The EVH2 Domain of the Vasodilator-stimulated Phosphoprotein Mediates Tetramerization, F-actin Binding, and Actin Bundle Formation*
- From the Institut für Klinische Biochemie und Pathobiochemie, Medizinische Universitätsklinik, Versbacher Straße 5, D-97078 Würzburg, Germany
Abstract
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.
Footnotes
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↵* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 176/TP A21.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed. Tel.: 49-931-201-3144; Fax: 49-931-201-3153; E-mail: Matthias.Reinhard@mail.uni- wuerzburg.de.
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↵2 C. Bachmann, L. Fischer, U. Walter, and M. Reinhard, our unpublished observations.
- Abbreviations:
- VASP
-
vasodilator-stimulated phosphoprotein
- Abl
-
Abelson tyrosine kinase
- EDAC
-
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
- EGS
-
ethylene glycol bis(succinimidylsuccinate)
- Ena
-
Enabled
- EVH
-
Ena-VASP homology
- Evl
-
Ena-VASP-like
- Mena
-
mammalian Enabled
- NHSS
-
N-hydroxysulfosuccinimide
- PBS
-
phosphate-buffered saline
- PAGE
-
polyacrylamide gel electrophoresis
- GST
-
glutathioneS-transferase
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- Received March 31, 1999.
- Revision received May 24, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
