(Received for publication, June 20, 1996, and in revised form, October 30, 1996)
From the Department of Pharmacological Sciences and
Institute for Cell and Developmental Biology, Stony Brook Health
Sciences Center, Stony Brook, New York 11794-8651, the
Department of Biochemistry, Gifu University School of
Medicine, Tsukasamachi-40, Gifu 500, Japan, § Onyx
Pharmaceuticals, Richmond, California 94806, and the
¶ Department of Chemistry, State University of New
York, Stony Brook, New York 11794-3400
We previously reported the cloning of
a cDNA encoding human phosphatidylcholine-specific phospholipase D1
(PLD1), an ADP-ribosylation factor (ARF)-activated
phosphatidylcholine-specific phospholipase D (Hammond, S. M., Tsung,
S., Autschuller, Y., Rudge, S. A., Rose, K., Engebrecht, J., Morris, A. J., and Frohman, M. A. (1995) J. Biol. Chem. 270, 29640-29643). We have now identified an evolutionarily conserved
shorter splice variant of PLD1 lacking 38 amino acids (residues
585-624) that arises from regulated splicing of an alternate exon.
Both forms of PLD1 (PLD1a and 1b) have been expressed in Sf9 cells
using baculovirus vectors and purified to homogeneity by detergent
extraction and immunoaffinity chromatography. PLD1a and 1b have very
similar properties. PLD1a and 1b activity is Mg2+dependent but insensitive to changes in free
Ca2+ concentration. Phosphatidylinositol
4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate
activate PLD1a and 1b but a range of other acidic phospholipids are
ineffective. PLD1a and 1b are highly responsive to activation by
GTP-S-liganded ADP-ribosylation factor-1 (ARF-1) and can also be
activated to a lesser extent by three purified RHO family monomeric
GTP-binding proteins, RHO A, RAC-1, and CDC42. Activation of PLD1a and
1b by the RHO family monomeric GTP-binding proteins is
GTP-dependent and synergistic with ARF-1. Purified protein
kinase C-
activates PLD1a and 1b in a manner that is stimulated by
phorbol esters and does not require ATP. Activation of PLD1a and 1b by
protein kinase C-
is synergistic with ARF and with the RHO family
monomeric GTP-binding proteins, suggesting that these three classes of
regulators interact with different sites on the enzyme.
Phosphatidylcholine (PC)1-specific phospholipase D enzymes (PLD) are emerging as key components of pathways of cell regulation leading to transduction of extracellular signals, regulation of intracellular protein trafficking and secretion, and control of meiosis in budding yeast (see Refs. 1, 2 for review). The common reaction catalyzed by these enzymes is hydrolysis of PC to form phosphatidic acid (PA) and choline. PA has a number of biological activities including direct regulation of a range of cell-specific target proteins in vitro (3, 4). Dephosphorylation of PA by phosphatidate phosphohydrolase produces diacylglycerol that activates members of the Ca2+- and phospholipid-dependent protein kinase C family (5). Other PA-derived molecules with regulatory properties include lyso-PA and arachidonic acid metabolites, both of which act on specific cell-surface receptors (6, 7).
PLD activities are present in prokaryotic and eukaryotic organisms
including many mammalian tissues and cell lines. cDNA sequences of
several bacterial and plant PLDs have been determined (8, 9). Mutation
or deletion of the Saccharomyces cerevisiae SPO14 gene
produces defects in meiosis, and the spo14 gene product has recently been identified as a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent PC-specific PLD (10-12).
We used sequence shared between this protein and the plant enzymes to
clone a human cDNA encoding a PC-specific PLD which we termed PLD1.
Heterologous expression of this cDNA generated a
PI(4,5)P2-dependent PLD activity that was
stimulated by GTPS-activated ARF-1 (13). The plant, yeast, and human
PLDs share four regions of homology. Two of these are present in
bacterial PLDs from Streptomyces species and presumably contain regions essential for catalysis. Apart from these regions, the
sequences of the proteins diverge widely. Although the PLD enzymes
catalyze the same reactions, these differences in primary structure
suggest that they are regulated in different ways and have
different functions (2, 9).
Regulation of PLD activities in mammalian systems is complex. In intact
cells, a range of agonists acting through G-protein-coupled receptors
and receptor tyrosine kinases stimulate PLD-catalyzed hydrolysis of PC.
In many systems, receptor-mediated activation of PLD appears to be
dependent on a prior activation of inositol lipid-specific
phospholipase C (PLC) and mediated by a protein kinase C
(PKC)-dependent process (14-16). In vitro
studies suggest that PKC regulates PLD by two different mechanisms.
Addition of a purified PKC preparation to membranes from rat liver
produced a modest PLD activation that was independent of ATP; in
addition, a partially purified PLD preparation from porcine brain was
strongly activated by PKC- (and its isolated lipid-binding
regulatory domain) in an ATP-independent manner (17, 18). PKC-
and, to a lesser extent, PKC-
also activate an HL-60 membrane PLD activity in an ATP-independent manner (19). In contrast, similar studies using human neutrophil membranes and a number of purified PKC
isoforms indicate that PKC-catalyzed phosphorylation of a membrane
component is required for PLD activation (20).
