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Thiol-Modifying Phenylarsine Oxide Inhibits Guanine Nucleotide Binding of Rho but Not of Rac GTPases

Institut für Toxikologie, Medizinische Hochschule Hannover, Hannover, Germany (R.G., I.J.); IPF PharmaCeuticals GmbH, Hannover, Germany (H.J.); Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany (K.A.)

Phenylarsine oxide (PAO) is a phosphotyrosine phosphatase inhibitor that cross-links vicinal thiol groups, thereby inactivating phosphatases possessing XCysXXCysX motifs. The RhoA-GTPase, but not the Rac1-GTPase, also possesses vicinal cysteines within the guanine nucleotide-binding region (aa 13–20) and the phosphohydrolase activity site. Treatment of Caco-2 cells with PAO showed a dose-dependent reorganization of the actin cytoskeleton, indicating involvement of Rho GTPases. As tested by pull-down experiments, RhoA, but not Rac1, from cell lysates was inactivated by PAO in a concentration-dependent manner. Modification of RhoA by PAO resulted in altered mobility on SDS-polyacrylamide gel electrophoresis, and PAO-modified RhoA was no longer substrate for C3-catalyzed ADP-ribosylation. Furthermore, RhoA treated with PAO, but not Rac1 treated with PAO, lost its property to bind to guanine nucleotides. Matrix-assisted laser desorption ionization-mass analysis of PAO-modified RhoA showed a mass shift according to an adduction of a single PAO molecule per molecule RhoA. Further analysis of Glu-C-generated RhoA peptides confirmed binding of PAO to a peptide harboring the guanine nucleotide binding region. Thus, PAO does not exclusively inhibit phosphotyrosine phosphatases but also inactivates RhoA by alteration of nucleotide binding.

Address correspondence to: Ralf Gerhard, Institut für Toxikologie, Medizinische Hochschule, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. E-mail: gerhard.ralf{at}mh-hannover.de

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