Direct Modulation of Phospholipase D Activity by G??
A. M. Preininger, L. G. Henage, W. M. Oldham, E. J. Yoon, H. E. Hamm, and
H. A. Brown
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee
Received December 4, 2005; accepted April 25, 2006
Phospholipase D-mediated hydrolysis of phosphatidylcholine
is stimulated by protein kinase C and the monomeric G proteins
Arf, RhoA, Cdc42, and Rac1, resulting in complex regulation of
this enzyme. Using purified proteins, we have identified a novel
inhibitor of phospholipase D activity, G?? subunits of hetero-
trimeric G proteins. G protein-coupled receptor activation alters
affinity between G? and G?? subunits, allowing subsequent
interaction with distinct effectors. G?1?1inhibited phospho-
lipase D1 and phospholipase D2 activity, and both G?1?1and
G?1?2inhibited stimulated phospholipase D1 activity in a dose-
dependent manner in reconstitution assays. Reconstitution as-
says suggest this interaction occurs through the amino termi-
nus of phospholipase D, because G?1?1is unable to inhibit
an amino-terminally truncated phospholipase D construct,
PLD1.d311, which like full-length phospholipase D isoforms,
requires phosphatidylinositol-4,5-bisphosphate for activity.
Furthermore, a truncated protein consisting of the amino-ter-
minal region of phospholipase D containing the phox/pleckstrin
homology domains was found to interact with G?1?1, unlike the
PLD1.d311 recombinant protein, which lacks this domain. In
vivo, expressed recombinant G?1?2was also found to inhibit
phospholipase D activity under basal and stimulated conditions
in MDA-MB-231 cells, which natively express both phospho-
lipase D1 and phospholipase D2. These data demonstrate that
G?? directly regulates phospholipase D activity in vitro and
suggest a novel mechanism to negatively regulate phospho-
lipase D signaling in vivo.
Phospholipase D (PLD) mediates the regulated hydrolysis
of phosphatidylcholine (PC), producing the second messenger
phosphatidic acid (PA). PA can be further metabolized to two
other signaling lipids, lysophosphatidic acid (LPA) and diac-
ylglycerol, through actions of a phospholipase A and lipid
phosphate phosphohydrolase, respectively. There are two
mammalian PLD isoforms, PLD1 and PLD2, both with splice
variants. PLD1 is found at plasma membranes and intracel-
lular membranes, including Golgi and nuclear membranes
(Liscovitch et al., 1999). PLD2 is predominantly localized to
the plasma membrane and is found in membrane fractions
containing caveolin (Xu et al., 2000). PLD1 is known to be
regulated by PKC, Arf, and Rho family proteins. Although
PLD2 was initially described as constitutively active, stimu-
lation by Arf and PKC have been reported (Lopez et al., 1998;
Chen and Exton, 2004) as well as modulation by inhibitory
factors (Jenco et al., 1998; Lee et al., 2001). Both PLD1 and
PLD2 share a common domain structure, consisting of
PX/PH and catalytic domains. The PX/PH domains in the
amino terminus contribute to membrane and lipid binding,
and this region is also known to participate in PKC-mediated
activation of PLD, although carboxyl-terminal regions have
been implicated as well. The amino-terminal region is also
thought to be autoinhibitory, because removal of this domain
increases the basal activity of the protein (Sung et al.,
1999a,b). The catalytic domains contain characteristic His-
Lys-Asp motifs and encompass regions implicated in lipid
binding. The lipid binding regions are important to both PLD
isoforms, because both PLD1 and PLD2 are membrane-asso-
ciated, use PC as substrate, and depend on PIP2for their
activity. The carboxyl terminus of PLD is implicated in in-
teractions with RhoA, and this well conserved region con-
tains residues critical for the enzymatic activity of the pro-
tein (Sung et al., 1999b).
A relatively small number of negative regulators of PLD
have been identified, including synucleins (Jenco et al.,
This work was supported by National Institutes of Health grant GM58516.
Article, publication date, and citation information can be found at
ABBREVIATIONS: PLD, phospholipase D; PC, phosphatidylcholine; PA, phosphatidic acid; LPA, lysophosphatidic acid; PKC, protein kinase C;
PX, phox; PH, pleckstrin homology; PIP2, phosphatidylinositol-4,5-bisphosphate; PLC, phospholipase C; GPCR, G protein-coupled receptor;
SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; h, human; Sf, Spodoptera frugiperda; DTT, dithiothreitol; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; MBP, maltose binding protein; N-PLD, amino-terminal phospholipase D; MIANS,
M8, 2-(4?-maleimidylanilino)naphthalene-6-sulfonic acid; PMA, phorbol 12-myristate 13-acetate; r, recombinant; mPLD, membranes from Sf21
cells expressing phospholipase D; GRK, G protein-coupled receptor kinase; SNAP, soluble N-ethylmaleimide-sensitive factor attachment protein.
Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 70:311–318, 2006
Vol. 70, No. 1
Printed in U.S.A.
1998), amphiphysins, clathrin assembly protein (Lee et al.,
2000), and munc-18-1 (Lee et al., 2004). In this work, we
identify G?? as a novel inhibitor of PLD activity, using pu-
rified proteins to demonstrate this direct effect on PLD. G??
is known to regulate a number of effector proteins, including
adenylyl cyclase, PLC-?, phosphatidylinositol 3-kinase, and
potassium and calcium channels, among others. The reduced
affinity of G?? for activated G? subunits upon receptor acti-
vation reveals new and distinct sites for protein-protein in-
teraction on G? and G?? subunits, which regulate GPCR
signaling cascades. PLD is the downstream target of a num-
ber of GPCRs, including LPA receptors (Kim et al., 2004),
endothelial differentiation gene/sphingosine 1-phosphate re-
ceptors (Meacci et al., 2003), and M3muscarinic receptors
(Nieto et al., 1994). In recent years, the number of G??
effectors has grown to include those involved in neurotrans-
mitter release (SNAREs; Blackmer et al., 2005), guanine
nucleotide exchange factor activity (P-Rex1, a guanine nucle-
otide exchange factor for Rac1; Hill et al., 2005), and clathrin-
mediated endocytosis (tubulin; Popova and Rasenick, 2004).
We now extend that list to include PLD, which has been
shown to play roles in vesicle transport (Roth et al., 1999),
trafficking and exocytosis (for review, see Jones et al., 1999),
cell migration, proliferation, and tumor formation (for re-
view, see Foster and Xu, 2003; Buchanan et al., 2005).
Materials and Methods
Protein Expression. Expression and purification of recombinant
hPLD1, Cdc42, PKC?, RhoA, and Arf were carried out as described
previously (Walker et al., 2000). Recombinant PLD isoforms from
membranes were obtained from monolayers of Spodoptera frugi-
perda (Sf) 21 cells infected with baculovirus encoding human PLD1
and PLD2, respectively, as described previously (Gidwani et al.,
G?1?1subunits were prepared from holotransducin as described
previously (Mazzoni et al., 1991). Purified proteins were stored at
?80°C in a buffer containing 10 mM Tris, pH 7.5, 100 mM NaCl, 5
mM ?-mercaptoethanol, and 10% glycerol. G?1?2subunits were ob-
tained from recombinant baculovirus expression in Sf9 cells as de-
scribed previously (Ford et al., 1998) and stored in 50 mM Tris, 100
mM NaCl, 1 mM EDTA, 3 mM DTT, and 0.03% CHAPS. Before use,
detergent was removed, and G?? subunits were concentrated in
buffer containing 50 mM Tris, 50 mM NaCl, and 1 mM DTT, pH 7.5,
by ultrafiltration (Vivascience, Stonehouse, UK).
PLD1.d311 consisting of amino acids 312 to 1036 of rat PLD1b
(with N-terminal MBP affinity tag) was expressed in baculovirus
infected Sf21 cells. Recombinant PLD1.d311 protein was extracted
in detergent and purified by fast-performance liquid chroma-
tography as described previously (Henage et al., 2006). In brief,
sequential metal-chelating chromatography, size exclusion chroma-
tography, and ultrafiltration steps were used to purify PLD1.d311 to
Purified PLD1.d311, amino-terminal PLD (N-PLD) domains, G??
subunits, and Arf, Cdc42, and RhoA proteins were shown to be ?85%
pure by SDS-polyacrylamide gel electrophoresis followed by Coomas-
sie staining, as shown in Henage et al. (2006) for various proteins
used in this study, and Fig. 1C for G?? subunits, PLD.d311, and
N-PLD. The protein concentrations of recombinant proteins were
determined with Bradford reagent (Pierce Chemical, Rockford, IL)
using bovine serum albumin as a standard. PLD1 and PKC? en-
zymes were prepared as described previously (Brown et al., 1995;
Henage et al., 2006). Given the low concentrations of these enzymes
in purified form, estimated from immunoblot analysis, their suitabil-
ity for use in exogenous assays was further evaluated by their ability
Fig. 1. G?? inhibits full-length PLD1 basal activity. A, G?1?1effect on basal
PLD activity in an exogenous substrate assay (Brown et al., 1995). Left
columns, 1 ?M G?? and 1.6 nM purified PLD1, mean basal PLD1 activity
was 1.9 pmol of PC hydrolyzed/30 min. Right columns, 2 nM PLD1.d311
(D311 PLD), 5 ?M G??; mean basal PLD1.d311 activity was 10.5 pmol of PC
hydrolyzed/30 min. Data are mean ? S.E.M. from three independent exper-
N-PLD. C, G??, PLD1.d311, and N-PLD proteins used in this study were
sie Blue. Left, G?? proteins (5 ?g of total protein) were analyzed using a 10%
Tris-glycine gel. Middle, PLD1.d311 and N-PLD with N-terminal MBP af-
finity tags (see Materials and Methods) results in molecular masses of ?130
and 90 kDa, respectively. PLD1.d311 (2 ?g of total protein) was analyzed
Preininger et al.
