JOURNAL OF CELLULAR PHYSIOLOGY 182:77– 87 (2000)

Expression and Regulation of Phospholipase D

Isoforms in Mammalian Cell Lines
TERRA C. GIBBS AND KATHRYN E. MEIER*
Department of Pharmacology, Medical University of South Carolina,
Charleston, South Carolina

Phospholipase D (PLD) is activated in mammalian cells in response to diverse

stimuli that include growth factors, activators of protein kinase C, and agonists
binding to G-protein-coupled receptors. Two forms of mammalian PLD, PLD1
and PLD2, have been identified. Expression of mRNA and protein for PLD1 and
PLD2 was analyzed in the following cell lines: A7r5 (rat vascular smooth muscle);
EL4 (mouse thymoma); HL-60 (human myeloid leukemia); Jurkat (human leuke-
mia); PC-3 (human prostate adenocarcinoma); PC-12K (rat phaeochromocytoma);
and Rat-1 HIR (rat fibroblast). All, with the exception of EL4, express agonist-
activated PLD activity. PLD1 is expressed in A7r5, HL-60, PC-3, and Rat-1, while
PLD2 is expressed in A7r5, Jurkat, PC12K, PC-3, and Rat-1. Neither isoform is
expressed in EL4. Guanine nucleotide-independent PLD activity is present in
membranes from all cells expressing PLD2. In PC12K cells, which express only
PLD2, treatment with nerve growth factor causes neurite outgrowth and increases
expression of PLD2 mRNA and protein within 6 –12 h. A corresponding increase
is observed in membrane PLD activity and in phorbol-12-myristate-13-acetate
(PMA)-stimulated PLD activity in intact cells. These results show that PLD2 can be
regulated both pretranslationally and posttranslationally by agonists. J. Cell.
Physiol. 182:77– 87, 2000. © 2000 Wiley-Liss, Inc.

Phospholipase D (PLD) is activated in mammalian
cells in response to growth factors and agonists binding
to G-protein-coupled receptors (Exton, 1997). Roles for
PLD in mitogenic signal transduction and vesicle traf-
ficking have been proposed. PLD regulation is complex,
in that mammalian PLDs can be activated in vitro via
protein kinase C (PKC), small guanine nucleotide-
binding proteins, and phosphatidylinositol-4,5-bis-
phosphate (PIP2).
Two isoforms of PLD, PLD1, and PLD2, have been
identified in mammalian cells. PLD1 was originally
cloned on the basis of homology to a yeast PLD (yPLD1)
(Ella et al., 1996; Rose et al., 1995; Waksman et al.,
1996). Sequences for PLD1 from human (Hammond et
al., 1995), rat (Park et al., 1997), and mouse (Colley et
al., 1997b), and for PLD2 from human (Lopez et al.,
1998), rat (Kodaki and Yamashita, 1997), and mouse
(Colley et al., 1997a) have been published. Mammalian
PLD1 can be regulated by PKC, PIP2, and ARF (or
other small GTPases) in vitro (Hammond et al., 1995;
Min et al., 1998). PLD1 is expressed as two alterna-
tively spliced forms, PLD1a and PLD1b, which appear
to be similarly regulated (Hammond et al., 1997).
PLD2 has approximately 50% overall sequence identity
with PLD1 at the amino acid level (Colley et al., 1997a;
Kodaki and Yamashita, 1997). PLD2 can be activated
by PIP2 in vitro, but is constitutively active when over-
expressed in COS cells (Colley et al., 1997a). PLD2 is
not ARF-dependent, but may be subject to regulation
by ARF (Lopez et al., 1998; Sung et al., 1999). In
© 2000 WILEY-LISS, INC.
transfected COS cells, a phorbol ester that activates
PKC stimulates overexpressed PLD1 to a greater ex-
tent than PLD2 (Colley et al., 1997a). In the same
study, PLD1 and PLD2 had distinct subcellular distri-
butions. However, compartmental localization of PLD
isoforms appears to vary between cell types (Brown et
al., 1998; Czarny et al., 1999; Iyer and Kusner, 1999;
Kim et al., 1999a, 1999b; Millar et al., 1999; Toda et al.,
1999). PLD1 and PLD2 are differentially expressed in
mammalian tissues. Although these studies point to
distinct roles for PLD1 and PLD2, the involvement of
individual isoforms in agonist-mediated responses has
not yet been delineated.
Although the ability of extracellular signals to regu-
late PLD activity have been extensively studied, fac-
tors regulating PLD expression are only beginning to
be examined. Upregulations of mRNA for both PLD1
(Ohguchi et al., 1997) and PLD2 (Nakashima et al.,
Contract grant sponsor: University Research Committee, Medical
University of South Carolina; Contract grant sponsor: National
Science Foundation; Contract grant number: EPS-9630167; Con-
tract grant sponsor: U.S. Department of Defense; Contract grant
number: DAMD17-98-8524; Contract grant sponsor: UNCF/
Merck Science Research Dissertation Fellowship.
*Correspondence to: Kathryn E. Meier, Department of Pharma-
cology, Medical University of South Carolina, 171 Ashley Avenue,
Charleston, SC 29425-2251. E-mail: meierke@musc.edu
Received 9 September 1998; Accepted 30 July 1999
78 GIBBS AND MEIER
1998) have been reported in differentiating HL-60
cells. Regulated transcription of PLD1 and/or PLD2
has likewise been observed in C6 glioma cells (Yo-
shimura et al., 1996) and differentiating keratinocytes
(Griner et al., 1999). The pathways mediating these
responses have not been defined. Studies of the regu-
lation of PLD expression will provide further informa-
tion concerning the roles of PLD isozymes in cell pro-
liferation and differentiation.
In this report, we analyzed expression of PLD1 and
PLD2 in mammalian cell lines in which PLD activity
has been previously characterized. The cell lines are:
A7r5 (rat vascular smooth muscle); EL4 (mouse thy-
moma); HL-60 (human myeloid leukemia); Jurkat (hu-
man leukemia); PC12 (rat pheochromocytoma); PC3
(human prostate adenocarcinoma); and Rat-1 (rat fi-
broblast). All cells, except EL4, were previously shown
to express agonist-activated PLD. EL4 expresses no
PLD activity. The regulation of PLD2 expression in
differentiating PC12 cells was also examined. The re-
sults show that mammalian cells can express one, both,
or neither PLD isoform, and that PLD2 is regulated
both pretranslationally and posttranslationally by ago-
nists.
MATERIALS AND METHODS
Cell lines
Methods for cell culture have been previously de-
scribed for A7r5 (Jones et al., 1994), EL4 (Bradshaw et
al., 1996), HL-60 (Bradshaw et al., 1996), Jurkat (Brad-
shaw et al., 1996), PC12 (Ella et al., 1997), PC-3 (Qi et
al., 1998), and Rat-1 HIR (Knoepp et al., 1996). PC12
cells (PC12K strain) were maintained on Primaria
(Falcon); other cell lines were maintained on standard
tissue culture plastic. HL-60 cells were provided by Dr.
