Cellular stability of Rho‐GTPases glucosylated by Clostridium difficile toxin B

Abstract

Mono-glucosylation of Rho, Rac, and Cdc42 by Clostridium difficile toxin B (TcdB) induces changes of actin dynamics and apoptosis. When fibroblasts were treated with TcdB, an apparent decrease of the cellular Rac1 level was observed when applying anti-Rac1(Mab 102). This decrease was not based on degradation as inhibition of the proteasome by lactacystin did not stabilise cellular Rac1 levels. The application of anti-Rac1 (Mab 23A8) showed that the cellular Rac1 level slightly increased in TcdB-treated fibroblasts; thus, the apparent loss of cellular Rac1 was not due to degradation but due to impaired recognition of glucosylated Rac1 by anti-Rac1 (Mab 102). In contrast, recognition of RhoA by anti-RhoA (Mab 26C4) and Cdc42 by anti-Cdc42 (Mab 44) was not altered by glucosylation; a transient decrease of cellular RhoA and Cdc42 in TcdB-treated fibroblasts was indeed due to proteasomal degradation, as inhibition of the proteasome by lactacystin stabilised both cellular RhoA and Cdc42 levels. The finding that the apparent decrease of Rac1 reflects Rac1 glucosylation offers a valuable tool to determine Rac1 glucosylation.

Rac1-dependent recruitment of PAK2 to G2phase centrosomes and their roles in the regulation of mitotic entry

“…2B). 18 The apparent loss of Rac1 detection reflected Rac1 glucosylation (not Rac1 degradation), as the alternative (glucosylation-insensitive) Rac1-mAb(23A8) confirmed the presence of Rac1 at G 2 -phase centrosomes (Fig. 2B).…”

“…4A). 18 The loss of Rac1 detection upon TcdBF treatment was due to glucosylation and not due to Rac1 degradation, as the cellular level of Rac1 was unchanged if the alternative Rac1-mAb(23A8) was applied (Fig. 4A).…”

Activation of Rac1 and the exchange factor Vav3 are involved in NPM-ALK signaling in anaplastic large cell lymphomas

“…Different types of natural molecules and synthetic drugs that inactivate GTPases or their effectors are available, some of them show striking antineoplastic or antimetastatic activity (Aznar et al, 2004;Fritz and Kaina, 2006). We have tested Clostridium toxins (Petit et al, 2003;Genth et al, 2006). Both Toxin B from Clostridium difficile (affects Rac, RhoA and Cdc42) and LT-IP82 from Clostridium sordellii (specific for Rac, Ras, Ral and Rap) affected NPM-ALK-induced migration but also blocked proliferation, demonstrating that the role of GTPases downstream of NPM-ALK is broader than migration and invasion (Colomba, A and Gaits-Iacovoni, F., unpublished).…”

Application of Mutated Clostridium difficile Toxin A for Determination of Glucosyltransferase-Dependent Effects

“…1C). To check the extent of intracellular glucosylation of GTPases, unmodified GTPases were detected by either sequential 14 C-glucosylation (RhoA/B and Rac1) (12), [ 32 P]ADP ribosylation (RhoA/B) (1), or Western blot analysis (with antiRac1, which exclusively recognizes unmodified Rac1) (8). There was a concentration-dependent decrease in sequential 14 C-glucosylation ( Fig.…”