to support PLD hydrolysis in the exogenous PLD assay described
below; in the case of PLD1, this value ranges from 1 to 10 pmol of PC
hydrolyzed per 30 min without addition of any activators (basal
activity). For activator PKC?, the ability to stimulate PLD-mediated
PC hydrolysis was 30 to 40 times greater than basal PLD measured
without activator stimulation.
The amino-terminal 331-amino acid region of PLD1 (N-PLD) con-
taining the PX/PH domain of PLD1 was expressed as a hexahisti-
dine-tagged MBP fusion protein in Escherichia coli, purified by nick-
el-chelating chromatography, and stored in buffer containing 50 mM
Tris, 150 mM NaCl, and 2 mM ?-mercaptoethanol. The N-terminal
sequence from rat PLD1 (residues 3–330) was amplified by polymer-
ase chain reaction using the following primers: 5?-GACTTGCGCAC-
CAGTGCTTCTGGATGAAC-3?. The amplified PCR product was
digested with FspI and HindIII and cloned in frame into the psV282
expression vector (courtesy of Laura Mizoue, Vanderbilt Center for
Structural Biology, Nashville, TN) to generate a PLD1 fragment
corresponding to amino acids 3 to 330 with N-terminal hexahistidine
and maltose-binding protein purification tags. The N-terminal PLD1
protein was expressed in BL21 (DE3) Rosetta E. coli (Novagen,
Madison, WI) and purified by nickel-chelating chromatography. One-
liter cultures were grown in Luria broth (30 ?g/ml kanamycin and 34
?g/ml chloramphenicol). Expression was induced at optical densi-
ty600? 0.9 with 0.3 mM isopropyl-1-thio-?-D-galactopyranoside. Cul-
tures were grown overnight at 25°C and harvested by centrifugation.
Cells were resuspended in 10 ml of lysis buffer [50 mM phosphate
buffer, pH 7.5, 250 mM NaCl, 1 mM MgCl2, 0.5 mM DTT, 2 mM
phenylmethylsulfonyl fluoride, 1% (w/v) n-octyl-?-D-glucopyrano-
side, and complete protease inhibitor cocktail (Roche Diagnostics,
Indianapolis, IN)] and lysed by incubation on ice with 10 mg/ml
lysozyme and sonication (6 ? 20-s pulses at 6W RMS). Insoluble
material was removed by centrifugation (40,000g for 30 min at 4°C),
and lysate was further clarified by centrifugation (100,000g for 1 h at
4°C). Clarified lysate was applied to a 1-ml HiTrap Chelating HP
column (GE Healthcare, Little Chalfont, Buckinghamshire, UK) in
25 mM phosphate buffer, pH 7.5, 150 mM NaCl, 0.2 mM DTT, and
0.2% (w/v) n-octyl-?-D-glucopyranoside. Purified (?90%) N-PLD
eluted at 220 mM imidazole in a linear imidazole gradient. Detergent
and DTT were removed from pooled fractions, and protein was con-
centrated to 1.5 mg/ml by ultrafiltration (Vivascience). Protein ali-
quots were frozen in liquid nitrogen and stored at ?80°C.
Exogenous PLD Activity Assay. PLD activity was measured in
an exogenous substrate assay as described previously (Brown et al.,
1995). The hPLD1 source for the exogenous assays was generated
with an SP-Sepharose-purified fraction of Sf21 cytosol expressing
hPLD1 and is described in detail in Walker et al. (2000). Reactions
were incubated for 30 min at 37°C with 10 ?M guanosine 5?-O-(3-
thio)triphosphate in the presence of lipid vesicles containing [3H]di-
palmitoylphosphatidylcholine as well as phosphatidyletholamine,
unlabeled phosphatidylcholine, cholesterol, and PIP2as described
previously (Brown et al., 1995). Activity was measured by scintilla-
tion counting of the soluble [3H]choline released as a result of PLD-
mediated PC hydrolysis.