Art Frankel (Medical University of South Carolina).
For some experiments, PC12 cells were treated with
100 ng/ml NGF (7S, Upstate Biotechnologies, Inc.,
Lake Placid, NY), 50 ng/ml EGF (Upstate Biotechnol-
ogies, Inc.), or 100 nM PMA (Calbiochem, La Jolla, CA)
in complete growth medium.
Reverse transcription-polymerase
chain reaction
Total RNA (2 mg) was reverse-transcribed and am-
plified by PCR using Taq PCR Core Kit (Qiagen, Va-
lencia, CA) according to the manufacturer’s instruc-
tions. Primers for hPLD1, rPLD1, and rPLD2 were
based on the following studies: Hammond et al. (1997),
Park et al. (1997), and Kodaki and Yamashita (1997),
respectively. Primers were synthesized by the MUSC
oligonucleotide synthesis facility. Conditions for re-
verse transcription (RT) were as follows: at 94°C for 0.5
min, 60°C for 1 min, and 72°C for 1 min for 27 cycles
(hPLD1); at 94°C for 1 min, 60°C for 1 min, and 72°C
for 1.5 min for 30 cycles (hPLD2); at 94°C for 1 min,
56°C for 1.5 min, and 72°C for 2 min for 30 cycles
(rPLD1); at 94°C for 1 min, 56°C for 1 min, and 60°C for
1.5 min for 40 cycles (rPLD2). The following PLD-
specific primers were used: hPLD1: 59-TGGGCTCAC-
CATGAGAA-39, (nucleotides 1475–1491) and 59-GT-
CATGCCAGGGCATCCGGGG-39 (nucleotides 2133–
2113); rPLD1: 59-GGGGGACACAGGATACCAGG-39
(nucleotides 871– 891) and 59-GGATGGAGCCGGTGT-
TGGAG-39 (nucleotides 1949 –1929); hPLD2: 59-TCC-
ATCCAGGCCATTCTGCAC-39 (nucleotides 2091–2112)
and 59-CTATGTCCACATTTCTAGGGGGAT-39 (nucleo-
tides 2802–2778); rPLD2: 59-TCAAGGCCAGATACA-
AGATACC-39 (nucleotides 2042–2063) and 59-CACGTA-
GACTCGGAAACACTGC-39 (nucleotides 2373–2352).
Northern blots
Total RNA (5 mg) was isolated from mammalian cell
lines (Qiagen RNeasy Mini Kit) and then loaded onto
an ethidium bromide-stained 1.2% agarose gel under
formaldehyde denaturing conditions. Gels were run at
4 V/cm for 4 h. RNA was transferred to nylon mem-
branes (Duralon-UV, Stratagene) in 103 SSC buffer by
capillary action, then crosslinked by UV light. RT-PCR-
generated fragments were verified by sequencing
(MUSC Sequencing Facility) and used as probes in
Northern blotting. Probes specific for hPLD1, hPLD2,
rPLD1, and rPLD2 were approximately 633-, 710-,
1080-, and 330-bp fragments, respectively. An approx-
imate 1000-bp probe specific for glyceraldehyde 3-phos-
phate dehydrogenase (GAPDH), kindly provided by Dr.
Gian Re (Department of Pathology, MUSC), was used
to normalize for loading. Probes were radiolabeled (a-
32
P-dCTP) using Prime-It II Random Labeling Kit
(Stratagene). Hybridization (Quickhybe, Stratagene)
was carried out at 68°C for 2 h. Membranes were suc-
cessively washed for 30 min in 23 SSC, 0.1% sodium
dodecyl sulfate (SDS) at room temperature followed by
two 15-min washes in 0.13 SSC, 0.1% SDS at 55°C.
Membranes were exposed overnight at 280°C with
Kodak XOmat/AR film.
Immunoblotting
Cell membranes were prepared as previously de-
scribed (Qi et al., 1998). Membrane proteins (20 mg)
were separated by SDS polyacrylamide gel electro-
phoresis (PAGE) on 12.5% gels and transferred to a
polyvinylidene difluoride (PVDF) membrane. Anti-
PLD antibody was a gift of Jae Ho Kim and colleagues
(Pohang University of Science and Technology, South
Korea). This antibody, raised against a C-terminal
PLD1 peptide (Lee et al., 1997), recognizes both PLD1
and PLD2. Blots were developed using ECL reagents
(Amersham) and quantitated by densitometry.
PLD assays
PLD assays were performed in vitro using BODIPY-
glycerophosphocholine (BPC), 2-decanoyl-1-(O-(11-(4,4-
difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-
propionyl)amino)undecyl)sn-glycero-3-phospho-choline,
as substrate, as described previously (Ella et al., 1994;
Meier and Gibbs, 1999; Qi et al., 1998). BPC was obtained
from Molecular Probes, Inc. (BODIPY-FL C3C11-C10-
PAF, catalog #D-3771). BODIPY is a trademark of Mo-
lecular Probes, Inc. An aliquot of 1 mM BPC in ethanol
was dried under nitrogen and briefly sonicated in 500 mM
octylglucoside, 400 mM NaCl, 66 mM HEPES (pH 7.0)
prior to use. The final reaction (12.5 ml) contained 0.1 mM
BPC, 150 mM NaCl, 200 mM octylglucoside, 25 mM
HEPES (pH 7.0), 5 mM EGTA, 1 mM EDTA, 40 mM
b-glycerophosphate, 1 mM dithiothreitol, and 1% (v/v)
n-butanol. The reaction, initiated by the addition of 5 mg
of membrane protein, was allowed to proceed for 60 min
at 30°C. For some experiments, cabbage PLD (0.5 mg/ml;
Sigma, St. Louis, MO) was used as a standard. A 5-ml
 PLD1 AND PLD2 EXPRESSION IN MAMMALIAN CELLS
Fig. 1. RT-PCR amplication of phospholipase D (PLD) isoforms from
mammalian cell lines. Total RNA (2 mg), prepared from the indicated
cell lines, was subjected to RT-PCR; all samples were analyzed in
parallel in the same experiment. PCR reactions (10 ml) were loaded
onto 1% agarose gels stained with ethidium bromide. Products were
imaged by a FluorImager. The following fragments were expected:
hPLD1, 638 and 533 bp, respectively, corresponding to hPLD1a and
hPLD1b; hPLD2, 710-bp; rPLD1, 1080 bp; rPLD2, 330 bp.
aliquot of the reaction was applied to a plastic-backed
silica gel G60 plate without fluorescent indicator (Merck,
Damstadt, Germany). The plates were developed in chlo-
roform: methanol:water:acetic acid (45:45:10:2, v/v), im-
aged using a Molecular Dynamics FluorImager, and
quantitated using a Helena Laboratories scanning fluo-
rescent densitometer. Protein concentrations were deter-
mined using a Coomassie binding assay (Pierce) with
bovine serum albumin standards.
