Synergistic activation of rat brain phospholipase D by ADP-ribosylation factor and rhoA p21, and its inhibition by Clostridium botulinum C3 exoenzyme.
An activator of rat brain phospholipase D (PLD) that is distinct from the already identified PLD activator, ADP-ribosylation factor (ARF), was partially purified from bovine brain cytosol by a series of chromatographic steps. The partially purified preparation contained a 22-kDa substrate for Clostridium botulinum C3 exoenzyme ADP-ribosyltransferase, which strongly reacted with anti-rhoA p21 antibody, but not with anti-rac1 p21 or anti-cdc42Hs p21 antibody. Treatment of the partially purified PLD-activating factor with both C3 exoenzyme and NAD significantly inhibited the PLD-stimulating activity. These results suggest that rhoA p21 is, at least in part, responsible for the PLD-stimulating activity in the preparation. Recombinant isoprenylated rhoA p21 expressed in and purified from Sf9 cells activated rat brain PLD in a concentration- and GTP gamma S (guanosine 5'-O-(3-thiotriphosphate))-dependent manner. In contrast, recombinant non-isoprenylated rhoA p21 (fused to glutathione S-transferase) expressed in Escherichia coli failed to activate the PLD. This difference cannot be explained by a lower affinity of non-isoprenylated rhoA p21 for GTP gamma S, as the rates of [35S]GTP gamma S binding were very similar for both recombinant preparations and the GTP gamma S-bound form of non-isoprenylated rhoA p21 did not induce PLD activation. Interestingly, recombinant isoprenylated rhoA p21 and ARF synergistically activated rat brain PLD; a similar pattern was seen with the partially purified PLD-activating factor. The synergistic activation was inhibited by C3 exoenzyme-catalyzed ADP-ribosylation of recombinant isoprenylated rhoA p21 in a NAD-dependent manner. Inhibition correlated with the extent of ADP-ribosylation. These findings suggest that rhoA p21 regulates rat brain PLD in concert with ARF, and that isoprenylation of rhoA p21 is essential for PLD regulation in vitro.
Kuribara
H
H
Department of Life Science, Tokyo Institute of Technology, Yokohama, Japan.
Tago
K
K
Yokozeki
T
T
Sasaki
T
T
Takai
Y
Y
Morii
N
N
Narumiya
S
S
Katada
T
T
Kanaho
Y
Y
D036002:ADP Ribose Transferases / Q000378:metabolism*; D020727:ADP-Ribosylation Factors; D000818:Animals; D001905:Botulinum Toxins; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D004357:Drug Synergism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D009243:NAD / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D017368:Protein Prenylation; D051381:Rats; D011994:Recombinant Proteins; D020742:rhoA GTP-Binding Protein
D011994:Recombinant Proteins; D009243:NAD; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D010739:Phospholipase D; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D020742:rhoA GTP-Binding Protein