Fluorescent Labeling of Proteins and Spectrofluorometric
Assays. G?1?1subunits were exchanged into labeling buffer (50 mM
Tris and 150 mM NaCl, pH 7.5) before modification with fluorescent
probe. MIANS (M8) [2-(4?-maleimidylanilino)naphthalene-6-sulfonic
acid] was used to label G?1?1subunits for fluorescent assays. In
brief, G?? was incubated with a 5-fold molar excess of M8 at 4°C for
4 h before quenching with 5 mM ?-mercaptoethanol, followed by
ultrafiltration to remove unreacted probe from labeled protein. Be-
cause of the presence of multiple Cys residues, stoichiometry of
labeling for G?? was between 1.5 and 3.2 mol M8/mol G?? (M8
extinction coefficient 27,000 cm?1M?1, excitation 320 nm, and Brad-
ford assay, respectively). Fluorescence assays were conducted in 50
mM Tris, 50 mM NaCl, and 5 mM ?-mercaptoethanol, pH 7.5, at
excitation/emission wavelengths 320/420 nm, respectively. Emission
increases upon binding of M8-G?1?1to MBP-tagged N-PLD was
compared with basal emission of M8-G?1?1in the absence of inter-
acting proteins, less contributions from MBP-tagged N-PLD alone in
Cell Culture and Transfection. MDA-MB-231 cells (American
Type Culture Collection, Manassas, VA) were maintained in Dulbec-
co’s modified Eagle’s medium supplemented with 10% fetal bovine
serum. Cells were seeded in six-well tissue culture plates (2 ? 105
cells/well) the day before transfection with either empty vector
pcDNA3.1 (control) or the combination of pcDNA 3.1 encoding G?1
and the vector encoding G?2(Chen et al., 2004). Transfections were
performed with a total of 2 ?g of DNA/well and 3 ?l of Lipo-
fectAMINE (Invitrogen, Carlsbad, CA) per microgram of DNA in
0.5% fetal bovine serum. Cells were labeled for in vivo PLD analysis
30 h after transfection.
PLD Activity Measurements in Intact Cells. PLD activity in
cells was measured as described previously (Walker and Brown,
2004). In brief, cells were incubated for 16 h in serum-free media
containing [3H]oleic acid, washed, and then incubated with 0.4%
1-butanol in serum-free Dulbecco’s modified Eagle’s medium for 1 h
at 37°C in 5% CO2to allow formation of phosphatidylbutanol. For
stimulated PLD activity, this incubation was reduced to 15 min
under stimulated conditions. Products were separated by thin layer
chromatography, and bands comigrating with nonradioactive phos-
phatidylbutanol standards were scraped, counted by scintillation,
and counts (less any background measured in the absence of butanol)
were compared with total radioactivity in the extract. For PMA- and
LPA-stimulated PLD activity, the percentage of phosphatidylbuta-
nol formed was compared with basal levels in control (vector-trans-
Immunoblot Analysis. Transient expression of G?1?2was deter-
mined by protein immunoblot analysis. Whole-cell radioimmunopre-
cipitation assay buffer lysates were denatured by boiling cells in
Laemmli sample buffer and then resolved on a 10% Tris-glycine gel
(5 ?g of total protein lysate/lane) and transferred to polyvinylidene
difluoride membranes. Membranes were probed with rabbit anti-G?
antibody and mouse anti-actin antibody to control for protein load-
ing, followed by incubation with appropriate secondary antibody
conjugated to horseradish peroxidase. Detection was performed with
chemiluminescence (GE Healthcare) and imaging with Fluor-S (Bio-
Rad, Hercules, CA).
To determine the effect of G?1?1(G??) on basal, unstimu-
lated PLD1 activity, G?? was incubated with soluble, puri-
fied hPLD1 in complex lipid vesicles containing PIP2, and
PLD activity was measured in this reconstituted system.
[3H]PC was cleaved over 30 min by PLD, and soluble [3H]cho-
line released as a result of PLD activation was recovered and
measured by scintillation counting. As seen in Fig. 1A, G??
inhibited basal activity of purified full-length PLD1. To de-
termine the role of the amino-terminal domain of PLD in this
interaction, which contains the PX/PH domains of PLD, we
used a truncated rPLD1 isoform lacking residues 1 to 311 of
full-length PLD (PLD1.d311) (Fig. 1B). PLD1.d311, like full-
length PLD1, requires PIP2for activity and is regulated by
Arf and Rho family proteins Cdc42, RhoA, and Rac1 (Henage
et al., 2006). This truncated rPLD1 isoform, expressed as a
MBP fusion protein, demonstrates high expression (?100-
fold compared with wild-type hPLD) and robust enzymatic
activity in vitro and in vivo. PLD1.d311 retains sensitivity to
monomeric G proteins, with activation kinetics similar to
that of wild type, but it demonstrates a reduced sensitivity to
PKC?, because of elimination of one of two putative PKC
interaction sites located within the amino terminus. We
Direct Modulation of Phospholipase D Activity by G??
tested the ability of G?? to inhibit both full-length and the
N-terminally truncated PLD isoform PLD1.d311, which are
both PIP2-dependent (Henage et al., 2006). Although full-
length PLD1 was inhibited by a relatively modest G?1?1
concentration (1 ?M), PLD1.d311 was not inhibited by even
higher levels of G?1?1, compared with their respective basal
activities in the absence of G?? (Fig. 1A), suggesting G?1?1
inhibits full-length PLD1.