Other work has identified roles for monomeric GTP-binding proteins in PLD regulation. Brain extracts appear to contain two PLD activities that can be separated by column chromatography and further distinguished by their differential dependences on PI(4,5)P2 and fatty acids for activity (21, 22). PI(4,5)P2-dependent PLD activities in brain and HL-60 monocytic cells are stimulated by members of the ADP-ribosylation factor (ARF) family of monomeric GTP-binding proteins and several RHO family monomeric GTP-binding proteins including RHO A, RAC-1, and CDC42 (23-28). The ARF proteins are key regulators of protein trafficking that control the assembly of the coatomer complex on the surface of Golgi membranes (29). ARF-activated PLD is enriched in Golgi membranes, and PLD may play a role in ARF-dependent coatomer assembly (30). The RHO proteins control both changes in organization of the actin cytoskeleton and activation of protein kinase cascades leading to gene transcription (31). The role of PLD in these processes is untested. Studies using bacterial toxins implicate the RHO proteins in receptor regulation of PLD in some systems (32, 33). Increases in PLD activity have also been reported in cells transformed by members of the ras oncogene family and by the src nonreceptor tyrosine kinase (34, 35).
The complex regulation of PLD activities reported in intact cells and
the variety of lipid and protein factors capable of PLD activation
in vitro has led to the proposal that distinct mammalian
PLDs exist (21-28). Purification of a 180-kDa oleate-activated PLD
from porcine lung has been described (21), but no mammalian monomeric
GTP-binding protein-regulated PLD has been purified to homogeneity.
During studies of the regulation of PLD1 mRNA levels in HL-60
cells, we identified a second form of PLD1 that arises by alternate
splicing of a 38-amino acid exon. We designate the "long" and
"short" forms of PLD1 as PLD1a and 1b, respectively. The present
study was undertaken to investigate the catalytic and regulatory
properties of PLD1a and 1b. Both proteins have been expressed in insect
cells using baculovirus vectors and purified to homogeneity. Our
results demonstrate that PLD1a and 1b can be activated by
PI(3,4,5)P3, PI(4,5)P2, ARF-1, RHO A, RAC-1,
CDC42, and protein kinase C-. The simplest explanation of our
findings is that this diverse range of regulators exert their effects
by direct interaction with the PLD1 catalyst.
Unless otherwise stated, reagents were from previously noted sources (13). Unlabeled phospholipids were obtained from Avanti Polar Lipids. PI(4,5)P2 was purified from a lipid extract of bovine brain as described (13). Di-C16 PtdIns(3,4,5)P3 was synthesized as described (36).
RNA Extraction and Reverse Transcriptase-PCR Analysis of PLD1 Human, Rat, and Mouse PLD1 SequencesTotal RNA was isolated from HL-60 cells by the acid guanidine thiocyanate method and reverse transcribed using random hexamer mixed primers. Primers used for amplification of hPLD1 were synthesized as follows: primer A (forward), nucleotides 1475-1491 TGGGCTCACCATGAGAA; primer B (reverse), nucleotides 2133-2113 GTCATGCCAGGGCATCCGGGG. Amplification conditions used were 94 °C for 30 s, 60 °C for 1 min, and 72 °C for 1 min at 25-27 cycles. The rat PLD1 sequences were obtained by PCR amplification from a reverse-transcribed rat brain RNA template. The mouse PLD1 sequences were obtained from cDNA clones isolated from a mouse embryo cDNA library.2 The genomic sequence of PLD1 was obtained from an expressed sequence tag corresponding to a partially processed mRNA (accession number R97756[GenBank]).
Purification of Monomeric GTP-binding ProteinsSome of our
experiments also employed human ARF-1 that was bacterially expressed
and purified as described (37) with a final step of hydroxylapatite
chromatography (Bio-Rad, Bio-Gel HTP). Human RHO A, RAC-1, and CDC42
were modified to contain the sequence MEEEEYMPME at the amino terminus.
Human ARF-1 was modified to contain this sequence at the carboxyl
terminus, and these proteins were expressed in Sf9 cells using
baculovirus vectors and purified by affinity chromatography using an
immobilized monoclonal antibody (38). All preparations of monomeric
GTP-binding proteins were estimated to be greater than 90% pure by
SDS-PAGE. The purified monomeric GTP-binding proteins were concentrated
to 1-10 mg/ml using an Amicon pressure concentrator with a PM-10
membrane and stored in aliquots at 80 °C. In some cases, the
monomeric GTP-binding proteins were preactivated by treatment with 5 mM EDTA on ice for 15 min followed by incubation with 10 mM MgCl2, 1 mM GTP
S at 37 °C
for 15 min.
Human PKC- was
expressed in Sf9 cells using a baculovirus vector provided by David
Burns (Parke-Davis) and purified using minor modifications from
published procedures (39). In brief, a 1-liter culture of Sf9 cells
(106 cells/ml) was grown in supplemented Grace's medium
containing 10% fetal bovine serum, antibiotic/antimycotic agents and
infected with recombinant baculovirus at a multiplicity of 10. The
cells were cultured for 48 h, harvested, washed once in
phosphate-buffered saline (PBS), and disrupted by nitrogen cavitation
in buffer containing 25 mM Tris, pH 7.5, 1 mM
dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine (Buffer A). PKC-
was purified from the
supernatant obtained after ultracentrifugation of this extract by
sequential chromatography on Source 15 Q (Pharmacia Biotech Inc.),
threonine-Sepharose 4BCL (prepared as described in Ref. 38), and
phenyl-Superose (Pharmacia). PKC-
was followed during this procedure
by measurements of calcium- and phospholipid-dependent protein kinase activity using histone as substrate. The final PKC-
preparation was homogeneous as determined by SDS-PAGE and silver
staining. The purified PKC-
was concentrated to approximately 0.1 mg/ml using an Amicon pressure concentrator with a PM30 membrane and
stored in aliquots at
80 °C.
