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D-Phosphatidyl-Myo-Inositol 3-Phosphate

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D-Phosphatidyl-Myo-Inositol 3-Phosphate Biochem. J. (1989) 259, 267-276 (Printed in Great Britain) 267 Metabolic and structural evidence for the existence of a third species of polyphosphoinositide in cells: D-phosphatidyl-myo-inositol 3-phosphate Leonard STEPHENS,* Phillip T. HAWKINSt and C. Peter DOWNES Smith Kline & French Research Ltd., The Frythe, Welwyn, Herts. AL6 9AR, U.K. When human 1321 NI astrocytoma cells were labelled to steady state with [3H]inositol and briefly with [32P]orthophosphate, a compound which contained both radiotracers and which co-migrated with phosphatidylinositol-myo-inositol 4-phosphate during t.l.c. could be extracted in acidic chloroform/ methanol. Treatment with methylamine under conditions which lead to deacylation of conventional glycerophospholipids yielded a water-soluble moiety which was labelled with both radioisotopes and was eluted from an anion-exchange h.p.l.c. column with a retention time similar to, but distinct from, that of glycerophosphoinositol 4-phosphate. Experiments using sodium periodate and selective phosphatase enzymes to degrade this compound systematically generated a series of products which suggested the structure of the parent phospholipid was phosphatidyl-myo-inositol 3-phosphate (PtdIns3P). PtdIns3P is metabolically closely related to the pool(s) of inositol phospholipid(s) that serves as substrate(s) for an agonist-sensitive phosphoinositidase C, as the levels of PtdIns3P fell significantly when 1321 NI cells were stimulated with carbachol. The relative rate of turnover of the inositol moiety of PtdIns3P is similar to that of both of the major polyphosphoinositides and significantly higher than that of total cellular phosphatidyl- myo-inositol. This suggests that all three polyphosphoinositides are synthesized from a common, rapidly metabolized, pool of phosphatidyl-myo-inositol. INTRODUCTION as described previously (Meeker & Harden, 1982). [3H]Inositol labelling was carried out using inositol- An inositol tetrakisphosphate that lacks a D- - depleted cells according to the following protocol. Con- phosphate has been identified in both mammalian and fluent cell monolayers were washed once with inositol- avian cells (Stephens et al., 1988a). The metabolic origin free Dulbecco's modified Eagle's medium. The cells were of this compound is unknown. Possible sources include dissociated with trypsin/EDTA (Flow Laboratories), inositol phospholipids that either lack a D-1-phosphate diluted into inositol-free Dulbecco's modified Eagle's or are hydrolysed by a phospholipase D activity. The medium containing dialysed 5 % (v/v) foetal-calf serum phospholipids demanded by either of these possibilities (Gibco) and plated out at 20 % of their original density have not been described, although their existence has in either 3 cm- or 10 cm-diameter Petri dishes. After been speculated upon a number of times (Batty et al., 12 h the medium was aspirated and replaced with fresh 1985; Heslop et al., 1985). These considerations, com- medium. After 5 days the inositol-free medium was bined with the recent description of a Ptdlns kinase replaced with a similar solution containing 2-25 ,uCi of which exclusively makes PtdIns3P (Whitman et al., [3H]inositol (New England Nuclear)/ml or I ,uCi of 1988), at least in vitro, led us to investigate the identity of ["4C]Ins (Amersham International)/ml, and the cells the inositol phospholipids found in [3H]Ins-prelabelled were used after 48-60 h incubation with tracer. human astrocytoma cells. The experiments reported here During this labelling period, both 'inositol-depleted' characterize a cellular inositol phospholipid whose and 'normal' cultures of 1321 NI cells expanded approxi- properties are consistent with it possessing the structure mately linearly with time, although 'inositol-depleted' PtdIns3P. cells did so at only 60 + 6 of the rate for 'normal' cells. After 48-60 h, the incorporation of label into phospho- lipids of inositol-depleted cells reached a constant value MATERIALS AND METHODS approx. 20-22 times (per culture plate) greater than that in normal cells at the same time. Moreover, at this time Radiotracer labelling and extraction of astrocytoma-cell the incorporation of tracer into inositol phospholipids of phospholipids normal cells was still rising. For experiments involving Human astrocytoma cells (1321 NI) were cultured hormonal stimulation the labelling medium was removed Abbreviations used: Ins, myo-inositol; InsP, InsP2, InsP3, InsP4 and InsP5, myo-inositol mono-, bis, tris, tetrakis and pentakis-phosphates; the positions of the phosphates on a given inositol phosphate are denoted by numbering from the position of the phosphate in D-InsP; Ptdlns, phosphatidyl-myo-inositol; PtdlnsP and PtdInsP2, phosphatidyl-myo-inositol phosphate and phosphatidyl-myo-inositol bisphosphate; PtdOH, phosphatidic acid; GroPIns, glycerophosphoinositol; GroPInsP, glycerophosphoinositol phosphate; GroPInsP2, glycerophosphoinositol bis- phosphate; BSA, bovine serum albumin; Cho, choline; PDGF, platelet-derived growth factor. * Present address.and address for correspondence and reprint requests: Biochemistry Department, AFRC, Babraham, Cambridge CB2 4AT, U.K. t Present address: MRC Molecular Neurobiology Unit, MRC Centre, University of Cambridge Medical School, Cambridge CB2 2QH, U.K. Vol. 259 268 L. Stephens, P. T. Hawkins and C. P. Downes and replaced with Dulbecco's modified Eagles medium 32P-labelled phospholipids were localized autoradio- containing 0.5% (w/v) bovine serum albumin (BSA; re- graphically using Kodak X-Omat RP film. crystallized fraction V; Sigma), NaHCO3 (3 g/l) and Glycerophosphoinositol phosphates were separated 25 mM-Hepes (pH approx. 