PLD1 activity is stimulated by PKC?, Arf, and RhoA fam-
ily proteins, and PKC? acts synergistically to activate PLD in
the presence of monomeric G proteins. To determine the
effect of G?? on stimulated PLD activity, PLD1 was incu-
bated with indicated activators (Fig. 2) in the presence and
absence of G?1?1, and PC hydrolysis was measured as indi-
cated above. We found that G?? inhibited PLD-mediated PC
hydrolysis in the presence of each activator tested (Fig. 2),
whereas using boiled G??, G?? storage buffer, or bacterial
PLD had no effect on PC hydrolysis, nor did G?? or activators
alone in the absence of PLD (data not shown). In addition,
synergistic PLD1 activation was also strongly inhibited by
G?1?1(Fig. 2, far right).
Because G?1?1was found to inhibit both basal and stimu-
lated PLD1 activity, we next examined the isoform specificity
of the interaction. Because PLD1 is more sensitive to regu-
lation by monomeric G proteins and PKC activators, PLD2
stimulation by selected activators has also been reported,
albeit to a lesser extent than PLD1 (Lopez et al., 1998; Chen
and Exton, 2004). Membranes from Sf21 cells expressing
PLD1 or PLD2 (mPLD1and mPLD2) were incubated with a
combination of G?? and activators PKC? and Arf. PLD-
mediated PC hydrolysis was measured, relative to basal PLD
activity, for each isoform in the absence of G?? and activa-
tors. We found G?1?1inhibited both PLD1 and PLD2 activity
(Fig. 3) stimulated by Arf and PKC?. Despite higher basal
activity of PLD2 (results normalized to basal), only modest
increases were seen upon activation of PLD2 by PKC and Arf,
consistent with the observation that this isoform is less sen-
sitive to activators (Lopez et al., 1998). Although G?1?1in-
hibited both stimulated PLD1 and PLD2 activity, G?1?1re-
duced stimulated PLD2 activity to a level below that of basal,
an effect not seen with mPLD1, nor with soluble PLD1 stim-
ulated by activators (Fig. 2), suggesting a strong inhibition of
PLD2 by G?? subunits. The G??-mediated inhibition of
PLD1 in membrane preparations is less complete than that
seen using purified PLD1 (compare Fig. 2 with Fig. 3), which
may reflect the accessibility of G?? to the PLD enzyme in a
To determine the potency of inhibition of PLD1 by G??,
increasing concentrations of G?? were used to inhibit PKC-
stimulated PLD1 activity. Because G?? inhibited both basal
and stimulated PLD activity, G??-mediated inhibition of
PLD was measured after stimulation with PKC? (to increase
signal amplitude in these assays). We found that increasing
amounts of G?1?1inhibited stimulated PLD1 activity in a
dose-dependent manner, compared with the maximal activa-
tion in the absence of G?? (Fig. 4). For comparison, G?1?2
was also examined for its ability to inhibit PLD. G?1?2was a
more potent inhibitor of PLD activation than G?1?1(Fig. 4);
Fig. 2. G?? inhibits stimulated PLD1 activities. Comparison of effect of 5
?M G?1?1on 1.6 nM PLD1-mediated PC hydrolysis (mean basal PLD
activity, 3.4 pmol of PC hydrolyzed/30 min) in the presence and absence
of PLD activators 166 nM Arf1, 650 nM PKC?, 65 nM RhoA, and 50 nM
Cdc42, measured by release of soluble [3H]choline from [3H]PC after a
30-min incubation of the indicated proteins at 37°C. Data are mean ?
S.E.M. from three independent experiments. Basal activity is defined as
the amount of PC hydrolysis in the presence of PLD alone.
Fig. 3. G?? inhibits PLD1 and PLD2 activities in membranes. PC hydro-
lysis was measured in the presence and absence of purified G?1?1added
to membranes expressing either PLD1 or PLD2 (26 and 10 ng of total
membrane protein, respectively), amounts chosen to normalize basal
activity (set to 1.0) measured in the absence of activators or G??. For
membrane-derived PLD1, mean basal activity was 9.5 pmol of PC hydro-
lyzed/30 min, and for membrane-derived PLD2, mean basal activity was
21.1 pmol of PC hydrolyzed/30 min. Activities compared with basal upon
addition of activators 18 nM Arf and 1 ?M PKC? and after addition of 4.4
?M G?1?1. Data are the mean ? S.E.M. (n ? 3).
Preininger et al.
