Bead-based protein-protein interaction assays for the analysis of Rho GTPase signaling.

Abstract

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.

• Authors: Stefanie Rimmele, Peter Gierschik, Thomas O Joos, Nicole Schneiderhan-Marra
• Journal: Journal of molecular recognition : JMR (J Mol Recognit) 2010 Nov-Dec Vol. 23 Issue 6 Pg. 543-50 ISSN: 1099-1352 [Electronic] England
• PMID: 21031432 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2010 John Wiley & Sons, Ltd.

Chemical References

• Bacterial Proteins
• Guanine Nucleotide Dissociation Inhibitors
• YopT protein, Yersinia
• rho-Specific Guanine Nucleotide Dissociation Inhibitors
• p21-Activated Kinases
• Cysteine Endopeptidases
• rho GTP-Binding Proteins

Topics

• Bacterial Proteins (pharmacology)
• Cysteine Endopeptidases (pharmacology)
• Guanine Nucleotide Dissociation Inhibitors (chemistry, metabolism)
• Humans
• Immunoprecipitation (methods)
• Microspheres
• Models, Biological
• Protein Binding (drug effects, physiology)
• Protein Interaction Domains and Motifs (drug effects)
• Protein Processing, Post-Translational (physiology)
• Signal Transduction (physiology)
• p21-Activated Kinases (chemistry, metabolism)
• rho GTP-Binding Proteins (chemistry, metabolism)
• rho-Specific Guanine Nucleotide Dissociation Inhibitors