Journal of Cell Science, Vol 110, Issue 12 1361-1371, Copyright © 1997 by Company of Biologists

JOURNAL ARTICLES

Ligand recruitment by vinculin domains in transfected cells

P Bubeck, S Pistor, J Wehland and BM Jockusch
Cell Biology, Zoological Institute, Technical University of Braunschweig, Germany.

Vinculin, a prominent protein component of microfilament-membrane attachment sites, consists of three major domains: an N-terminal, compact head and a C-terminal rod-like tail that are connected by a flexible, proline-rich hinge. In vitro, the protein has been shown to interact with numerous ligands, including other components of the microfilament system. To characterize the ligand recruitment ability of the different vinculin domains in a cellular environment, we used a novel approach of comprising chimeric proteins of either the vinculin head, hinge or tail regions, fused to the membrane anchor sequence of ActA, a surface protein of the intracellular bacterial pathogen Listeria monocytogenes. When PtK2 cells were transfected with the corresponding constructs, the ActA membrane anchor directed the chimeric polypeptides to mitochondrial membranes. In this position, they accumulated microfilament proteins, as seen by immunofluorescence analysis. A chimera comprising the full length vinculin clone recruited a substantial amount of the cellular F-actin, the vasodilator stimulated phosphoprotein (VASP) and paxillin, but little alpha-actinin and talin. The presence of only the vinculin head directed some of the fusion protein to focal contacts, and alpha-actinin recruitment was still ineffective. Prominent recruitment of F-actin and of VASP required the presence of the tail and proline-rich hinge, respectively. Reducing the vinculin tail to short pieces harboring only one of the two F-actin binding sequences, which were defined by in vitro experiments, resulted in loss of activity, possibly by incorrect polypeptide folding. The proline-rich hinge domain could be exchanged for the analogous region of the ActA protein, and the number of such proline-clusters, containing an FPPPP motif, correlated with the extent of VASP recruitment. The results show that this system can be used to analyze in vivo the activity of vinculin domains responsible for the assembly of various cytoskeletal ligands.

This article has been cited by other articles:

				P. R. J. Bois, R. A. Borgon, C. Vonrhein, and T. Izard Structural Dynamics of {alpha}-Actinin-Vinculin Interactions Mol. Cell. Biol., July 15, 2005; 25(14): 6112 - 6122. [Abstract] [Full Text] [PDF]

				D. W. Lawrence and K. B. Pryzwansky The Vasodilator-Stimulated Phosphoprotein Is Regulated by Cyclic GMP-Dependent Protein Kinase During Neutrophil Spreading J. Immunol., May 1, 2001; 166(9): 5550 - 5556. [Abstract] [Full Text] [PDF]

				R Massoumi and A Sjolander Leukotriene D(4) affects localisation of vinculin in intestinal epithelial cells via distinct tyrosine kinase and protein kinase C controlled events J. Cell Sci., January 5, 2001; 114(10): 1925 - 1934. [Abstract] [PDF]

				M. M. R. Petit, J. Fradelizi, R. M. Golsteyn, T. A.Y. Ayoubi, B. Menichi, D. Louvard, W. J. M. Van de Ven, and E. Friederich LPP, an Actin Cytoskeleton Protein Related to Zyxin, Harbors a Nuclear Export Signal and Transcriptional Activation Capacity Mol. Biol. Cell, January 1, 2000; 11(1): 117 - 129. [Abstract] [Full Text]

				M. Reinhard, J. Zumbrunn, D. Jaquemar, M. Kuhn, U. Walter, and B. Trueb An alpha -Actinin Binding Site of Zyxin Is Essential for Subcellular Zyxin Localization and alpha -Actinin Recruitment J. Biol. Chem., May 7, 1999; 274(19): 13410 - 13418. [Abstract] [Full Text] [PDF]

				M Perez-Moreno, A Avila, S Islas, S Sanchez, and L Gonzalez-Mariscal Vinculin but not alpha-actinin is a target of PKC phosphorylation during junctional assembly induced by calcium J. Cell Sci., January 12, 1998; 111(23): 3563 - 3571. [Abstract] [PDF]

				D. A. Calderwood, S. J. Shattil, and M. H. Ginsberg Integrins and Actin Filaments: Reciprocal Regulation of Cell Adhesion and Signaling J. Biol. Chem., July 21, 2000; 275(30): 22607 - 22610. [Full Text] [PDF]