G?1?2(1.2 ?M) was sufficient to inhibit PLD1 activation by
50%, compared with 2.4 ?M G?1?1required to mediate the
same level of PLD inhibition.
The higher potency of G?1?2is not surprising; in other
signaling systems, G?1?2has been shown to interact with
higher affinity to effectors than G?1?1. For example, G?1?2
more potently stimulated PLC? (Ueda et al., 1994) and in-
hibited SNARE fusion machinery (Blackmer et al., 2005)
than G?1?1. This may be due largely to post-translation
modifications of these G?? isoforms. G?1?1is farnesylated,
whereas G?1?2is geranylgeranylated, and these modifica-
tions influence membrane association. Geranylgeranylation
of G?1?2may allow this isoform to more effectively bind
phospholipids, thus conferring greater potency toward mem-
brane associated effectors such as PLD.
The ability of G?? ability to inhibit full-length PLD is in
residues of full-length PLD1 have been ablated (Fig. 1). To
further investigate this result, residues 3 to 311 encompassing
the PX/PH of PLD1 domain were expressed and purified as
MBP-fusion proteins (N-PLD), as was PLD1.d311. Because the
PX/PH domain itself is relatively insoluble, creation of a MBP
fusion protein allows for a significant improvement in solubility
and moderate levels of protein expression. Binding of purified
N-PLD to G?? was measured using fluorescently labeled G??.
G?1?1was labeled with M8, a thiol-reactive, environmentally
sensitive fluorescent probe that increases its emission upon a
binding event as a result of an increase in the hydrophobicity of
the environment of the probe. Binding is detected as an in-
creased emission from the labeled protein compared with emis-
sion in the absence of the interacting protein. M8-G?1?1bound
N-PLD in a dose-dependent manner (Fig. 5A), in contrast to
quenched label alone (data not shown). Both the amino-termi-
nally truncated PLD1.d311 and the N-PLD proteins contain an
MBP tag; however, the PLD1.d311 protein did not interact with
labeled G?? (Fig. 5B). Only the N-PLD protein demonstrated
increases in fluorescence upon incubation with M8-G??. These
results indicate the amino-terminal region of PLD is necessary
and sufficient for interaction with G?1?1subunits. PLD1 con-
structs lacking this domain do not interact with G?1?1, consis-
tent with the inability of G?? to regulate PLD1.d311 activity
The ability of G?? to inhibit full-length PLD activity in
vitro, both basal and stimulated (in contrast to the N-termi-
nally truncated PLD1.d311), suggests G?? directly inhibits
PLD. This interaction is likely to be mediated through the
amino-terminal 311-amino acid stretch of PLD1, which binds
to G?? with a dose-response relationship consistent with its
effect on PC hydrolysis as measured in reconstitution assays.
To determine the effect of G?? on PLD activity in cells, we
overexpressed G?1?2in MDA-MB-231 cells (Fig. 6), which
express both PLD1 and PLD2 (Meier et al., 1999). G?? re-
duced basal PLD activity in these cells significantly com-
pared with control (Fig. 6B). Furthermore, G?? modestly
reduced PLD activation in PMA and LPA stimulated MDA-
MB-231 cells (Fig. 6C), indicating G?? may play a modula-
tory role in PLD activation in vivo.
PLD activity is highly regulated by a number of factors,
which together combine to determine signaling output to
Fig. 4. G?1?2inhibits PLD1 with greater potency than G?1?1. PKC? (654
nM) and increasing concentrations of indicated G?? subtypes were incu-
bated with purified PLD1 (1.6 nM) in PC vesicles containing PIP2for 30
min at 37°C in an exogenous substrate assay. Mean basal PLD1 activity,
2.26 pmol of PC hydrolyzed/30 min; maximal PKC?-stimulated PLD1
activity, 95.01 pmol of PC hydrolyzed/30 min. Maximum PKC?-stimu-
lated PLD activation measured in the absence of G??. Data are expressed
as the mean ? S.E.M. (n ? 3).
Fig. 5. G?? binds to the amino-terminal domain of PLD1. Fluorescence of
MIANS-labeled G?1?1(100 nM), excitation/emission 320/420 nm, upon
interaction with increasing concentrations of N-PLD, encompassing the
PX/PH domain of PLD (A) or 2.0 ?M PLD1.d311 (D311) (B), which lacks
the first 311 residues of PLD. Data are expressed as the mean ? S.E.M.
(n ? 3).
Direct Modulation of Phospholipase D Activity by G??
PLD. Although PKC, monomeric G proteins, and PIP2are
well known activators, less is known about the negative
regulation of PLD activity. Because PLD is known to be
involved in a myriad of processes from tumor formation to
exocytosis to membrane remodeling, activation of PLD is
likely to be tightly regulated. Increases in PLD1 expression
and activity were demonstrated in colorectal tumors, com-
pared with normal adjacent tissues, and the same study
identified PLD1 as a downstream effector of oncogenic Ras
transformation (Buchanan et al., 2005).
