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Originally published In Press as doi:10.1074/jbc.M201391200 on April 11, 2002

J. Biol. Chem., Vol. 277, Issue 25, 22974-22979, June 21, 2002
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Detecting Protein-Phospholipid Interactions

EPIDERMAL GROWTH FACTOR-INDUCED ACTIVATION OF PHOSPHOLIPASE D1b IN SITU*

William E. HughesDagger §, Banafshé Larijani||, and Peter J. ParkerDagger ||**

From the Dagger  Protein Phosphorylation Laboratory and || Cell Biophysics Laboratory, Cancer Research United Kingdom London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom

Received for publication, February 11, 2002, and in revised form, March 29, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
Conclusion
REFERENCES

Phospholipase D (PLD) proteins have been identified in secretory and endocytic vesicles, consistent with their proposed role in regulating membrane traffic. However, their sites of catalytic action remain obscure. We have developed here a novel, analytical approach to monitor PLD activation in intact cells employing lifetime imaging microscopy to measure fluorescence resonance energy transfer between protein and membrane phospholipid. Verification and application of this technique demonstrates a dispersed endosomal, epidermal growth factor-induced activation of the PLD1b isoform. Application of this approach will facilitate the spatial resolution of many protein-phospholipid interactions that are key events in the regulation of cellular processes.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
Conclusion
REFERENCES

For many proteins, there is a need to integrate spatial data with information on catalytic function (1). This is a particular concern with many membrane-associated proteins where the sites of action and consequences are compartmentalized. To address this problem, we have sought to develop the means of detecting activity/activation of membrane-associated proteins in a spatially resolved manner. The enzyme phospholipase D (PLD)1 has a well described catalytic activity and has been assigned numerous cellular functions (2, 3). The various isoforms have been described in a number of disparate membranous cellular locations (2, 3). Although these distributions reflect the steady state situation, the predicted involvement of PLD in membrane traffic demands that the critical issue of function relates to where PLD becomes activated. This typifies the compartmentation problem and serves here as the model for developing the means to resolve it.

PLD catalyzes the hydrolysis of the principal membrane lipid phosphatidylcholine (PtdCho) to phosphatidic acid (PtdOH) and choline. In mammalian cells, two PLD genes have been cloned, which in human cells consist of PLD1, with four possible splice variants (4, 5), and PLD2 (6). In addition, cytosolic and 90-kDa PLDs have been described; however, the genes encoding these activities have yet to be identified (7-9). Constitutive activities of cellular PLDs are normally low but increase on treatment of cells by a number of agonists including many growth factors (3). Such activity changes have been readily characterized as PLDs exhibit the unique ability to transphosphatidylate primary alcohols producing phosphatidylalcohol (2).

Extensive in vitro characterization of recombinant PLD isoforms has identified many lipid and protein cofactors that can modulate the activity of PLDs, including phosphatidylinositol bisphosphates, protein kinase C isoforms, and the small GTPases of the ARF, Rho, and Ral families (2). PLD1 isoforms have also been shown to be resident in a number of cellular locations, plasma membrane (10), secretory granules (11), nuclear membranes (12), Golgi (13, 14), perinuclear vesicles (15, 16), caveolin-enriched membranes (17, 18), secretory vesicles and lysosomes (19), and endosomal vesicles (5, 20), and in different cell types, it is also evident that different PLD isoforms localize to different cellular compartments (5, 8, 21, 22). Treatment of HeLa cells with epidermal growth factor (EGF) dramatically increases cellular PLD activity (3) and, as we have recently demonstrated (5), results in the down-regulation of the EGF receptor (EGF-R) kinase via PLD1-containing endosomal vesicles. We therefore speculated that EGF may activate these endosomal PLD1 isoforms, PLD1a and PLD1b.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
Conclusion
REFERENCES