PLD assays in intact cells were performed as de-
scribed previously (Ella et al., 1994; Meier and Gibbs,
1999). Briefly, PC12 cells were grown to approximately
70% confluence in 35-mm plates. Fresh medium with or
without 100 ng/ml NGF was then added. [3H]-palmitic
acid (10 mCi/ml) was added 12 h before the assay was
performed. Triplicate or quadruplicate dishes were pre-
pared for each experimental condition. At the end of
the incubation, the medium was removed. Cells were
rinsed twice with warm Dulbecco’s Modified Eagle Me-
dium containing 10 mM HEPES, pH 7.5 (DMEM/H).
The cells were then incubated for 30 min at 37°C in
DMEM/H in the absence and presence of 0.5% (v/v)
ethanol and 100 nM phorbol-12-myristate-13-acetate
(PMA). Cellular lipids were extracted and separated by
thin-layer chromatography. Areas containing phos-
phatidylethanol (PEt) and phosphatidic acid (PA) were
scraped from the plate, along with the remainder of
each lane, and quantitated as a percent of the total
radioactivity recovered from each sample.
RESULTS
RT-PCR for PLD isoforms in mammalian cells
RT-PCR was used to amplify fragments of PLD1 and
PLD2 from cDNA prepared from mammalian cell lines.
In our previous work, this approach was used to estab-
79
Fig. 2. Northern blots for phospholipase D (PLD) isoforms in mam-
malian cell lines. Total RNA (5 mg), prepared from the indicated cell
lines, was resolved on a 1.2% agarose gel under formaldehyde dena-
turing conditions and transferred to a nylon membrane. Probes were
prepared from RT-PCR fragments generated as shown in Figure 1.
The blot was sequentially probed with radiolabeled fragments specific
for PLD1, PLD2, and GAPDH. The autoradiographs of the blots are
shown.
lish that PLD1 was expressed in PC-3 cells (Qi et al.,
1998). In subsequent experiments, we found that many
of the cell lines in which we had characterized PLD
activity expressed both PLD1 and PLD2. These results
are shown in Figure 1. hPLD1-specific primers ampli-
fied two sequences of 638 and 533 bp from PC-3 cells.
These correspond to the sizes of the alternatively
spliced fragments of human PLD 1a and 1b, respec-
tively. hPLD1 was also amplified from HL-60, a cell
line that has previously been shown to express this
isoform (Marcil et al., 1997; Ohguchi et al., 1997; Saqib
and Wakelam, 1997). Similar results were seen with
alternative primers designed to amplify larger frag-
ments of hPLD1 (data not shown). Primers designed
for rPLD1 amplified a fragment of 1080 bp from A7r5
and Rat-1 cells. No rPLD1 was amplified from PC12
cells.
RT-PCR reactions using primers specific for PLD2
revealed that most of the cell lines expressed this
isozyme (Fig. 1). hPLD2 was amplified from PC-3 and
Jurkat cells. The highest levels of rPLD2 amplification
were observed in PC12 and Rat-1 cells, with A7r5 also
expressing this isoform. rPLD2 was also amplified from
RG20, a PC12 cell line over-expressing the a2D-adren-
ergic receptor (data not shown). The lack of detectable
PLD2 mRNA in undifferentiated HL-60 cells is consis-
tent with previous reports (Colley et al., 1997b; Marcil
et al., 1997; Saqib and Wakelam, 1997).
80 GIBBS AND MEIER
Fig. 3. Immunoblots for phospholipase D (PLD) isoforms in mam-
malian cell lines. Membrane protein (50 mg) was separated by SDS-
PAGE and transferred to nitrocellulose membrane. PLD isozymes
were detected using a primary antibody specific for PLD1/PLD2. The
blot was developed using the ECL detection system.
Northern blot analysis for PLD isoforms in
mammalian cells
Northern blot analysis was performed to verify the
results obtained by RT-PCR. An oligonucleotide probe
specific for hPLD1 detected two species of mRNA in
PC-3 and HL-60 cells (Fig. 2). The difference in size
between these transcripts was larger than the pre-
dicted difference between PLD1a and PLD1b. The RNA
separation was repeated in the presence of higher con-
centrations of formaldehyde, with similar results (data
not shown). Reprobing with a different probe for
hPLD1 (2.3 kb) likewise gave similar results (data not
shown). We therefore concluded that one of the bands
represents messages for PLD1a/b, while the other rep-
resents a related or alternatively spliced transcript.
Using a rPLD1 probe, mRNA was detected in A7r5 and
Rat-1 cells but not in PC12 cells.
mRNA for PLD2 was detected in all cell lines except
HL-60 and EL4. PC12 and Rat-1 cells expressed par-
ticularly high levels, while Jurkat expressed low levels.
The observed differences in transcript size between
human and rat cells (e.g., PC-3 vs. PC12) were ex-
pected, because sequence length differs between these
species. The human and rat probes did not detect
mRNA for PLD1 or PLD2 in EL4, a murine cell line,
despite high homology between the rat and murine
genes. Murine-specific probes were not generated, be-
cause our previous work had likewise indicated that
EL4 cells lack PLD expression (Bradshaw et al., 1996).
In summary, Northern blotting data were consistent
with results obtained by RT-PCR.
Western blot analysis for PLD isoforms
in mammalian cells
Immunoblotting was used to examine expression of
PLD proteins. The anti-PLD antibody recognized
bands of approximately 120 kDa (hPLD1 and rPLD1),
106 kDa (hPLD2), and 115 kDa (rPLD2) (Fig. 3). Both
PLD1 and PLD2 were expressed in A7r5, PC-3, and
Rat-1 membranes. Jurkat and PC12 cell membranes
contained only PLD2, while HL-60 expressed only
PLD1. EL4 cells expressed neither protein, consistent
with the apparent lack of expression of mRNA and with
previous data indicating lack of PLD activity and pro-
tein (Bradshaw et al., 1996). Thus, immunoblotting
verified expression of the expected isozymes in cells
expressing mRNA for PLD1 and/or PLD2.
PLD activity in mammalian cell membranes
An in vitro assay was used to assess PLD activity in
membrane preparations. This assay, which utilizes a
fluorescent glycerophosphocholine as substrate, has
been previously used in all of the cell lines tested here.
