Mechanism of Redox-mediated Guanine Nucleotide Exchange on Redox-active Rho GTPases

Abstract

Rho GTPases regulate multiple cellular processes including actin cytoskeletal rearrangements, transcriptional regulation, and oxidant production. The studies described herein demonstrate that small molecule redox agents, in addition to protein regulatory factors, can regulate the activity of redox-active Rho GTPases. A novel (GXXXXGK(S/T)C) motif, conserved in a number of Rho GTPases, appears critical for redox-mediated guanine nucleotide dissociation in vitro. A detailed molecular mechanism for redox regulation of GXXXXGK(S/ T)C motif-containing Rho GTPases is proposed.Rho GTPases comprise a large branch of the Ras superfamily of small guanine nucleotide-binding proteins and include the well characterized family members Rac1, RhoA, and Cdc42 (1). They are involved in regulation of a plethora of cellular processes, including cell morphology, movement, and proliferation (2).The guanine nucleotide bound state of most Rho GTPase family members is regulated by three distinct types of protein modulatory agents, which alter GTPase activity by regulating cycling of the GTPase between inactive GDP and active GTPbound forms (2-4). In particular, guanine nucleotide exchange factors (GEFs) 1 facilitate exchange of GDP with GTP to promote GTPase activation, whereas GTPase-activating proteins deactivate the GTPase protein by stimulating hydrolysis of bound GTP to GDP. Deactivation can also be achieved by association with guanine nucleotide dissociation inhibitors, which prevent membrane association and GDP dissociation. Similar to Ras GTPases, exchange of GDP for GTP leads to a conformational change in the GTPase that greatly enhances affinity to downstream effectors. The interaction between the GTPase and effector leads to activation of GTPase effectormediated signal transduction pathways.A fourth distinct type of modulatory agent has been shown to regulate Ras GTPase activity (5-9). Similar to the action of GEFs, various redox agents, including both reactive oxygen species (ROS) and reactive nitrogen species (RNS), have been shown to stimulate Ras guanine nucleotide dissociation in vitro and up-regulate Ras function in vivo. We have recently elucidated the molecular mechanism by which certain redox agents modulate Ras activity (10 -12). Intriguingly, in addition to Ras, Rho GTPase signaling is sensitive to the presence of ROS and RNS and to the redox state of the cell (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) EXPERIMENTAL PROCEDURESPreparation of Chemicals and Proteins-The chemicals used for all experiments were of the highest grade unless otherwise noted. Radiolabeled guanine nucleotides ([ 3 H]GDP and [ 3 H]GTP) were diluted with unlabeled guanine nucleotides prior to use, giving ϳ1000 dpm/ M nucleotide. Rac1-(1-177), Rac1 F28L, Rac1 C18S, RhoA-(1-181), and Cdc42-(1-188) proteins were expressed and purified as described previously (34). The final proteins were Ͼ95% pure as determined by SDS-PAGE. The protein concentration was determined by the Bradford method (35). XO was purchased from Sigma.Experimental Conditions ...

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