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Mol Biol Cell. Apr 2002; 13(4): 1252–1262.
PMCID: PMC102266

Antigen-stimulated Activation of Phospholipase D1b by Rac1, ARF6, and PKCα in RBL-2H3 Cells

Joan S. Brugge, Monitoring Editor

Abstract

Phospholipase D (PLD) activity can be detected in response to many agonists in most cell types; however, the pathway from receptor occupation to enzyme activation remains unclear. In vitro PLD1b activity is phosphatidylinositol 4,5-bisphosphate dependent via an N-terminal PH domain and is stimulated by Rho, ARF, and PKC family proteins, combinations of which cooperatively increase this activity. Here we provide the first evidence for the in vivo regulation of PLD1b at the molecular level. Antigen stimulation of RBL-2H3 cells induces the colocalization of PLD1b with Rac1, ARF6, and PKCα at the plasma membrane in actin-rich structures, simultaneously with cooperatively increasing PLD activity. Activation is both specific and direct because dominant negative mutants of Rac1 and ARF6 inhibit stimulated PLD activity, and surface plasmon resonance reveals that the regulatory proteins bind directly and independently to PLD1b. This also indicates that PLD1b can concurrently interact with a member from each regulator family. Our results show that in contrast to PLD1b's translocation to the plasma membrane, PLD activation is phosphatidylinositol 3-kinase dependent. Therefore, because inactive, dominant negative GTPases do not activate PLD1b, we propose that activation results from phosphatidylinositol 3-kinase–dependent stimulation of Rac1, ARF6, and PKCα.

INTRODUCTION

Phosphatidic acid (PtdOH) is a lipid-signaling molecule (Hodgkin et al., 1998 blue right-pointing triangle) implicated in the regulation of various cellular processes including the oxidative burst in neutrophils (McPhail et al., 1995 blue right-pointing triangle), mitogenesis (Inui et al., 1994 blue right-pointing triangle; Ghosh et al., 1996 blue right-pointing triangle), vesicle trafficking/exocytosis (Bi et al., 1997 blue right-pointing triangle; Chen et al., 1997 blue right-pointing triangle; Brown et al., 1998 blue right-pointing triangle; Siddhanta and Shields, 1998 blue right-pointing triangle; Siddhanta et al., 2000 blue right-pointing triangle), and actin cytoskeletal rearrangement (Ha and Exton, 1993 blue right-pointing triangle; Cross et al., 1996 blue right-pointing triangle; Hastie et al., 1998 blue right-pointing triangle). The hydrolysis of phosphatidylcholine to generate PtdOH and free choline is catalyzed by PLD in response to a range of agonists in most cell types (Hodgkin et al., 1998 blue right-pointing triangle; Exton et al., 1999 blue right-pointing triangle). Two PLD genes have been cloned (PLD1 and PLD2), each capable of generating two splice variants (PLD1a, PLD1b, and PLD2a, PLD2b; Colley et al., 1997a blue right-pointing triangle, 1997b blue right-pointing triangle; Hammond et al., 1997 blue right-pointing triangle). So far there is little understanding of the pathways involved in PLD activation after receptor occupation. In contrast to phospholipase C (PLC), there is no evidence for the activation of PLD through direct interaction with heterotrimeric G-protein–coupled receptors or tyrosine kinase–coupled receptors. Thus, the regulation of this phospholipase differs from PLC; indeed PLD activation is often downstream of PLC activation (Exton, 1999 blue right-pointing triangle). In vitro studies have revealed that PLD1b activity is stimulated in the presence of any one Rho family member, any one ADP-ribosylation family (ARF) member, and protein kinase C (PKC) family members that contain a C2 domain (Hodgkin et al., 1999 blue right-pointing triangle). Combinations of single members from each of these activator families result in a cooperative increase in PLD1 activity (Hodgkin et al., 1999 blue right-pointing triangle). We have also recently shown that PLD1 has an essential requirement for phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) through interaction with an N-terminal PH domain (Hodgkin et al., 2000 blue right-pointing triangle). These data suggest that in vivo PLD1b can form a multicomponent complex involving PtdIns(4,5)P2, possibly as a membrane tether, a Rho family member, an ARF family member, and a C2 domain-containing PKC family member.

Exocytosis in the rat basophilic leukemic cell line, RBL-2H3, is controlled by a signal transduction cascade after cross-linking of the high-affinity immunoglobulin E receptors (FcεRI) as a result of multivalent antigen binding (Scharenberg and Kinet, 1994 blue right-pointing triangle) and is mediated through the activation of PI3-kinase and PLCγ1 (Barker et al., 1998 blue right-pointing triangle; Djouder et al., 2001 blue right-pointing triangle; Hong-Geller et al., 2001 blue right-pointing triangle). Similarly, antigen stimulation also initiates activation of other proteins essential to exocytosis including Rho (Guillemot et al., 1997 blue right-pointing triangle; Hong-Geller and Cerione, 2000 blue right-pointing triangle; Djouder et al., 2001 blue right-pointing triangle; Hong-Geller et al., 2001 blue right-pointing triangle) and ARF family GTPases (Way et al., 2000 blue right-pointing triangle), members of the PKC family (Apgar, 1991 blue right-pointing triangle; Hong-Geller and Cerione, 2000 blue right-pointing triangle), and PLD (Brown et al., 1998 blue right-pointing triangle; Frigeri and Apgar, 1999 blue right-pointing triangle; Field et al., 2000 blue right-pointing triangle). We have previously shown that antigen stimulation promotes the translocation of PLD1b from the secretory granules and secretory lysosomes to the plasma membrane along with these secretory compartments (Brown et al., 1998 blue right-pointing triangle). Because this translocation was independent of PLD activity, we concluded that PLD-dependent exocytosis was likely to proceed after PLD1b's activation at the plasma membrane (Brown et al., 1998 blue right-pointing triangle).