Butanol is included as substrate for production of phos-
phatidylbutanol (PBt) by the transphosphatidylation
reaction. The assay is capable of detecting basal PLD
activity in membrane preparations in the absence of
added guanine nucleotides, PIP2, or GTP-binding pro-
teins. Moreover, as illustrated in Figure 4A for PC12
cells, in vitro PLD activity is enhanced in membranes
prepared from PMA-treated cells. In the experiment
shown, membrane PLD activity was maximal 10 min
after addition of PMA. Production of both PBt and
lyso-phosphatidylbutanol (LPBt), products of the
PLA2-mediated hydrolysis of PBt (Ella et al., 1994),
was increased in membranes from PMA-treated cells.
As reported previously (Ella et al., 1997), the PMA-
stimulated increase in activity is relatively modest (i.e.,
33%). This is due, in part, to the high level of basal PLD
activity in PC12 membranes.
PLD activity was assayed in membranes prepared
from the cell lines tested. PLD activity was indicated
by production of PBt and LPBt (Fig. 4B). The only cell
lines lacking basal PLD activity were HL-60 and EL4.
To test for latent PLD activity that was dependent on
GTP-binding proteins (e.g., ARF, rho), we added gua-
nine nucleotide to the assay. Addition of Mg-GTPgS
resulted in the detection of a low level of PLD activity
in HL-60 membranes. This activity was not seen in the
presence of magnesium alone or with Mg-GDP. It
should be noted that the conditions used here were not
optimal for detecting activity of PLD1, because exoge-
nous ARF and PIP2 were not added to the assay. EL4
membranes, as previously reported (Bradshaw et al.,
1995), had no detectable PLD activity in the absence or
presence of GTPgS. GTPgS was not required for PLD
activity in the other cell lines tested, indicating that
the major activity detected was guanine nucleotide-
independent. Stimulation of PLD activity by Mg-
GTPgS in cells coexpressing PLD1 and PLD2 (e.g.,
A7r5, PC-3, Rat-1 HIR) probably reflects partial acti-
vation of PLD1. In summary, levels of guanine nucle-
otide-independent membrane PLD activity are gener-
ally consistent with levels of PLD2 protein (compare
Figs. 3 and 4).
Effects of nerve growth factor on PC12 cells
In view of the fact that PC12K cells express only
PLD2, we addressed whether this isoform was respon-
sible for the PLD activity observed in membranes and
intact cells. Nerve growth factor (NGF) induces neurite
outgrowth and neuronal differentiation in PC12 cells.
We showed previously that NGF induces acute activa-
tion of PLD in PC12 cells but that membrane PLD
activity is not increased in fully differentiated cells
incubated with NGF for 3 days (Ella et al., 1997).
However, early signaling events are involved in initi-
ating PC12 differentiation. For example, neurite out-
growth is observed within 6 h after NGF addition (Fig.
5). We therefore examined the time course of the effects
of NGF on mRNA levels for PLD1 and PLD2.
Northern blotting was used to measure mRNA levels
for PLD isozymes in NGF-treated cells. Two separate
experiments, examining different time courses, are
shown in Figure 6. NGF induced an increase in PLD2
mRNA that was detectable by 6 h, and maximal by
 PLD1 AND PLD2 EXPRESSION IN MAMMALIAN CELLS
Fig. 4. Phospholipase D (PLD) activity in membranes prepared from
mammalian cell lines. A: membranes were prepared from PC12 cells
incubated for the indicated times with or without 100 nM phorbol-12-
myristate-acetate (PMA). Membranes (5 mg) were added to a reaction
mixture containing 0.1 mM BPC and 1% butanol. Reaction products
were separated by TLC and imaged using a FluorImager. The prod-
ucts generated via the PLD reaction, phosphatidylbutanol (PBt) and
lyso-phosphatidylbutanol (LPBt), are shown along with additional
metabolites (MG, monoglyceride; DG, diglyceride; LPC, lyso-BPC).
12 h. After 24 h, PLD2 mRNA decreased below control
levels. In untreated cells, PLD2 mRNA levels were
maintained throughout the time course of the experi-
ments (data not shown). In a larger series of experi-
ments (data not shown), NGF treatment for 12 h in-
creased PLD2 message levels (normalized to GAPDH)
to 142.1% 6 18.7% (mean 6 SD, n 5 7) that seen in
control cells. rPLD1 mRNA was not induced by NGF at
any time point tested (data not shown). Epidermal
growth factor (EGF) (50 ng/ml), which typically induces
proliferation but not differentiation of PC12 cells
(Nguyen et al., 1993), did not alter PLD mRNA levels
(data not shown).
81
PBt production was calculated as a percent of total fluorescence for
each lane, and then expressed as a percent of the value obtained for
untreated cells. B: membranes (5 mg) prepared from the indicated cell
lines were incubated with 0.1 mM BPC and 1% butanol in the absence
(control) or presence of 125 mM MgCl2, 10 mM GDP, and/or 10 mM
GTPgS. Products were separated by TLC and imaged using a Fluo-
rImager. Only products generated via the PLD reaction, PBt, and
LPBt, are shown. These results were from a single experiment in
which all cell lines were analyzed and imaged together.
The signaling pathways responsible for NGF-in-
duced upregulation of PLD2 mRNA were examined
(data not shown). NGF-induced neuronal differentia-
tion in PC12 cells has been shown to require activation
of both ERK and p38 mitogen-activated protein kinase
(MAPKs) (Leppa et al., 1998; Morooka and Nishida,
1998). Because MAPKs regulate transcription, we
asked whether these kinases were involved in the ef-
fects of NGF on PLD2 mRNA levels. PD98059 (50 mM),
an inhibitor of the ERK phosphorylation cascade, did
not block the effects of NGF (12 h) on PLD2 expression.
The response to NGF in the presence of 50 mM
PD98059 was 90.4% 6 11.6% (mean 6 SD, n 5 10
82 GIBBS AND MEIER

Fig. 5. Effects of nerve growth factor (NGF) on neurite outgrowth in photographed using interference contrast microscopy. Untreated cells
PC12 cells. PC12 cells, grown on Primaria tissue culture plates to (not shown) were similar in morphology to the cells seen on the upper
approximately 70% confluency, were incubated in complete medium left side of the 6-h photograph.
containing 100 ng/ml NGF for the indicated times. The cells were

experiments) of that seen without inhibitor. This result

is consistent with the observed inability of EGF, which
induces prolonged activation of ERKs in PC12 cells
(Nguyen et al., 1993), to induce PLD2 upregulation.
SB203580, an inhibitor of the p38 pathway, likewise
had no significant effect on the NGF response (89.3% 6
6.7% of NGF alone, n 5 5 experiments). Thus, neither
ERKs nor p38 appear to play major roles in mediating
the effects of NGF on PLD2 expression.