We found that G?? inhibits PLD activation, and reconsti-
tution assays suggest the inhibition is mediated by the N-
terminal region of PLD. The N terminus of PLD contains
PX/PH domains, which contain palmitoylation sites that,
along with these domains, are important for membrane as-
sociation (Ktistakis et al., 2003). This region is also critical
for interaction with G??, as shown by our direct binding
data. A number of G?? effectors contain PH domains, such as
protein kinase D (Jamora et al., 1999), PLC? (Wang et al.,
2000), and GRK proteins (Carman et al., 2000), to name a
few. PH domains may serve to localize G?? to effector pro-
teins and position distinct sites on G?? for interactions with
other regions on effector molecules to regulate their activity.
A recent study demonstrates that the amino terminal coiled-
coil region of G?? mediates inhibitory contacts with PLC?
catalytic domains, whereas the switch II binding regions on
G?? mediate stimulatory contacts with distinct regions on
PLC? (Chen et al., 2004; Bonacci et al., 2005). Likewise, the
N terminus of PLD may bind G?? and position it for inhibi-
tory interaction with residues important for the catalytic
activity of PLD on a distinct surface of G??. Mutational
studies of residues on G?? mediating the interaction with
PLD will shed light on the molecular determinants of this
interaction. Such studies have revealed that the residues on
G?? mediating effector interactions vary, with some overlap
between effectors (Ford et al., 1998). It will therefore be of
interest to determine which effector proteins can compete
with PLD for binding to G??, or whether simultaneous bind-
ing can occur, resulting in a scaffold of interacting proteins.
The structure of GRK2 in complex with G?1?2(Lodowski et
al., 2003) suggests a signaling scaffold of receptor, GRK2,
and G?? (and G? proteins, which may interact with RGS
domains of GRK2). These interactions lead to fast, efficient
signal termination by localizing players involved in signal
down-regulation into one complex. In this case, G?1?2binds
the PH domain of GRK2, which enhances receptor phosphor-
ylation and assists in orienting the complex toward the mem-
brane and membrane bound effectors.
Because almost all agonists that activate PLD also stimu-
late phosphatidylinositol hydrolysis, and because G?? has
been shown to mediate activation of PLC? isoforms leading
to PKC stimulation, a potent activator of PLD, we used
purified proteins to demonstrate the direct effect of G?? on
PLD. Findings from these in vitro studies are consistent with
our results in MDA-MB-231 cells, which express both PLD1
and PLD2. Overexpression of G?1?2reduces PLD activity in
these cells, suggesting G?? modulates PLD signaling in vivo.
Although the effect seems modest in this cell line, even small
changes in the enzymatic activity of PLD may result in large
downstream effects, as a result of signal amplification, re-
quiring tight regulation of the enzymatic activity of PLD. The
role of G?? may be to fine-tune PLD signaling and maintain
membrane homeostasis under a variety of conditions. G??
may also play a greater modulatory role in other cell types
and under conditions that remain to be tested.
Previous studies by other groups have also suggested some
interaction between PLD and G??. In myometrial homoge-
nates containing PLD, Arf, and heterotrimeric G proteins,
reduction in PLD activity was measured upon treatment
with the heterotrimeric G protein activator AlF4
Fig. 6. Inhibition of PLD activity in vivo. MDA-MB-231 cells were tran-
siently transfected with plasmid DNA encoding either empty vector (con-
trol) or cotransfected with plasmid DNA encoding G?1and G?2. A, trans-
fected MDA-MB-231 cells were analyzed by Western blotting as described
under Materials and Methods. G?1?1protein, 1 ?g, is shown as standard
in lane 3. B and C, PLD activity in transfected cells was measured by the
transphosphatidylation of radiolabeled endogenous substrate to form
phosphatidylbutanol, measured by scintillation counting. Results are the
mean (? S.E.M.) for two separate experiments performed in triplicate.
Asterisk denotes values significantly lower than control. Student’s t test
was used to determine significant differences (two-tailed: ?, p ? 0.02; ???,
p ? 0.001). B, transfected cells were assayed for basal PLD activity over
a 1-h period in the presence of primary butanol. C, PLD activity in
transfected cells measured after a 15-min stimulation by either 1 ?M
PMA or 5 ?M LPA in the presence of primary butanol, compared with
vector control (average 0.15% of total).
