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-microM range (0.3 and 0.9 microM Ca2+, respectively), whereas PI-PLDa exhibits an optimal [Ca2+] at 20 microM. 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.
Highlights
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 [3,4,5]. In view of their physiological importance, the metabolism of these lipid second messengers has been the focus of many recent investigations
We report the identification of three distinct cytosolic phosphoinositide-specific phospholipase D (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
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
Summary
[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]. 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 ϫ 10 cm) previously equilibrated with buffer A. 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 ϫ 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. 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) 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)
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