Preparation of Endosomal Vesicle Fractions-- HeLa Ohio cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. HEK-293 cells, also grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, were transiently transfected with GFP-PLD constructs using a CaPO4 technique (5). After 48 h, cells were washed with cold (4 °C) PBS and resuspended in cold (4 °C) buffer (250 mM sucrose, 10 mM HEPES-KOH, pH 7.2, 1 mM EDTA 1 mM MgOAc, complete protease inhibitors; Roche Molecular Biochemicals) before disruption using a stainless steel ball homogenizer (18-µm clearance, EMBL Workshop) as described (23). Postnuclear supernatants were prepared by centrifugation in a Sorvall TechnoSpin at 3200 rpm for 8 min at 4 °C. This sample was separated through a linear 14-ml velocity sucrose gradient prepared from 0.3 and 1.2 M sucrose, as described (23, 24), in a Beckman SW40 rotor for 15 min at 25,000 rpm, and 1-ml fractions enriched for light vesicles and containing PLD1 and endosomal markers were pooled and further separated overnight on a 14-ml equilibrium sucrose step gradient (1 ml; 1.6 M, 2 ml; 1.4, 1.2, 1.0, and 0.8 M sucrose, 10 mM HEPES-KOH, pH 7.2, 1 mM EDTA, 1 mM MgOAc) in a Beckman SW40 for a minimum of 6 h at 25,000 rpm as described. For fluorescent lifetime imaging microscopy (FLIM) analysis (see below), endosomal fractions were pooled and pelleted at 40,000 × g for 15 min in membrane wash buffer (250 mM sucrose, 50 mM KCl, 2.5 mM MgCl2, 50 mM HEPES, pH 7.5, 1 mM dithiothreitol, 1 mM ATP, 1 mM fresh phenylmethylsulfonyl fluoride, pH 7.5, as described (25)).

Western Blot Analysis-- Proteins from the equilibrium sucrose step gradient fractions were incubated in 10% trichloroacetic acid at 4 °C overnight, pelleted, washed in cold (4 °C) ethanol/ether (1:1 v/v), and resuspended in urea sample buffer (8 M urea, 1% SDS, 100 mM Tris (unbuffered), 150 mM NaCl, 50 mM EDTA, 1% beta -mercaptoethanol) before separation overnight by SDS-PAGE. Proteins were transferred "wet" to polyvinylidene difluoride membranes (Millipore) at 4 °C in 25 mM TRIS (unbuffered), 193 mM glycine, 20% methanol buffer for 2 h at 600 mA using a Bio-Rad TransBlot apparatus. Antisera to PLD1s and Rab7 (Santa Cruz Biotechnology), PLD1a/b (5), and EGF-R (ICRF) were incubated at 1/500 with blocked (PBS, 0.1% Tween 20, 1% milk powder, 25 °C, 1 h) membranes in PBS, 0.1% Tween 20 overnight at 4 °C. Western blots using anti-PRK1/PKN (Transduction Labs) and Rab5B (Santa Cruz Biotechnology) were carried out as above except that incubation with the primary antibody (1/1000) was carried out at room temperature for 1 h. For detection of antigen by chemiluminescence, membranes were washed (1× PBS, 0.1% Tween 20, 5 min; 1× PBS, 0.1% Tween 20, 0.5 M NaCl, 5 min; 1× PBS, 0.1% Tween 20, 5 min), and an appropriate horseradish peroxidase-linked antibody (Amersham Biosciences) second layer was employed for 1 h before visualization with ECL (Amersham Biosciences).

Preparation of Fluorescently Labeled Phospholipids Liposomes-- For labeling cells, liposomes containing 20 µM PtdCho (dipalmitoyl, Sigma) and 20 µM fluorescent phospholipid, labeled on the acyl chain (BODIPY 530/550 C5 HPtdCho or 530/550 C12 human phosphatidylethanolamine (HPtdEtn), Molecular Probes), were prepared on resuspension in PBS by probe sonication (MSE Microsonicator) for 3 × 10-s cycles at 20 watts. Liposomes for labeling endosomal fractions were prepared in the same way but with 50 µM PtdCho and 50 µM fluorescent phospholipid. Cells prepared on glass coverslips were incubated at 37 °C for 15 min with liposomes before washing in PBS and mounting (as described above). Endosomal fractions were incubated at 37 °C for 15 min with liposomes before washing twice in membrane wash buffer (see above) and resuspension in a minimum volume of membrane wash buffer before mounting on slides in Mowiol 1:1, endosomal fractions:Mowiol (5).