Most of the data published with regard to the regulation of PLD1b at the molecular level has been produced in vitro. In the present study, using the RBL-2H3 cell line, we present the first in vivo evidence for a pathway from receptor occupation through to the direct, molecular activation of PLD1b.

MATERIALS AND METHODS

Cell Culture and Transfection

RBL-2H3 cells were cultured at 37°C in 5% CO2 in DMEM (Life Technologies) with 10% fetal calf serum. FcεRI were sensitized by the addition of 1 μg/ml antidinitrophenol (DNP) IgE (Sigma, St. Louis, MO) for 16 h. Where applicable, LY294002 (Calbiochem, La Jolla, CA) was added 10 min before stimulation. Stimulation followed the addition of 50 ng/ml DNP-HSA (Sigma) in stimulation medium (125 mM NaCl, 5 mM KCl, 1.8 mM MgCl2, 0.5 mM Na2PO4, 5 mM NaHCO3, 10 mM glucose, 0.1% BSA, and 10 mM HEPES, pH 7.2). Cells (1 × 107) were transfected by electroporation with 3 pmol of each construct (equivalent to ~20 μg of pHA-PLD1b, a level that alone seems to have little morphological effect upon the nonstimulated cell) in a Bio-Rad Gene Pulser II (250 V, 950 μF; Richmond, CA). Experiments were performed 16 h posttransfection.

PLD Activity Assay

Cells were grown for 16 h in the presence of 5 μCi/ml [3H]palmitate (Amersham PharmaciaBiotech, Piscataway, NJ) and 1 μg/ml anti-DNP IgE. Five minutes before stimulation cells were washed and incubated in stimulation medium containing 0.3% (vol/vol) butan-1-ol. Stimulation proceeded after the addition of 50 ng/ml DNP-HSA. Lipids were extracted with chloroform-methanol and [3H]PtdBut isolated by TLC as previously described (Wakelam et al., 1995 blue right-pointing triangle).

BIAcore Experiments

Purified proteins (glutathione S-transferase [GST], GST-PLD1b, GST-JNK2, carbonic anhydrase, and BSA) were covalently immobilized to CM5 surfaces (BIAcore Ltd., Stevenage, United Kingdom) by amine coupling using N-hydroxysuccinamide and ethyl-dimethylaminopropyl carbodiamide. Similar-sized proteins were immobilized to equal densities. After ethanolamine treatment, surfaces were equilibrated in buffer (HEPES-buffered saline) and cycled in 10 mM HCl before challenging with 100 μM BSA to assess surface integrity and nonspecific interactions. Binding events were initiated by injection of the required protein such as the small G-proteins or carbonic anhydrase in HBS flowing over the chip surface at 10 μl/min. Surfaces could be regenerated effectively with glycine, pH 2.

Confocal Immunofluorescence Microscopy

Transfected cells grown on poly-l-lysine–coated glass coverslips were fixed for 7 min with ice-cold 4% paraformaldehyde and permeabilized using 0.5% CHAPS (Sigma) for 2 min. After blocking for 1 h with 20% heat-inactivated goat serum in PBS the hemaglutinin (HA) tag was detected with clone 12CA5 antibody (Roche, Indianapolis, IN), endogenous Rac1 was detected using clone 23A8 antibody (Upstate Biotechnology, Lake Placid, NY), endogenous PKCα was detected using clone 3 antibody (BD Transduction Laboratories, Lexington, KY), and ARF6 was detected using a specific antibody supplied by Julie Donaldson (NIH, Bethesda, MD), each for 1 h. Subsequently, subclass specific Texas Red– (Southern Biotechnology Associates, Birmingham, AL) or TRITC (Sigma)-conjugated secondary antibodies were added for 1 h to fluorescently label primary antibody-bound proteins. All antibodies were diluted in 0.2% saponin/20% heat-inactivated goat serum. Coverslips were mounted onto slides in Prolong (Molecular Probes, Eugene, OR). Images in Figures Figures1,1, ,2,2, and and55 are confocal sections acquired using a Nikon TE 300 microscope/PCM2000 system (Garden City, NY) and the accompanying EZ2000 software. Images in Figure Figure33 were acquired using a Zeiss Axioskop 50 microscope (Thornwood, NY) and a Hamamatsu Orca camera (Bridgewater, NJ). These were subsequently deconvolved to confocal images using Openlab 2.2 software (Improvision, Coventry, United Kingdom). Z-series were obtained using a step size of 0.4 μm.