Peptides corresponding to residues 1-15 and 525-541
of the sequence of human PLD1 were prepared, conjugated to keyhole
limpet hemocyanin, suspended in saline, and emulsified with Freund's adjuvant. Two rabbits were immunized with the peptide corresponding to
residues 1-15, and two different rabbits were immunized with the
peptide corresponding to residues 525-541 by subcutaneous injection.
After a second immunization, serum was obtained from the animals, and
antibody titers were determined by enzyme-linked immunosorbent assay
using the individual peptide antigens as the solid phase. Antibodies
were affinity purified from serum obtained from the animals with the
highest antibody titers by affinity chromatography using immobilized
peptide antigens as the solid phase as described (40). The purified
antibodies were adjusted to approximately 1 mg/ml and stored as
aliquots in buffer containing 10 mM
NaPO42, 20 mM NaCl, and
0.1 mM NaN3 at
80 °C. These antibodies
recognize PLD1a and 1b by Western blotting and can immunoprecipitate
their antigens under denaturing and nondenaturing conditions. The
peptide antigens were chosen to generate antibodies that can
distinguish PLD1 from a structurally related mammalian PLD enzyme,
PLD2.3
1 mg of a mixture of the two affinity purified antibodies was adsorbed to 0.5 ml of protein A coupled to Sepharose CL4B (Sigma) in phosphate-buffered saline (PBS) for 1 h at room temperature. The resin was washed with 0.2 M Na+ borate, pH 9.0, and the antibodies were covalently linked to the immobilized protein A by reaction with 20 mM dimethylpimelimidate in 0.2 M Na+ borate, pH 9.0, for 30 min at room temperature with constant agitation. The reaction was quenched by washing the resin in 0.2 M ethanolamine, pH 8, after which the resin was washed in 100 mM glycine, pH 3.0, to remove antibodies that had not been covalently attached. The resin was then washed extensively and stored in PBS containing 0.1% NaN3.
Baculovirus Expression and Purification of PLD1Recombinant baculoviruses for expression of PLD1a and 1b were generated, selected, purified, and propagated using methods described in Ref. 41. In brief the PLD1a and 1b cDNAs were inserted into the multiple cloning sites of the pAcHLT and PVL1392 transfer vectors, respectively (Pharmingen and Invitrogen Inc.). Monolayers of Sf9 cells were transfected with mixtures of the PLD1a and 1b transfer vectors and linearized wild-type baculovirus DNA. Recombinant baculoviruses were plaque-purified from media removed from these transfected cells. The pure viruses were amplified by infection of suspension cultures of Sf9 cells, and the high titer virus stocks were stored at 4 °C. All of our studies used Sf9 cells grown at 27 °C in complete Grace's medium supplemented with lactalbumin, yeastolate, and 10% fetal bovine serum containing antibiotic antimycotic agents.
For expression of PLD1a and 1b, monolayers of exponentially growing Sf9
cells (3 × 107 cells/225-cm2 flask,
generally two flasks of cells were used for each purification) were
infected with recombinant baculoviruses viruses at a multiplicity of 10 for 1 h with gentle rocking. The virus-containing medium was
removed and replaced with fresh supplemented Grace's medium. The
infected cells were grown for 48 h; the medium was removed, and
the cells were washed once with ice-cold phosphate-buffered saline. The
cells were lysed on ice by addition of 5 ml/225-cm2 flask
of ice-cold lysis buffer containing 150 mM NaCl, 50 mM Tris, pH 8.0, 1% Nonidet P-40, 1 mM EGTA,
0.1 mM benzamidine, 0.1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml pepstatin A, 10 µg/ml leupeptin. After 30 min on
ice, the cells were scraped up, and the suspension was centrifuged at
50,000 × g for 30 min at 4 °C. The supernatant
obtained (10 ml) was mixed with 0.5 ml of the immunoaffinity resin and
kept at 4 °C with constant agitation for 1 h. The resin was
sedimented by gentle centrifugation, and the supernatant containing
unbound proteins was removed. The resin was washed three times with 25 volumes of lysis buffer. After the final wash, the resin was
resuspended in 5 ml of lysis buffer and placed in a 10-ml Bio-Rad
disposable chromatography column. The resin was washed with 10 ml of 10 mM phosphate buffer, pH 6.8, containing 1%
-D-octylglucoside (
-DOG). Bound protein was eluted
with 100 mM glycine, pH 3.0, containing 1%
-DOG as
3 × 0.5-ml fractions. The eluant was collected on ice into tubes
containing 0.075 ml of 1 M phosphate buffer, pH 8.0. ARF-stimulated PLD activity in these fractions was determined as
described below, and the fractions were also analyzed by SDS-PAGE on
7.5% gels and Western blotting with detection by alkaline
phosphatase-conjugated secondary antibody. We prepared two batches of
immunoaffinity resin and found that these could be re-used many times
with little deterioration in performance.