FLIM-- A detailed description of the FLIM apparatus, which measures phase (tau p) and modulation depth (tau m) of emitted fluorescence in the frequency domain, can be found elsewhere (26, 27). Lifetime, <tau >, is presented as the average phase shift and relative modulation depth ((tau m + tau p)/2), and the fluorescence resonance energy transfer (FRET) efficiency is defined by EFF = 1 - tau da/<tau d>, where tau da is the lifetime map of the donor in the presence of the acceptor, and <tau d> is the average lifetime of the donor in the absence of the acceptor (26-30). For each experiment, the average GFP lifetime without acceptor (<tau d>) was calculated from eight cells, and the average GFP lifetime with acceptor (<tau da>) was deduced from six cells. Each experiment was repeated three times. The GFP lifetime measurements from vesicles were calculated from four different experiments. The cumulative lifetimes of GFP-PLD1 alone and that measured with acceptor fluorophore are plotted on two-dimensional histograms. The population variation, a concomitant decrease in tau p and tau m, indicates a reduction in GFP lifetime due to FRET.

All images were taken using a Zeiss Plan-APOCHROMAT ×100/1.4NA phase 3 oil objective with images recorded at a modulation frequency of 80.218 MHz. The donor (GFP-PLD1) was excited using the 488-nm line of an argon/krypton laser, and the resultant fluorescence was separated using a combination of dichroic beam splitter (Q 505 LP; Chroma Technology Corp.) and narrow band emitter filter (BP514/10; Lys and Optik). Acceptor images (BODIPY-PtdCho and BODIPY-PtdEtn) were taken using a 100-watt Mercury arc lamp (Zeiss Attoarc 2) as a source of sample illumination combined with a high Q Cy3 filter set (exciter, HQ 535/50; dichroic, Q 565 LP; emitter, HQ 610/75 LP; Chroma Technology Corp.).

Phospholipase D Assays-- Phospholipase D activity was assessed by prelabeling cells with 2 µCi/ml myristic acid ([9,10-3H(N)], PerkinElmer Life Sciences) for 7 h and then adding ethanol to a final concentration of 2% with or without EGF (100 ng/ml) for 30 min. Cells were fractionated (see above), and lipids extracted as below for phospholipid analysis. In vitro assays were performed on fractions using lipid-labeled PtdCho (L-alpha -dipalmitoyl-[2-palmitoyl-9,10-3H(N)], PerkinElmer Life Sciences) substrate (1 µCi/assay) presented in PtdCho (dipalmitoyl, Sigma), PtdEtn (dipalmitoyl, Sigma), PtdIns(4,5)P2 (dipalmitoyl, Echelon) liposomes prepared as described in Ref. 31. This was added to cell fractions in 2% (final) ethanol with or without 100 µM GTPgamma S or myristoylated human ARF1 (produced from bacteria containing human ARF1 and N-myristoyltransferase using a method based on that previously described (32)). Assays were terminated by phospholipid extraction using chloroform:methanol:aqueous (8:4:3) before separation by thin layer chromatography and quantitation as described (5).

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
Conclusion
REFERENCES

To demonstrate that EGF could activate endosomal PLD1, we sought to detect PLD-substrate interactions in vivo and whether this changed on EGF treatment (i.e. activating conditions). Recently, we have successfully detected specific interactions between proteins in vivo by using FLIM (1, 23). This technique allows the detection of FRET, which is a photophysical phenomenon whereby under the appropriate conditions, energy is transferred non-radiatively between fluorophores and typically occurs only at distances of between 1 and 10 nm (1, 29). Although we had used FLIM to detect interactions between proteins in vivo by using GFP fusion proteins and fluorophore-labeled antibodies, we postulated that it may be possible to detect FRET between PLD and its lipid substrate using GFP-PLD1b (where the GFP acts as the donor) and PtdCho substrate, labeled with a fluorescent BODIPY moiety on an acyl chain (acting as the acceptor). We therefore separated and purified PLD1-containing vesicular fractions from untreated and EGF-treated HeLa cells and determined that endogenous PLD1 co-fractionated with the endosomal markers, Rab5B, Rab7, PRK1/PKN, and only after EGF treatment, EGF-R (Fig. 1).