Figure 1
Antigen stimulation of RBL-2H3 cells induces a PI3-kinase–dependent increase in PLD activity. PLD activity was determined in the absence (A) and presence (B) of increasing concentrations of LY294002. [3H]palmitate–labeled ...
Figure 2Figure 2
Antigen-stimulated colocalization of PLD1b with Rac1, ARF6, and PKCα. The FcεRIs of RBL-2H3 cells were sensitized by the addition of 1 μg/ml anti-DNP IgE for 16 h. Confocal images were obtained from cells before receptor cross-linking ...
Figure 3Figure 3
Cooperative activation of PLD1b in RBL-2H3 cells. [3H]palmitate–labeled RBL-2H3 cells, transfected with HA-tagged PLD1b, GFP-tagged Rac1, ARF6, PKCα, Rac1T17N, or ARF6T27N as stated (A–C), were preincubated for ...
Figure 5Figure 5
The majority of PLD1b is Triton insoluble. (A) HA-tagged PLD1b was immunoprecipitated from antigen-stimulated RBL-2H3 cells cotransfected with HA-tagged PLD1b and GFP-tagged ARF6. After electrophoresis the gel was probed with an anti-ARF6 antibody. WCL, ...

Immunoprecipitation and Western Blotting

Cells (1 × 107) were lysed for 10 min in ice-cold 50 mM HEPES, pH 7.4, 0.5% Triton X-100, 2 mM sodium orthovanadate, 50 mM NaF, PMSF, and protease inhibitor tablets (Roche). The Triton-insoluble fraction was removed by centrifugation at 16,000 × g for 5 min. Antibody, 1 μg, was added to the lysates, incubated with mixing at 4°C for 2 h, and captured with 25 μl bed-volume of Protein G Sepharose (Amersham PharmaciaBiotech) for 2 h. The Protein G Sepharose was washed five times in lysis buffer, and bound proteins were eluted into 25 μl of 2× Lamelli buffer. Proteins were separated on 4–20% gradient gels (Novex, Encinitas, CA) and transferred to PVDF and blocked in 5% Milk (Marvel, Richmond, IN; in PBS and 0.1% Tween 20). Proteins were blotted and detected as described previously (Hodgkin et al., 1999 blue right-pointing triangle). Antibodies used were the same as used for immunofluorescence.

RESULTS

PLD Activity Is PI3-kinase Dependent

In the presence of a short-chain primary alcohol PLD preferentially catalyzes the production of a stable phosphatidylalcohol rather than rapidly metabolized PtdOH. Therefore, we measured PLD activity in the presence of butan-1-ol by quantifying the production of phosphatidylbutan-1-ol (PtdBut). After antigen stimulation of RBL-2H3 cells, PLD activity increased rapidly up to 5 min after which point it began to level off (Figure (Figure1A).1A). We have previously shown that this activity is essential for exocytosis (Brown et al., 1998 blue right-pointing triangle), a process that it also known to be PI3-kinase dependent in RBL-2H3 cells (Barker et al., 1998 blue right-pointing triangle). Stimulation of the RBL-2H3 cells in the presence of the PI3-kinase inhibitor, LY294002, reduced PLD activity, indicating that its regulation is also PI3-kinase dependent (Figure (Figure1B).1B). Coincident with PLD-dependent exocytosis from the RBL-2H3 cells, PLD1b translocates to the plasma membrane, indicating that PLD1b activation at the plasma membrane may control exocytosis (Brown et al., 1998 blue right-pointing triangle). An inactive mutant of PLD1b still translocates to the plasma membrane, suggesting that PLD activity is not necessary for its translocation (Brown et al., 1998 blue right-pointing triangle). Similarly, here we find that in the presence of LY294002, upon stimulation (+DNP), a HA-tagged version of PLD1b still translocates to the plasma membrane (Figure (Figure1C).1C). Because in vitro studies indicate that PLD1b alone has little basal activity (Hodgkin et al., 1999 blue right-pointing triangle), it is therefore likely that it becomes activated upon its arrival at the plasma membrane and that these subsequent steps are PI3-kinase dependent.

Antigen–stimulated Colocalization of PLD1b with Rac1, ARF6, and PKCα

It has been shown in vitro that PLD1b is cooperatively activated by a Rho family member, an ARF family member and PKCα or δ (Hodgkin et al., 1999 blue right-pointing triangle). Considerable evidence points to the activation of Rho, ARF, and PKC family proteins through PI3-kinase (Apgar, 1991 blue right-pointing triangle; Guillemot et al., 1997 blue right-pointing triangle; Stam et al., 1997 blue right-pointing triangle; Hong-Geller and Cerione, 2000 blue right-pointing triangle; Venkateswarlu and Cullen, 2000 blue right-pointing triangle; Way et al., 2000 blue right-pointing triangle; Djouder et al., 2001 blue right-pointing triangle; Hong-Geller et al., 2001 blue right-pointing triangle). In antigen-stimulated RBL-2H3 cells, it is therefore possible that PI3-kinase–dependent PLD activity (Figure (Figure1)1) results from PLD1b's association with PI3-kinase–dependent regulators.

