This article is published in Journal of Molecular Recognition as a special issue on Affinity 2009, edited by Gideon Fleminger, Tel-Aviv University, Tel-Aviv, Israel and George Ehrlich, Hoffmann-La Roche, Nutley, NJ, USA.
Bead-based protein–protein interaction assays for the analysis of Rho GTPase signaling†
Version of Record online: 28 OCT 2010
Copyright © 2010 John Wiley & Sons, Ltd.
Journal of Molecular Recognition
Special Issue: Affinity 2009 – The 18th biennial meeting of the International Society for Molecular Recognition
Volume 23, Issue 6, pages 543–550, November/December 2010
How to Cite
Rimmele, S., Gierschik, P., Joos, T. O. and Schneiderhan-Marra, N. (2010), Bead-based protein–protein interaction assays for the analysis of Rho GTPase signaling. J. Mol. Recognit., 23: 543–550. doi: 10.1002/jmr.1051
- Issue online: 28 OCT 2010
- Version of Record online: 28 OCT 2010
- Manuscript Accepted: 7 APR 2010
- Manuscript Revised: 19 MAR 2010
- Manuscript Received: 4 DEC 2009
- bead-based assays;
- protein–protein interaction;
- Rho GTPases;
- Rho GDI;
- p21-activated kinase;
- post-translational modification
Bead-based interaction assays are excellently suited to study protein–protein interactions, as they require only minimal amounts of sample material. Miniaturized protein–protein interaction assays were designed to analyze Rho GTPase activation based on its interaction with Rho GDI or p21-activated kinase (PAK).
Rho GDI plays a key role in the regulation of a variety of cellular functions through its interaction with Rho GTPases. Rho GDI is frequently overexpressed in many human cancers. Therefore, there is a growing and as yet unfulfilled demand for screening assays to identify biologically active compounds that may inhibit the Rho GTPase–Rho GDI interaction. Bead-based interaction assays provide an interesting alternative that facilitate such assays to be performed faster with only small amounts of material compared to routinely used co-immunoprecipitation followed by Western Blot analysis.
Bead-based protein interaction assays for overexpressed HA-tagged Rho GTPases were established to study the GTPγS-dependent interaction of five different Rho GTPases with the regulatory protein Rho GDIα and the downstream effector PAK1. In addition, it was demonstrated that the ability of Rho GTPases to interact with Rho GDI in this experimental system was markedly, but differentially sensitive to post-translational modification of their carboxyl terminus. Importantly, this modification also notably affected the ability of Rac1 and Rac2, but not of Cdc42, to interact with PAK1. Copyright © 2010 John Wiley & Sons, Ltd.