7.4 at 37°C in air). Agonist using a Partisphere SAX anion-exchange h.p.l.c column additions were made in 0.5 ml of prewarmed medium. (Whatman), which was eluted with a gradient based Reactions were terminated by rapidly aspirating the on buffers A (water)/B [1.25M-(NH4)2HP04 (adjusted medium and replacing it with 1.5 ml of ice-cold 20 % to pH 3.8 with H3P04 at 25 °C)] at a flow rate of (w/v) trichloroacetic acid. 1.0 ml min-': 0 min, 0% B; 5 min, 00 B; 45 min, 12 % For [32P]Pi labelling experiments, astrocytoma cells B; 52min, 20% B; 64min, 100% B; 70min, 100% B; were first labelled with [3H]inositol as described above 71 min, 000 B. for 2 days in 10 cm-diameter Petri dishes with a final Inositol bisphosphates were separated by using the concentration of [3H]Ins of 2,tCi/ml in a total volume of same column, buffers and flow rate, with the following 20 ml. The cells were then washed twice with phosphate gradient: 0min, 0 B; 5min, 0% B; 6min, IO% B; and inositol-free Dulbecco's-modified Eagle's medium 60min, 10% B; 61min, 1000% B; 70min, 1000% B; containing recrystallized 0.5% (w/v) BSA, 25 mM-Hepes 71 min, 000 B. and 1 % (v/v) antibiotics (penicillin/streptomycin; Flow In some preparative situations a weak anion-exchange Laboratories) which had been equilibrated with 5 % h.p.l.c. column (Partisphere WAX, Whatman) was util- CO2 at 37 'C. After 1 h the cells were washed once more ized (see below). It was eluted with the following gradient with the above medium before recommencing incubation based on A (water) and B [2.0 M-triethylamine (pH 3.8 in 10 ml of the same medium, containing 1 mCi of with formic acid at 25 0C)], at a flow rate of 1 ml min-1: [32P]PI (Amersham). The medium was then rapidly 0 min, 00 B; 5 min, 00 B; 45 min, 1000 B; 50 min, aspirated and the cells were quenched with 5 ml of 100 00 B; 51 min, 0 00 B. This procedure had the ice-cold 200 (w/v) trichloroacetic acid. advantage that the eluate could be freeze-dried directly. Phospholipid extraction was carried out by quantita- The dephosphorylation products of GroPInsP, tively transferring the cell debris and supernatants from GroPInsP2 and InsP2 species (see below) were separated trichloroacetic acid-precipitated cell lysates to poly- by using a Partisil 10-SAX anion-exchange h.p.l.c. propylene test tubes and sedimenting the precipitates in column which was eluted with a gradient based on buffer a refrigerated bench-top centrifuge. The supernatants A (water) and B [1.7 M-ammonium formate (pH 3.7 were removed and the pellets washed once with 500 with H3PO4 at 25 °C)] at a flow rate of 1.25 ml * min-': trichloroacetic acid/2 mM-EDTA and then solubilized 0 min, 000 B; 5 min, 000 B; 45 min, 29 % B; 46 min, by vigorous mixing in 1 ml of (0.1 M-HCl/0.1 mM- 70 % B; 48 min, 70O B; 50 min, 00 B. inositol/ 10 mM-EDTA)/methanol/chloroform (4:9:5, Polyols contained in 10,1 aliquots of water were by vol.). Two phases were obtained by the addition of separated with a Brownlee-Polypore cation-exchange 370,tl of chloroform and 370,l of a solution containing h.p.l.c. column in the Pb2+ mode (Anachem), maintained 0.1 M-HCl, 0.1 mM-inositol and 10 mM-EDTA. After at 25 °C by a thermostatically controlled jacket and mixing and centrifugation the upper phases were removed eluted with water at a flow rate of 0.2 ml * min-'. Polyols and the lower and interphases were washed with 1.33 ml were detected in the eluate from the column by liquid- of 'synthetic' upper phase. The remaining cell debris and scintillation counting of individual fractions or by an lower phase were dried under vacuum and either re- on-line differential refractometer (Waters model 410). suspended in chloroform (if they were to be subjected to t.l.c.) or deacylated (see below). Desalting fractions of the h.p.l.c. eluate Compounds in triethylammonium formate or am- Preparation of rat brain cytosol fractions monium formate were dried under vacuum. Fractions Rat brain homogenates were prepared in 0.25 M- containing H3PO4 were neutralized with triethyl- sucrose/50 mM-Hepes/2 mM-EGTA/ 15 mM-2-mercapto- amine, diluted 10-fold with water, and applied to a ethanol/SO,tM-phenylmethanesulphonyl fluoride/ 2cmxO.6cm column of Bio-Rad AG 1X8 (200-400 antipain (1 ,ug/ml)/leupeptin (1 ug/ml)/pepstatin A mesh, formate form) resin.
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