cells derived from these Tg mice. WASP-interacting protein binding to the EVH1 Domain of WASP was also inhibited in these Tg mice T cells. Furthermore, tyrosine phosphorylation of WASP and nuclear translocation of nuclear factor of activated T cells following TCR stimulation was severely inhibited by over-expression of the WASP N-terminal Domain. These observations strongly suggest that the WASP N-terminal Domain plays a pivotal role in the TCR signaling cascade by binding to Fyn.
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Impaired LPS-induced signaling in microglia overexpressing the Wiskott-Aldrich syndrome protein N-terminal Domain.
International immunology, 2007Co-Authors: Mitsuru Sato, Kazumasa Ogihara, Ryoko Sawahata, Kenji Sekikawa, Hiroshi KitaniAbstract:Wiskott–Aldrich syndrome protein (WASP) plays important roles in TCR signaling, but its roles in signal transduction in innate immune cells have not been well characterized. As microglia are the primary immune effector cells in the brain, WASP may possibly have important roles in microglial activation, such as production of inflammatory and anti-inflammatory cytokines and neurotoxic factors. Here, we established a microglial cell line from WASP dominant-negative transgenic (Tg) mice overexpressing the N-terminal enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) Domain. WASP Tg microglia were impaired in production of inflammatory cytokines such as tumor necrosis factor-a, IL-6 and IL-1b upon LPS stimulation, whereas anti-inflammatory IL-10 production was significantly enhanced. Also, LPS-induced phosphorylation of nuclear factor kB was reduced in WASP Tg microglia. Furthermore, WASP Tg microglia exhibited less cytotoxicity against co-cultured neurons after stimulation by LPS and IFN-g, with a concordant decrease in nitric oxide production. These results strongly suggest that WASP may have pivotal roles through the EVH1 Domain in the LPS signaling cascade, either directly or indirectly, and modulates inflammatory immune responses in microglia.
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Intrabodies against the EVH1 Domain of Wiskott-Aldrich syndrome protein inhibit T cell receptor signaling in transgenic mice T cells.
The FEBS journal, 2005Co-Authors: Mitsuru Sato, Ryo Iwaya, Kazumasa Ogihara, Ryoko Sawahata, Hiroshi Kitani, Joe Chiba, Yoshikazu Kurosawa, Kenji SekikawaAbstract:Intracellularly expressed antibodies (intrabodies) have been used to inhibit the function of various kinds of protein inside cells. However, problems with stability and functional expression of intrabodies in the cytosol remain unsolved. In this study, we show that single-chain variable fragment (scFv) intrabodies constructed with a heavy chain variable (V(H)) leader signal sequence at the N-terminus were translocated from the endoplasmic reticulum into the cytosol of T lymphocytes and inhibited the function of the target molecule, Wiskott-Aldrich syndrome protein (WASP). WASP resides in the cytosol as a multifunctional adaptor molecule and mediates actin polymerization and interleukin (IL)-2 synthesis in the T-cell receptor (TCR) signaling pathway. It has been suggested that an EVH1 Domain in the N-terminal region of WASP may participate in IL-2 synthesis. In transgenic mice expressing anti-EVH1 scFvs derived from hybridoma cells producing WASP-EVH1 mAbs, a large number of scFvs in the cytosol and binding between anti-EVH1 scFvs and native WASP in T cells were detected by immunoprecipitation analysis. Furthermore, impairment of the proliferative response and IL-2 production induced by TCR stimulation which did not affect TCR capping was demonstrated in the scFv transgenic T cells. We previously described the same T-cell defects in WASP transgenic mice overexpressing the EVH1 Domain. These results indicate that the EVH1 intrabodies inhibit only the EVH1 Domain function that regulates IL-2 synthesis signaling without affecting the overall Domain structure of WASP. The novel procedure presented here is a valuable tool for in vivo functional analysis of cytosolic proteins.
Ulrich Walter - One of the best experts on this subject based on the ideXlab platform.
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Identification of SPRED2 (Sprouty-related Protein with EVH1 Domain 2) as a Negative Regulator of the Hypothalamic-Pituitary-Adrenal Axis
The Journal of biological chemistry, 2011Co-Authors: Melanie Ullrich, Ulrich Walter, Karin Bundschu, Peter M. Benz, Marco Abesser, Ruth Freudinger, Tobias Fischer, Julia Ullrich, Thomas Renné, Kai SchuhAbstract:Sprouty-related proteins with EVH1 (enabled/vasodilator-stimulated phosphoprotein homology 1) Domain (SPREDs) are inhibitors of MAPK signaling. To elucidate SPRED2 in vivo function, we characterized body homeostasis in SPRED2(-/-) mice. They showed a doubled daily water uptake, induced by elevated serum osmolality, originating from increased blood salt load. Accordingly, serum aldosterone was doubled, accompanied by augmented adrenal aldosterone synthase (AS) expression. Surprisingly, serum vasopressin (AVP) was unaltered, and, as evidenced by halved angiotensin II (Ang II) levels, the renin angiotensin system (RAS) was down-regulated. Adrenocorticotropic hormone (ACTH) was significantly elevated in SPRED2(-/-) mice, together with its secretagogue corticotropin-releasing hormone (CRH) and its downstream target corticosterone. ERK phosphorylation in brains was augmented, and hypothalamic CRH mRNA levels were elevated, both contributing to the increased CRH release. Our data were supported by CRH promoter reporter assays in hypothalamic mHypoE-44 cells, revealing a SPRED-dependent inhibition of Ets (ERK/E-twenty-six)-dependent transcription. Furthermore, SPRED suppressed CRH production in these cells. In conclusion, our study suggests that SPRED2 deficiency leads to an increased MAPK signaling, which results in an augmented CRH promoter activity. The subsequent CRH overproduction causes an up-regulation of downstream hypothalamic-pituitary-adrenal (HPA) hormone secretion. This constitutes a possible trigger for the observed compulsive grooming in SPRED2(-/-) mice and may, together with hyperplasia of aldosterone-producing cells, contribute to the hyperaldosteronism and homeostatic imbalances.