In nonstimulated cells (−DNP), confocal sections revealed that PLD1b primarily localized to vesicular compartments, which we have previously identified as secretory granules and secretory lysosomes (Brown et al., 1998 blue right-pointing triangle; Figure Figure2,2, A, B, E, G, H, K, M, and N). At this time, both endogenous (Figure (Figure2A)2A) and transfected (Figure (Figure2B)2B) Rac1 were predominantly detected at a diffuse intracellular localization, distinct from these secretory compartments. After antigen stimulation (+DNP), however, PLD1b colocalized with both endogenous (Figure (Figure2C)2C) and transfected (Figure (Figure2D)2D) Rac1 at the plasma membrane within structures resembling lamellipodia and membrane ruffles, as the cells began to flatten and spread out, and also within intracellular vesicle-like structures. In contrast little colocalization was observed with another Rho family member, RhoA, before (Figure (Figure2E)2E) or after antigen stimulation (Figure (Figure2F).2F).

Previous studies have highlighted a close functional relationship between Rac1 and ARF6 in the control of actin-rich structures at the plasma membrane (D'Souza-Schorey et al., 1997 blue right-pointing triangle; Franco et al., 1999 blue right-pointing triangle; Honda et al., 1999 blue right-pointing triangle; Radhakrishna et al., 1999 blue right-pointing triangle; Tolias et al., 2000 blue right-pointing triangle; Santy and Casanova, 2001 blue right-pointing triangle). In the present study, confocal sections revealed that the distribution of ARF6 in nonstimulated cells (−DNP) was distinct from that of PLD1b, being detectable at both the plasma membrane and an intracellular compartment (Figure (Figure2,2, G and H). However, after antigen stimulation (+DNP) as these cells began to flatten and spread out, both endogenous (Figure (Figure2I)2I) and transfected (Figure (Figure2J)2J) ARF6 colocalized with PLD1b at the plasma membrane, within lamellipodia and membrane ruffles and also intracellularly. The colocalization of PLD1b with ARF6 is not unique to this cell line because we have observed similar results, for example, in HT1080 fibrosarcoma cells (our unpublished results). Equivalent experiments performed with ARF1 highlighted little colocalization with PLD1b before (Figure (Figure2K)2K) or after antigen stimulation (Figure (Figure2L)2L) either within the cell or at the plasma membrane.

As with Rac1 and ARF6, the PKC family of PLD1b regulators have also been shown to regulate actin cytoskeletal reorganizations (Apgar, 1991 blue right-pointing triangle; Frank et al., 1998 blue right-pointing triangle; Job and Lagnado, 1998 blue right-pointing triangle). In correlation with these observations, the PKC family activating compound, phorbol 12-myristate 13-acetate, initiated translocation of PLD1b to the plasma membrane and subsequent exocytosis by the RBL-2H3 cells (Brown et al., 1998 blue right-pointing triangle; Hong-Geller and Cerione, 2000 blue right-pointing triangle; Hong-Geller et al., 2001 blue right-pointing triangle). Similarly to both Rac1 and ARF6, in confocal sections little colocalization was observed between PLD1b and endogenous (Figure (Figure2M)2M) or transfected (Figure (Figure2N)2N) PKCα in nonstimulated cells (−DNP). However, after antigen stimulation (+DNP) as the cells began to flatten and spread out, both enzymes colocalized at the plasma membrane, within structures resembling lamellipodia and membrane ruffles and also intracellularly (Figure (Figure2,2, O and P).

Control experiments performed in the absence of transfected PLD1b showed that Rac1, ARF6, and PKCα still translocated to lamellipodia and membrane ruffles at the plasma membrane, indicating that this phenomenon was not induced by overexpression of PLD1b (our unpublished results). In contrast, transfection of PLD1b and either empty vector did not result in phospholipase and tag colocalization at the plasma membrane, indicating that the colocalization of PLD1b with Rac1, ARF6, and PKCα was a specific event (our unpublished results).

Cotransfection of PLD1b and either Rac1, ARF6, or PKCα Results in Cooperative Increases in Activity

The results in Figure Figure22 show that after antigen stimulation of RBL-2H3 cells PLD1b colocalizes with its activators at the plasma membrane. Colocalization is predominant within actin-rich structures, such as lamellipodia and membrane ruffles, as the cell begins to flatten and spread out. Because each of PLD1b and these activators have been shown to control actin cytoskeletal reorganizations, it appears that colocalization may be functionally significant to the flattening and spreading of the cell. Accordingly, increased PLD activity is coincident with this colocalization, suggesting that it may result from the direct, molecular stimulation of PLD1b by these activators (Figure (Figure1A).1A). Because PLD1b colocalized with transfected as well as endogenous regulators, we therefore hypothesized that transfection of PLD1b and/or its regulators would result in increased, stimulatable PLD activity. Figure Figure33 shows that in comparison to control cells, PLD1b transfected cells exhibited increased activity, whereas cells transfected with either Rac1, ARF6, or PKCα alone exhibited no such increase. In contrast, antigen stimulation of cells cotransfected with PLD1b and either Rac1, ARF6, or PKCα resulted in a further, cooperative increase in PLD activity, above that seen for cells transfected with PLD1b alone.

