Neosynthesis and activation of Rho by Escherichia coli cytotoxic necrotizing factor (CNF1) reverse cytopathic effects of ADP-ribosylated Rho.
Clostridium botulinum exoenzyme C3 inactivates the small GTPase Rho by ADP-ribosylation. We used a C3 fusion toxin (C2IN-C3) with high cell accessibility to study the kinetics of Rho inactivation by ADP-ribosylation. In primary cultures of rat astroglial cells and Chinese hamster ovary cells, C2IN-C3 induced the complete ADP-ribosylation of RhoA and concomitantly the disassembly of stress fibers within 3 h. Removal of C2IN-C3 from the medium caused the recovery of stress fibers and normal cell morphology within 4 h. The regeneration was preceded by the appearance of non-ADP-ribosylated RhoA. Recovery of cell morphology was blocked by the proteasome inhibitor lactacystin and by the translation inhibitors cycloheximide and puromycin, indicating that intracellular degradation of the C3 fusion toxin and the neosynthesis of Rho were required for reversal of cell morphology. Escherichia coli cytotoxic necrotizing factor CNF1, which activates Rho by deamidation of Gln(63), caused reconstitution of stress fibers and cell morphology in C2IN-C3-treated cells within 30-60 min. The effect of CNF1 was independent of RhoA neosynthesis and occurred in the presence of completely ADP-ribosylated RhoA. The data show three novel findings; 1) the cytopathic effects of ADP-ribosylation of Rho are rapidly reversed by neosynthesis of Rho, 2) CNF1-induced deamidation activates ADP-ribosylated Rho, and 3) inhibition of Rho activation but not inhibition of Rho-effector interaction is a major mechanism underlying inhibition of cellular functions of Rho by ADP-ribosylation.
Barth
H
H
Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strasse 5, D-79104 Freiburg, Germany.
Olenik
C
C
Sehr
P
P
Schmidt
G
G
Aktories
K
K
Meyer
D K
DK
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D001905:Botulinum Toxins / Q000633:toxicity*; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D003014:Clostridium botulinum / Q000378:metabolism*; D006224:Cricetinae; D003513:Cycloheximide / Q000494:pharmacology; D003603:Cytotoxins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D005973:Glutamine; D007700:Kinetics; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011691:Puromycin / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000633:toxicity; D014162:Transfection
D001427:Bacterial Toxins; D003603:Cytotoxins; D029968:Escherichia coli Proteins; D020690:GTPase-Activating Proteins; D011993:Recombinant Fusion Proteins; C085379:rho GTPase-activating protein; D005973:Glutamine; C060296:cytotoxic necrotizing factor type 1; D000246:Adenosine Diphosphate Ribose; D011691:Puromycin; D003513:Cycloheximide; D011065:Poly(ADP-ribose) Polymerases; D001905:Botulinum Toxins; D020558:GTP Phosphohydrolases; C096324:botulinum toxin type C