Abstract
RhoA, -B, and -C are ADP-ribosylated by Clostridium botulinum exoenzyme C3 to induce redistribution of the actin filaments in intact cells, a finding that has led to the notion that the ADP-ribosylation blocks coupling of Rho to the downstream effectors. ADP-ribosylation, however, does not alter nucleotide binding, intrinsic, and GTPase-activating protein-stimulated GTPase activity. ADP-ribosylated Rho is even capable of activating the effector protein ROK in a recombinant system. Treatment of cells with a cell-permeable chimeric C3 toxin led to complete localization of modified Rho to the cytosolic fraction based on the complexation of ADP-ribosylated Rho with the guanine-nucleotide dissociation inhibitor-1 (GDI-1). The modified complex turned out to be resistant to phosphatidylinositol 4,5-bisphosphate- and GTPgammaS-induced release of Rho from GDI-1. Thus, ADP-ribosylation leads to entrapment of Rho in the GDI-1 complex. The increased stability of the GDI complex prevented binding of Rho to membrane-associated players of the GTPase cycle such as the activating guanine nucleotide exchange factors and effector proteins.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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3T3 Cells
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ADP Ribose Transferases / metabolism*
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Animals
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Botulinum Toxins / metabolism*
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Drug Stability
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Electrophoresis, Polyacrylamide Gel
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GTP Phosphohydrolases / metabolism
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Glutathione Transferase / genetics
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Guanine Nucleotide Dissociation Inhibitors / genetics
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Guanine Nucleotide Dissociation Inhibitors / metabolism*
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Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
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Mice
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Phosphatidylinositol 4,5-Diphosphate / metabolism
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Recombinant Fusion Proteins
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Recombinant Proteins
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ras Proteins
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rho GTP-Binding Proteins / genetics
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rho GTP-Binding Proteins / metabolism*
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rhoA GTP-Binding Protein / genetics
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rhoA GTP-Binding Protein / metabolism*
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rhoB GTP-Binding Protein / genetics
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rhoB GTP-Binding Protein / metabolism*
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rhoC GTP-Binding Protein
Substances
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GDP dissociation inhibitor 1
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Guanine Nucleotide Dissociation Inhibitors
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Phosphatidylinositol 4,5-Diphosphate
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Recombinant Fusion Proteins
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Recombinant Proteins
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Guanosine 5'-O-(3-Thiotriphosphate)
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ADP Ribose Transferases
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exoenzyme C3, Clostridium botulinum
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Glutathione Transferase
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Botulinum Toxins
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GTP Phosphohydrolases
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Rhoc protein, mouse
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ras Proteins
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rho GTP-Binding Proteins
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rhoA GTP-Binding Protein
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rhoB GTP-Binding Protein
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rhoC GTP-Binding Protein