The basic PLD assay was performed as described
previously using headgroup-labeled PC (13). In some experiments, the
[Ca2+] (using Ca2+/EGTA buffers) and
[Mg2+] of the assay medium were varied. For some assays
the lipid component of the vesicles was altered as indicated. Purified
monomeric GTP-binding proteins and protein kinase C- were included
in some of the experiments. In certain experiments, the monomeric
GTP-binding proteins were pre-activated with GTP
S as described
above. In other cases 50 µM GTP
S was included in the
assays. Reagents were mixed on ice and reactions initiated by transfer
to a 37 °C water bath.
We
designed PCR primer sets to amplify a central fragment of the coding
region of PLD1 and attempted to use these to investigate the regulation
of PLD1 mRNA in HL-60 cells using quantitative reverse
transcriptase-PCR. In addition to a PCR product of the expected size,
we also amplified a smaller fragment. Both fragments were cloned and
sequenced, and the results indicated that the larger product
corresponded to the PLD1 sequence reported (13) and the shorter product
corresponded to an altered form of PLD1 in which 114 nucleotides (38 amino acids) were missing (Fig. 1A). Synthesis of appropriately chosen junction primers permitted
amplification of each product independently (data not shown).
These results could arise from regulated splicing of an optional
38-amino acid exon or from partial processing of an intron which would
imply that our original report described a partially processed mRNA
(13). Examination of the PLD1 genomic sequence at codon corresponding
to amino acid 623 (the 3 junction of the missing sequence) revealed
that an acceptable splice donor site (and 11 nucleotide T/C-rich
sequence followed by any nucleotide and AG) is present and that the
adjacent genomic sequence does not encode the nucleotide sequence found
in either of the PCR products detected (Fig. 1B). We
therefore conclude that the originally reported sequence of PLD1
includes an alternately spliced exon. Subsequent analysis of cDNAs
obtained by screening a HeLa cell cDNA library with a PLD1
nucleotide probe (13) revealed that two clones obtained encoded the
"long" form of PLD1 and three the short form (hereafter designated
PLD1a and PLD1b, respectively). PCR analysis of reverse-transcribed rat
brain mRNA using these primer sets also led to the amplification of
fragments corresponding to PLD1a and 1b. Partial length cDNA clones
corresponding to PLD1a and PLD1b have also been isolated from a mouse
embryo cDNA library using human PLD1-derived probes (Fig.
1C). The position of this alternately spliced region of PLD1
and its relationship to sequences conserved among other PLD enzymes is
shown in Fig. 1D.
We have expressed
and purified PLD1a and PLD1b using baculovirus vectors and investigated
their regulation by divalent cations, phospholipids, monomeric
GTP-binding proteins, and PKC-. Baculovirus-mediated expression of
both forms of PLD1 is considerably better when monolayers (as opposed
to suspension cultures) of insect cells are used. In both cases, large
quantities of insoluble proteins accumulate. The active fraction of
these recombinantly expressed enzymes is predominantly
membrane-associated. Despite considerable efforts, we were unable to
purify PLD1a and 1b to homogeneity from Sf9 cell extracts using
conventional chromatographic techniques. The major problems were
instability of the enzymes during purification and a pronounced
tendency to behave heterogenously or "smear" during column
chromatography presumably due to interactions with other proteins in
the extracts and/or nonspecific interactions with the chromatography
resins used. We surmise that similar problems coupled with low starting
levels of the proteins have hampered attempts to purify these enzymes
from tissue sources by other investigators. We therefore developed an
immunoaffinity procedure for isolation of the proteins that uses
immobilized affinity purified anti-peptide antibodies. This procedure
is extremely rapid and produces essentially homogeneous preparations of
the proteins in reasonable yield. For the experiment shown in
Fig. 2, two 225-cm2 flasks each containing
3 × 107 cells were infected with baculoviruses for
expression of PLD1a, 1b, or an irrelevant control protein
(PLC-
3). Proteins were extracted from PBS-washed
monolayers of cells with 1% Nonidet P-40 as described above.
Supernatants obtained after ultracentrifugation of these extracts
(approximately 45 mg of protein) were adsorbed to 0.5 ml of
antibody-coupled Protein A-Sepharose 4B(CL), and the resin was washed
as described above. Bound proteins were released with elution buffer at
pH 3. Rapid neutralization of the eluant was essential for preservation
of enzymatic activity. Fig. 2 shows a silver-stained 7.5% SDS gel of
purified PLD1a, PLD1b, and material obtained from a purification using
an extract from cells infected with an irrelevant control baculovirus
and a Western blot of the same fractions using a mixture of the two
anti-peptide antibodies. We obtained approximately 10 µg each of
PLD1a and 1b from two 225-cm2 flasks which corresponded to
a 20-30% yield of the ARF-stimulated PLD activity present in the
starting Sf9 cell detergent extract (PLD activity in the detergent
extracts was determined after detergent removal by gel filtration
chromatography). Identical purifications from Sf9 cells infected with
an irrelevant baculovirus control produced no detectable protein by
SDS-PAGE, Western blotting, or activity measurement so presumably the
protein(s) responsible for endogenous Sf9 cell PLD activity are present
at extremely low levels compared with the recombinantly expressed human
proteins and/or are not bound by the immunoaffinity resin. We have
purified PLD1a and 1b over 20 times using two separate preparations of immunoaffinity resin with similar results. As described below, purified
PLD1a and 1b are strongly activated by a range of lipid and protein
factors. Purified PLD1a and 1b have an ARF-stimulated specific activity
of approximately 0.3 µmol/min/mg under our standard assay conditions.
