Glucosylation and ADP Ribosylation of Rho Proteins:  Effects on Nucleotide Binding, GTPase Activity, and Effector Coupling

Abstract

We studied the effects of glucosylation of RhoA, Rac1, and Cdc42 at threonine-35 and -37 by Clostridium difficile toxin B on nucleotide binding, GTPase activity, and effector coupling and compared these results with the ADP ribosylation of RhoA at asparagine-41 catalyzed by Clostridium botulinum C3 transferase. Whereas glucosylation and ADP ribosylation had no major effects on GDP release from RhoA, Rac1, and Cdc42, the rate of GTPgammaS release from Rho proteins was increased 3-6-fold by glucosylation. ADP ribosylation decreased the rate of GTPgammaS release by about 50%. Glucosylation reduced the intrinsic activities of the GTPases by 3-7-fold and completely blocked GTPase stimulation by Rho-GAP. In contrast, ADP ribosylation slightly increased GTPase activity ( approximately 2-fold) and had no major effect on GAP stimulation of GTPase. Whereas ADP ribosylation did not affect the interaction of RhoA with the binding domain of protein kinase N, glucosylation inhibited this interaction. Glucosylation of Rac1 markedly diminished its ability to support the activation of the superoxide-generating NADPH oxidase of phagocytes. Glucosylated Rac1 did not interfere with NADPH oxidase activation by unmodified Rac1, even when present in marked molar excess, indicating that it was incapable of competing for a common effector. The data indicate that the functional inactivation of small GTPases by glucosylation is mainly caused by inhibition of GTPase-effector protein interaction.

Entrapment of Rho ADP-ribosylated by Clostridium botulinum C3 Exoenzyme in the Rho-Guanine Nucleotide Dissociation Inhibitor-1 Complex

“…However, it is still unclear how ADP-ribosylation causes inactivation of the Rho GTPases. Studies on the functional consequences of ADP-ribosylation on the GTPase cycle of Rho do not reveal remarkable changes in nucleotide binding (35,36), intrinsic GTPase cycle (36,37), and GTPase-activating proteinstimulated GTPases activity (36,37). Furthermore, ADP-ribosylated Rho is still capable of binding to effector proteins: nucleotide independently to the lipid kinases phospholipase D1 (38) and phosphatidylinositol 4-phosphate 5-kinase (39) but nucleotide dependently to rhotekin (38), Rho kinase (38), and protein kinase N (36).…”

C3 exoenzymes, novel insights into structure and action of Rho-ADP-ribosylating toxins

“…The mechanism underlying the inactivation of Rho by C3-mediated ADPribosylation was analyzed in great detail. First, it was shown that the ADP-ribosylation does not influence nucleotide-binding and hydrolysis (Sehr et al 1998). This finding is in line with the crystal structure of RhoA-GTP, where RhoA N41 is surface located and does not interact with the nucleotide.…”

“…It was thus reasonable to assume that ADP-ribosylation inactivates RhoA by inhibition of downstream signaling (i.e., blocking Rho-effector interaction) or by affecting the Rho-regulator interaction. From the follow-up studies it turned out that ADPribosylation inactivates RhoA by at least two steps: first by inhibition of the GEF-mediated nucleotide exchange of RhoA (e.g., Lbc; Sehr et al 1998) and second by the trapping of ADP-ribosylated RhoA in the inactive RhoARhoGDI complex in the cytosol ( Fig. 3; Genth et al 2003a;Fujihara et al 1997).…”

“…CNF1 deamidates RhoA at RhoA Q63 , which is located in the switch II region, and thereby constitutively activates RhoA (Barth et al 1999). In contrast, no evidence was found for the hypothesis that ADP-ribosylation prevents binding and activation of downstream effectors (Sehr et al 1998;Genth et al 2003b) or influences the GAP-mediated Rho inactivation (Sehr et al 1998). …”

Recognition of RhoA by Clostridium botulinum C3 Exoenzyme

“…The finding, that C3-catalyzed ADP-ribosylation of RhoA in intact cells causes depolymerization of the actin filaments, led to the notion that ADP-ribosylation at Asn 41 inactivates Rho. ADPribosylation has no significant influence on nucleotide exchange, intrinsic and GTPase-activating protein-stimulated GTPase activity but clearly decreases the exchange activity of Lbc (17)(18)(19). Furthermore, the guanine nucleotide dissociation inhibitor-driven cycling between cytosol and membranes is blocked leading to sequestration of ADP-ribosylated Rho in the inactive guanine nucleotide dissociation inhibitor complex.…”