SYNERGISTIC ACTIVATION OF RAT-BRAIN PHOSPHOLIPASE-D BY ADP-RIBOSYLATION FACTOR AND RHOA P21, AND ITS INHIBITION BY CLOSTRIDIUM-BOTULINUM C3EXOENZYME

An activator of rat brain phospholipase D (PLD) that is distinct from the already identified PLD activator, ADP-ribosylation factor (ARF), w as partially purified from bovine brain cytosol by a series of chromat ographic steps. The partially purified preparation contained a 22-kDa substrate for Clostridium botulinum C3 exoenzyme ADP-ribosyltransferas e, which strongly reacted with anti-rhoA p21 antibody, but not with an ti-rac1 p21 or anti-cdc42Hs p21 antibody. Treatment of the partially p urified PLD-activating factor with both C3 exoenzyme and NAD significa ntly inhibited the PLD-stimulating activity. These results suggest tha t rhoA p21 is, at least in part, responsible for the PLD-stimulating a ctivity in the preparation. Recombinant isoprenylated rhoA p21 express ed in and purified from Sf9 cells activated rat brain PLD in a concent ration- and GTP gamma S (guanosine 5'-O-(3-thiotriphosphate))-dependen t manner. In contrast, recombinant non-isoprenylated rhoA pal (fused t o glutathione S-transferase) expressed in Escherichia coil failed to a ctivate the PLD. This difference cannot be explained by a lower affini ty of non-isoprenylated rhoA p21 for GTP gamma S, as the rates of [S-3 5]GTP gamma S binding were very similar for both recombinant preparati ons and the GTP gamma S-bound form of non-isoprenylated rhoA p21 did n ot induce PLD activation. Interestingly, recombinant isoprenylated rho A p21 and ARF synergistically activated rat brain PLD; a similar patte rn was seen with the partially purified PLD-activating factor. The syn ergistic activation was inhibited by C3 exoenzyme-catalyzed ADP-ribosy lation of recombinant isoprenylated rhoA p21 in a NAD-dependent manner . Inhibition correlated with the extent of ADP-ribosylation. These fin dings suggest that rhoA pal regulates rat brain PLD in concert with AR F, and that isoprenylation of rhoA p21 is essential for PLD regulation in vitro.