QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online September 22, 2005
doi: 10.1242/10.1242/jcs.02564

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lee, J. S. Articles by Ryu, S. H. Search for Related Content PubMed PubMed Citation Articles by Lee, J. S. Articles by Ryu, S. H.

Phosphatidylinositol (3,4,5)-trisphosphate specifically interacts with the phox homology domain of phospholipase D1 and stimulates its activity

¹ Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang 790-784, Korea
² Schepens Eye Research Institute, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA
³ Graduate School of Life Science, University of Hyogo, Harima Science Garden City, Hyogo 678-1297, Japan

View larger version (36K): [in a new window]   Fig. 1. The PX domain of PLD1 specifically binds to PtdIns(3,4,5)P3. (A) The sequence alignment of regions from several PX domains. The conserved Arg residue mutated in this study is marked as . (B) Specificities of PLD1 PX, PLD1 PX(R179K), PLD1 PX(R179A) and PLD2 for a range of phosphatidylinositols were analyzed by liposome-binding assay. Binding of GST-PLD PX domains to liposomes containing PE (phosphatidylethanolamine), PC (phosphatidylcholine) and indicated PtdIns (phosphatidylinositol) were analyzed. Liposomes (see Materials and Methods) were incubated with 10 ng of purified GST-PLD PX for 15 minutes at 37°C. The reaction mixtures were then centrifuged and the resulting supernatant (S) and pellets (P) were subjected to SDS-PAGE. Western blotting was performed with anti-GST antibody. A representative result of three individual experiments is shown. (C) The selectivity of PtdIns(3,4,5)P3 for the PLD1 PX domain was confirmed by competition analysis. Water-soluble inositolphosphates were added to the reaction mixture as described in (B). (D) The affinity of PLD1 PX for PtdIns(3,4,5)P3 was compared with those of PH domains from Btk and PDK-1.

View larger version (56K): [in a new window]   Fig. 2. The PLD1 PX domain interacts with PtdIns(3,4,5)P3 in NIH-3T3 cells. NIH-3T3 cells grown on cover slips were transfected with pEGFP-PLD1 PX, pEGFP-PLD1 PX(R179K), pEGFP-PLD1 PX(R179A), or pEGFP-Btk PH. (A) After 24 hours of serum starvation, cells were treated with PDGF (25 ng/ml). After 5 minutes, cells were fixed and the localization of PLD1 PX domains was visualized by confocal microscopy. Bars, 10 µm. (B) Cells as described in (A) were cultured in dishes and fractionated into cytosolic (Cyt) and membrane (Mem) fractions. Western blotting was performed with anti-EGFP antibody and show that only the wild-type PLD1 PX domain showed membrane localization after PDGF treatment.

View larger version (45K): [in a new window]   Fig. 3. Inhibition of PI3K attenuates the plasma membrane localization of PLD1 PX. NIH-3T3 cells transfected with pEGFP-PLD1 PX were serum starved for 24 hours. PDGF (25 ng/ml) was applied for 5 minutes, after the pre-incubation of cells with DMSO or 50 µM of the PI3K inhibitor LY 294002, for 20 minutes at 37°C. (A) Subcellular PLD1 PX localization was visualized by confocal microscopy. Bars, 10 µm. (B) Biochemical fractionation study showing localization of PLD1 PX in the cytosol (Cyt) or the plasme membrane (Mem), in the absence or presence of LY 294002.

View larger version (23K): [in a new window]   Fig. 4. PtdIns(3,4,5)P3 activates PLD1 in vitro. (A) The effects of a range of phosphatidylinositols on the in-vitro activity of PLD1 were analyzed as described in Materials and Methods. Liposomes, reconstituted with phosphatidylethanolamine (PE), phosphatidylcholine (PC) and the indicated phosphatidylinositols, were used as substrates. Error bars indicate standard deviations. Data are representative of three independent experiments. Immunoblot in the inset indicates that the amount of PLD1 preparation used in this study were similar. (B) The dose-dependency of stimulation was checked using liposomes containing PE, PC, PtdIns and PtdIns(3,4,5)P3. The sum of the concentrations of PtdIns and PtdIns(3,4,5)P3 was maintained at 4.6 µM.

View larger version (22K): [in a new window]   Fig. 5. PtdIns(3,4,5)P3-induced activation of PLD1 promotes ERK phosphorylation. (A) 5x105 HEK 293 cells depleted of endogenous PLD2, were transfected with 1 µg of wild-type PLD1 DNA. 24 hours after transfection, cells were starved for 24 hours followed by an incubation with [3H]myristic acid for 3 hours. Phosphatidylbutanol (PBt) accumulation was measured in the presence of 0.4% butanol after 2 minutes. Cells were incubated with LY 294002 (50 µM) or DMSO for 20 minutes before PDGF treatment. Error bars indicate standard deviations. Data are representative of three separate experiments. To check expression levels of PLD, the same amounts of cell lysates (20 µg) were subjected to SDS-PAGE and then immunoblotted with anti-C-terminal PLD antibody (data not shown). (B) Activation of ERK1 and ERK2 induced by the stimulation of PLD were compared. (Upper panel) The phosphorylation status of ERK1/2 was analyzed by western blotting with an anti-phosphorylated-ERK antibody. (Lower panel) Expression levels of ERK1/2. The fold-increases of ERK2 phosphorylation were calculated from the ratio of phosphorylated ERK2 to unphosphorylated ERK2 (pERK2:ERK2) determined by densitometric analysis.

View larger version (20K): [in a new window]   Fig. 6. Interaction with PtdIns(3,4,5)P3 is crucial for PDGF-induced activation of PLD1. HepG2 cells stably expressing mutant Y40/51 PDGFßR were transiently transfected as indicated. PLD activity was measured for 2 minutes in the presence of PDGF (25 ng/ml). To inhibit PI3K, cells were treated with 50 µM LY 294002 for 20 minutes before treatment with PDGF. Data are representative of three separate experiments. Error bars indicate standard deviations. Immunoblots in the inset show that the expression levels of PLD were similar.