Immunoblotting was used to determine whether the
effects of NGF on PLD2 mRNA levels were reflected by
changes in PLD2 protein levels (Fig. 7). NGF increased
levels of PLD2 protein in PC12 membranes. PLD2 pro-
tein was upregulated by 12 h after NGF addition, and
decreased thereafter. NGF did not induce expression of
PLD1 protein. These results are consistent with the
changes in mRNA expression discussed above. The
magnitude of the maximal increase in PLD2 protein
(58%) was comparable to the increase of PLD2 mRNA
(42%), indicating that increased mRNA levels were
correlated with increased protein expression under
these conditions.
The effects of NGF on membrane PLD activity were
examined. PLD activity was increased after NGF ad-
dition, with maximal response observed at 12 h (Fig.
8A). The increase in membrane activity was approxi-
mately 30% (Fig. 8B). EGF (50 ng/ml) did not alter
membrane PLD activity over a similar time course
(data not shown).
The effects of NGF on PLD activity in intact PC12
cells were studied (Fig. 9). As previously noted (Ella et
al., 1997), basal PLD activity was not detectable in
untreated PC12 cells under the conditions used (Fig.
9A). In other words, radioactivity migrating with PEt Fig. 6. Effects of nerve growth factor (NGF) on PLD2 mRNA expres-
was not higher in cells incubated with ethanol as com- sion in PC12 cells. PC12 cells were incubated with 100 ng/ml NGF for
the indicated times. Expression of mRNA for PLD1 and PLD2 was
pared to the background radioactivity measured in assessed by Northern blotting, as described for Figure 2. A: Autora-
cells incubated without ethanol. Treatment of cells diographs from two separate experiments are shown; note that differ-
with NGF for 12 h did not significantly increase basal ent time intervals were tested in the two experiments. B: Results from
PLD activity. PMA treatment resulted in PEt produc- two separate experiments were quantitated. PLD2 mRNA was nor-
malized to glyceraldehyde 3-phosphate dehydrogenase (GADPH)
tion, indicating PLD activation. PMA-induced PEt ac- mRNA, then expressed as a percent of the value for untreated con-
cumulation was maximal by 30 min (data not shown). trols. At least two control dishes were used in each experiment. Each
Treatment of PC12 cells with NGF for 12 h caused an value represents the mean 6SD from the duplicate experiments.
 PLD1 AND PLD2 EXPRESSION IN MAMMALIAN CELLS
Fig. 7. Effects of nerve growth factor (NGF) on PLD2 protein expres-
sion in PC12 cells. Membrane protein (20 mg) was separated by
SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) mem-
brane. Immunoblotting was performed using an antibody recognizing
PLD1 and PLD2. The blot was developed using ECL Plus reagents.
Results were quantitated by densitometry and expressed relative
those obtained for untreated cells (control).
enhancement of PMA-induced PLD activation. PLD
activation was increased by 46.7% 6 17.2% (mean 6
SEM, n 5 3 experiments) in NGF-treated cells, as
calculated after subtraction of assay background. An
increase in PMA-stimulated production of PA was also
noted (Fig. 9B). In view of the fact that PLD2 mRNA
declined after 24 h (Fig. 6), the effects of 12- and 36-h
treatments with NGF were compared (Fig. 9C). PMA
responsiveness of cells treated with NGF for 36 h was
similar to that of untreated cells. Thus, the magnitude
of the effects of NGF on PMA-stimulated PLD activity
in intact cells are consistent with its effects on PLD2
mRNA, PLD2 protein, and membrane PLD activity.
These data suggest that PLD2 is largely responsible for
the PMA-activated PLD activity detected in intact
PC12K cells.
DISCUSSION
The data presented here demonstrate the PLD iso-
forms are expressed in a cell-specific manner. The re-
sults are summarized in Table 1. Although PLD1 and
PLD2 probably have distinct roles in cellular regula-
tion, it is notable that mammalian cells can survive
and proliferate with expression of one, both, or neither
isozyme.
Previous work in our laboratory has focused on the
characterization of agonist-activated PLDs. A7r5 cells
activate PLD in response to PMA or vasopressin (Ella
et al., 1994; Jones et al., 1995), Rat-1 HIR cells activate
PLD in response to PMA or insulin (Knoepp et al.,
1996), PC12K cells activate PLD in response to PMA
and NGF (Ella et al., 1997), PC-3 cells activate PLD in
response to PMA or lysophosphatidic acid (LPA) (Qi et
al., 1998), and Jurkat and HL-60 cells activate PLD in
response to PMA (Bradshaw et al., 1995). EL4 cells do
not express basal or PMA-activated PLD activity
(Bradshaw et al., 1995). Thus, although their expres-
sion of PLD isoforms vary, all cell lines except EL4 can
activate PLD in response to PMA.
Previous studies using a polyclonal anti-PLD anti-
body raised in our laboratory established that an im-
83
Fig. 8. Effects of nerve growth factor (NGF) on membrane phospho-
lipase D (PLD) activity in PC12 cells. Membrane extracts (5 mg) were
prepared from PC12 cells incubated with or without 100 ng/ml NGF
for the indicated times. Membrane PLD activity was assessed as
described for Figure 4. A: Reaction products were imaged using a
FluorImager. B: Results from three separate experiments were quan-
titated. Production of phosphatidylbutanol (PBt) plus lyso-phosphati-
dylbutanol (LPBt) was calculated as a percent of total fluorescence for
each sample, then expressed as a percent of the value for untreated
cells in the same experiment. Each value represents the mean 6SD
from triplicate experiments.
munoreactive band of 120 kDa was expressed in PC-3
and in LNCaP, another prostate cancer cell line (Qi et
al., 1998). As previously noted, this band represents
PLD1. An approximate 100-kDa band, possibly repre-
senting PLD2, was also observed. Immunoreactive
bands of 100 –120 kDa were also observed in Jurkat
and HL-60 (Bradshaw et al., 1996). For the current
study, we obtained another anti-PLD antibody that
recognizes both PLD1 and PLD2. Immunoblotting of
membranes prepared from the various cell lines con-
firmed the pattern of isoform expression predicted by
RT-PCR and Northern blotting, and showed that PLD2
protein was upregulated in PC12K cells treated for
12 h with NGF. RT-PCR, Northern blotting, and West-
ern blotting therefore provide complementary ap-
proaches to examine expression of PLD isoforms at the
mRNA and protein levels.