Although the most effective extraction of the proteins required
detergent treatment, approximately 50% of the membrane-bound PLD
activity could be extracted with 0.5 M NaCl (not shown).
However, in the absence of detergents, enzyme activity was less stable.
The purified proteins were kept at 4 °C in buffer containing 1%
-DOG and were stable for up to 2 days. PLD1a and 1b show a
pronounced tendency to aggregate during SDS-PAGE, and this problem is
exacerbated by boiling. We used sample buffer containing 8 M urea at room temperature to denature the proteins for
SDS-PAGE. As we reported for studies of PLD1a using membranes from
baculovirus-infected Sf9 cells as a source of activity (13), purified
PLD1a and 1b are both PC-specific PLD enzymes that catalyze hydrolysis
and (in the presence of a primary alcohol) transphosphatidylation reactions (not shown).
Regulation of PLD1 Activity by Lipids, Metal Ions, Monomeric GTP-binding Proteins, and PKC-
Other workers (10-13, 16-28)
have described mammalian PLD activities that can be modulated by a
variety of protein and lipid factors. We investigated the dependence of
PLD1a and 1b activity on divalent cations, phospholipids, monomeric
GTP-binding proteins, and PKC-.
We previously reported
that PLD1 activity was dependent on PI(4,5)P2 (13). We have
now studied the effects of a range of acidic phospholipids on the
activity of purified PLD1a and 1b using our standard assay conditions.
Vesicles contained 7% acidic lipid in a background of
phosphatidylethanolamine (PE)/phosphatidylserine (PS). Of the lipids
tested, only PI(4,5)P2 and PI(3,4,5)P3
stimulated the activity of PLD1a and 1b significantly, and the two
forms of PLD1 showed a similar dependence on these lipids
(Fig. 3B). Activity of PLD1a and 1b depends
on the molar fraction of polyphosphoinositide in the vesicles. Maximal
activation was observed with approximately 7% PI(4,5)P2 or
PI(3,4,5)P3, and PI(4,5)P2 was a more effective activator (Fig. 3A). At concentrations of up to 100 µM, soluble inositol 1,4,5-trisphosphate or
glycerophosphoinositol 4,5-bisphosphate neither activated PLD1a or 1b
nor blocked activation by PI(4,5)P2 or
PI(3,4,5)P3 (data not shown).
Dependence on Ca2+ and Mg2+
ARF-stimulated PLD1a and 1b activity was
determined as the free concentrations of these ions were varied in the
assay medium. PLD1a and 1b activity was insensitive to changes in
[Ca2+] over a wide range
(<108-10
2 M). By contrast,
ARF-stimulated activity of both PLD1a and 1b was dependent on
Mg2+ with half-maximal activity observed at approximately
10
4 M (Fig. 4).
Activation by ARF and RHO Family Monomeric GTP-binding Proteins
Purified PLD1b was incubated with increasing
concentrations of purified GTPS-activated ARF-1, RHO A, RAC-1, and
CDC42. Half-maximal activation was observed with approximately 0.2 µM ARF-1. The three RHO family monomeric GTP-binding
proteins were somewhat less potent activators of PLD1 with half-maximal
effects observed at approximately 1 µM. ARF-1 was the
most effective activator producing an approximately 50-fold stimulation
of the enzyme. RHO A and RAC-1 stimulated the enzyme approximately 10- and 13-fold, respectively, and CDC42 produced an approximately 5-fold
activation. Effects of ARF and RAC-1 were clearly saturable and,
although the concentrations of purified RHO A and CDC42 we obtained
limited the final concentrations achieved in the assay, at the highest
concentrations used, effects of these activators also appeared to be
approaching saturation (Fig. 5A). We compared
the effects of maximal concentrations of these monomeric GTP-binding
proteins on activity of purified PLD1a and 1b. ARF-1, RHO A, RAC-1, and
CDC42 activated PLD1a and 1b to similar extents, and in all cases the
activation observed was strictly dependent on GTP
S (Fig.
5B).
Activation by Protein Kinase C-
Regulation of PLD
activities by PKC is complex, and phosphorylation-dependent
and -independent mechanisms for PKC-dependent PLD
activation have been reported. We investigated the effects of purified
PKC- on purified PLD1b activity and found that this protein could
stimulate the enzyme in a concentration-dependent and
saturable manner. Half-maximal effects of PKC-
were observed with
approximately 10 nM protein, and the maximal effect of this activator (25-fold stimulation) was approximately 50% that observed with a maximally effective concentration of ARF-1. Inclusion of 100 nM phorbol myristate acetate (PMA) in the assay medium
increased the potency with which PKC-
stimulated PLD1b by
approximately 10-fold and the maximal effect by approximately 1.5-fold
(Fig. 6A). A maximally effective
concentration of PKC-
activated purified PLD1a and 1b to a similar
extent. Activation of PLD1a and 1b by PKC-
was ATP-independent
irrespective of the inclusion of PMA in the assay medium (Fig.
6B). Inclusion of apyrase in the assays to degrade any ATP
that might have contaminated our purified protein preparations had no
effect on activation of PLD1a and 1b by PKC-
(not shown). In fact,
when 0.1 mM ATP was included in the assays, PKC-stimulated
PLD activity was somewhat lower than observed in the absence of ATP,
and the effects of PKC on PLD1a and 1b activity appeared to be more
strongly dependent on PMA. This inhibitory effect of ATP was
concentration-dependent and half-maximal at approximately
10 µM (not shown). Activation of PLD1 by ARF was unaffected by the inclusion of ATP in the assay (data not shown).
