Functional association between Arf and RalA in active phospholipase D complex.
Activation of phospholipase D1 (PLD1) by Arf has been implicated in vesicle transport and membrane trafficking. PLD1 has also been shown to be associated with the small GTPase RalA, which functions downstream from Ras in a Ras-RalA GTPase cascade that facilitates intracellular signal transduction. Although PLD1 associates directly with RalA, RalA has no effect upon the activity of PLD1. However, PLD1 precipitated from cell lysates with immobilized glutathione S-transferase-RalA fusion protein is active. This suggests the presence of an additional activating factor in the active RalA-PLD1 complexes. Because Arf stimulates PLD1, we looked for the presence of Arf in the active RalA-PLD1 complexes isolated from v-Src- and v-Ras-transformed cell lysates. Low levels of Arf protein were detected in RalA-PLD1 complexes; however, if guanosine 5'-[gamma-thio]triphosphate was added to activate Arf and stimulate translocation to the membrane, high levels of Arf were precipitated by RalA from cell lysates. Interestingly, deletion of 11 amino-terminal amino acids unique to Ral GTPases, which abolished the ability of RalA to precipitate PLD activity, prevented the association between RalA and Arf. Brefeldin A, which inhibits Arf GDP-GTP exchange, inhibited PLD activity in v-Src- and v-Ras-transformed cells but not in the nontransformed cells, suggesting that the association of Arf with RalA is required for the increased PLD activity induced by v-Src and v-Ras. These data implicate Arf in the transduction of intracellular signals activated by v-Src and mediated by the Ras-RalA GTPase cascade. Because both Arf and PLD1 stimulate vesicle formation in the Golgi, these data raise the possibility that vesicle formation and trafficking may play a role in the transduction of intracellular signals.
Luo
J Q
JQ
Department of Biological Sciences, Hunter College of The City University of New York, New York, NY 10021, USA.
Liu
X
X
Frankel
P
P
Rotunda
T
T
Ramos
M
M
Flom
J
J
Jiang
H
H
Feig
L A
LA
Morris
A J
AJ
Kahn
R A
RA
Foster
D A
DA
D016475:3T3 Cells; D020727:ADP-Ribosylation Factors; D000818:Animals; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D010739:Phospholipase D / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D020725:ral GTP-Binding Proteins
D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D020725:ral GTP-Binding Proteins