Purification and characterization of an 85 kDa talin-binding fragment of vinculin

Cell Motility and the Cytoskeleton
M E Groesch, J J Otto

Abstract

Vinculin and talin are adhesion plaque proteins which have been shown to interact with each other in vitro. In order to begin to investigate where the talin-binding domain is in vinculin, vinculin was digested with Staphylococcus aureus V8 protease to generate two major fragments of 85 and 30 kDa, and these fragments were purified. Nitrocellulose overlays with 125I-talin and the 125I-85 kDa vinculin fragment and sucrose density gradient centrifugation demonstrated that the talin-binding domain was localized to the 85 kDa vinculin fragment. Quantification of 125I-talin binding in the overlays showed that four times more talin bound to the 85 kDa fragment as compared to intact vinculin. Competitive immunoprecipitation experiments demonstrated that unlabeled 85 kDa fragment was about three-fold more effective at competing for 125I-85 kDa binding to talin than was unlabeled vinculin. These results suggest that the 30 kDa fragment inhibits the vinculin-talin interaction even though the talin-binding domain is localized in the 85 kDa fragment.

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Citations

Jan 1, 1997·Cell Motility and the Cytoskeleton·S Lee, J J Otto
Jan 1, 1990·Cell Motility and the Cytoskeleton·J J Otto
Dec 27, 2008·The Journal of Biological Chemistry·Sean M PalmerSharon L Campbell
Sep 1, 1990·The Journal of Cell Biology·C E TurnerK Burridge
Jul 1, 1993·American Journal of Respiratory Cell and Molecular Biology·S M HallS G Haworth
Oct 24, 2015·Journal of Molecular Biology·Laura Y KimGregory M Alushin
Jun 1, 1995·Microscopy Research and Technique·M H Stromer
May 21, 1999·Archives of Biochemistry and Biophysics·J M SchmidtR M Robson
Apr 13, 2017·Cellular and Molecular Life Sciences : CMLS·Jennifer L Bays, Kris A DeMali
Jan 2, 2001·The Journal of Biological Chemistry·G J MillerE H Ball
Nov 1, 2006·The Journal of Biological Chemistry·Hui ChenSusan W Craig
May 31, 2001·The Journal of Biological Chemistry·G J Miller, E H Ball
Jun 22, 1999·The Journal of Biological Chemistry·P A SteimleS W Craig
Feb 25, 2005·The Journal of Biological Chemistry·Daniel M CohenSusan W Craig
Jan 5, 2000·The Journal of Biological Chemistry·R P Johnson, S W Craig
Jan 11, 2001·European Journal of Cell Biology·R Palovuori, S Eskelinen

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