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 ChenDagger

From the Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536-0082

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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[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 PLCdelta -1, PLCgamma -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.

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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-gamma 1, recombinant PLC-delta 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.

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.

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.

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.

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).

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.

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.

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-delta 1, PLC-gamma 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.

Dagger 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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DISCUSSION
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