Ubiquitin-mediated proteasomal degradation of Rho proteins by the CNF1 toxin

Methods Enzymol. 2006:406:447-56. doi: 10.1016/S0076-6879(06)06033-2.

Abstract

The CNF1 toxin is produced by uropathogenic and meningitis-causing Escherichia coli. CNF1 catalyzes the constitutive activation of Rho proteins by deamidation. The threshold of activation of Rho proteins by CNF1 is, however, attenuated because of a concomitant decrease of their cellular levels. Depletion of activated-Rac1 is catalyzed by ubiquitin-mediated proteasomal degradation. Consequently, we show by effector-binding pull-down that co-treatment of intoxicated cells with the MG132 proteasome-inhibitor results in a higher level of activation of Rac, as well as RhoA and Cdc42. We show that CNF1 induces the transient recruitment of Rho proteins to cellular membranes. Interestingly, at the difference of Rac and Cdc42, the inhibition of the proteasome during CNF1 treatment does not result in a significant accumulation of RhoA to cellular membranes. Using an in vivo ubiquitylation assay, we evidence that mutation of the geranylgeranyl acceptor cysteine of Rac1 (Rac1C189G) abolished the sensitivity of permanently activated-Rac1 to ubiquitylation, whereas Rac1C189G remained able to bind to the effector-binding domain of p21-PAK. Collectively, these results indicate that association with the cellular membranes is a necessary step for activated-Rac1 ubiquitylation.

MeSH terms

  • Bacterial Toxins / isolation & purification
  • Bacterial Toxins / metabolism*
  • Cell Membrane / metabolism
  • Cells, Cultured
  • Endothelium, Vascular
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / isolation & purification
  • Escherichia coli Proteins / metabolism*
  • Humans
  • Proteasome Endopeptidase Complex / metabolism*
  • Ubiquitin-Protein Ligase Complexes / metabolism
  • cdc42 GTP-Binding Protein / metabolism
  • rho GTP-Binding Proteins / metabolism*

Substances

  • Bacterial Toxins
  • Escherichia coli Proteins
  • cytotoxic necrotizing factor type 1
  • Ubiquitin-Protein Ligase Complexes
  • Proteasome Endopeptidase Complex
  • cdc42 GTP-Binding Protein
  • rho GTP-Binding Proteins