Direct activation of RhoA by reactive oxygen species requires a redox-sensitive motif.
BACKGROUND
Rho family GTPases are critical regulators of the cytoskeleton and affect cell migration, cell-cell adhesion, and cell-matrix adhesion. As with all GTPases, their activity is determined by their guanine nucleotide-bound state. Understanding how Rho proteins are activated and inactivated has largely focused on regulatory proteins such as guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). However, recent in vitro studies have indicated that GTPases may also be directly regulated by redox agents. We hypothesized that this redox-based mechanism occurs in cells and affects cytoskeletal dynamics, and in this report we conclude this is indeed a novel mechanism of regulating the GTPase RhoA.


RESULTS
In this report, we show that RhoA can be directly activated by reactive oxygen species (ROS) in cells, and that this requires two critical cysteine residues located in a unique redox-sensitive motif within the phosphoryl binding loop. First, we show that ROS can reversibly activate RhoA and induce stress fiber formation, a well characterized readout of RhoA activity. To determine the role of cysteine residues in this mechanism of regulation, we generated cysteine to alanine RhoA mutants. Mutation of these cysteines abolishes ROS-mediated activation and stress fiber formation, indicating that these residues are critical for redox-regulation of RhoA. Importantly, these mutants maintain the ability to be activated by GEFs.


CONCLUSIONS
Our findings identify a novel mechanism for the regulation of RhoA in cells by ROS, which is independent of classical regulatory proteins. This mechanism of regulation may be particularly relevant in pathological conditions where ROS are generated and the cellular redox-balance altered, such as in asthma and ischemia-reperfusion injury.
Aghajanian
Amir
A
Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina, United States of America. amir@med.unc.edu
Wittchen
Erika S
ES
Campbell
Sharon L
SL
Burridge
Keith
K
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002448:Cell Adhesion; D003599:Cytoskeleton / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D051381:Rats; D017382:Reactive Oxygen Species; D015427:Reperfusion Injury; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D017382:Reactive Oxygen Species; D020742:rhoA GTP-Binding Protein