GLUCOSYLATION AND ADP-RIBOSYLATION OF RHO-PROTEINS - EFFECTS ON NUCLEOTIDE-BINDING, GTPASE ACTIVITY, AND EFFECTOR COUPLING

We studied the effects of glucosylation of RhoA, Rac1, and Cdc42 at th reonine-35 and -37 by Clostridium difficile toxin B on nucleotide bind ing, GTPase activity, and effector coupling and compared these results with the ADP ribosylation of RhoA at asparagine-41 catalyzed by Clost ridium botulinum C3 transferase. Whereas glucosylation and ADP ribosyl ation had no major effects on GDP release from RhoA, Rac1, and Cdc42. the rate of GTP gamma S release from Rho proteins was increased 3-6-fo ld by glucosylation. ADP ribosylation decreased the rate of GTP gamma S release by about 50%. Glucosylation reduced the intrinsic activities of the GTPases by 3-7-fold and completely blocked GTPase stimulation by RhoGAP. In contrast, ADP ribosylation slightly increased GTPase act ivity (similar to 2-fold) and had no major effect on GAP stimulation o f GTPase. Whereas ADP ribosylation did not affect the interaction of R hoA with the binding domain of protein kinase N, glucosylation inhibit ed this interaction. Glucosylation of Rac1 markedly diminished its abi lity to support the activation of the superoxide-generating NADPH oxid ase of phagocytes. Glucosylated Rac1 did not interfere with NADPH oxid ase activation by unmodified Rac1, even when present in marked molar e xcess, indicating that it was incapable of competing for a common effe ctor. The data indicate that the functional inactivation of small GTPa ses by glucosylation is mainly caused by inhibition of GTPase-effector protein interaction.