The specificity of this activation was confirmed using dominant negative mutants of Rac1 and ARF6. Members of the Rho and ARF GTP binding proteins are predicted to be active in the GTP-bound state and inactive in the GDP-bound state. Transfection of PLD1b with either Rac1 or ARF6 dominant negative mutants trapped in the inactive, GDP-bound conformation (Rac1T17N and ARF6T27N) did not result in the same cooperative increases in PLD activity as seen with their wild-type counterparts; indeed, an inhibition was observed with Rac1T17N (Figure (Figure3A).3A). Analysis of these cells using confocal microscopy revealed that after antigen stimulation, PLD1b translocated to the plasma membrane where it partially colocalized with Rac1T17N (Figure (Figure3D),3D), in keeping with the apparent dominant negative effects of Rac1T17N (Figure (Figure3A)3A) but to a lesser extent with ARF6T27N (Figure (Figure3D).3D). Although ARF6T27N-transfected cells exhibited pseudopodia-like extensions, cells transfected with either dominant negative mutant lacked lamellipodia or membrane ruffles and did not flatten and spread to the same extent as wild-type cells after antigen stimulation (Figure (Figure3D).3D). Transfections in the presence of PLD1b and constitutively active mutants of Rac1 and ARF6 (Rac1Q61L and ARF6Q67L) resulted in a higher degree of cell death than other transfections such that it was impossible to accurately assess their effects.

PLD1b Binds Directly and Independently to Rho, ARF, and PKC Family Proteins

Colocalization of PLD1b with each of Rac1, ARF6, and PKCα was coincident with cooperatively increasing PLD activity (Figures (Figures22 and and3).3). Because in vitro studies using purified recombinant proteins have also shown activation of PLD1b by single Rho and ARF family members and PKCα or δ (Hodgkin et al., 1999 blue right-pointing triangle), we hypothesized that this activation is mediated through physical interaction between PLD1b and each regulator. Therefore, the interactions of purified, recombinantly expressed proteins were assessed by surface plasmon resonance (SPR). Figure Figure4A4A shows that recombinant Rac1 (1 μM) bound specifically to immobilized PLD1b in the presence of GTPγS (30 μM). Rac1 did not interact with recombinant GST or GST-JNK2, a GST-fusion protein of a size similar to PLD1b. The binding of Rac1 to PLD1b was observed at flow rates of between 5 and 20 μl/min and varying concentrations of Rac1. BSA (10 μM) did not interact with any of the immobilized protein surfaces. Analysis of the kinetics of the interaction between Rac1 and PLD1b using Biaevaluation 3.0 software (Bioevaluation 3, Stevenage, U.K.) gave an apparent KD of ~0.15 μM (Figure (Figure4F).4F). Both Cdc42 and RhoA showed very similar specificity for PLD1b compared with control proteins and similar kinetic parameters (our unpublished results). Figure Figure4B4B shows that the binding of PLD1b to control surfaces and ARF1 in the presence of GTPγS was specific. Measurement affinity of the interaction between Arf1 and PLD1 over a range of concentrations gave an apparent KD of ~0.7 μM (Figure (Figure4F).4F). PLD1b did not interact with immobilized carbonic anhydrase (a protein of similar size to ARF1) or with BSA. Neither GST nor GST-JNK2 interacted with the immobilized ARF1 or the control proteins. The binding of PKCα to PLD1b was observed in the absence of exogenous phorbol ester, ATP, or GTPγS and was independent of changes in flow rate. Figure Figure4C4C illustrates that recombinant PKCα (500 nM) bound specifically to the PLD1b surface but did not interact with GST or GST-JNK2 surfaces. Kinetic analysis of the binding of PKCα to PLD1b over a range of concentrations gave an apparent KD of ~50 nM. Both specificity data and kinetic data were confirmed at a range of flow rates.

Figure 4
The formation of a multicomponent complex consisting of PLD1b, ARF1, Rac1, and PKCα. The interactions between Rac1 (A), ARF1 (B), and PKCα (C) in the presence of GTPγS and PLD1b were quantified using SPR. In A and C PLD1b was immobilized ...

PLD1b is cooperatively activated by single members from each of the Rho, ARF, and PKC families. In subsequent SPR experiments, purified, recombinant GTPases (1 μM) and purified, recombinant PKCα (100 nM) were sequentially injected across both PLD1b and GST surfaces and interactions monitored by SPR. Under these conditions, the activating regulatory proteins bound directly and independently to PLD1b and a multicomponent complex was generated (Figure (Figure4D).4D). Figure Figure4E4E illustrates that the order of addition of each activating protein does not affect their binding to PLD1b during the formation of this complex, and Figure Figure4F4F indicates the apparent affinities of the interactions between each regulator and PLD1b.

