REGULATION OF PHOSPHOLIPASE-D BY LOW-MOLECULAR-WEIGHT GTP-BINDING PROTEINS

Phospholipase D (PLD) is believed to play an important role in cell si gnal transduction: PLD catalyzes the hydrolysis primarily of phosphati dylcholine (PC) to produce phosphatidic acid that may serve as a lipid second messenger. Although the mechanism of PLD activation has not ye t been fully understood, a member of the low molecular weight GTP-bind ing protein (small G protein) superfamily, ADP-ribosylation factor (AR F), has been identified as a PLD-activating factor. In addition to ARF , we found that RhoA, another member of the small G proteins, activate d rat brain PLD, and that ARF and RhoA synergistically stimulated the enzyme activity. When proteins of bovine brain cytosol were subjected to anion exchange column chromatography and then reconstituted with ra t brain PLD partially purified from the membranes, fractions eluted at 60 mM NaCl, where ARF was not detected, activated the enzyme in a gua nosine 5'-O-(3-thiotriphosphate)-dependent manner. This PLD-stimulatin g activity seemed to be attributed to a small G protein RhoA. Evidence provided includes the findings that: (1) the partially purified prepa ration of the PLD-activating factor by subsequent column chromatograph ies contained a 22 kDa substrate for botulinum C3 exoenzyme ADP-ribosy ltransferase; (2) the 22 kDa protein strongly reacted with anti-RhoA a ntibody; (3) the treatment of the partially purified PLD-activating fa ctor with C3 exoenzyme and NAD together, but not individually, signifi cantly inhibited the PLD-stimulating activity; and (4) recombinant iso prenylated RhoA activated the PLD. On the contrary, recombinant noniso prenylated RhoA failed to activate the PLD. Interestingly, the partial ly purified PLD-activating factor and ARF synergistically activated ra t brain PLD, and recombinant isoprenylated RhoA could substitute for t he partially purified preparation. These results conclude that rat bra in PLD is regulated by RhoA in concert with ARF, and that the post-tra nslational modification of RhoA is essential for its function as the P LD activator.