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  		Fig. 1.   Fractionation of HeLa cell extracts. Postnuclear supernatants from untreated or EGF-treated (100 ng/ml, 30 min) HeLa cells were separated through a linear sucrose gradient, and the fractions were characterized by Western blot with Rab5B, Rab7, PRK1/PKN, and PLD1 antibodies to identify Golgi, plasma membrane, and endosomal and endogenous PLD1a and PLD1b antigens (not shown). The PLD1-containing fractions, also enriched for predominantly endosomal markers, were then pooled and further separated over a stepped sucrose gradient yielding 11 fractions in which PLD1 antigen and endosomal markers were identified (as shown in the figure).

These data confirm our microscopic identification of PLD1 in endosomal vesicles (and are consistent with our observations that PLD isoforms do not change localization upon EGF stimulation) but also indicated that it should be possible to isolate GFP-PLD1-containing endosomal vesicles to use in FLIM analysis. Indeed, similarly characterized endosomal fractions from transfected HEK-293 cells (which readily express GFP-PLD1b) were pooled and demonstrated, by Western blot, to contain full-length GFP-PLD1b and endosomal markers. Additionally, these fractions exhibited considerably higher levels of PLD activity relative to fractions from untransfected cells when assayed in the presence of GTPgamma S and exogenously added ARF1 (data not shown; see below). PtdCho, the substrate for PLD, or a fluorescently labeled PtdCho, BODIPY-PtdCho, was introduced into samples of the GFP-PLD1b-containing endosomal fractions by incubation with liposomes. After examination by confocal microscopy, it was clear that incubation with the BODIPY-PtdCho liposomes labeled the vesicles in the preparation, as significant overlap between GFP-PLD1b and BODIPY-PtdCho could be seen on vesicular structures (data not shown). This provided the context in which to assess interactions between GFP-PLD1b and its fluorescently labeled substrate.

By fluorescence lifetime imaging microscopy (FLIM), the lifetime measured for GFP from GFP-PLD1b in untreated endosomal vesicles or those treated with unlabeled PtdCho liposomes was unchanged (Fig. 2A). However, in endosomal vesicles that had incorporated BODIPY-PtdCho, the measured lifetime of GFP decreased, as indicated by an increase in red, shorter lifetime, pixels (Fig. 2A). This suggests that the GFP and BODIPY fluorophores are closely interacting to allow energy transfer (FRET) and reduction in measured lifetime. This is confirmed as the images showing pseudocolor scales for FRET efficiency (a relationship between the average donor lifetime in the absence of acceptor and the donor in the presence of the acceptor) also demonstrate that FRET efficiency is higher in the presence of the labeled phospholipid.


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  		Fig. 2.   FRET detected in endosomal vesicles containing GFP-PLD1b and BODIPY-PtdCho. GFP-PLD1b-containing endosomal vesicles prepared from HEK-293 cells were incubated with liposomes containing PtdCho, (upper panels), BODIPY-PtdCho (middle panels), or BODIPY-PtdEtn (lower panels). The vesicle-like structures contain detectable signals for GFP and (when added) BODIPY-phospholipid (A). The lifetime (<tau >) of GFP is shown with pseudocolor scales from red (1.70 ns) to blue (2.10 ns), and the FRET efficiency, also shown with pseudocolor scales, is from blue (0%) to red (30%). The cumulative lifetimes of GFP-PLD1b from endosomal fractions treated with PtdCho liposomes (green), BODIPY-PtdCho liposomes (red, upper), and BODIPY-PtdEtn liposomes (red, lower) from four experiments are plotted on the two-dimensional histograms, showing both the phase (tau p) and modulation (tau m) measurements for lifetime for all recorded pixels (B). Cumulative histograms of all the FRET efficiencies at each pixel, measured in six to ten experiments, are shown represented as the number of pixel counts versus FRET efficiency (C).

To confirm that the FRET observed could only be detected when labeled substrate for PLD was added to cells, we assessed GFP-PLD1b lifetime in experiments using BODIPY-PtdEtn, representing a phospholipid for which PLD1 has no catalytic activity (4). This was also introduced into purified vesicles, and as shown in Fig. 2A, the lifetime of GFP-PLD remains unchanged in the presence of BODIPY-PtdEtn, indicating that the two fluorophores are not in close proximity despite colocalization in the vesicular membrane. The data collected from four independent experiments with PtdCho, labeled PtdCho, or labeled PtdEtn are shown in the two-dimensional histograms in Fig. 2B. These represent the cumulative lifetimes of all pixels recorded for GFP-PLD1b alone and in the presence of BODIPY-PtdCho or BODIPY-PtdEtn. The figure shows the lifetimes measured by both parameters assessed using the FLIM apparatus, phase and modulation, and a concomitant decrease in both indicates a reduction in GFP lifetime due to FRET; this is only observed with BODIPY-PtdCho. The variation in the lifetime populations shown is representative of that seen in the images of Fig. 2A and is indicative of a reduction of average GFP-PLD1b lifetime from 2.1 to 1.85 ns due to FRET with BODIPY-PtdCho. The cumulative FRET efficiencies at each pixel, measured in all experiments, is shown in Fig. 2C, indicating the number of pixel counts versus FRET efficiency and these results again demonstrate that efficient FRET is only seen between GFP-PLD1b and BODIPY-PtdCho.

Having characterized this technique in vitro using isolated endosomal GFP-PLD1b-containing vesicles, we examined whether we could detect these specific interactions in vivo using HeLa cells transfected with GFP-PLD1b. Examination by confocal microscopy indicated that BODIPY-phospholipid-containing liposomes could be used to label whole cells and that colocalization between BODIPY-PtdCho and GFP-PLD1b on endosomal vesicles could be detected (see below). Similar lipid loading approaches have been used recently to label cellular phospholipids and study the transport of PtdCho itself in HepG2 cells (33). In transfected cells, GFP-PLD1b is seen on vesicular structures, and the lifetime of GFP is unchanged in untreated cells (data not shown) and in cells incubated with unlabeled PtdCho (Fig. 3A). However, in cells incubated with BODIPY-PtdCho, the lifetime of GFP decreases apparently on vesicular structures within the cell, coincident with the detected GFP-PLD1b. We also measured the average lifetime of GFP within defined regions of the cell. Regions representative of GFP-PLD1 vesicles showed an average lifetime of 1.98 ± 0.13 ns; however, in the presence of BODIPY-PtdCho, similar vesicular regions show a reduction in average lifetime to 1.71 ± 0.08 ns. These data confirm that a reduction of GFP lifetime occurs in the presence of the BODIPY-PtdCho on vesicular structures and that FRET is occurring between the fluorophores. In parallel experiments, the lifetime measure for GFP-PLD1b in the presence of BODIPY-PtdEtn remains unchanged, confirming the specificity of the FRET detected between GFP-PLD1b and BODIPY-PtdCho (Fig. 3). The cumulative data for all experiments for variation in the lifetime populations (Fig. 3B, indicative of a reduction of average GFP-PLD1b lifetime from 2.1 to 1.85 ns) and the cumulative FRET efficiencies (Fig. 3C) confirm that efficient FRET is only seen between GFP-PLD1b and BODIPY-PtdCho. These data indicate that using FLIM, it is possible to detect, in vivo, specific interactions between vesicular PLD1b and PtdCho.


