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 biological settings including the regulation of membrane vesicular trafficking. PLD1 is activated by the small G proteins, ADP-ribosylation factor (ARF) and RhoA, and by protein kinase C-alpha (PKC-alpha). This stimulation has been proposed 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 of ARF and PKC-alpha with PLD1 was not achieved, but a C-terminal fragment of human PLD1 (denoted "D4") interacted with the active mutant of RhoA, RhoAVal-14. Deletion of the CAAX box from RhoAVal-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 validated by showing that the PLD1 D4 fragment interacts with glutathione S-transferase-RhoA in vitro in a GTP-dependent manner and that it associates with RhoAVal-14 in COS-7 cells, whereas the N-terminal two-thirds of PLD1 does not. Finally, we show that recombinant D4 peptide inhibits RhoA-stimulated PLD1 activation but not ARF- or PKC-alpha-stimulated PLD1 activation. These results conclusively demonstrate that the C-terminal region of PLD1 contains the RhoA-binding site and suggest that the ARF and PKC interactions occur elsewhere in the protein.
Yamazaki
M
M
Department of Life Science, Tokyo Institute of Technology, Yokohama 226-8501, Japan.
Zhang
Y
Y
Watanabe
H
H
Yokozeki
T
T
Ohno
S
S
Kaibuchi
K
K
Shibata
H
H
Mukai
H
H
Ono
Y
Y
Frohman
M A
MA
Kanaho
Y
Y
D001665:Binding Sites; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D010739:Phospholipase D / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D020742:rhoA GTP-Binding Protein
D010739:Phospholipase D; C106801:phospholipase D1; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein