Identification of Multiple Phosphoinositide-specific
Phospholipases D as New Regulatory Enzymes for Phosphatidylinositol
3,4,5-Trisphosphate*
Tsui-Ting
Ching,
Da-Sheng
Wang,
Ao-Lin
Hsu,
Pei-Jung
Lu, and
Ching-Shih
Chen
From the Division of Pharmaceutical Sciences, College of Pharmacy,
University of Kentucky, Lexington, Kentucky 40536-0082
 |
ABSTRACT |
In the course of delineating the regulatory
mechanism underlying phosphatidylinositol 3,4,5-trisphosphate
(PI(3,4,5)P3) metabolism, we have
discovered three distinct phosphoinositide-specific phospholipase D
(PI-PLD) isozymes from rat brain, tentatively designated as PI-PLDa,
PI-PLDb, and PI-PLDc. These enzymes convert
[3H]PI(3,4,5)P3 to generate a novel inositol
phosphate, D-myo-[3H]inositol
3,4,5-trisphosphate ([3H]Ins(3,4,5)P3) and
phosphatidic acid. These isozymes are predominantly associated with the
cytosol, a notable difference from phosphatidylcholine PLDs. They are
partially purified by a three-step procedure consisting of DEAE,
heparin, and Sephacryl S-200 chromatography. PI-PLDa and PI-PLDb
display a high degree of substrate specificity for PI(3,4,5)P3, with a relative potency of
PI(3,4,5)P3
phosphatidylinositol 3-phosphate (PI(3)P)
or phosphatidylinositol 4-phosphate (PI(4)P) > phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) > phosphatidylinositol 3,4-bisphosphate
(PI(3,4)P2). In contrast, PI-PLDc preferentially utilizes
PI(3)P as substrate, followed by, in sequence, PI(3,4,5)P3, PI(4)P, PI(3,4)P2, and PI(4,5)P2. Both
PI(3,4)P2 and PI(4,5)P2 are poor substrates for
all three isozymes, indicating that the regulatory mechanisms
underlying these phosphoinositides are different from that of
PI(3,4,5)P3. None of these enzymes reacts with
phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine.
All three PI-PLDs are Ca2+-dependent. Among
them, PI-PLDb and PI-PLDc show maximum activities within a
sub-µM range (0.3 and 0.9 µM
Ca2+, respectively), whereas PI-PLDa exhibits an optimal
[Ca2+] at 20 µM. In contrast to PC-PLD,
Mg2+ has no significant effect on the enzyme activity. All
three enzymes require sodium deoxycholate for optimal activities; other
detergents examined including Triton X-100 and Nonidet P-40 are,
however, inhibitory. In addition, PI(4,5)P2 stimulates
these isozymes in a dose-dependent manner. Enhancement in
the enzyme activity is noted only when the molar ratio of
PI(4,5)P2 to PI(3,4,5)P3 is between 1:1 and
2:1.
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INTRODUCTION |
PI(3,4,5)P31and
PI(3,4)P2 are produced by PI 3-kinase in response to a wide
array of external stimuli (1, 2). These two phosphoinositides and their
downstream effector Akt constitute the key component of a major
signaling pathway that acts both to stimulate cell growth and to
prevent apoptosis (3-5). In view of their physiological importance,
the metabolism of these lipid second messengers has been the focus of
many recent investigations. Evidence indicates that they are not
susceptible to hydrolysis by any known phospholipase C (6) and that
different types of phosphatases mediate the major degradative pathway
via dephosphorylation. For example, there exist multiple inositide
polyphosphate 5-phosphatases that transform PI(3,4,5)P3 to
PI(3,4)P2, through which the ratio of these two lipid
second messengers is controlled. These enzymes include
PI(3,4,5)P3 5-phosphatases (7, 8), SHIP (SH2-containing inositol 5-phosphatase), (9-11) or SIP (signaling inositol
polyphosphate 5-phosphatase) (12), and synaptojanin (13). Especially
noteworthy is the discovery that the PTEN tumor suppressor displays a
PI(3,4,5)P3 3-phosphatase activity (14), thus terminating
the second messenger activities of PI(3,4,5)P3 by
converting it to PI(4,5)P2. This finding provides an
intricate link between PI(3,4,5)P3 regulation and
tumorigenesis (15). Tumor cells with mutant forms of PTEN lack such an
off-switch mechanism for PI 3-kinase, thereby containing high levels of
PI(3,4,5)P3 and PI(3,4)P2 and high endogenous
Akt activity (16, 17). Loss of PTEN function has been found in a
variety of common human cancers, including breast, prostate, and brain
cancer (18), and may attribute to the inability of cancer cells to
undergo apoptosis (17).
In an effort to gain insight into the complex machinery that regulates
PI(3,4,5)P3, we have synthesized
[1-3H]PI(3,4,5)P3 to examine its metabolite
fate in rat brain extracts. Here we report the identification of three
distinct cytosolic PI-PLD isozymes that convert
[1-3H]PI(3,4,5)P3 to a novel inositol
phosphate [1-3H]Ins(3,4,5)P3 and PA. The
present data raise a possibility that these PI-PLDs act as a regulator
of PI(3,4,5)P3 in vivo. This premise connotes
physiological implications conforming to that of the PTEN tumor
suppressor. Furthermore, because PA is generated, these isozymes may
provide a putative link between PI 3-kinase and other signaling
pathways mediated by PA or its metabolites.
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MATERIALS AND METHODS |
[1-3H]PI(3,4,5)P3 (specific
activity 13.5 mCi/mmol), [1-3H]PI(3,4)P2
(38.8 mCi/mmol), [1-3H]PI(4,5)P2 (11.7 mCi/mmol), [1-3H]PI(4)P (16.3 mCi/mmol),
[1-3H]PI(3)P (40 mCi/mmol),
[palmitoyl-14C(U)]PI(3,4,5)P3 (1.6 mCi/mmol),
[1-3H]Ins(3,4,5)P3,
[1-3H]Ins(3,4)P2, and
[1-3H]Ins(4,5)P2 were synthesized according
to a modification of the synthetic methods described previously for the
respective nonradioactive counterparts (19, 20). All synthetic inositol
lipids were di-C16 derivatives. Phosphatidylethanolamine
(PE), phosphatidylserine (PS), phosphatidylcholine (PC), protease
inhibitors (leupeptin, pepstatin-A, and AEBSF), porcine pancreas
PLA2, Bacillus cerus PI-specific PLC, cabbage
PLD, heparin-agarose (type 1; H-6508), and Sephacryl S-200-HR were
products of Sigma. Toyopearl DEAE-650M was purchased from Tosohaas.
[3H]PC, [3H]Ins(1,4,5)P3, and
[3H]Ins(1,3,4,5)P4 were obtained from NEN
Life Science Products. Recombinant PLC
-1, PLC
-1, and PLD1 were
kind gifts of Drs. Jon Lomasney (Northwestern University Medical
School), Sue Goo Rhee (NHLBI, National Institutes of Health), and John
H. Exton (Vanderbilt University Medical School), respectively.
Unstripped rat brain was purchased from Pel-Freez Biologicals. Enzyme
inhibitors such as aristolochic acid, ET-18-OCH3, and
dihydro-D-erythro-sphingosine were products of Calbiochem.
Assay of PI(3,4,5)P3-metabolizing or PI-PLD
Activity
During purification, PI(3,4,5)P3-metabolizing or
PI-PLD activity in all enzyme preparations was assayed by monitoring
the liberation of the 3H-labeled phosphoinositol head group
from [1-3H]PI(3,4,5)P3 into the medium.
[1-3H]PI(3,4,5)P3 (0.8 µg; total
radioactivity, 0.2 µCi), PE (40 µg), and PS (5 µg) were suspended
in 1 ml of 20 mM Hepes, pH 7, containing 120 mM
KCl, 10 mM NaCl, 2 mM EGTA, and 0.8 mM sodium deoxycholate. Various amounts of
CaCl2 were added to the mixture before assays, and the free
Ca2+ concentration was calculated by a computer program
developed by Karl-Josef Foehr and programmed by Wokciech Warchei (1990, version 2.1). The suspension was sonicated in a water bath-type sonicator for 5 min, and mixed vigorously with a vortex mixer before
assays. Various enzyme preparations (10 µl) were incubated with 40 µl of the aforementioned Hepes buffer. The reaction was initiated by
adding 50 µl of the phospholipid mixture, incubated at 37 °C
for 30 min, and stopped by adding 200 µl of 10% trichloroacetic acid
and 150 µl of 10 mg/ml bovine serum albumin. The mixture was
centrifuged at 15,000 × g for 5 min, and the
radioactivity in the supernatant was measured by liquid scintillation.
The composition of the control was identical to that mentioned above
except that the enzyme preparation was replaced by an equal amount of
distilled H2O.
Partial Purification of PI-PLD Isozymes from Rat Brain
Cytosol
Rat brain was minced and suspended in 8 volumes of ice-cold 10 mM Tris/HCl, pH 7.4, containing 0.25 M sucrose,
1 mM dithiothreitol, 20 µg/ml leupeptin, 2 µg/ml
pepstatin A, and 1 mM AEBSF. The small pieces were
homogenized in a Dounce homogenizer with six strokes up and down. The
homogenate was centrifuged at 1,000 × g to remove cell
debris and intact nuclei. The supernatant was centrifuged at
100,000 × g for 1 h to prepare the cytosolic fraction.
Step 1. DEAE Chromatography--
After being dialyzed against 50 mM Tris/HCl, pH 7.4, containing 1 mM
dithiothreitol (buffer A) for 12 h, the cytosolic fraction (1.8 g
of protein; 0.039 nmol/mg/min PIP3-metabolizing activity) was loaded onto a Toyopearl DEAE-650M column (3 × 10 cm)
previously equilibrated with buffer A. The column was washed with 400 ml of buffer A followed by 300 ml of a linear NaCl gradient of 0-200 mM in the same buffer. Fractions of 2.25 ml were collected
after the gradient started. PI(3,4,5)P3-metabolizing
activity was analyzed as described above at two different free
Ca2+ concentrations, 0.3 and 20 µM,
respectively. Two different activity profiles were noted under these
two Ca2+ levels (Fig. 1), indicating the presence of more
than one PI(3,4,5)P3-metabolizing enzyme. Fractions
271-289 and 295-312, designated as D1 and D2, were pooled separately,
concentrated over a molecular weight 10,000 cutoff ultrafiltration
membrane (Filtron Omega unit), and dialyzed against buffer A for
12 h.
Step 2. Heparin Chromatography--
The dialyzed D1 and D2
samples (76.7 and 79.7 mg of protein; 0.6 and 0.58 nmol of
PIP3/min/mg specific activity, respectively) from step 1 were individually applied to a heparin-agarose column (1 × 10 cm)
equilibrated with buffer A. The column was washed with 85 ml of buffer
A, and the adsorbed proteins were eluted with 150 ml of a linear
gradient of 0-400 mM NaCl. Fractions of 1 ml were
collected. As shown in Fig. 2, A and B, both D1
and D2 were resolved into two PI(3,4,5)P3-metabolizing
activity peaks. For D1, fractions 145-160 (designated as D1/H1) and
fractions 177-192 (designated as D1/H2) were pooled separately and
concentrated by ultrafiltration. For D2, fractions 125-139 (designated
as D2/H) were pooled and concentrated by ultrafiltration.
Step 3. Sephacryl S-200-HR Chromatography--
The concentrated
D1/H1 (4.5 mg; 2.3 nmol of PIP3/mg/min specific activity),
D1/H2 (2.3 mg; 9.1 nmol of PIP3/mg/min specific activity),
and D2/H (3.84 mg; 2.6 nmol of PIP3/mg/min specific activity) from step 2 were loaded individually onto a Sepharose S-200-HR column (1 × 100 cm). The equilibration and eluting
buffer was buffer A, and fractions of 2 ml were collected (see Fig. 3, A-C). The collected fractions were: the D1/H1 column,
fractions 76-79 (designated as D1/H1/S or PI-PLDa; 0.18 mg; 13.7 nmol
of PIP3/mg/min specific activity); the D1/H2 column,
fractions 71-78 (designated as D1/H2/S or PI-PLDb; (0.077 mg; 40.9 nmol PIP3/mg/min sp. act.); the D2/H column, fractions
71-77 (designated as D2/H/S or PI-PLDc; 0.24 mg; 39.8 nmol
PIP3/mg/min specific activity).
Identification and Quantitation of the Radiolabeled Inositol
Phosphates by HPLC
For the substrate specificity study, the identity and
quantity of the radioactive phosphoinositols liberated by the enzymatic hydrolysis of [3H]PI(3,4,5)P3,
[3H]PI(3,4)P2,
[3H]PI(4,5)P2, [3H]PI(3)P, and
[3H]PI(4)P were determined by HPLC. The enzyme
incubation, under the same conditions as described above for
[3H]PI(3,4,5)P3, was terminated by adding 100 µl of HClO4/CHCl3 (v/v, 1:0.33) followed by
100 µl of 10 mg/ml bovine serum albumin. The mixture was centrifuged
at 13,000 × g for 10 min. The supernatant was
collected and was extracted immediately with 300 µl of
tri-n-octylamine/Freon (v/v, 1:1) twice to remove
HClO4. The neutralized solution was transferred to a new
vial, and the radioactive inositol phosphate was analyzed by HPLC on an
Adsorbosphere Sax column (5 µm; 4.6 × 200 mm) equilibrated with
H2O. The phosphoinositol was eluted with a linear gradient
of 0-0.9 M NH4H2PO4 in
60 min at a flow rate of 1 ml/min. Fractions were collected every 1 ml,
and their radioactivity was measured by liquid scintillation.
Synthetic [3H]Ins(1,3,4,5)P4,
[3H]Ins(3,4,5)P3,
[3H]Ins(1,4,5)P3,
[3H]Ins(4,5)P2,
[3H]Ins(3,4)P2, [3H]Ins(4)P,
and [3H]Ins(3)P were used as standards. The respective
retention times were 60, 50, 48, 43, 41, 31, and 29 min, respectively.
Stoichiometric Formation of Ins(3,4,5)P3 and
PA
A mixture of [1-3H]PI(3,4,5)P3 (15 µM; 20,000 cpm) and
[palmitoyl-14C(U)]PI(3,4,5)P3 (117 µM; 35,000 cpm) was exposed to fraction D1/H2/S
(PI-PLDb), in a final volume of 200 µl, in the presence of 0.3 µM Ca2+. Reaction was stopped at the
indicated times (1, 5, 20, 40, and 60 min) by extracting the mixture
with 200 µl of HClO4/CHCl3. After a brief centrifugation,
the two phases were separated. The aqueous phase was treated as
described above for the HPLC analysis of Ins(3,4,5)P3. The
organic phase was transferred to a new vial and dried by a stream of
N2. The residue was dissolved in CHCl3, spotted
onto 1% oxalic acid-treated TLC plate, and developed with n-propyl alcohol and 2 M acetic acid (13: 7)
overnight. After drying, spots were located by autoradiography and
compared with standards. The autoradiograms were scanned by a photodyne
image system. The spots corresponding to [14C]PA and
[14C]PI(3,4,5)P3 were scraped off the plate,
and the associated radioactivity was measured by liquid scintillation.
Periodate Oxidation of
[1-3H]Ins(3,4,5)P3
The HPLC-purified [1-3H]Ins(3,4,5)P3
with a total radioactivity of approximately 20,000 cpm was oxidized
with 0.1 M periodic acid, pH 2.0, and reduced with 1 M NaBH4 as described (21). Such treatments
would oxidize inositol phosphates at vicinal hydroxyls. It was found
that the resulting polyphosphate lost all radioactivity, indicating the
adjacent 1-, 2-, and 6-hydroxyls were unsubstituted.
 |
RESULTS |
To investigate the metabolic fate of D-3 phosphoinositides,
[1-3H]PI(3,4,5)P3 was synthesized and exposed
to the cell lysate of rat brain. The incubation mixture was extracted
with CHCl3/CH3OH to isolate the
phosphoinositide metabolites. However, substantial radioactivity
appeared in the aqueous phase in a time- and protein concentration-dependent manner, suggesting that the
3H-labeled head group was released from
[1-3H]PI(3,4,5)P3 via phospholipase
hydrolysis. HPLC analysis of the aqueous fraction revealed that the
liberated radioactivity was associated with free inositol and trace
amounts of inositol mono- and bisphosphates (data not shown). This
result showed that the inositol phosphate generated from
PI(3,4,5)P3 was rapidly metabolized in the crude extract.
Other rat tissues examined including the liver, the kidney, and
platelets also contained such PI(3,4,5)P3-metabolizing activity.
As part of our effort to verify the identity of the
PI(3,4,5)P3-metabolizing enzyme(s), purified phospholipase
preparations from different sources were examined for the activity
toward [1-3H]PI(3,4,5)P3. These included
porcine pancreas PLA2, recombinant PLC-
1, recombinant
PLC-
1, B. cerus PI-specific PLC, recombinant PLD1, and
cabbage PLD. However, none of these enzymes showed appreciable hydrolysis of [1-3H]PI(3,4,5)P3. With regard
to enzyme inhibition, neomycin (1 mM), which binds
phosphoinositides with high affinity (22), completely blocked the
hydrolysis by rat brain extracts. Other known phospholipase inhibitors
such as aristolochic acid for PLA2, ET-18-OCH3
for PI-PLC, and dihydro-D-erythro-sphingosine
for PLD gave no or only partial inhibition of the enzyme activity even
at concentrations 20 times over the corresponding IC50
values (data not shown).
These data prompted us to identify the enzyme(s) responsible for
[3H]PI(3,4,5)P3 hydrolysis. Subcellular
fractionation indicated that more than 85% of the
[3H]PI(3,4,5)P3-metabolizing activity resided
in the cytosolic fraction (100,000 × g supernatant).
The rest of the activity was associated with various membrane fractions
including the plasma membrane and microsomes. Thus, the rat brain
cytosol was subjected to chromatographic purification by DEAE ion
exchange, heparin-agarose, and Sephacryl S-200-HR columns, in tandem.
To explore the possibility that there might exist multiple responsible
enzymes with different Ca2+ requirements,
PI(3,4,5)P3-metabolizing activity was monitored at low and
high Ca2+ concentrations, represented by 0.3 and 20 µM, respectively, throughout the purification. As shown
in the DEAE chromatographic profile (Fig.
1), a broad peak exhibiting
PI(3,4,5)P3-metabolizing activity was detected at each
Ca2+ level (0.3 µM, closed
triangles; 20 µM, open squares).

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Fig. 1.
Separation of two
PI(3,4,5)P3-metabolizing activities by DEAE
chromatography. Rat brain cytosol, after dialysis, was applied to
a Toyopearl DEAE-650M column and eluted as described under "Materials
and Methods." Closed circles denote
A280 readings. Every third eluted fraction was
assayed for the release of radioactivity from
[1-3H]PI(3,4,5)P3 into medium at 20 µM (open squares) and 0.3 µM
(closed triangles) Ca2+, separately. Only the
region after the gradient started (at 200th fraction) is shown because
no enzyme activity was found in prior fractions. The thick
lines above each activity peak indicate the fractions that were
pooled for fractions D1 and D2, respectively.
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Nevertheless, the activity peaks detected at 0.3 and 20 µM Ca2+ showed different migration patterns
in the elution profile. This finding suggested that there were two or
more PI(3,4,5)P3-metabolizing enzymes with different
Ca2+ dependence among these active fractions. Thus,
fractions 271-289 and 295-312, designated as D1 and D2, were pooled
separately. The subsequent heparin chromatography provided definite
evidence that D1 and D2 each contained two
PI(3,4,5)P3-metabolizing activities (Fig.
2, A and B).

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Fig. 2.
Heparin chromatography of fraction D1
(panel A) and fraction D2 (panel
B). The concentrated, dialyzed D1 and D2 samples were
applied to heparin-agarose columns and eluted as described under
"Materials and Methods." Closed circles denote
A280 readings. Every third fraction was assayed
for [1-3H]PI(3,4,5)P3-metabolizing activity
at 20 µM (open squares) and 0.3 µM (closed triangles), separately. The
thick lines above each activity peak indicate the
fractions that were pooled for fractions D1/H1, D1/H2, and D2/H,
respectively.
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As shown, fraction D1 gave two well resolved activity peaks between
fractions 150 and 210 (Fig. 2A). Among these active
fractions, the enzyme activity of the first peak increased with higher
[Ca2+] (0.3 µM, closed
triangles; 20 µM, open squares), whereas
the activity of the second peak was unaffected by Ca2+
change. Accordingly, fractions 145-160 and 177-192 were pooled separately and were designated as D1/H1 and D1/H2, respectively. Similarly, two PI(3,4,5)P3-metabolizing activity peaks were
also noted for fraction D2 after the heparin column (Fig.
2B; 0.3 µM, closed triangles; 20 µM, open squares). In contrast, the first peak
exhibited a higher activity at low Ca2+ compared with high
Ca2+, whereas the second, smaller peak showed no
significant difference in the Ca2+ requirement. Considering
that fractions D1 and D2 were juxtaposed in the DEAE elution profile,
it was possible that the second peak from fraction D2 was identical to
fraction D1/H2 in light of their elution times and Ca2+
dependence. Thus, only the first activity peak was collected, which was
designated as D2/H. Fractions D1/H1, D1/H2, and D2/H were
chromatographed further on a Sephacryl S-200-HR column (Fig. 3, A-C).

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Fig. 3.
Sephacryl S-200 chromatography of fraction
D1/H1 (panel A), fraction D1/H2 (panel
B), and fraction D2/H (panel C). The
concentrated, dialyzed D1/H1, D1/H2, and D2/H samples were purified
further by gel filtration columns (see "Materials and Methods").
Closed circles denote A280 readings.
Every third fraction was assayed for
[1-3H]PI(3,4,5)P3-metabolizing activity at
either 20 µM (open squares) or 0.3 µM (closed triangles). The thick
lines above each activity peak indicate the fractions that were
pooled for fractions D1/H1/S (PI-PLDa), D1/H2/S (PI-PLDb), and D2/H/S
(PI-PLDc), respectively.
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The resulting active fractions were designated as D1/H1/S, D1/H2/S, and
D2/H/S for further discussion. This three-step procedure resulted in
354-, 1,056- and 1,027-fold purification with specific activities of
13.7, 40.9, and 39.8 nmol of PI(3,4,5)P3/mg of protein/min ([Ca2+] = 2 µM) for fractions D1/H1/S,
D1/H2/S, and D2/H/S, respectively. Concerning
PI(3,4,5)P3-metabolizing activity, fraction D2/H/S was
highly unstable. Up to 80% of the enzyme activity was lost after
storing at 0 °C for 2 days.
The Ca2+ dependence of these enzyme preparations was
examined. Fig. 4 indicates that the
PI(3,4,5)P3-metabolizing activity of these enzymes was
inhibited by EDTA, and the inhibition could be overcome by adding
Ca2+ in a concentration-dependent manner. Both
D1/H2/S and D2/H/S showed a similar Ca2+ requirement, with
maximum PI(3,4,5)P3-metabolizing activities at a
sub-µM range (0.3 and 0.9 µM,
respectively), whereas D1/H1/S displayed an optimal
[Ca2+] at 20 µM. The difference in the
Ca2+ requirement underscored a potential distinction in the
roles of these enzymes in PI(3,4,5)P3 metabolism. Moreover,
Mg2+ had no appreciable effect by itself on the activity of
or on the Ca2+ dependence for any of these enzymes (data
not shown).

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Fig. 4.
Effect of Ca2+ on
PI(3,4,5)P3-metabolizing activities of fractions D1/H1/S
(PI-PLDa; closed circles), D1H2/S (PI-PLDb; open
circles), and D2/H/S (PI-PLDc; closed
triangles). Each enzyme preparation was incubated with
[1-3H]PI(3,4,5)P3 in the presence of the
indicated concentrations of Ca2+, and the
PI(3,4,5)P3-metabolizing activity was assayed as described
under "Materials and Methods." The incubation medium contained 2 mM EGTA, and the concentrations of free Ca2+
were calculated from the added Ca2+ concentrations. Each
data point represents the mean of three determinations.
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In an effort to gain insight into the catalytic behaviors of these
enzymes, we also synthesized [1-3H]PI(3,4)P2,
[1-3H]PI(4,5)P2,
[1-3H]PI(3)P, and [1-3H]PI(4)P for
examinations. These phospholipids were exposed to individual enzymes,
and the released water-soluble products were analyzed by reverse-phase
HPLC, aiming at both product identification and substrate specificity
determination. Representative HPLC profiles of the
[1-3H]phosphoinositol products from incubations of
the respective substrates with fraction D1/H2/S are shown in Fig.
5.

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Fig. 5.
HPLC profiles of 3H-labeled
inositol phosphates generated from
[1-3H]PI(3,4,5)P3,
[1-3H]PI(3,4)P2,
[1-3H]PI(4,5)P2, [1-3H]PI(3)P,
and [1-3H]PI(4)P metabolism by D1/H2/S (PI-PLDb) (see
"Materials and Methods"). The arrows indicate the
retention times for the standards of (a)
[3H]Ins(1,3,4,5)P4 (60 min), (b)
[3H]Ins(3,4,5)P3 (50 min), (c)
[3H]Ins(1,4,5)P3 (48 min), (d)
[3H]Ins(4,5)P2 (43 min), (e)
[3H]Ins(3,4)P2 (41 min), (f)
[3H]Ins(4)P (31 min), and (g)
[3H]Ins(3)P (29 min).
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These HPLC profiles revealed two important findings. First, D1/H2/S
displayed a high degree of substrate specificity for
PI(3,4,5)P3. The relative potency for the substrates
examined was PI(3,4,5)P3
PI(4)P > PI(3)P
PI(4,5)P2 and PI(3,4)P2. The utilization of the
latter two, especially PI(3,4)P2, accounted for less than 5% of that of PI(3,4,5)P3 (Fig.
6, panel A). These HPLC
results were consistent with those obtained by measuring
[3H]phosphoinositol release from the respective
substrates by liquid scintillation.

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Fig. 6.
Substrate specificity of D1/H2/S (PI-PLDb)
(panel A), D1/H1/S (PI-PLDa) (panel
B), and D1/H/S (PI-PLDc) (panel C).
The enzyme assay was performed by HPLC analysis of
[3H]phosphoinositol formation from the respective
substrates. Each data point represents the means of three
determinations.
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Second, the retention times of the hydrolysis products from
[3H]PI(3,4,5)P3,
[3H]PI(3,4)P2,
[3H]PI(4,5)P2, [3H]PI(3)P, and
[3H]PI(4)P coincided with those of
[3H]Ins(3,4,5)P3,
[3H]Ins(3,4)P2,
[3H]Ins(4,5)P2, [3H]Ins(3)P,
and [3H]Ins(4)P, respectively. These data clearly
indicated that D1/H2/S displayed a PLD activity. In principle,
should it be a PLC, the respective degradative products would have been
[3H]Ins(1,3,4,5)P4,
[3H]Ins(1,3,4)P2,
[3H]Ins(1,4,5)P3,
[3H]Ins(1,3)P2, and
[3H]Ins(1,4)P2, of which the retention times
would differ from the respective experimental data by almost 10 min
because of an additional phosphate moiety in the PLC products.
Moreover, it is worthy to note that after periodate
oxidation/NaBH4 reduction, the radioactivity associated
with the hydrolysis product of PI(3,4,5)P3 was completely lost, confirming that the adjacent 1-, 2-, and 6-hydroxyls were unsubstituted (data not shown).
Additional evidence that the responsible enzyme was a PLD was obtained
from the stoichiometry of product formation. A mixture of
palmitoyl-[14C(U)]PI(3,4,5)P3 and
[1-3H]PI(3,4,5)P3 was exposed to fraction
D1/H2/S, and the formation of [14C]PA and
[3H]Ins(3,4,5)P3 was analyzed by TLC and
HPLC, respectively, at different time intervals (Fig.
7). The amounts of
[3H]Ins(3,4,5)P3 formed at the indicated
times were consistent with those of [14C]PA.

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Fig. 7.
Time course of
[3H]Ins(3,4,5)P3 (panel
A) and palmitoyl-[14C(U)]PA formation
(panel B). A mixture of
[1-3H]PI(3,4,5)P3 and
palmitoyl-[14C(U)PI(3,4,5)P3 was incubated
with D1/H2/S (PI-PLDb). The reaction was stopped at the indicated times
and subjected to solvent extraction to separate
[3H]phosphoinositol and [14C]PA for
HPLC and TLC analyses, respectively (see "Material and Methods").
The lines beside the right panel indicate the
migration patterns of the authentic standards on TLC, including
diacylglycerol (DAG), PA, lyso-PA, PI(3,4)P2, and
PI(3,4,5)P3.
|
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Similar analytical data from HPLC and TLC were obtained with fractions
D1/H1/S and D2/H/S, confirming that these two enzymes were also of the
type of PLD. However, their substrate specificities were slightly
different from that of D1/H2/S (Fig. 6). For further discussion,
D1/H1/S, D1/H2/S, and D2/H/S were tentatively designated as PI-PLDa,
PI-PLDb, and PI-PLDc. As shown, PI-PLDa (Fig. 6, panel B)
and PI-PLDb (panel A) displayed high degree of specificity for PI(3,4,5)P3. The activities toward other substrates
examined accounted for 1% (PI(3,4)P2) to 30% (PI(3)P or
PI(4)P) of that of PI(3,4,5)P3. In contrast, PI-PLDc
preferentially hydrolyzed PI(3)P (relative activity, 100%), followed
by, in sequence, PI(3,4,5)P3 (60%), PI(4)P (37%),
PI(3,4)P2 (12%), and PI(4,5)P2 (3%). Evidence showed that none of these PI-PLDs utilized PC, PS, or PE as substrates. First, prolonged exposure of
N-[methyl-3H]PC to any of these
isozymes did not give rise to appreciable release of radioactivity into
the milieu. Second, the rate of [3H]PI(3,4,5)P3 hydrolysis was not affected
by the addition of excess amounts of PE or PS to the incubation mixture.
The effect of detergents on the PI-PLD activity was investigated. In
all of the aforementioned assays, the reaction mixture contained 0.8 mM sodium deoxycholate. Removal of the detergent or
replacement with 0.1-1% Nonidet P-40 or Triton X-100 resulted in
substantial loss of enzyme activity for all three isozymes, indicating
the stringent requirement of sodium deoxycholate for PI-PLD activity.
This dependence might be attributable to the effect of detergent on
PI(3,4,5)P3 packaging in lipid vesicles, which affected the
substrate availability and/or enzyme accessibility.
Earlier studies have shown that PC-PLDs were strongly stimulated by
PI(4,5)P2 and PI(3,4,5)P3 with an equal potency
(23, 24). Fig. 8 depicts the effect of
PI(4,5)P2 on the PI(3,4,5)P3-metabolizing activity of three PI-PLD isozymes. The individual enzymes were incubated with [3H]PI(3,4,5)P3 in the
presence of increasing amounts of PI(4,5)P2 without sodium
deoxycholate. As shown, PI(4,5)P2 enhanced the basal enzyme
activity up to 2.5-fold. However, this stimulating effect occurred only
within a narrow range of PI(4,5)P2/PI(3,4,5)P3 molar ratios between 1:1 and 2:1. Excess amounts of
PI(4,5)P2 either inhibited or had no effect on the
PI(3,4,5)P3-metabolizing activity. It is plausible that
because PI(4,5)P2 was a poor substrate, it might compete
with PI(3,4,5)P3 for enzyme binding, thereby counteracting
its stimulating effect.

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Fig. 8.
Dose-dependent effect of
PI(4,5)P2 on PI(3,4,5)P3-metabolizing activity
of PI-PLDs. The PI(3,4,5)P3-metabolizing activity of
individual isozymes was determined at the respective optimal
[Ca2+] as described under "Materials and Methods."
However, sodium deoxycholate (0.8 mM) was replaced by
increasing amounts of PI(4,5)P2.
|
|
 |
DISCUSSION |
PI 3-kinase activation leads to a transient accumulation of
PI(3,4,5)P3 and PI(3,4)P2, of which the
concentrations rise from 0.05-0.2 µM at resting states
to 1-2 µM upon agonist stimulation (25). The prevailing
levels of these PI 3-kinase lipid products are regulated by a delicate
balance between its rates of synthesis and metabolism. This study
presents the first evidence that there exist at least three distinct
cytosolic Ca2+-dependent PLD isozymes that may
take part in PI(3,4,5)P3 regulation in vivo.
Taken together, the metabolism of PI(3,4,5)P3 consists of
three pathways. These include dephosphorylation by multiple PI(3,4,5)P3 5-phosphatases to form PI(3,4)P2
(7-13), which is further metabolized to PI(3)P or PI(4)P (2),
dephosphorylation by a 3-phosphatase (e.g. PTEN) to form
PI(4,5)P2 for recycling by PI 3-kinase (14), and hydrolysis
by PI-PLDs to form Ins(3,4,5)P3. Among these products,
PI(3,4)P2 and PI(4,5)P2 act as lipid second messengers involved in different cellular processes (2, 26), whereas
Ins(3,4,5)P3, a novel inositol phosphate with unknown function, diffuses into the cytosol to generate other inositol phosphates and eventually free inositol for recycling. Considering the
underlying difference in the functions of these metabolic products, the
relative fluxes of PI(3,4,5)P3 through these pathways are
likely to be controlled stringently. The present data on substrate preference suggest that the PLD pathway may represent a major pathway
for PI(3,4,5)P3 metabolism. This mechanism is unique to PI(3,4,5)P3 because other phosphoinositides examined,
especially PI(3,4)P2 and PI(4,5)P2, are poor
substrates for all three PI-PLDs.
