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

Abstract

Phenylarsine oxide (PAO) is a phosphotyrosine phosphatase inhibitor that cross-links vicinal thiol groups, thereby inactivating phosphatases possessing XCysXXCysX motifs. The RhoAGTPase, but not the Rac1-GTPase, also possesses vicinal cysteines within the guanine nucleotide-binding region (aa [13][14][15][16][17][18][19][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.

Nitric Oxide Block of Outward-Rectifying K+ Channels Indicates Direct Control by Protein Nitrosylation in Guard Cells

“…7 and 8). PAO preferentially reacts with closely spaced (vicinal) thiol groups in proteins to form stable, sulfur-metal ring complexes, and its targets include protein Tyr phosphatases (Carballo et al, 1999), vesicle trafficking, and associated proteins (Arien et al, 2003;Gerhard et al, 2003), as well as inducible NO synthase (Oda et al, 2000). A feature of PAO complexes is their lability to reduction by BAL.…”

“…In mammalian tissues, PAO targets protein Tyr phosphatases (Carballo et al, 1999) through its ability to oxidatively bridge critical Cys, and it also affects other protein-signaling intermediates, including human syntaxin 1A (Arien et al, 2003) and Rho GTPases (Gerhard et al, 2003). In guard cells, PAO has been reported to counter the effects of ABA, dark, and H 2 O 2 in closing stomata and to promote stomatal opening, at least in part through its effect on tonoplast ion transport (MacRobbie, 2002), and to affect ABA-mediated gene expression (Heimovaaradijkstra et al, 1996).…”

Oxidative challenges sensitize the capsaicin receptor by covalent cysteine modification

“…PAO forms cyclic dithiaarsenane adducts with thiols in X-C-X-X-C-X motifs to irreversibly inactivate many cellular signaling molecules (30)(31)(32)(33)(34). We thus exploited its membrane permeability and reactivity on vicinal cysteines (while sparing free monothiols) to dissect TRPV1 modulation.…”

Rho GTPases, oxidation, and cell redox control

“…PAO can crosslink vicinal thiol groups and is a known phosphatase inhibitor. 135 Treatment of human colonic Caco-2 cells with PAO exhibited a dose-dependent reduction in actin stress fibers, a phenotype consistent with reduced RhoA activity. Moreover, pull-down experiments revealed a PAO concentration-dependent decrease in the levels of GTPbound RhoA.…”

“…The authors performed nucleotide dissociation and association assays in vitro and observed a loss of nucleotide binding in RhoA but not Rac1 upon exposure to PAO. 135 Mass spectrometry revealed that PAO crosslinked 2 vicinal cysteines in RhoA to occlude nucleotide binding. These effects were specific to RhoA but not Rac1 as only Rho GTPases (RhoA, RhoB, RhoC, and RhoE) possess vicinal cysteines (Cys 16 and Cys 20 ) within the guanine nucleotide-binding region.…”