ACTIVATION OF RAT-LIVER PHOSPHOLIPASE-D BY THE SMALL GTP-BINDING PROTEIN RHOA

Stimulation of phospholipase D by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in rat liver plasma membranes indicates the involvement of GTP-binding proteins. We used RhoGDI, an inhibitor of GDP dissociat ion from small GTP-binding proteins of the Rho family, to determine th e involvement of these proteins. Incuba tion, and subsequent washing, of plasma membranes with RhoGDI dose-dependently diminished GTP gamma S-stimulated phospholipase D activity, as determined by accumulation o f phosphatidylethanol in the presence of ethanol. Incubation with RhoG DI also caused a rapid and dose dependent appearance of RhoA in the wa sh, which was associated with the inhibition of phospholipase D. RhoGD I also rapidly extracted Cdc42 from membranes, but Rad was not extract ed. Full reconstitution of GTP gamma S-stimulated phospholipase D in R hoGDI-washed membranes was achieved with recombinant RhoA. There was p artial reconstitution with Rac1 and no enhancement with Cdc42 or ADP-r ibosylation factor. The response to RhoA was dose-dependent (EC(50) = 0.5 mu M). ADP-ribosylation of RhoA by Clostridium botulinum C3 exoenz yme did not affect its ability to recover GTP gamma S-stimulated phosp holipase D activity in RhoGDI-washed membranes. These findings support a role for GTP-binding proteins of the Rho family in the activation o f membrane-associated phospholipase D and implicate RhoA as the major protein involved.