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, Sung Ho Ryu

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

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 PKCα 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.

Original languageEnglish
Pages (from-to)3976-3988
Number of pages13
JournalMolecular Biology of the Cell
Volume13
Issue number11
DOIs
Publication statusPublished - 2002 Nov 1

Fingerprint

Lipoylation
Caveolins
Epidermal Growth Factor
Phosphorylation
Membranes
Caveolin 1
Phospholipase D
COS Cells
Epidermal Growth Factor Receptor
phospholipase D1
Point Mutation
Adenoviridae
Transfection

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Cite this

Han, Jung Min ; Kim, Yong ; Lee, Jun Sung ; Lee, Chang Sup ; Lee, Byoung Dae ; Ohba, Motoi ; Kuroki, Toshio ; Suh, Pann Ghill ; Ryu, Sung Ho. / Localization of phospholipase D1 to caveolin-enriched membrane via palmitoylation : Implications for epidermal growth factor signaling. In: Molecular Biology of the Cell. 2002 ; Vol. 13, No. 11. pp. 3976-3988.
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abstract = "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 PKCα 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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Localization of phospholipase D1 to caveolin-enriched membrane via palmitoylation : Implications for epidermal growth factor signaling. / Han, Jung Min; Kim, Yong; Lee, Jun Sung; Lee, Chang Sup; Lee, Byoung Dae; Ohba, Motoi; Kuroki, Toshio; Suh, Pann Ghill; Ryu, Sung Ho.

In: Molecular Biology of the Cell, Vol. 13, No. 11, 01.11.2002, p. 3976-3988.

Research output: Contribution to journalArticle

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T1 - Localization of phospholipase D1 to caveolin-enriched membrane via palmitoylation

T2 - Implications for epidermal growth factor signaling

AU - Han, Jung Min

AU - Kim, Yong

AU - Lee, Jun Sung

AU - Lee, Chang Sup

AU - Lee, Byoung Dae

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AU - Kuroki, Toshio

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AU - Ryu, Sung Ho

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