It appears likely that the guanine nucleotide-inde-
pendent PLD activity detected in membrane prepara-
tions from A7r5, PC-3, PC12K, Rat-1, and Jurkat cells
by our in vitro assay represents PLD2 activity. First,
the relative level of membrane PLD activity generally
84 GIBBS AND MEIER
Fig. 9. Effects of nerve growth factor (NGF) on phospholipase D
(PLD) activity in intact PC12 cells. Intact PC12 cells were incubated
in the absence or presence of 100 ng/ml NGF for 12 or 36 h. Cells were
metabolically labelled with [3H]-palmitic acid during the last 12 h of
incubation. The cells were then washed and incubated with and
without 100 nM phorbol-12-myristate-acetate (PMA) and 0.5% (v/v)
ethanol. Levels of phosphatidylethanol and phosphatidic acid, as-
correlates well with the mRNA and protein levels for
PLD2. Second, several groups have reported that PLD2
activity is independent of GTP-binding proteins. Thus,
detection of this activity in the absence of GTP is not
unexpected. In contrast, the lack of GTP-independent
PLD activity in HL-60 membranes (Fig. 4B) confirms
that PLD1 activity cannot be detected in these cells
without activation of small GTP-binding proteins.
Work by others has established that PLD in HL60 is
activated in intact cells by fMLP (Pai et al., 1988), in
sessed as described in the text, were expressed as a percent of total
radioactivity recovered. A, B: Cells were incubated for 12 h with and
without NGF. Each data point represents the mean 6SD of values
obtained from 2– 4 dishes of cells; 4 dishes were used for all PMA
incubations. C: Cells were incubated for 12 and 36 h with and without
NGF. Each data point represents the mean 6 SD of values obtained
from three dishes of cells.
permeabilized cells by guanine nucleotides (Xie and
Dubyak, 1991), and in vitro by ARF and PIP2 (Brown
et al., 1993; Cockcroft et al., 1994). The current study
confirms that undifferentiated HL-60 cells express pre-
dominantly PLD1 (Colley et al., 1997b; Marcil et al.,
1997; Ohguchi et al., 1997; Saqib and Wakelam, 1997).
Thus, the lack of GTP-independent activity observed
here for HL-60 is consistent with published data.
PC12K, which expresses only PLD2, has abundant
guanine nucleotide-independent PLD activity (Fig.
 PLD1 AND PLD2 EXPRESSION IN MAMMALIAN CELLS
TABLE 1. Summary of phospholpase D expression1
Cell line Species Cell type
A7r5 rat vascular smooth muscle
EL4 mouse T-lymphocyte
HL60 human myeloid
Jurkat human T-lymphocyte
PC3 human prostate epithelium
PC12 rat neuronal
Rat-1 rat fibroblast
1
PMA, phorbol-12-myristate-acetate; PLD, phospholipase D; GTP.
4B). These data do not rule out roles for GTP-binding
proteins in modulating PLD2 activity.
Potential differences between strains of the PC12
cell line, and between culture conditions, remain to be
explored. Significant differences in morphology and
protein expression occur between sublines of PC12
(Swanson et al., 1998). The PC12K cell line used here is
notable for its highly adherent phenotype, which is
further accentuated by culture on Primaria plastic.
One study of PC12 cells described only oleate-depen-
dent, guanine nucleotide-independent activity (Banno
et al., 1996). Calcium-activated PLD activity was re-
ported in intact PC12 cells (Ito et al., 1997). Expression
of PLD1 in PC12 cells was reported by one group (Park
et al., 1997), while rho-dependent PLD activity was
described in these cells by others (Jinsi-Parimoo and
Deth, 1997). Recently, PLD1a, PLD1b, and PLD2 were
all cloned from a PC12 cDNA library (Nakashima et al.,
1997). Together, these data indicate that some PC12
cell lines express both PLD1 and PLD2. Variations in
PLD isozyme expression probably explain some of the
differences in PLD regulation reported by different in-
vestigators.
Our accumulated data suggest that PLD2 can be
regulated by agonists in a variety of cell types. While
both PLD1 and PLD2 confer PMA-activated PLD ac-
tivity when overexpressed in COS cells, the effect of
PMA is less pronounced for PLD2 (Colley et al., 1997a).
Colley and coworkers (1997a) reported that overex-
pressed PLD2 is constitutively active in their model
system, and suggested that PLD2 might be regulated
by endogenous inhibitors. In subsequent work, this
group established that PLD2 can be inhibited by
synucleins, proteins isolated from brain (Jenco et al.,
1998). In our hands, A7r5, Rat-1, and PC12 cells do not
express detectable basal PLD activity in intact cells.
The increase in PLD2 levels induced by NGF does not
increase basal activity in intact cells, even though
PMA-stimulated activity is enhanced. However, both
basal and PMA-stimulated activity are present in
membranes from PC12 and other PLD2-expressing cell
lines. Whether PLD2 activity is suppressed in intact
cells by an endogenous inhibitor(s), or whether agonist-
induced activation is required for manifestation of
PLD2 activity in intact cells at endogenous levels of
expression, remains to be elucidated.
The data presented here indicate that guanine nu-
cleotide-independent PLD activity in cell membranes
represents PLD2. An alternative consideration is that
some of the cell lines examined here could express a
85
Intact cell: Membrane: PLD
PMA-stimulated PLD GTP-independent PLD Isoform(s)
1 1 PLD1, PLD2
2 2 2
1 2 PLD1
1 1 PLD2
1 1 PLD1, PLD2
1 1 PLD2
1 1 PLD1, PLD2
form of PLD that has not yet been identified. Although
the PCR primers used in this study amplified alterna-
tive products in some cell lines, subcloning and se-
quencing of these products did not reveal additional
PLD isoforms (data not shown). Nonetheless, the find-
ings that plants (Pappan et al., 1997) and yeast (Waks-
man et al., 1997) express apparently novel forms of
PLD suggest that additional mammalian isozymes
may remain to be characterized. While the novel yeast
PLD and plant PLDs require calcium for activity, we
have not detected calcium-dependent PLD activity in
mammalian membranes using our in vitro assay sys-
tem. Biochemical evidence for the existence of ARF-
independent/oleate-dependent PLD activity has also
been presented (Massenburg et al., 1994). Although we
have been unable to obtain significant enhancement of
membrane PLD activity upon addition of oleate to our
in vitro assay system, it remains possible that oleate-
dependent activity represents PLD2. Until additional
PLD isoforms are sequenced, it will be difficult to an-
alyze their contributions to cellular regulation.
Factors regulating the expression of PLD isoforms
have not been extensively studied. Previous data from
our laboratory have shown that PC12 expresses gua-
nine nucleotide-independent PLD activity, and that
this activity can be inhibited via a Gi-dependent path-
way (Ella et al., 1997). The results presented here show
that these cells express PLD2 exclusively. PLD2
mRNA and protein are increased when PC12 cells are
induced to differentiate. PLD activity in membranes,
as well as PMA-stimulated PLD activity in intact cells,
are increased when PLD2 is up-regulated. PLD2 is
proposed to participate in cytoskeletal reorganization
(Colley et al., 1997a). Thus, a role for increased PLD2
expression in the initiation of neurite outgrowth can be
envisioned. However, because the increases noted are
transient and relatively small in magnitude, it remains
to be determined whether this upregulation plays a
critical role in NGF-induced differentiation.
