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Vol. 13, Issue 11, 3976-3988, November 2002

Localization of Phospholipase D1 to Caveolin-enriched Membrane via Palmitoylation: Implications for Epidermal Growth Factor Signaling

Jung Min Han,^* Yong Kim,^* Jun Sung Lee,^* Chang Sup Lee,^* Byoung Dae Lee,^* Motoi Ohba, Toshio Kuroki, Pann-Ghill Suh,^* and Sung Ho Ryu^*§

From the  ^*Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Korea, and the  Institute of Molecular Oncology, Showa University, Tokyo, 142-8555, Japan.

Phospholipase D (PLD) has been suggested to mediate epidermal growth factor (EGF) signaling. However, the molecular mechanism of EGF-induced PLD activation has not yet been elucidated. We investigated the importance of the phosphorylation and compartmentalization of PLD1 in EGF signaling. EGF treatment of COS-7 cells transiently expressing PLD1 stimulated PLD1 activity and induced PLD1 phosphorylation. The EGF-induced phosphorylation of threonine147 was completely blocked and the activity of PLD1 attenuated by point mutations (S2A/T147A/S561A) of PLD1 phosphorylation sites. The expression of a dominant negative PKC mutant by adenovirus-mediated gene transfer greatly inhibited the phosphorylation and activation of PLD1 induced by EGF in PLD1-transfected COS-7 cells. EGF-induced PLD1 phosphorylation occurred primarily in the caveolin-enriched membrane (CEM) fraction, and the kinetics of PLD1 phosphorylation in the CEM were strongly correlated with PLD1 phosphorylation in the total membrane. Interestingly, EGF-induced PLD1 phosphorylation and activation and the coimmunoprecipitation of PLD1 with caveolin-1 and the EGF receptor in the CEM were significantly attenuated in the palmitoylation-deficient C240S/C241S mutant, which did not localize to the CEM. Immunocytochemical analysis revealed that wild-type PLD1 colocalized with caveolin-1 and the EGF receptor and that phosphorylated PLD1 was localized exclusively in the plasma membrane, although some PLD1 was also detected in vesicular structures. Transfection of wild-type PLD1 but not of C240S/C241S mutant increased EGF-induced raf-1 translocation to the CEM and ERK phosphorylation. This study shows, for the first time, that EGF-induced PLD1 phosphorylation and activation occur in the CEM and that the correct localization of PLD1 to the CEM via palmitoylation is critical for EGF signaling.

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Present address: The Rockefeller University, Laboratory of Molecular and Cellular Neuroscience, P.O. Box 296, 1230 York Avenue, New York, NY 10021.

^§ Corresponding author. E-mail address: sungho{at}postech.ac.kr.

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Molecular Biology of the Cell
Vol. 13, 3976-3988, November 2002
Copyright © 2002 by The American Society for Cell Biology

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