Synergistic Effects of Monomeric GTP-binding Proteins and PKC-
As described above, the ARF and RHO
family monomeric GTP-binding proteins and PKC- activate PLD1a and 1b
independently. We examined interactions between these regulators as
activators of purified PLD1. The results obtained are shown in Fig. 7.
Consistent with the data shown in Figs. 3 and 4 above, in this
experiment maximally effective concentrations of ARF-1 (4.7 µM), RHO A (3.8 µM), RAC-1 (5.3 µM), CDC42 (5.5 µM), and PMA-activated
PKC-
(0.043 µM) produced approximately 50-, 13-, 13-, 10- and 30-fold activations of PLD1a and 1b, respectively
(Fig. 7A). When this experiment was repeated
including 4.7 µM GTP
S-activated ARF-1 in each set of
assays, the response to additional ARF-1 was unchanged as expected, but
substantial increases in response to the RHO proteins were observed. In
the presence of ARF-1, activation was increased to an approximately
140-fold stimulation over basal activity. Similarly, responses to RAC-1
and CDC42 were both increased to approximately 100-fold of basal.
Combination of PKC-
with the RHO family monomeric GTP-binding
proteins also produced a substantial activation of PLD1. When combined
with RHO A, RAC-1, or CDC42, PKC-
-stimulated PLD1 activity was
increased approximately 60-, 70-, and 70-fold of basal, respectively,
whereas ARF-1 increased the response to PKC-
to an approximately
140-fold stimulation. By contrast, combinations of the three RHO
proteins did not result in greater PLD1a or 1b activity than observed
with each of the proteins alone. For example, RHO A alone produced a
13-fold activation of PLD1, and PLD activity was not further increased
by addition of concentrations of RAC-1 or CDC42 that were sufficient to
cause a maximal activation of PLD1a or 1b when added alone. Similar
observations were made for combinations of RAC-1 and CDC42. Finally,
combination of maximally effective concentrations of ARF-1 and PKC-
with each of the RHO family monomeric GTP-binding proteins produced a
dramatic increase in PLD1a and 1b activity. As discussed above
combination of ARF-1 and PKC-
stimulated PLD1 activity to a level
145-fold over basal. In the presence of RHO A, RAC-1, and CDC42 this
was increased to approximately 280-, 270-, and 250-fold of basal
activity (Fig. 7B).
The complex regulation of PLD activities in intact cells and cell extracts coupled with a failure to isolate these enzymes from tissue sources has led to considerable speculation about the number and nature of PLD enzymes present in mammalian cells. Recent advances in identification of plant and yeast PLD genes provided an important insight into the structure of a new multigene family, and we reported the cloning and expression of the first mammalian PLD enzyme, PLD1. We describe an alternately spliced form of PLD1 in this paper. Our initial description of the properties of PLD1 used Sf9 cell membranes from baculovirus-infected cells as a source of enzyme activity (13). Even though increases in PLD activity in PLD1 virus-infected cells are substantial, the membranes themselves contain endogenous PLD activities, phospholipids, G-proteins, protein kinases, and possibly other factors that may influence activity of the recombinant PLD enzymes. Definitive characterization of PLD1a and 1b therefore required isolation of the protein to homogeneity, and the experiments reported in this manuscript were designed to address this issue.
Purification of PLD1Recombinant PLD1 was purified from detergent extracts of baculovirus-infected Sf9 cells. Our results demonstrate that, as reported for crude preparations of the enzyme, the isolated PLD1 protein functions as a PC-specific PLD enzyme that catalyzes both the hallmark hydrolysis and trans-phosphatidylation reactions that characterize this class of phospholipases.
Although our purification of PLD1 used detergent extraction and the purified protein appeared more stable when maintained in detergent-containing solution, we believe the enzyme to be a tightly associated extrinsic membrane protein rather than an integral membrane protein. Approximately half of the membrane-associated recombinant PLD1a and 1b activity of Sf9 cells can be extracted with 0.5 M NaCl and PLD1 can be purified under detergent-free conditions using the immunoaffinity procedure. Consistent with these findings, the PLD1 sequence does not contain large stretches of hydrophobic residues indicative of regions involved in membrane insertion. PLD1a and 1b do not contain pleckstrin homology domains or C2 domains, protein motifs known to be involved in protein phospholipid interactions.
Dependence of PLD1a and 1b Activity on PI(4,5)P2 and PI(3,4,5)P3In assays employing exogenously provided
substrates, PLD1a and 1b activity is stimulated by
PI(4,5)P2 and PI(3,4,5)P3. This effect is
highly selective for these two lipids as a variety of other acidic
phospholipids and phosphoinositides with different positional phosphate
group substitutions were ineffective. The mechanism by which
PI(4,5)P2 activates PLD1a and 1b is unclear. It is possible
that the presence of a low molar fraction of PI(4,5)P2 alters the substrate-containing phospholipid surface in a manner that
renders the PC substrate more readily hydrolyzed by the enzyme. Not all
PLD activities are stimulated by PI(4,5)P2 (21, 22), and
the high degree of phospholipid headgroup selectivity coupled with our
observation that PI(4,5)P2 and PI(3,4,5)P3
activate the purified PLD1 isoenzymes suggest that activation involves
a direct interaction between PLD1a and 1b and PI(4,5)P2.
