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J Biol Chem, Vol. 274, Issue 10, 6035-6038, March 5, 1999

COMMUNICATION
Interaction of the Small G Protein RhoA with the C Terminus of Human Phospholipase D1

Masakazu Yamazaki, Yue Zhang^§, Hiroshi Watanabe, Takeaki Yokozeki, Sigeo Ohno, Kozo Kaibuchi^**, Hideki Shibata, Hideyuki Mukai, Yoshitaka Ono, Michael A. Frohman^§, and Yasunori Kanaho

From the  Department of Life Science, Tokyo Institute of Technology, Yokohama 226-8501, the  Department of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236-0004, the ^** Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, the  Department of Biology, Faculty of Science, Kobe University, Kobe 657-8501, Japan and the ^§ Department of Pharmacological Sciences and the Institute for Cell and Developmental Biology and the Program for Molecular and Cellular Biology, State University of New York at Stony Brook, Stony Brook, New York 11794-8651

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- (PKC-). 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- with PLD1 was not achieved, but a C-terminal fragment of 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 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 RhoA^Val-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--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.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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