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. 1996 Sep 15;318(Pt 3):753–757. doi: 10.1042/bj3180753

The focal-adhesion vasodilator-stimulated phosphoprotein (VASP) binds to the proline-rich domain in vinculin.

N P Brindle 1, M R Holt 1, J E Davies 1, C J Price 1, D R Critchley 1
PMCID: PMC1217682  PMID: 8836115

Abstract

In mammalian cells vasodilator-stimulated phosphoprotein (VASP) is localized to focal adhesions and areas of dynamic membrane activity where it is thought to have a role in actinfilament assembly. The proteins responsible for recruiting VASP to these sites within the cell are not known. The bacterial protein ActA binds VASP via a proline-rich motif that is very similar to a sequence in the proline-rich region of the focal-adhesion protein vinculin. We have examined the ability of VASP, synthesized using an in vitro transcription/translation system, to bind to a series of vinculin peptides expressed as glutathione S-transferase fusion proteins, and have shown that it binds specifically to the proline-rich region in vinculin. Using immobilized peptides corresponding to the two proline-rich motifs within this domain, the VASP-binding site was localized to proline-rich motif-l (residues 839-850). Binding to this motif was not affected by the phosphorylation state of VASP. The C-terminal region of VASP, which is known to be important in targeting VASP to focal adhesions, was shown to be required for binding. These results identify vinculin as a VASP-binding protein likely to be important in recruiting VASP to focal adhesions and the cell membrane.