Phosphatidylinositol-specific phospholipase C-1
(PLC-
1) is activated by PI(4,5)P2, which binds to an
NH2-terminal noncatalytic site (a pleckstrin homology
domain) anchoring the enzymes to the membrane and allowing them to
function in a scooting mode of catalysis (42). Although inspection of the primary sequence of PLD1a and 1b, or of the S. cerevisiae SPO14 protein which is also a
PI(4,5)P2-dependent PLD, does not reveal
homologies to other proteins known to interact with inositol lipids and
phosphates, it is possible that an analogous mechanism underlies the
PI(4,5)P2-stimulated increase in catalytic activity. At
present, the physiological role of PI(4,5)P2 and/or
PI(3,4,5)P3 in PLD1a and 1b regulation is not known. Since
PI(4,5)P2 and PI(3,4,5)P3 are approximately
equipotent activators of PLD1 in vitro
(PI(4,5)P2 is approximately 1.5-fold more effective), given
the relative abundance of these two lipids in mammalian cells it is
reasonable to speculate that PI(4,5)P2 is the most likely
candidate for a physiologic PLD activator. Studies using permeabilized
U937 cells suggest a role for stimulated PI(4,5)P2
synthesis in controlling monomeric GTP-binding protein-regulated PLD
activity (43). On the other hand, in general, agonist-promoted changes
in PI(4,5)P2 levels in stimulated cells are modest, whereas
PI(3,4,5)P3 levels can increase dramatically (44).
As we reported for crude preparations of the enzyme expressed in Sf9 or COS-7 cells, purified PLD1a and 1b are strongly activated by ARF-1. These results firmly establish PLD1a and 1b as direct effectors for ARF and further emphasize the importance of defining the role of PLD in ARF function. The ARF proteins are central regulators of protein trafficking (29). Several independent lines of evidence suggest a role for PLD in ARF-dependent protein trafficking and secretion. The most specific proposal is that ARF-dependent PLD activation plays a role in coated vesicle formation in the endoplasmic reticulum and Golgi apparatus, and experimental evidence in support of this idea has been presented (30, 45).
Protein kinase C- is also an effective activator of PLD1a and 1b and
does so in an ATP-independent manner. Phorbol esters increase both the
potency and efficacy with which PKC-
activates PLD1a and 1b. This
unexpected mode of PLD regulation has been reported by others using
crude or partially purified PLD preparations (17, 18), and our results
make it likely that this effect results from a direct interaction
between PLD1a and 1b and PKC-
. Phorbol esters are effective stimuli
of PLD activities in many cells, but since in vitro
activation of PLD1 by PKC-
is not absolutely dependent on phorbol
esters, at present it is unclear if the phosphorylation-independent regulatory mechanism that operates in vitro underlies
PKC-dependent PLD activation in vivo. The
observation that, in several systems, inhibitors that block the
catalytic activity of PKC inhibit PLD activation by cell-surface
receptors is also suggestive of a phosphorylation-dependent mechanism for PKC-mediated PLD activation (20).
Although the effects are modest by comparison with the responses to
ARF-1 and PKC-, PLD1a and 1b are also activated by three RHO family
monomeric GTP-binding proteins, RHO A, RAC-1, and CDC42. One component
of the signal generated by the RHO-type monomeric GTP-binding proteins
is mediated by protein kinase cascades ultimately leading to
transcriptional activation. Microinjection studies indicate that RHO,
RAC, and CDC42 can also promote changes in cell morphology, motility,
and organization of the actin cytoskeleton on a time scale that is
clearly too rapid to involve alterations in gene expression so they
must control other regulatory pathways (31). Our finding that PLD1a and
1b are directly activated by these monomeric GTP-binding proteins
focuses attention on this enzyme as a mediator of these processes.
Although appropriate accessory proteins (guanine
nucleotide-dissociation inhibitors and guanine nucleotide exchange
factors) have been identified, the mechanisms controlling activation of
the RHO proteins are not fully defined (31). One possibility
suggested by two recent studies is that the RHO proteins play a role in
coupling cell-surface receptors to PLD activation (32, 33).
As described above,
three classes of protein factors, the ARF and RHO family monomeric
GTP-binding proteins, and PKC- activate PLD1a and 1b independently.
We also investigated the effects of combinations of these factors on
PLD1a and 1b activity. Whereas combinations of maximally effective
concentrations of RHO A, RAC-1, and CDC42 did not increase PLD1
activity further than observed when each of these monomeric GTP-binding
proteins were included in the assays alone, we found that combinations
of the ARF and RHO family monomeric GTP-binding proteins and PKC-
increased PLD1a and 1b activity to levels that were considerably
greater than would be observed if their effects on PLD1a and 1b
activity were additive. It therefore appears that these three classes
of activators interact synergistically to increase PLD1a and 1b
activity.