Our studies establish that PLD2 is regulated both
pretranslationally and posttranslationally in PC12, a
neuronal cell line. Strategies designed to explore the
role of PLD2 in agonist-mediated responses should pro-
vide useful information concerning the involvement of
this enzyme in signal transduction.
ACKNOWLEDGMENTS
The authors thank Dr. Chen Qi, Dr. Hsun Ku, Mr.
Stewart Knoepp, and Mr. Jin-Hyouk Park for helpful
advice, and Drs. Sung Ho Ryu and Jae Ho Kim for
86 GIBBS AND MEIER
providing anti-PLD antibody. The UNCF/Merck Sci-
ence Research Dissertation Fellowship was awarded to
T.G.
LITERATURE CITED
Banno Y, Ito Y, Oijo K, Kanoh H, Nakashima S, Nozawa Y. 1996.
Membrane-associated phospholipase D activity in neural cell line
PC12. J Lipid Mediat Cell Signal 14:237–243.
Bradshaw CD, Ella KM, Qi C, Turnquist HM, Wisehart-Johnson AE,
Meier KE. 1996. Effects of phorbol ester on phospholipase D and
mitogen-activated protein kinase activities in T-lymphocyte cell
lines. Immunol Letts 53:69 –76.
Brown H, Gutowski S, Moomaw CR, Slaughter C, Sternweis PC. 1993.
ADP-ribosylation factor, a small GTP-dependent regulatory pro-
tein, stimulates phospholipase D activity. Cell 75:1137–1144.
Brown FD, Thompson N, Saqib KM, Clark JM, Pownder D, Thompson
NT, Solari R, Wakelam MJ. 1998. Phospholipase D1 localises to
secretory granules and lysosomes and is plasma-membrane trans-
located on cellular stimulation. Curr Biol 8:835– 838.
Cockcroft S, Thomas GMH, Fensome A, Geny B, Cunningham E, Gout
I, Hiles I, Totty NF, Truong O, Hsuan JJ. 1994. Phospholipase D: A
downstream effector of ARF in granulocytes. Science 263:523–2526.
Colley WC, Sung T-C, Roll R, Jenco J, Hammond SM, Altshuller Y,
Bar-Sagi D, Morris AJ, Frohman MA. 1997a. Phospholipase D2, a
distinct phospholipase D isoform with novel regulatory properties
that provokes cytoskeletal reorganization. Curr Biol 7:191–201.
Colley WC, Altshuller YM, Sueling CK, Copeland NG, Gilbert DJ,
Jenkins NA, Branch KD, Tsirka SE, Bollag RJ, Bollag WB,
Frohman MA. 1997b. Cloning and expression analysis of murine
phospholipase D. Biochem J 326:745–753.
Czarny M, Lavie Y, Fiucci G, Liscovitch M. 1999. Localization of
phospholipase D in detergent-insoluble, caveolin-rich membrane
domains. Modulation by caveolin-1 expression and caveolin-182-
101. J Biol Chem 274:2717–2724.
Ella KM, Meier GP, Bradshaw CD, Huffman KM, Spivey EC, Meier
KE. 1994. A fluorescent assay for agonist-activated phospholipase D
in mammalian cell extracts. Anal Biochem 218:136 –142.
Ella KM, Dolan JW, Qi C, Meier KE. 1996. Characterization of Sac-
charomyces cerevisiae deficient in expression of phospholipase D.
Biochem J 314:15–19.
Ella KM, Qi C, McNair AF, Park J-H, Wisehart-Johnson AE, Meier
KE. 1997. Phospholipase D activity in PC12 cells: effects of overex-
pression of alpha2A-adrenergic receptors. J Biol Chem 272:12909 –
12912.
Exton JH. 1997. New developments in phospholipase D. J Biol Chem
272:15579 –15582.
Griner RD, Qin F, Jung E, Sue-Ling CK, Crawford KB, Mann-Blak-
eney R, Bollag RJ, Bollag WB. 1999. 1,25-Dihydroxyvitamin D3
induces phospholipase D-1 expression in primary mouse epidermal
keratinocytes. J Biol Chem 274:4663– 4670.
Hammond SM, Altshuller YM, Sung T-C, Rudge SA, Rose K, Enge-
brecht J, Morris AJ, Frohman MA. 1995. Human ADP-ribosylation
factor-activated phosphatidylcholine-specific phospholipase D de-
fines a new and highly conserved gene family. J Biol Chem 270:
29640 –29643.
Hammond SM, Jenco JM, Nakashima S, Cadwallader K, Gu Q, Cook
S, Nozawa Y, Prestwich GD, Frohman MA, Morris AJ. 1997. Char-
acterization of two alternately spliced forms of phospholipase D1.
J Biol Chem 272:3860 –3868.
Ito Y, Nakashima S, Kanoh H, Nozawa Y. 1997. Implication of
Ca(21)-dependent protein tyrosine phosphorylation in carbachol-
induced phospholipase D activation in rat pheochromocytoma PC12
cells. J Neurochem 68:419 – 425.
Iyer SS, Kusner DJ. 1999. Association of phospholipase D activity
with the detergent-insoluble cytoskeleton of U937 promonocytic
leukocytes. J Biol Chem 274:2340 –2359.
Jenco JM, Rawlingson A, Daniels B, Morris AJ. 1998. Regulation of
phospholipase D2: selective inhibition of mammalian phospholipase
D isoenzymes by a- and b-synucleins. Biochemistry 37:4901– 4909.
Jinsi-Parimoo A, Deth RC. 1997. Reconstitution of alpha2D-adrener-
gic receptor coupling to phospholipase D in a PC12 cell lysate. J Biol
Chem 272:14556 –14561.
Jones LG, Ella KM, Bradshaw CD, Gause KC, Dey M, Wisehart-
Johnson A, Spivey EC, Meier KE. 1994. Activations of mitogen-
activated protein kinases and phospholipase D in A7r5 vascular
smooth muscle cells. J Biol Chem 269:23790 –23799.
Kim Y, Kim JE, Lee SD, Lee TG, Kim JH, Park JB, Han JM, Jang SK,
Suh PG, Ryu SH. 1999a. Phospholipase D1 is located and activated
by protein kinase Ca in the plasma membrane in 3Y1 fibroblast cell.
Biochim Biophys Acta 1436:319 –330.
Kim JH, Han JM, Lee S, Kim Y, Lee TG, Park JB, Lee SD, Suh PG,
Ryu SH. 1999b. Phospholipase D1 in caveolae: regulation by protein
kinase Ca and caveolin-1. Biochemistry 38:3763–3769.