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Characterization of a Novel Interaction Between Vasodilator-Stimulated Phosphoprotein and Abelson Interactor 1 in Human Platelets: A Concerted Computational and Experimental Approach
Arteriosclerosis thrombosis and vascular biology, 2010Co-Authors: Marcus Dittrich, Ulrich Walter, Verena Strassberger, Marc Fackler, Piet W.l. Tas, Urs Lewandrowski, Albert Sickmann, Thomas Dandekar, Ingvild BirschmannAbstract:The goal of this study was systematic profiling of vasodilator-stimulated phosphoprotein (VASP)-Ena/VASP homology 1 (EVH1) interactors in human platelets using a combined in silico and in vitro approach. Exploiting the information of the comprehensive proteome catalogue in the PlateletWeb database (http://plateletweb.bioapps.biozentrum.uni-wuerzburg.de/PlateletWeb.php), we performed a motif search of all sequences and identified potential target sites of class I EVH1 Domains in human platelet proteins. Performing affinity purification with VASP-EVH1 Domain and the lysates of platelets, we examined complex partners by mass spectrometry. Combining the results of both analyses, we identified Abelson interactor 1 (Abi-1) as a novel EVH1 Domain-specific interaction partner of VASP in human platelets and investigated this interaction by yeast 2-hybrid mutational studies and immunoprecipitation. Immunofluorescence microscopy indicated colocalization of both proteins at the lamellipodia of spread human platelets, suggesting a role in reorganizing the cytoskeleton during spreading. The combination of experimental and computational interactome research has emerged as a valuable tool for the analysis of protein-protein interaction networks and facilitates the discovery and characterization of novel interactions as detailed here for Abi-1 and VASP in human platelets. System biological approaches can be expected to play an important role in basic and clinical platelet research, as they offer the potential to analyze signal transduction beyond the scope of established pathways.
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The VASP-Spred-Sprouty Domain Puzzle
The Journal of biological chemistry, 2006Co-Authors: Karin Bundschu, Ulrich Walter, Kai SchuhAbstract:Sprouty-related proteins with an EVH1 Domain (Spreds) belong to a new protein family harboring a conserved N-terminal EVH1 Domain, which is related to the VASP (vasodilator-stimulated phosphoprotein) EVH1 Domain (Enabled/VASP homology 1 Domain) and a C-terminal Sprouty-related Domain, typical for Sprouty proteins. Spreds were, like Sproutys, initially discovered as inhibitors of the Ras/MAPK pathway, and the SPR (Sprouty-related) Domains of both protein families seem to be very important for many protein interactions and cellular processes. VASP was initially characterized as a proline-rich substrate of protein kinases A and G in human platelets and later shown to be a scaffold protein, regulating both signal transduction pathways and the actin filament system. The VASP-EVH1 Domain is known to bind specifically to a FP(4) binding motif, which is, for example, present in the focal adhesion proteins vinculin and zyxin. In this review we give a structural and functional overview on these three protein families and ask whether nature plays a modular protein Domain puzzle with stable exchangeable elements or if these closely related Domains have various functions when pasted in a different protein context.
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Design of N-substituted peptomer ligands for EVH1 Domains.
The Journal of biological chemistry, 2003Co-Authors: Jürgen Zimmermann, Ronald Kuhne, Ulrich Walter, Thomas Jarchau, Rudolf Volkmer-engert, Hartmut Oschkinat, Linda J BallAbstract:Ena/VASP proteins are implicated in cytoskeletal reorganization during actin-dependent motility processes. Recruitment to subcellular sites of actin polymerization is mediated by the highly conserved N-terminal EVH1 Domain, which interacts with target proteins containing proline-rich motifs. The VASP EVH1 Domain specifically binds peptides with the consensus motif FPPPP present in all its binding partners, including the Listerial ActA protein. Previous studies have shown that the Phe and first and final Pro residues are highly conserved and cannot be substituted with any other natural amino acid without significant loss of binding affinity. We have incorporated peptoid building blocks (sarcosine derived, non-natural amino acids) into the peptide SFEFPPPPTEDEL from the Listerial ActA protein and were able to substitute the most highly conserved residues of this motif while maintaining binding to the VASP EVH1 Domain with affinities in the range of 45-180 microm. We then used NMR chemical shift perturbations to locate specific Domain residues involved in particular interactions. These studies may open up the way for designing selective modulators of VASP function for biological studies and for the development of novel therapeutics for diseases involving pathologically altered cell adhesion or cell motility.
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dual epitope recognition by the vasp EVH1 Domain modulates polyproline ligand specificity and binding affinity
The EMBO Journal, 2000Co-Authors: Linda J Ball, Ronald Kuhne, Berit Hoffmann, Angelika Hafner, Peter Schmieder, Rudolf Volkmerengert, Martin Hof, M Wahl, Jens Schneidermergener, Ulrich WalterAbstract: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 specifically recognize ‘FPPPP’ motifs found in the mammalian zyx in and vinculin proteins, and ActA protein of the intracellular bacterium Listeria monocytogenes . Here, evidence is provided that the affinities of these EVH1–peptide interactions are strongly dependent on the recognition of residues flanking the core FPPPP motifs. Determination of the VASP EVH1 Domain solution structure, together with peptide library screening, measurement of individual K d s by fluorescence titration, and NMR chemical shift mapping, revealed a second affinity‐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 affinity 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.