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  		Fig. 3.   FRET detected in cells containing GFP-PLD1b and BODIPY-PtdCho. HeLa cells grown on coverslips and transfected with GFP-PLD1b were incubated with liposomes containing PtdCho, (upper panels), BODIPY-PtdCho (middle panels), or BODIPY-PtdEtn (lower panels). The vesicle-like structures seen within the cells contain detectable signals for GFP and, where added, BODIPY (A). (The quality of the images is not well resolved as the detector used here for FLIM has an intrinsic limit of resolution.) The lifetime (<tau >) of GFP is shown with pseudocolor scales from red (1.70 ns) to blue (2.10 ns), and the FRET efficiency, also shown with pseudocolor scales, is from blue (0%) to red (30%). The cumulative lifetimes of GFP-PLD1b from transfected cells treated with PtdCho liposomes (green), BODIPY-PtdCho liposomes (red, upper), and BODIPY-PtdEtn liposomes (red, lower) from four experiments are plotted on the two-dimensional histograms, showing both the phase (tau p) and modulation (tau m) measurements for lifetime for all recorded pixels (B). Cumulative histograms of all the FRET efficiencies at each pixel, measured in all experiments, are shown represented as the number of pixel counts versus FRET efficiency (C).

It is likely that in these experiments, the FRET detected between the GFP and BODIPY moieties may be representative of either substrate, BODIPY-PtdCho, or product, BODIPY-PtdOH, interacting with the catalytic site of GFP-PLD1b. Therefore we speculated that catalytically inactive point mutants of GFP-PLD1b used in similar experiments would result in the loss of detected FRET (or at least reduced FRET). To test this hypothesis, we transfected HeLa cells with catalytically inactive PLD mutants GFP-PLD1b-K466E and GFP-PLD1b-K860E, which, while exhibiting no catalytic activity, continue to localize to endosomal vesicles (5). Cells were analyzed for FRET between the mutants and BODIPY-PtdCho. The lifetime of catalytically inactive mutants GFP-PLD1b-K466E or GFP-PLD1b-K860E is unchanged in the presence of BODIPY-PtdCho, indicating that the two fluorophores are not interacting; in control experiments, GFP-PLD1b showed lifetime decreases, and an increase in FRET efficiency is observed (Fig. 4A). The cumulative data for all experiments for variation in the lifetime populations (Fig. 4B) and the cumulative FRET efficiencies (Fig. 4C) confirm that no FRET is seen with the two catalytically inactive PLD mutants. Similar results were obtained for isolated endosomal fractions containing catalytically inactive PLD1 isoforms (data not shown). Thus, the FRET detected for GFP-PLD1b is not only specific for PtdCho but is also specific for active PLD1. Whether this distinction reflects the loss of interaction between the inactive point mutants and BODIPY-PtdCho, a change in conformation that reduces FRET (e.g. via a distancing of the acceptor-donor couple) or implies that it is principally a product (BODIPY-PtdOH)-based effect, cannot be resolved at present. However, we would emphasize that the mutant PLDs remain localized to the same vesicular compartment as the wild-type protein (5), suggesting that these mutations do not provoke gross misfolding.


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  		Fig. 4.   FRET is specific for catalytically active PLD. HeLa cells, transfected with GFP-PLD1b (upper panels), GFP-PLD1b-K466E (middle panels), or GFP-PLD1b-K860E (lower panels), were incubated with liposomes containing BODIPY-PtdCho. The vesicle-like structures seen within the cells contain detectable signals for GFP and BODIPY-PtdCho (A). The lifetime (<tau >) of GFP is shown with pseudocolor scales from red (1.70 ns) to blue (2.10 ns), and the FRET efficiency, also shown with pseudocolor scales, is from blue (0%) to red (30%). The cumulative lifetimes of GFP-PLD1b (upper), PLD1bK466E (middle), and PLD1K860E (lower) with BODIPY-PtdCho (red) or alone (green) from four experiments are plotted on the two-dimensional histograms, showing both the phase (tau p) and modulation (tau m) measurements for lifetime for all recorded pixels (B). Cumulative histograms of all the FRET efficiencies at each pixel, measured in all experiments, are shown represented as the number of pixel counts versus FRET efficiency (C).