Knoepp SM, Wisehart-Johnson AE, Buse MG, Ella KM, Bradshaw
CD, Meier KE. 1996. Synergistic effects of insulin and phorbol ester
on mitogen-activated protein kinase in Rat-1 HIR cells. J Biol Chem
271:1678 –1686.
Kodaki T, Yamashita S. 1997. Cloning, expression, and characteriza-
tion of a novel phospholipase D complementary DNA from rat brain.
J Biol Chem 272:11408 –11413.
Lee TG, Park JB, Lee SD, Hong S, Kim JH, Kim Y, Yi KS, Bae S,
Hannun YA, Obeid LM, Suh P-G, Ryu SH. 1997. Phorbol myristate
acetate-dependent association of protein kinase Ca with phospho-
lipase D1 in intact cells. Biochim Biophys Acta 1347:199 –204.
Leppa S, Saffrich R, Ansorge W, Bohmann D. 1998. Differential
regulation of c-Jun by ERK and JNK during PC12 cell differentia-
tion. EMBO J 17:4404 – 4413.
Lopez I, Arnold RS, Lambeth JD. 1998. Cloning and initial charac-
terization of a human phospholipase D2 (hPLD2). J Biol Chem
273:12846 –12852.
Marcil J, Harbour D, Naccache PH, Bourgoin S. 1997. Human phos-
pholipase D1 can be tyrosine-phosphorylated in HL-60 granulo-
cytes. J Biol Chem 272:20660 –20664.
Massenburg D, Han J-S, Liyange M, Patton WA, Rhee SG, Moss J,
Vaughan M. 1994. Activation of rat brain phospholipase D by ADP-
ribosylation factors 1, 5, and 6: separation of ADP-ribosylation
factor-dependent and oleate-dependent enzymes. Proc Natl Acad
Sci USA 91:11718 –11722.
Meier KE, Gibbs TC. 1999. Phospholipase D and phosphatidylcholine
metabolism. In: Milligan G, editor. Signal transduction: a practical
approach. Oxford, UK: Elsevier. p. 301–330.
Millar CA, Jess TJ, Saqib KM, Wakelam MJ, Gould GW. 1999.
3T3–L1 adipocytes express two isoforms of phospholipase D in
distinct subcellular compartments. Biochem Biophys Res Commun
254:734 –738.
Morooka T, Nishida E. 1998. Requirement of p38 mitogen-activated
protein kinase for neuronal differentiation in PC12 cells. J Biol
Chem 273:24285–24288.
Nakashima S, Matsuda Y, Akao Y, Yoshimura S, Sakai H, Hayakawa
K, Andoh M, Nozawa Y. 1997. Molecular cloning and chromosome
mapping of rat phospholipase D genes, Pld1a, Pld1b and Pld2.
Cytogenet Cell Genet 79:109 –113.
Nakashima S, Ohguchi K, Frohman MA, Nozawa Y. 1998. Increased
mRNA expression of phospholipase D (PLD) isozymes during gran-
ulocytic differentiation of HL-60 cells. Biochim Biophys Acta 1389:
173–177.
Nguyen TT, Scimeca JC, Filloux C, Peraldi P, Carpentier JL, Van
Obberghen E. 1993. Co-regulation of the mitogen-activated protein
kinase, extracellular signal-regulated kinase 1, and the 90-kDa
ribosomal S6 kinase in PC12 cells. Distinct effects of the neurotro-
phic factor, nerve growth factor, and the mitogenic factor, epider-
mal growth factor. J Biol Chem 268:9803–9810.
Ohguchi K, Nakashima S, Tan Z, Banno Y, Dohi S, Nozawa Y. 1997.
Increased activity of small GTP-binding protein-dependent phos-
pholipase D during differentiation in human promyelocytic leuke-
mic HL60 cells. J Biol Chem 272:1990 –1996.
Pai J-K, Siegel MI, Egan RW, Billah MM. 1988. Activation of phos-
pholipase D by chemotactic peptide in HL60 granulocytes. Biochem
Biophys Res Commun 150:355–364.
Pappan K, Qin W, Dyer JH, Zheng L, Wang X. 1997. Molecular
cloning and functional analysis of polyphosphoinositide-dependent
phospholipase D, PLDb, from Arabidopsis. J Biol Chem 272:7055–
7061.
Park S-K, Provost JJ, Bae CD, Ho W-T, Exton JH. 1997. Character-
ization of rat brain phospholipase D isozyme. J Biol Chem 272:
29263–29271.
Qi C, Park J-H, Gibbs TC, Shirley DW, Bradshaw CD, Ella KM, Meier
KE. 1998. Lysophosphatidic acid stimulates phospholipase D activ-
ity and cell proliferation in PC-3 human prostate cancer cells. J Cell
Physiol 174:261–272.
Rose K, Rudge SA, Frohman MA, Morris AJ, Engebrecht JA. 1995.
Phospholipase D signaling is essential for meiosis. Proc Natl Acad
Sci USA 92:12151–12155.
Saqib KM, Wakelam MJO. 1997. Differential expression of human
phospholipase D genes. Biochem Soc Trans 25:S586.
Sung TC, Altshuller YM, Morris AJ, Frohman MA. 1999. Molecular
analysis of mammalian phospholipase D2. J Biol Chem 274:494 –
502.
Swanson KD, Reigh C, Landreth GE. 1998. ATP-stimulated activa-
tion of the mitogen-activated protein kinases through ionotrophic
P2X2 purinoreceptors in PC12 cells. J Biol Chem 273:19965–19971.
 PLD1 AND PLD2 EXPRESSION IN MAMMALIAN CELLS
Toda K, Nogami M, Murakami K, Kanaho Y, Nakayama K. 1999.
Colocalization of phospholipase D1 and GTP-binding-defective mu-
tant of ADP-ribosylation factor 6 to endosomes and lysosomes.
FEBS Letts 442:221–225.
Waksman M, Eli Y, Liscovitch M, Gerst JE. 1996. Identification and
characterization of a gene encoding phospholipase D activity in
yeast. J Biol Chem 271:2361–2364.
Waksman M, Tang X, Eli Y, Gerst JE, Liscovitch M. 1997. Identifi-
cation of a novel Ca21-dependent, phosphatidylethanolamine-hy
87
drolyzing phospholipase D in yeast bearing a disruption in PLD1.
J Biol Chem 272:36 –39.
Xie M, Dubyak GR. 1991. Guanine-nucleotide- and adenine-nucleo-
tide-dependent regulation of phospholipase D in electropermeabi-
lized HL-60 granulocytes. Biochem J 278:81– 89.
Yoshimura S, Nakashima S, Ohguchi K, Sakai H, Shinoda J, Sakai N,
Nozawa Y. 1996. Differential mRNA expression of phospholipase D
(PLD) isozymes during cAMP-induced differentiation in C6 glioma
cells. Biochem Biophys Res Commun 225:494 – 499.