ExoS Rho GTPase-activating protein activity stimulates reorganization of the actin cytoskeleton through Rho GTPase guanine nucleotide disassociation inhibitor.
ExoS is a bifunctional Type III cytotoxin of Pseudomonas aeruginosa with N-terminal Rho GTPase-activating protein (RhoGAP) and C-terminal ADP-ribosyltransferase domains. Although the ExoS RhoGAP inactivates Cdc42, Rac, and RhoA in vivo, the relationship between ExoS RhoGAP and the eukaryotic regulators of Rho GTPases is not clear. The present study investigated the roles of Rho GTPase guanine nucleotide disassociation inhibitor (RhoGDI) in the reorganization of actin cytoskeleton mediated by ExoS RhoGAP. A green fluorescent protein-RhoGDI fusion protein was engineered and found to elicit actin reorganization through the inactivation of Rho GTPases. Green fluorescent protein-RhoGDI and ExoS RhoGAP cooperatively stimulated actin reorganization and translocation of Cdc42 from membrane to cytosol, and a RhoGDI mutant, RhoGDI(I177D), that is defective in extracting Rho GTPases off the membrane inhibited the actions of RhoGDI and ExoS RhoGAP on the translocation of Cdc42 from membrane to cytosol. A human RhoGDI small interfering RNA was transfected into HeLa cells to knock down 90% of the endogenous RhoGDI expression. HeLa cells with knockdown RhoGDI were resistant to the reorganization of the actin cytoskeleton elicited by type III-delivered ExoS RhoGAP. This indicates that ExoS RhoGAP and RhoGDI function in series to inactivate Rho GTPases, in which RhoGDI extracting GDP-bound Rho GTPases off the membrane and sequestering them in cytosol is the rate-limiting step in Rho GTPase inactivation. A eukaryotic GTPase-activating protein, p50RhoGAP, showed a similar cooperativity with RhoGDI on actin reorganization, suggesting that ExoS RhoGAP functions as a molecular mimic of eukaryotic RhoGAPs to inactivate Rho GTPases through RhoGDI.
Medical College of Wisconsin, Microbiology and Molecular Genetics, Milwaukee, Wisconsin 53226, USA.
Jianjun
J
Sun
Joseph T
JT
Barbieri
D036002:ADP Ribose Transferases / Q000378:metabolism / Q000502:physiology*; D000199:Actins / Q000737:chemistry; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003599:Cytoskeleton / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000378:metabolism; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011550:Pseudomonas aeruginosa / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013347:Subcellular Fractions; D013997:Time Factors; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000737:chemistry*; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
C569638:ARHGDIA protein, human; D000199:Actins; D001427:Bacterial Toxins; D020690:GTPase-Activating Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C085379:rho GTPase-activating protein; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D004247:DNA; D036002:ADP Ribose Transferases; C444820:exoenzyme S; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins