Thiol-modifying phenylarsine oxide inhibits guanine nucleotide binding of Rho but not of Rac GTPases.
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.
Gerhard
Ralf
R
Institut für Toxikologie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. gerhard.ralf@mh-hannover.de
John
Harald
H
Aktories
Klaus
K
Just
Ingo
I
D000199:Actins; D000209:Acute-Phase Proteins; D001152:Arsenicals / Q000494:pharmacology*; D018938:Caco-2 Cells; D003545:Cysteine / Q000378:metabolism; D003599:Cytoskeleton; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006150:Guanine Nucleotides / Q000378:metabolism*; D006801:Humans; D011487:Protein Conformation / Q000187:drug effects; D020744:rac GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000187:drug effects / Q000378:metabolism*; D020743:rhoB GTP-Binding Protein / Q000378:metabolism; D000075024:rhoC GTP-Binding Protein
D000199:Actins; D000209:Acute-Phase Proteins; D001152:Arsenicals; D004791:Enzyme Inhibitors; D006150:Guanine Nucleotides; C010781:acute-phase protein rho; C029341:oxophenylarsine; C066690:RHOC protein, human; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D020743:rhoB GTP-Binding Protein; D000075024:rhoC GTP-Binding Protein; D003545:Cysteine