The Majority of PLD1b Is Triton Insoluble

Because PLD1b can directly and independently interact with members from each of its regulator families (Figure (Figure4),4), the antigen-stimulated colocalization of PLD1b with each of Rac1, ARF6, and PKCα (Figure (Figure2)2) coupled to the coincident, cooperative increase in PLD activity (Figures (Figures1A1A and and3)3) indicates a direct interaction between PLD1b and its regulators in RBL-2H3 cells. To further examine this, RBL-2H3 cells were cotransfected with HA-tagged PLD1b and either of the GFP-tagged versions of Rac1, ARF6, or PKCα. After 5 min of antigen stimulation, the cells were lysed in Triton X-100–containing buffer, and PLD1b was immunoprecipitated from the Triton-soluble fraction using the anti-HA antibody, 12CA5. Western blotting of these samples using the respective Rac1-, ARF6-, or PKCα-specific antibodies revealed that only ARF6 coimmunoprecipitated with PLD1b in both its GFP-tagged and endogenous forms, indicating the possibility of a direct interaction in RBL-2H3 cells (Figure (Figure5A).5A).

Coimmunoprecipitation of PLD1b with Rac1 or PKCα may have been impossible because Western blotting revealed that the majority of PLD1b was present in the nonimmunoprecipitatable, Triton-insoluble fraction of the RBL-2H3 cells. In contrast, Rac1 and PKCα were found within both the Triton-soluble and -insoluble fractions (Figure (Figure5B).5B). In many hematopoetic cell lines, including RBL-2H3s, the actin cytoskeleton is a major constituent of the Triton-insoluble fraction (Frigeri and Apgar, 1999 blue right-pointing triangle; Hodgkin et al., 1999 blue right-pointing triangle; Iyer and Kusner, 1999 blue right-pointing triangle). Previously it has been shown that reorganizations of the actin cytoskeletal can be controlled by PLD (Ha and Exton, 1993 blue right-pointing triangle; Cross et al., 1996 blue right-pointing triangle; Hastie et al., 1998 blue right-pointing triangle) as well as each of Rac1, ARF6, and PKC (Apgar, 1991 blue right-pointing triangle; D'Souza-Schorey et al., 1997 blue right-pointing triangle; Frank et al., 1998 blue right-pointing triangle; Hall, 1998 blue right-pointing triangle; Job and Lagnado, 1998 blue right-pointing triangle; Franco et al., 1999 blue right-pointing triangle; Radhakrishna et al., 1999 blue right-pointing triangle; Tolias et al., 2000 blue right-pointing triangle). Furthermore, Figure Figure22 shows that as these cells begin to flatten and spread out, PLD1b colocalizes with Rac1, ARF6, and PKCα in structures resembling lamellipodia and membrane ruffles. These structures are rich in filamentous actin (F-actin), and consistent with these data, after antigen stimulation we found that in confocal sections PLD1b colocalized with F-actin at the cell cortex and also on the dorsal surface of the RBL-2H3 cells (Figure (Figure5C,5C, 1–2 and 5–8 min).

DISCUSSION

In this report we provide the first in vivo evidence for a pathway from receptor occupation at the cell surface to the direct, molecular activation of PLD1b. Antigen stimulation of RBL-2H3 cells promotes the selective colocalization of PLD1b with Rac1, ARF6, and PKCα in actin-rich structures at the plasma membrane as PLD activity increases. When PLD1b is cotransfected with each of these regulators, colocalization is coincident with a further specific, cooperative increase in PLD activity. Consequently, the demonstration here that PLD1b binds directly and independently to its regulators indicates that this activity increase results from PLD1b's physical interaction with and subsequent activation by Rac1, ARF6, and/or PKCα.

The results in Figure Figure44 illustrate that the interactions between PLD1b and its activators are not only specific and direct (Figure (Figure4,4, A–C) but also independent of each other (Figure (Figure4,4, D and E). These data highlight that conserved regions from each family interact with distinct but as yet incompletely defined domains of PLD1b; it has been suggested that Rho proteins interact between residues K946 and K962 and at I870 (Du et al., 2000 blue right-pointing triangle; Cai and Exton, 2001 blue right-pointing triangle), PKC proteins interact around residue E87 (Zhang et al., 1999 blue right-pointing triangle), whereas the location of the ARF binding site is unclear, but they do not interact within the N-terminal or the loop region (Sung et al., 1999 blue right-pointing triangle). Here we show that the propensity for such interactions to occur in vivo is determined by spatially directed and temporally coordinated targeting (Figure (Figure2).2). The specificity profile of interaction is consistent with previous experiments illustrating that PLD1 is stimulated by Rho, ARF, and PKC proteins (Hodgkin et al., 1999 blue right-pointing triangle; for review see Exton, 1999 blue right-pointing triangle). Between 1 and 5 μM of any Rho family member is required to maximally stimulate PLD1 activity in vitro, and our data regarding the affinity of the interactions between PLD1b and Rho family members are consistent with this (Hodgkin et al., 1999 blue right-pointing triangle; Figure Figure4).4). The binding of Rho and ARF proteins to PLD1b, detected by SPR, was surprisingly not dependent on their GTP loading (our unpublished results); however, the in vitro activation of PLD1b requires GTP (Hodgkin et al., 1999 blue right-pointing triangle). It is possible that the GTPases bind GTP when associated with PLD1b and this induces activation, mediated through a conformational change. The concept that the binding of a GTPase to its effector increases its GTP binding has been previously demonstrated for ARF proteins both in vivo and in vitro (Zhu et al., 2000 blue right-pointing triangle). The results in Figure Figure33 support this possibility because GDP-ligated Rac1 and ARF6 mutants inhibited PLD activation, presumably by binding to the phospholipase.