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Selected References

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  1. Abel K., Mieskes G., Walter U. Dephosphorylation of the focal adhesion protein VASP in vitro and in intact human platelets. FEBS Lett. 1995 Aug 21;370(3):184–188. doi: 10.1016/0014-5793(95)00817-s. [DOI] [PubMed] [Google Scholar]
  2. Butt E., Abel K., Krieger M., Palm D., Hoppe V., Hoppe J., Walter U. cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human platelets. J Biol Chem. 1994 May 20;269(20):14509–14517. [PubMed] [Google Scholar]
  3. Chakraborty T., Ebel F., Domann E., Niebuhr K., Gerstel B., Pistor S., Temm-Grove C. J., Jockusch B. M., Reinhard M., Walter U. A focal adhesion factor directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-based cytoskeleton of mammalian cells. EMBO J. 1995 Apr 3;14(7):1314–1321. doi: 10.1002/j.1460-2075.1995.tb07117.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Domann E., Wehland J., Rohde M., Pistor S., Hartl M., Goebel W., Leimeister-Wächter M., Wuenscher M., Chakraborty T. A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin. EMBO J. 1992 May;11(5):1981–1990. doi: 10.1002/j.1460-2075.1992.tb05252.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Draijer R., Vaandrager A. B., Nolte C., de Jonge H. R., Walter U., van Hinsbergh V. W. Expression of cGMP-dependent protein kinase I and phosphorylation of its substrate, vasodilator-stimulated phosphoprotein, in human endothelial cells of different origin. Circ Res. 1995 Nov;77(5):897–905. doi: 10.1161/01.res.77.5.897. [DOI] [PubMed] [Google Scholar]
  6. Dutartre H., Davoust J., Gorvel J. P., Chavrier P. Cytokinesis arrest and redistribution of actin-cytoskeleton regulatory components in cells expressing the Rho GTPase CDC42Hs. J Cell Sci. 1996 Feb;109(Pt 2):367–377. doi: 10.1242/jcs.109.2.367. [DOI] [PubMed] [Google Scholar]
  7. Gilmore A. P., Burridge K. Regulation of vinculin binding to talin and actin by phosphatidyl-inositol-4-5-bisphosphate. Nature. 1996 Jun 6;381(6582):531–535. doi: 10.1038/381531a0. [DOI] [PubMed] [Google Scholar]
  8. Gilmore A. P., Jackson P., Waites G. T., Critchley D. R. Further characterisation of the talin-binding site in the cytoskeletal protein vinculin. J Cell Sci. 1992 Nov;103(Pt 3):719–731. doi: 10.1242/jcs.103.3.719. [DOI] [PubMed] [Google Scholar]
  9. Gouin E., Dehoux P., Mengaud J., Kocks C., Cossart P. iactA of Listeria ivanovii, although distantly related to Listeria monocytogenes actA, restores actin tail formation in an L. monocytogenes actA mutant. Infect Immun. 1995 Jul;63(7):2729–2737. doi: 10.1128/iai.63.7.2729-2737.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Haffner C., Jarchau T., Reinhard M., Hoppe J., Lohmann S. M., Walter U. Molecular cloning, structural analysis and functional expression of the proline-rich focal adhesion and microfilament-associated protein VASP. EMBO J. 1995 Jan 3;14(1):19–27. doi: 10.1002/j.1460-2075.1995.tb06971.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Horstrup K., Jablonka B., Hönig-Liedl P., Just M., Kochsiek K., Walter U. Phosphorylation of focal adhesion vasodilator-stimulated phosphoprotein at Ser157 in intact human platelets correlates with fibrinogen receptor inhibition. Eur J Biochem. 1994 Oct 1;225(1):21–27. doi: 10.1111/j.1432-1033.1994.00021.x. [DOI] [PubMed] [Google Scholar]
  12. Johnson R. P., Craig S. W. F-actin binding site masked by the intramolecular association of vinculin head and tail domains. Nature. 1995 Jan 19;373(6511):261–264. doi: 10.1038/373261a0. [DOI] [PubMed] [Google Scholar]
  13. Kocks C., Gouin E., Tabouret M., Berche P., Ohayon H., Cossart P. L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein. Cell. 1992 Feb 7;68(3):521–531. doi: 10.1016/0092-8674(92)90188-i. [DOI] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Lucher L. A., Lego T. Use of the water-soluble fluor sodium salicylate for fluorographic detection of tritium in thin-layer chromatograms and nitrocellulose blots. Anal Biochem. 1989 May 1;178(2):327–330. doi: 10.1016/0003-2697(89)90647-7. [DOI] [PubMed] [Google Scholar]
  16. McGregor A., Blanchard A. D., Rowe A. J., Critchley D. R. Identification of the vinculin-binding site in the cytoskeletal protein alpha-actinin. Biochem J. 1994 Jul 1;301(Pt 1):225–233. doi: 10.1042/bj3010225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nobes C. D., Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell. 1995 Apr 7;81(1):53–62. doi: 10.1016/0092-8674(95)90370-4. [DOI] [PubMed] [Google Scholar]
  18. Pistor S., Chakraborty T., Walter U., Wehland J. The bacterial actin nucleator protein ActA of Listeria monocytogenes contains multiple binding sites for host microfilament proteins. Curr Biol. 1995 May 1;5(5):517–525. doi: 10.1016/s0960-9822(95)00104-7. [DOI] [PubMed] [Google Scholar]
  19. Price G. J., Jones P., Davison M. D., Patel B., Bendori R., Geiger B., Critchley D. R. Primary sequence and domain structure of chicken vinculin. Biochem J. 1989 Apr 15;259(2):453–461. doi: 10.1042/bj2590453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Reinhard M., Giehl K., Abel K., Haffner C., Jarchau T., Hoppe V., Jockusch B. M., Walter U. The proline-rich focal adhesion and microfilament protein VASP is a ligand for profilins. EMBO J. 1995 Apr 18;14(8):1583–1589. doi: 10.1002/j.1460-2075.1995.tb07146.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Reinhard M., Halbrügge M., Scheer U., Wiegand C., Jockusch B. M., Walter U. The 46/50 kDa phosphoprotein VASP purified from human platelets is a novel protein associated with actin filaments and focal contacts. EMBO J. 1992 Jun;11(6):2063–2070. doi: 10.1002/j.1460-2075.1992.tb05264.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Reinhard M., Jouvenal K., Tripier D., Walter U. Identification, purification, and characterization of a zyxin-related protein that binds the focal adhesion and microfilament protein VASP (vasodilator-stimulated phosphoprotein). Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7956–7960. doi: 10.1073/pnas.92.17.7956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sadler I., Crawford A. W., Michelsen J. W., Beckerle M. C. Zyxin and cCRP: two interactive LIM domain proteins associated with the cytoskeleton. J Cell Biol. 1992 Dec;119(6):1573–1587. doi: 10.1083/jcb.119.6.1573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sanger J. M., Sanger J. W., Southwick F. S. Host cell actin assembly is necessary and likely to provide the propulsive force for intracellular movement of Listeria monocytogenes. Infect Immun. 1992 Sep;60(9):3609–3619. doi: 10.1128/iai.60.9.3609-3619.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sudol M. The WW module competes with the SH3 domain? Trends Biochem Sci. 1996 May;21(5):161–163. [PubMed] [Google Scholar]
  26. Theriot J. A., Mitchison T. J. The three faces of profilin. Cell. 1993 Dec 3;75(5):835–838. doi: 10.1016/0092-8674(93)90527-w. [DOI] [PubMed] [Google Scholar]
  27. Theriot J. A., Mitchison T. J., Tilney L. G., Portnoy D. A. The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization. Nature. 1992 May 21;357(6375):257–260. doi: 10.1038/357257a0. [DOI] [PubMed] [Google Scholar]
  28. Waldmann R., Nieberding M., Walter U. Vasodilator-stimulated protein phosphorylation in platelets is mediated by cAMP- and cGMP-dependent protein kinases. Eur J Biochem. 1987 Sep 15;167(3):441–448. doi: 10.1111/j.1432-1033.1987.tb13357.x. [DOI] [PubMed] [Google Scholar]
  29. Weekes J., Barry S. T., Critchley D. R. Acidic phospholipids inhibit the intramolecular association between the N- and C-terminal regions of vinculin, exposing actin-binding and protein kinase C phosphorylation sites. Biochem J. 1996 Mar 15;314(Pt 3):827–832. doi: 10.1042/bj3140827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wood C. K., Turner C. E., Jackson P., Critchley D. R. Characterisation of the paxillin-binding site and the C-terminal focal adhesion targeting sequence in vinculin. J Cell Sci. 1994 Feb;107(Pt 2):709–717. [PubMed] [Google Scholar]

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