Localization of phospholipase D1 to caveolin-enriched membrane via palmitoylation: implications for epidermal growth factor signaling.
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 PKCalpha 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.
Han
Jung Min
JM
Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Korea.
Kim
Yong
Y
Lee
Jun Sung
JS
Lee
Chang Sup
CS
Lee
Byoung Dae
BD
Ohba
Motoi
M
Kuroki
Toshio
T
Suh
Pann-Ghill
PG
Ryu
Sung Ho
SH
D000818:Animals; D019556:COS Cells; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D051571:Protein Kinase C-alpha; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism
C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; C087857:protein kinase C gamma; D066246:ErbB Receptors; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D010739:Phospholipase D; C106801:phospholipase D1; D013755:Tetradecanoylphorbol Acetate