Although our data show PLD1b's initial regulation after FcεRI-ligation of RBL-2H3 cells, it is also possible that after activation of different signaling pathways in response to diverse stimuli, more prolonged stimuli or in other cell types, PLD1b is regulated by different regulators than those identified here. The activation of PLD1b by different regulatory proteins may help explain the many cellular functions attributed to this enzyme/activity. Consistent with this, in COS-7 cells PLD1b directly interacts with a constitutively active mutant of RhoA (Cai and Exton, 2001 blue right-pointing triangle).

In stimulated cells, transfected PLD1b colocalizes with endogenous Rac1, ARF6, and PKCα (Figure (Figure2,2, C, I, and O, respectively). Coincidently PLD activity increases to a level above that seen for control cells (Figure (Figure3).3). In contrast, the PLD activity of Rac1-, ARF6-, or PKCα-transfected cells was similar to that of control cells (Figure (Figure3).3). Because PLD1b has little basal activity, these results indicate that transfected PLD1b was accessible to endogenous activators, which must therefore be in excess of endogenous, available PLD1b. Accordingly, cotransfection of PLD1b with either Rac1, ARF6, or PKCα resulted in a further cooperative increase in PLD activity, above that seen for cells transfected with PLD1b alone (Figure (Figure3)3) and coincidently with their colocalization (Figure (Figure2D,2D, D,2J2J and and2P).2P). Thus the increase in PC hydrolysis results from specific activation of PLD1b by these transfected regulators as dominant negative versions of Rac1 and ARF6 (Rac1T17N and ARF6T27N) did not result in a similar increase (Figure (Figure3,3, A and B); indeed, Rac1T17N mediated a decrease in endogenous PLD activity (Figure (Figure3A).3A). From these findings we consider that PLD1b levels in the cell are a limiting factor controlling the magnitude of PLD activation, whereas the endogenous levels of the regulatory proteins could support further PLD activation. It is only when PLD1b is overexpressed and its levels are in excess of endogenous, available Rac1, ARF6, and PKCα that overexpression of these regulators can mediate a further increase in PLD activity. Therefore the excess level of endogenous regulators in RBL-2H3s perhaps suggests that under the appropriate conditions, they may be able to stimulate larger PLD responses after an increase in transcription/translation of PLD1b.

Although our experiments do not allow us to define the precise makeup of PLD1b/regulator complexes at a given time or location, SPR indicates that PLD1b can simultaneously bind a member from all three regulators (Figure (Figure3,3, D and E). In the presence of all three regulators in vitro, activation is 40-fold higher than in the presence of individual regulators (Hodgkin et al., 1999 blue right-pointing triangle). If the in vivo activation of PLD1b results in similar activity increases, the formation of different PLD1b/regulator complexes would provide a highly amplifiable mechanism for regulating the PLD1b activity necessary for different functions and may be one reason why cells normally only require such relatively low levels of PLD1b.

Previously we found that a catalytically inactive mutant of PLD1b was still able to translocate to the plasma membrane (Brown et al., 1998 blue right-pointing triangle). Similarly, although we find that PLD activity is diminished in the presence of the PI3-kinase inhibitor LY294002, PLD1b was still able to translocate to the plasma membrane (Figure (Figure1C).1C). Therefore, because activity is not required for translocation, PLD1b must be activated when at the plasma membrane. In RBL-2H3 cells, stimulation promotes the activation of Rho, ARF, and PKC family proteins (Apgar, 1991 blue right-pointing triangle; Guillemot et al., 1997 blue right-pointing triangle; Way et al., 2000 blue right-pointing triangle; Hong-Geller et al., 2001 blue right-pointing triangle). Here, subsequent to stimulation, PLD1b colocalizes with Rac1, ARF6, and PKCα as its activity increases (Figure (Figure2).2). In the case of Rac1 and ARF6, activation results from the exchange of GDP for GTP (Djouder et al., 2001 blue right-pointing triangle). Our data showing that PLD activity is not stimulated in the presence of GDP-ligated, mutants of Rac1 and ARF6 (Figure (Figure3,3, A and B) indicate that these GTPases must be able to become active by cycling through the GTP-bound conformation in order to activate PLD1b. Because the exchange of GDP for GTP on these GTPases has been shown to be PI3-kinase dependent, their stimulation presumably accounts for the PI3-kinase dependence of PLD1b activation.

Coimmunoprecipitation experiments in the RBL-2H3 cells revealed PLD1b in complex with ARF6 (Figure (Figure5A).5A). The proportion of total ARF6 immunoprecipitated with PLD1b was low, probably because the majority of PLD1b was present in the Triton-insoluble fraction of the RBL-2H3 cells such that it cannot be immunoprecipitated (Figure (Figure5B).5B). This may also be the reason that PLD1b did not coimmunoprecipitate with Rac1 and PKCα. As with PLD1b in RBL-2H3 cells, a Rho-, ARF-, and PKC-regulated PLD has been identified in the Triton-insoluble fraction of HL60 and U937 cells (Hodgkin et al., 1999 blue right-pointing triangle; Iyer and Kusner, 1999 blue right-pointing triangle). Rac1, ARF6, PKC, and PLD all have the ability to mediate actin reorganization, and therefore it is consistent for the majority of the F-actin to also be located within the Triton-insoluble fraction of these three cell lines (Frigeri and Apgar, 1999 blue right-pointing triangle; Hodgkin et al., 1999 blue right-pointing triangle; Iyer and Kusner, 1999 blue right-pointing triangle).