The simplest explanation of our findings is that the PLD1a and 1b
proteins contain separate sites for interaction with
PI(4,5)P2/PI(3,4,5)P3, ARF, the RHO family
monomeric GTP-binding proteins, and protein kinase C- and that
occupancy of these sites by their respective ligands results in a
cooperative increase in catalytic efficiency of the enzyme. Comparison
of the primary sequences of plant, yeast, and human PLD enzymes
identifies four regions of homology including two regions containing
sequences conserved among a family of related proteins that catalyze
phospholipid synthesis reactions. We and others (9, 46) have therefore
suggested that these sequences are important in catalysis, so it seems
reasonable to postulate that other regions of the protein are involved
in regulatory interactions with the lipid and protein factors. A more
detailed kinetic analysis of PLD1a and 1b activity will be required to
understand how these factors increase catalytic efficiency of the
enzyme and why cooperative interactions between combinations of the ARF
and RHO monomeric GTP-binding proteins and PKC-
result in
synergistic increases in PLD1 activity. Photoaffinity labeling studies
with PtdIns(4,5)P2 and PtdIns(3,4,5)P3
analogs (36) and mutagenesis studies of PLD1a and 1b promise to define
the sites of interaction between PLD1 and these lipid and protein
regulators and may lead to identification of mutant forms of the
enzymes with altered selectivities for activation by ARF and RHO family
monomeric GTP-binding proteins and PKC. These will clearly be of great
potential value for elucidating the roles played by the three classes
of regulators in PLD regulation in cells.
The studies reported in this
paper indicate that PLD1a and 1b have identical catalytic and
regulatory properties. The alternate splicing of PLD1 transcripts would
appear to be of biological importance since it is conserved across
three different mammalian species. The 38-amino acid sequence that
distinguishes PLD1a from PLD1b is located in a 150-amino acid region
that is unique to PLD1 (Fig. 1C). This 150-amino acid region
is located between conserved sequence domains I and II identified in
plant, yeast, and bacterial PLD enzymes that do not encompass conserved
sequences shared among a growing family of PLD-related proteins (9,
46). Our results indicate that the 38-amino acid alternately spliced region is not involved in catalysis or regulation by ARF, RHO family
monomeric GTP-binding proteins,
PI(4,5)P2/PI(3,4,5)P3, or PKC-. Further work
will be required to determine the role of this region in PLD1
function.
All of the experiments reported in this study employed in vitro assays with purified proteins so it will clearly be important to establish the roles played by the monomeric GTP-binding proteins and protein kinase C in regulation of PLD1 in cells. With this in mind, our findings suggest that PLD1a and 1b may be uniquely positioned to receive and integrate different kinds of extracellular signals, transducing them to generate lipid-derived molecules that, in turn, mediate cell-specific responses. Given the growing evidence for involvement of ARF-activated PLD in intracellular protein trafficking, clearly receptor-regulated secretion would be a good candidate for a PLD1-mediated response. Another possibility (not necessarily incompatible with the first idea) is that PLD1 is present in different membrane compartments of the cell where different modes of regulation predominate and different downstream effectors are present. For example, ARF-dependent activation of PLD1 in the Golgi apparatus might generate PA for coated vesicle formation, whereas PKC and/or RHO-dependent PLD activation of PLD1 in the plasma membrane could result in changes in cell morphology mediated by the actin cytoskeleton or lead to formation of diglyceride for PKC activation.
Relationship of PLD1 to Other Mammalian Monomeric GTP-binding Protein-regulated PLD ActivitiesSternweis and colleagues (18,
28, 47) have reported several studies on an extensively purified PLD
preparation from porcine brain. This activity is
PI(4,5)P2-dependent and activated by ARF and
RHO family monomeric GTP-binding proteins and PKC- in a synergistic
manner (18, 28, 47). It therefore seems likely that these workers were
studying a porcine homolog of PLD1a, 1b, or a closely related PLD
enzyme. Work from other laboratories has suggested that distinct ARF
and RHO-activated PLD enzymes exist (25-27). Given the pronounced
synergy between the three classes of protein regulators for activation
of PLD1a and 1b, it is possible that crude or partially resolved PLD
preparations containing PLD1a or 1b could exhibit different
sensitivities to activation by exogenously added ARF and RHO proteins
depending on the presence of endogenous monomeric GTP-binding proteins
or PKC isoenzymes. Cell and tissue extracts also contain a variety of
factors that exert inhibitory effects on monomeric GTP-binding
protein-regulated PLD activities measured using exogenously provided
substrates (48, 49). These include several as yet unidentified protein
inhibitors of PLD1a and 1b.4 Taken
together, these two considerations clearly complicate attempts to
classify crude PLD preparations on the basis of their responses to ARF
and RHO family monomeric GTP-binding proteins. The availability of
PLD1-selective antibodies should aid resolution of this issue.
In summary, we report the purification
and characterization of two closely related forms of PLD1. Our results
establish PLD1a and 1b as targets for direct regulation by ARF and RHO
family monomeric GTP-binding proteins and PKC-. Although further
work will be required to establish the roles played by these different factors in regulation of PLD1 in cells, our results focus attention on
PLD1a and 1b as mediators of processes of protein trafficking and
alterations in cell motility and morphology controlled by the ARF and
RHO family monomeric GTP-binding proteins. The purified proteins, PLD
isoenzyme-selective antisera, and PLD cDNAs now available should
prove effective tools for defining the role of PLD1a and 1b in cell
regulation.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U38545[GenBank].
We thank Dr. JoAnne Engebrecht for valuable discussions and critical reading of the manuscript; Dr. Yusef Hannun and Dr. David Burns for a variety of PKC reagents; and Dr. Richard Kahn for the ARF expression system.