As it appeared that FRET was dependent on PLD activity, we investigated whether the stimulation of cells resulted in increases in localized FRET between the GFP and BODIPY moieties. To address this, we chose initially to activate GFP-PLD1b in vivo by the addition of TPA, a potent activator of protein kinase C isoforms. TPA has been shown to activate cellular PLD in many cells types including HeLa cells, and in vitro protein kinase C isoforms are potent activators of recombinant PLD1b (2, 3). Therefore we measured the fluorescent lifetime of GFP-PLD1b and catalytically inactive PLD1b-K860E in cells pretreated with BODIPY-PtdCho liposomes that were then left untreated or further treated with TPA. The data (Fig. 5) indicated that TPA caused a significant additional increase in FRET between GFP-PLD1b and BODIPY-PtdCho on vesicular structures and had no effect on the lack of FRET detected for the catalytically inactive mutant. Thus, it appears that further decreases in GFP lifetime in the presence of BODIPY-PtdCho and PLD1 activators correlate with PLD activity changes.


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  		Fig. 5.   FRET detected in TPA-treated HeLa cells. HeLa cells, transfected with GFP-PLD1b or GFP-PLD1b-K860E, were incubated with liposomes containing BODIPY-PtdCho and then were untreated or treated with 400 nM TPA for 30 min. The vesicle-like structures seen within the cells contain detectable signals for GFP and BODIPY-PtdCho (A). The lifetime (<tau >) of GFP is shown with pseudocolor scales from red (1.6 ns) to blue (2.1 ns), and the FRET efficiency, also shown with pseudocolor scales, is from blue (0%) to red (30%). The cumulative lifetimes, from six experiments, of GFP-PLD1b or GFP-PLD1b-K860E are plotted on the two-dimensional histograms, showing both the phase (tau p) and modulation (tau m) measurements for lifetime for all recorded pixels (B). The lifetimes for GFP-PLD alone (blue), treated with BODIPY-PtdCho (green), or treated with BODIPY-PtdCho and TPA (red) are shown. Cumulative histograms of all the FRET efficiencies at each pixel, measured in all experiments, are shown represented as the number of pixel counts versus FRET efficiency (C).

We then investigated the response of PLD1b to the growth factor EGF. The results indicate that EGF treatment caused an increase in detected FRET on GFP-PLD1b vesicles in transfected, BODIPY-PtdCho-loaded cells and that FRET could not be detected in cells transfected with catalytically inactive GFP-PLD1b-K860E (Fig. 6). The average lifetimes measured within specific regions of the cell indicated that EGF could induce a further drop in lifetime from 1.75 ± 0.10 to 1.60 ± 0.10 ns in vesicular regions of the cell. The cumulative data shown in Fig. 6, B and C, confirm that significant FRET is seen in GFP-PLD1b-transfected cells treated with EGF. It is important to note that we did not observe FRET between GFP-PLD1b and BODIPY-PtdCho at the plasma membrane. Thus it appears that endosomal PLD1b can indeed be regulated by EGF/EGF-R in that compartment.


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  		Fig. 6.   FRET detected in EGF-treated HeLa cells. HeLa cells, transfected with GFP-PLD1b or GFP-PLD1b-K860E, were incubated with liposomes containing BODIPY-PtdCho and then treated or untreated with EGF (100 ng/ml, 30 min). The vesicle-like structures seen within the cells contain detectable signals for GFP and BODIPY-PtdCho (A). The lifetime (<tau >) of GFP is shown with pseudocolor scales from red (1.60 ns) to blue (2.10 ns), and the FRET efficiency, also shown with pseudocolor scales, is from blue (0%) to red (30%). The cumulative lifetimes, from six experiments, of GFP-PLD1b or GFP-PLD1b-K860E are plotted on the two-dimensional histograms, showing both the phase (tau p) and modulation (tau m) measurements for lifetime for all recorded pixels (B). The lifetimes for GFP-PLD alone (blue), treated with BODIPY-PtdCho (green), or treated with BODIPY-PtdCho and EGF (red) are shown. Cumulative histograms of all the FRET efficiencies at each pixel, measured in all experiments, are shown represented as the number of pixel counts versus FRET efficiency (C).