In RBL-2H3 cells, actin polymerization proceeds with kinetics similar to those of PLD1b activation (Frigeri and Apgar, 1999 blue right-pointing triangle; Figures Figures1A1A and and3).3). Coincidently, as the cells flatten and spread out, PLD1b colocalizes with F-actin at the plasma membrane within actin-rich lamellipodia and membrane ruffles (Figures (Figures22 and and5C).5C). These structures are synonymous with spreading and migration (Hall, 1998 blue right-pointing triangle), and here it was observable that PLD1b-transfected cells spread out more rapidly than vector alone or nontransfected cells. Consistent with this, cotransfection of PLD1b with dominant negative Rac1 or ARF6 caused a reduction in both PLD activity and the ability of these cells to flatten and spread out after stimulation (Figure (Figure3).3). Fittingly therefore, inhibition of PLD after the addition of butan-1-ol or the PI3-kinase inhibitor, LY294002, is also known to negatively regulate the spreading and migration processes (Powner and Wakelam, unpublished observations; Santy and Casanova, 2001 blue right-pointing triangle).

Previous studies have indicated that Rac1 and ARF6 control actin cytoskeletal rearrangements via distinct pathways (D'Souza-Schorey et al., 1997 blue right-pointing triangle; Boshans et al., 2000 blue right-pointing triangle; Santy and Casanova, 2001 blue right-pointing triangle). Because Rac1, ARF6, and PKCα can all independently activate PLD1b (Hodgkin et al., 1999 blue right-pointing triangle), this phospholipase may be a point of convergence for each of its regulators' respective abilities to control these rearrangements. Indeed, in the absence of a stimulus, the addition of PtdOH alone mediates actin reorganizations (Cross et al., 1996 blue right-pointing triangle). PtdIns(4,5)P2 is known to be a major factor governing actin cytoskeletal rearrangements and each of Rac1-, ARF6-, and PLD-produced PtdOH have been implicated in production of this lipid through the activation of type I α phosphatidylinositol 4-phosphate 5-kinase (Jenkins et al., 1994 blue right-pointing triangle; Divecha et al., 2000 blue right-pointing triangle; Tolias et al., 2000 blue right-pointing triangle). Hence, we hypothesize that the localized production of PtdIns(4,5)P2 may be responsible for at least some of Rac1, ARF6, PKCα, and PLD1b's effects on the actin cytoskeleton.

In conclusion, we formulate the first in vivo model for PLD1b activation. In RBL-2H3 cells, receptor cross-linking initiates a tyrosine kinase cascade, through Lyn and Syk leading to activation of class Ia PI3-kinase (Scharenberg and Kinet, 1994 blue right-pointing triangle; Barker et al., 1998 blue right-pointing triangle). The subsequent production of PtdIns(3,4,5)P3 contributes to the stimulation of PLCγ1 through its PH domains (Barker et al., 1998 blue right-pointing triangle), while also activating PH-domain–containing GEFs for both Rac1 and ARF6. The generation of diacylglycerol and release of calcium as a consequence of PLCγ1 stimulation leads to PKCα activation, whereas GEF stimulation catalyzes the GTP-loading and therefore activation of Rac1 and ARF6. PLD1b translocates to the plasma membrane and becomes associated with PtdIns(4,5)P2 via its PH domain. Spatially directed and temporally coordinated targeting of the activated regulators to the plasma membrane results in their binding to and activation of PLD1b.

ACKNOWLEDGMENTS

We thank M. Aggarwal for help with early experiments. ARF cDNAs were a gift from N. Thompson (GlaxoWellcome). PKCα cDNA was a gift from Peter Parker (ICRF). Rho cDNAs were a gift from N. Akhtar (Biosciences, Birmingham University). The ARF6 antibody was a gift from J.G. Donaldson (NIH). This study was supported by a grant from the Wellcome Trust. M.N.H. is a Beit Memorial Research Fellow.

Abbreviations used:

ARF
ADP-ribosylation factor
DNP
dinitrophenol
FcεRI
high-affinity immunoglobulin E receptor
F-actin
filamentous actin
HA
hemaglutinin
PtdOH
phosphatidic acid
PtdBut
phosphatidylbutan-1-ol
PKC
protein kinase C
PtdIns(4,5)P2
phosphatidylinositol 4,5-bisphosphate
PtdIns(3,4,5)P3
phosphatidylinositol 3,4,5-trisphosphate
PLC
phospholipase C
PLD
phospholipase D
RBL
rat basophilic leukemia

Footnotes

Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.01–05–0235. Article and publication date are at www.molbiolcell.org/cgi/doi/10.1091/mbc.01–05–0235.

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