RHO-PROTEIN-INHIBITION BLOCKS PROTEIN-KINASE-C TRANSLOCATION AND ACTIVATION

Small GTP-binding proteins of the Ras and Rho family participate in va rious important signalling pathways. Large clostridial cytotoxins inac tivate GTPases by UDP-glucosylation. Using Clostridium difficile toxin B-10463 (TcdB) for inactivation of Rho proteins (RhoA/Racl Cdc42) and Clostridium sordellii lethal toxin-1522 (TcsL) for inactivation of Ra s-proteins (Ras/Rac/Ral, Rap) the role of these GTPases in protein kin ase C (PKC) stimulation was studied. Phorbol-myristate-acetate (PMA) i nduced a rapid PKC translocation to and activation in the particulate cell fraction as determined by PKC-activity measurements and Western b lots for PKC alpha. These effects were blocked by TcdB inhibiting Rho proteins in endothelial cells, but not in TcsL-treated cells (i.e., ce lls without Ras activity), suggesting that Rho GTPases (RhoA and/or Cd c42) are the most likely GTP-binding proteins responsible for PKC acti vation. The Rho requirement for PKC activation/translocation was also verified for human epithelial cells and for lipopolysaccharide-stimula ted endothelial cells. In summary, the data presented indicate that Rh o protein inhibition blocked PKC translocation/activation in endotheli al and epithelial cells. (C) 1998 Academic Press.