Interaction of the small G protein RhoA with the C terminus of human phospholipase D1

Mammalian phosphatidylcholine-specific phospholipase D1 (PLD1) is a signal transduction-activated enzyme thought to function in multiple cell biologic al settings including the regulation of membrane vesicular trafficking. PLD 1 is activated by the small G proteins, ADP-ribosylation factor (ARF) and R hoA, and by protein kinase C-alpha (PKC-alpha). This stimulation has been p roposed to involve direct interaction and to take place at a distinct site in PLD1 for each activator. In the present study, we employed the yeast two -hybrid system to attempt to identify these sites. Successful interaction o f ARF and PKC-alpha with PLD1 was not achieved, but a C-terminal fragment o f human PLD1 (denoted "D4") interacted with the active mutant of RhoA, RhoA (Val-14) Deletion of the CAAX box from RhoA(Val-14) decreased the strength of the interaction, suggesting that lipid modification of RhoA is important for efficient binding to PLD1. The specificity of the interaction was vali dated by showing that the PLD1 D4 fragment interacts with glutathione S-tra nsferase-RhoA in vitro in a GTP-dependent manner and that it associates wit h RhoA(Val-14) in COS-7 cells, whereas the N-terminal two-thirds of PLD1 do es not. Finally, we show that recombinant D4 peptide inhibits RhoA-stimulat ed PLD1 activation but not ARF- or PKC-alpha-stimulated PLD1 activation. Th ese results conclusively demonstrate that the C-terminal region of PLD1 con tains the RhoA-binding site and suggest that the ARF and PKC interactions o ccur elsewhere in the protein.