To confirm these observations, we returned to endosomal fractionation and assessed the levels of PLD activity in the fractions by measuring ethanol-dependent [3H]phosphatidylethanol (PtdEtOH) production from [3H]PtdCho added to the fractions in PtdIns(4,5)P2-containing liposomes. The results (Fig. 7A) indicate that PLD activity in all fractions is very low but that in those fractions containing endosomal markers (Fig. 1, fractions 5-8), a PLD activity stimulated by GTPgamma S is apparent. The catalytic activity of PLD2 is dependent on PtdIns(4,5)P2 in vitro but is not GTPgamma S-stimulated, whereas PLD1 isoforms display a dependence on GTP-bound forms of ARF and Rho family small GTPases, some of which would co-fractionate with endosomal vesicles. Furthermore, when purified recombinant myristoylated ARF1 was added to the assays, PLD activity increased further, confirming the presence of PLD1-like activity in these fractions (data not shown). These results suggested that the predominant PLD in these endosomal fractions is likely to be a PLD1 isoform.


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  		Fig. 7.   PLD activity in HeLa cell fractions. Postnuclear supernatants from untreated HeLa cells were separated through a linear sucrose gradient, and fractions enriched for endosomal vesicles were then further separated through a stepped sucrose gradient into the 11 fractions shown. Fractions were assayed for PLD activity and GTPgamma S-stimulated PLD activity by determining the ability of extracts incubated with ethanol to produce [3H]PtdEtOH from exogenously added vesicles containing [3H]PtdCho, a reaction specific to PLD (A). HeLa cells were labeled with [3H]myristic acid for 7 h and then treated with ethanol (2.5% final) or ethanol and EGF (100 ng/ml) for 30 min (B). Fractions were assayed for PLD activity by determining the levels of [3H]PtdEtOH produced from [3H]myristate-labeled PtdCho. The data are representative of three independent experiments.

The fractionation procedure was then applied to extracts from cells prelabeled with [3H]myristic acid (to label endogenous cellular phospholipids, including PtdCho) and subsequently treated with ethanol or ethanol and EGF. In this way, we were able to monitor the compartment(s) in which PLD was activated by EGF. The data indicate that EGF induces an increase in PtdEtOH production in fractions 5-8 (Fig. 6B), those identified as containing PLD1 and endosomal proteins (Figs. 1 and 7A), and therefore confirmed that EGF activates PLD in this compartment, leading to the accumulation of this poorly metabolized product.

    Conclusion
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
Conclusion
REFERENCES

These studies demonstrate that PLD1 is not simply localized in endosomes in HeLa cells but that it is activated in these endosomal compartments after treatment with EGF. This is demonstrated by subcellular fractionation and analysis of the distribution of the accumulated, stable product PtdEtOH and also by exploiting fluorescent probes labeling proteins and lipids. Using these reagents, we were able to analyze protein-phospholipid interactions by measuring FRET through FLIM. The ability to explore protein-lipid interactions through such techniques will provide unrivalled opportunities in unraveling the controls operating in membrane subcompartments.

    ACKNOWLEDGEMENTS

We thank Tony Ng and Philippe I. H. Bastiaens for facilitating the FLIM analysis and Sharon Tooze for advice on fractionation.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a Royal Society Fellowship. Present address: The Garvan Institute of Medical Research, 384 Victoria Street, Sydney, NSW 2010, Australia.

These authors contributed equally to this work.

** To whom correspondence should be addressed. E-mail: peter.parker@cancer.org.uk.

Published, JBC Papers in Press, April 11, 2002, DOI 10.1074/jbc.M201391200

    ABBREVIATIONS

The abbreviations used are: PLD, phospholipase D; EGF, epidermal growth factor; EGF-R, EGF receptor; FLIM, fluorescent lifetime imaging microscopy; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; PBS, phosphate-buffered saline; TPA, 12-O-tetradecanoylphorbol-13-acetate; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; PtdCho, phosphatidylcholine; PtdOH, phosphatidic acid; PtdEtn, phosphatidylethanolamine; PtdEtOH, phosphatidylethanol; PtdIns, phosphatidylinositol.

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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
Conclusion
REFERENCES

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