CNF1-induced ubiquitylation and proteasome destruction of activated RhoA is impaired in Smurf1-/- cells.
Ubiquitylation of RhoA has emerged as an important aspect of both the virulence of Escherichia coli producing cytotoxic necrotizing factor (CNF) 1 toxin and the establishment of the polarity of eukaryotic cells. Owing to the molecular activity of CNF1, we have investigated the relationship between permanent activation of RhoA catalyzed by CNF1 and subsequent ubiquitylation of RhoA by Smurf1. Using Smurf1-deficient cells and by RNA interference (RNAi)-mediated Smurf1 knockdown, we demonstrate that Smurf1 is a rate-limiting and specific factor of the ubiquitin-mediated proteasomal degradation of activated RhoA. We further show that the cancer cell lines HEp-2, human embryonic kidney 293 and Vero are specifically deficient in ubiquitylation of either activated Rac, Cdc42, or Rho, respectively. In contrast, CNF1 produced the cellular depletion of all three isoforms of Rho proteins in the primary human cell types we have tested. We demonstrate that ectopic expression of Smurf1 in Vero cells, deficient for RhoA ubiquitylation, restores ubiquitylation of the activated forms of RhoA. We conclude here that Smurf1 ubiquitylates activated RhoA and that, in contrast to human primary cell types, some cancer cell lines have a lower ubiquitylation capacity of specific Rho proteins. Thus, both CNF1 and transforming growth factor-beta trigger activated RhoA ubiquitylation through Smurf1 ubiquitin-ligase.
Faculté de Médecine, Institut National de la Santé et de la Recherche Médicale, U627, 06107 Nice Cedex 2, France.
Laurent
L
Boyer
Laurent
L
Turchi
Benoit
B
Desnues
Anne
A
Doye
Gilles
G
Ponzio
Jean-Louis
JL
Mege
Motozo
M
Yamashita
Ying E
YE
Zhang
Jacques
J
Bertoglio
Gilles
G
Flatau
Patrice
P
Boquet
Emmanuel
E
Lemichez
D001427:Bacterial Toxins / Q000494:pharmacology*; D002460:Cell Line; D029968:Escherichia coli Proteins / Q000494:pharmacology*; D006801:Humans; D007668:Kidney; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025801:Ubiquitin / Q000187:drug effects / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000172:deficiency* / Q000235:genetics; D020742:rhoA GTP-Binding Protein / Q000187:drug effects / Q000378:metabolism*
D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; D025801:Ubiquitin; C060296:cytotoxic necrotizing factor type 1; C498342:SMURF1 protein, human; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; D020742:rhoA GTP-Binding Protein