Preininger et al.
et al., 2000), which liberates G?? upon activation of hetero-
trimeric G proteins. Dissociation of G? and G?? subunits
may reveal activation-dependent interaction sites on G?? for
PLD. Although these results do not definitively rule out G??
binding to Arf, preventing Arf-mediated activation of PLD,
we demonstrate G?? inhibits both basal PLD and PLD ac-
tivity stimulated by Arf (and other activators). This may
suggest a broader role for G?? in modulating PLD signaling
that does not require indirect regulation through Arf or
Subcellular localization of PLD may regulate its activity in
vivo, modulating interactions with regulators generated at
signaling nodes within the cell. G?? subunits are highly
localized to cell membranes and may well serve as physiolog-
ically relevant modulators of PLD activation. Redistribution
between intracellular membranes may be mediated by spe-
cific lipid binding or protein-protein interactions, which to-
gether regulate PLD activity. PLD1 activity has been de-
tected in multiple cellular membrane fractions, including
Golgi, endoplasmic reticulum, secretory vesicles, and plasma
membrane (for review, see Liscovitch et al., 1999). PLD1
activity is regulated through access to various activators, and
in the absence of such activators, it has a relatively lower
basal activity than PLD2. In PC12 cells, which express pre-
dominantly PLD2, overexpression of the ?2A-adrenergic re-
ceptor impaired PMA stimulation of PLD, which was re-
stored by treatment with either ?2-adrenergic antagonist or
pertussis toxin (Ella et al., 1997). Consistent with these re-
sults, we found that G?1?1inhibited PLD2 (and PLD1) in
vitro. Although PLD2 activity was reduced to a level below
that of basal upon addition of G?? in our reconstitution
assay, this may be due in part to the high affinity of both
PLD2 and G?? for membrane components, whereas PLD1 is
found in both membrane and cytosolic fractions. In vivo, G??
may dissociate from PLD to mediate activation, similar to
other identified PLD inhibitors, such as the cytoskeletal pro-
tein ?-actin (Lee et al., 2001), or neuronal proteins such as
synucleins (Pronin et al., 2000) and munc-18-1 (Lee et al.,
2004), initially identified through the use of reconstitution
assays to characterize their effects on PLD activity.
In a cellular context, G?? and PLD may work in concert to
regulate exocytosis. In Saccharomyces cerevisiae, a chimeric
SNAP-25 ortholog deficient in vesiculation was rescued by
up-regulation of a gene encoding a phosphatidylinositol 4?-
kinase ortholog responsible for the production of phosphati-
dylinositol-4,5-biphosphate, an essential cofactor for PLD
activity. Furthermore, the amino terminus of the SNAP or-
tholog was observed to bind PA, the direct product of PLD
activation (Coluccio et al., 2004), which has been shown to
stimulate vesicle formation (Kaldi et al., 2002). Exocytotic
processes may require PLD activation and PA production to
lower the energy barrier to membrane vesiculation. Modula-
tion of PLD activity may restrict membrane remodeling and
maintain membrane homeostasis, because sustained hydro-
lysis of cationic phosphatidylcholine to the anionic phospho-
lipid PA would be expected to have significant effects on
membrane electrostatics, architecture, and phospholipid con-
tent. G?? may play a role in fine tuning such activity. G?1?2,
highly enriched in brain tissue (Betty et al., 1998), has been
shown to interact with SNAP-25 and syntaxin in SNARE
complexes, inhibiting exocytosis (Blackmer et al., 2005). G??
may regulate both SNAREs and PLD to efficiently inhibit
exocytosis, or it may function to maintain a low level of basal
PLD activity until exocytosis is triggered. Relief of this inhi-
bition by liberation of G??, or in the case of SNARE binding,
by competitive binding of synaptotagmin, could allow fast,
efficient exocytosis to occur.
The data suggest G?? inhibits both basal and activator-
mediated PLD activity, consistent with a direct interaction
between PLD and G??, which our data suggest is mediated
by the N-terminal domain of PLD. This negative regulation
of PLD may play a role in vivo to fine-tune the outcome from
various signaling pathways that converge on PLD to activate
it, or G?? may function to modulate effects of multiple stim-
uli that would be detrimental to membrane homeostasis. It
may further define precisely which subcellular membranes
tolerate sustained activation of PLD. The effects of G?? on
PLD function is likely to be context-dependent and need to be
deconstructed in terms of regulatory molecules (identified in
biochemical assays) that have been shown to directly modu-
late PLD activity. The spatial and temporal distribution of
these regulatory molecules is likely to influence the eventual
output in terms of PLD signaling. Small changes in the
enzymatic activity of PLD may have large effects on signaling
downstream from PLD. Together, these data suggest a pre-
viously unappreciated role for direct G?? modulation of PLD
and present new opportunities for the intersection of these
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Address correspondence to: Dr. H. Alex Brown, Department of Pharmacol-
ogy: 442 RRB, Vanderbilt University School of Medicine, 23rd Ave. South and
Pierce, Nashville, TN 37232-6600. E-mail: email@example.com
Preininger et al.