This study reaffirms the notion that there are different PLD subtypes
in different subcellular compartments of mammalian cells, each of which
plays a distinct role in cellular signaling pathways leading to various
cellular processes (27-30). In contrast to mammalian PC-PLDs (PLD1 and
PLD2) which are predominantly membrane-associated enzymes (31), PI-PLD
isozymes were constitutively present in the cytosol. Cytosolic PLD
activities with varying substrate preference have been reported in the
literature (32-34). Attempts to purify these enzymes, however, have
not met with success. Moreover, PLD activity toward
[3H]phosphatidylinositol has been detected in the
postnuclear fraction of human neutrophils (35) and in the cytosol of
Madin-Darby canine kidney cells (33). These PLDs were reported to be
Ca2+-dependent (33, 35), and the cytosolic PLD
in these cells did not require detergent for its activity (33). Because
the properties of these PLDs are undescribed in the literature, their correlation to PI-PLDa, PI-PLDb, or PI-PLDc remains inconclusive.
These three PI-PLD isozymes, nevertheless, displayed common biochemical
and catalytic properties. They were all
Ca2+-dependent enzymes with somewhat similar
substrate specificity, required sodium deoxycholate for the activity,
and were stimulated by PI(4,5)P2 in a
dose-dependent manner. However, PI(4,5)P2
stimulation was noted only within a narrow
PI(4,5)P2/PI(3,4,5)P3 molar ratio range. It is
unclear whether this stimulatory effect is mediated by direct enzyme
activation or by affecting the PI(3,4,5)P3 packaging in
lipid vesicles, which remains to be investigated.
The biological utility of PI-PLD may be multifaceted. As demonstrated
by the tumorigenic consequence of PTEN mutations, modifications to any
of these PI(3,4,5)P3-metabolizing activities may also lead to pathological conditions. In light of the formation of PA, PI-PLD may
provide a putative link between D-3 phosphoinositides and signaling
pathways mediated by these PLD products. The physiological function of
PC-PLD has been largely attributed to the rapid and transient increases
in PA (27-30). Evidence indicates that PA and its metabolites such as
diacylglycerol and lysophosphatidic acid serve as regulators of key
cellular processes including vesicle trafficking and other
membrane-associated events. The premise that PI-PLD may contribute to
transient PA increase in a manner distinct from that of PC-PLD suggests
a potential mechanism for cross-communication between PI 3-kinase and
PA-dependent processes. With regard to
Ins(3,4,5)P3, it is a novel inositol phosphate whose
physiological function remains unknown. Recent evidence indicates that
its structural analog Ins(3,4,5,6)P4 is a potent activator
of Ca2+-dependent chloride channels (36).
In summary, this study provides definite evidence that there exist at
least three cytosolic PI-PLD isozymes. Although these isozymes need
further characterization, the present data suggest that they represent
new regulators of PI(3,4,5)P3 in vivo. It is
conceivable that additional regulating and membrane localization mechanisms are involved in the regulation of these PI-PLD activities, which is currently under investigation.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Jon Lomasney (Northwestern
University Medical School), Dr. Sue Goo Rhee (NHLBI, National
Institutes of Health), and Dr. John H. Exton (Vanderbilt University
Medical School) for kind gifts of recombinant PLC-
1, PLC-
1, and
PLD1, respectively.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant R01 GM53448.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: ASTeCC Facility, Rm.
323B, University of Kentucky, Lexington, KY 40506-0286. Tel.: 606-257-2300 (ext. 261); Fax: 606-257-2489; E-mail:
cchen1{at}pop.uky.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
PI(3, 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate;
PI(3, 4)P2, phosphatidylinositol 3,4-bisphosphate;
PI
3-kinase, phosphoinositide 3-kinase;
PLD, phospholipase D;
PI(3)P, phosphatidylinositol 3-monophosphate;
PI(4)P, phosphatidylinositol
4-monophosphate;
PI(4, 5)P2, phosphatidylinositol
4,5-bisphosphate;
PA, phosphatidic acid;
PC, phosphatidylcholine;
PS, phosphatidylserine;
PE, phosphatidylethanolamine;
Ins(3)P, D-myo-inositol 3-phosphate;
Ins(4)P, D-myo-inositol 4-phosphate;
Ins(1, 4,5)P3, D-myo-inositol
3,4,5-trisphosphate;
Ins(1, 3,4,5)P4,
D-myo-inositol 1,3,4,5-tetrakisphosphate;
PLA2, phospholipase A2;
PIP3, phosphatidylinositol trisphosphate;
AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride;
HPLC, high performance liquid
chromatography.
 |
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