JOURNAL OF VIROLOGY, Aug. 1990,
p.
Vol. 64, No. 8
3895-3904
0022-538X/90/083895-10$02.00/0 Copyright X) 1990, American Society for Microbiology
Phosphatidylinositol Metabolism in Cells Transformed by Polyomavirus Middle T Antigen EMIN T. ULUG,1 PHILLIP T. HAWKINS,2 MICHAEL R. HANLEY,2 AND SARA A. COURTNEIDGEl* Differentiation Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, 6900 Heidelberg, Federal Republic of Germany,' and Medical Research Council Molecular Neurobiology Unit, University of Cambridge Medical School, Cambridge CB2 2QH, United Kingdom2 Received 9 April 1990/Accepted 18 May 1990
Associated with the middle T antigen of polyomavirus is a novel phosphatidylinositol (Ptdlns) kinase activity which phosphorylates Ptdlns at the D-3 position of the inositol ring. We have undertaken an analysis of myo-[3H]inositol-containing compounds in a panel of NIH 3T3 cell lines stably transfected with transforming and nontransforming middle T antigen mutants. All cell lines from which PtdIns 3-kinase activity coprecipitated with middle T antigen exhibited modestly elevated levels of Ptdlns(3)P and compounds with predicted PtdIns(3,4)P2 and Ptdlns(3,4,5)P3 structures. Complex formation between middle T antigen and Ptdlns 3-kinase correlated not with an increase in total inositol phosphate levels but rather with elevated levels of InsP2 and InsP4. A specific increase in the level of an InsP2 species which comigrated in high-pressure liquid chromatography analysis with Ins(3,4)P2 was observed. These results suggest that association of the polyomavirus middle T antigen with Ptdlns 3-kinase activates a distinct inositol metabolic pathway.
with these middle T antigen-tyrosine kinase complexes is an 81-kilodalton phosphoprotein (6, 15; S.A.C., unpublished data) whose presence correlates with PtdIns 3-kinase activity and which is most likely identical to the putative 85kilodalton Ptdlns 3-kinase which binds the PDGF receptor (20). Ptdlns 3-kinase activity and p81 are associated in immune precipitates containing all transforming and certain nontransforming middle T antigen mutants (e.g., d11015) but not with other nontransforming middle T antigen mutants which retain the ability to bind pp6-src (6, 30). Association of PtdIns 3-kinase activity with middle T antigen, therefore, appears necessary but not sufficient for cell transformation. Although physical association between Ptdlns 3-kinase activity and middle T antigen-tyrosine kinase complexes has been demonstrated in immune complex kinase assays, it remains unclear whether this association affects Ptdlns metabolism in vivo. Dephosphorylation of p81 either in vitro or during extraction correlates with loss of Ptdlns 3-kinase activity, suggesting that phosphorylation of p81 by pp6Oc-src could then stimulate production of PtdIns(3)P (7). The PtdIns(3)P product, however, does not fit into any of the described pathways for inositol turnover, and conflicting reports about activation of inositol turnover in middle T antigen and v-src-transformed cells have appeared in the literature. We have therefore undertaken an analysis of inositol metabolites in middle T antigen-transformed cells to define whether the levels of Ptdlns 3-kinase products or other inositol-containing compounds are altered as a consequence of middle T antigen expression.
Activation of phosphatidylinositol (Ptdlns) turnover is a hallmark feature of many receptor-mediated signal transduction and cellular growth regulation pathways. A variety of growth factor receptors respond to agonist binding by stimulation of phosphoinositidase C activity, generating at least two molecules with second-message function: inosito/ (1,4,5)triphosphate [Ins(1,4,5)P3] and diacylglycerol (reviewed in reference 2 and 28). Further interest in the role of phosphoinositide signaling has been generated by the recent finding of PtdIns 3-kinase activity associated with retroviral oncogene protein-tyrosine kinases, including v-src, v-abl, v-ros, and v-erbB (12, 14, 15, 22, 27, 40, 41), and receptor protein-tyrosine kinases, including the platelet-derived growth factor (PDGF) receptor (20), the colony-stimulating factor 1 receptor (44), and the insulin receptor (11, 33). This PtdIns 3-kinase activity, however, phosphorylates PtdIns at the D-3 position of the inositol ring to form phosphatidylinositol(3)phosphate [Ptdlns(3)P] rather than the D-4 position seen in the phospholipase C-dependent signal transduction pathway (39, 45). This novel type 1 Ptdlns 3-kinase (46) has been recently demonstrated also to phosphorylate phosphatidylinositol(4)phosphate [Ptdlns(4)P] and phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P2] to form products with predicted phosphatidylinositol(3,4)bisphosphate [Ptdlns(3,4)P2] and phosphatidylinositol(3,4,5)triphosphate [PtdIns(3,4,5)P3] structures, respectively (1). Although no correlation between stimulation of Ptdlns 3-kinase activity and Ptdlns signaling pathways has been established, the association of PtdIns 3-kinase activity with oncogene products suggests that it plays an important role in growth control. Among the first oncogene products shown to have associated Ptdlns 3-kinase activity was the middle T antigen of polyomavirus (47). Middle T antigen, which is both sufficient and necessary for transformation of established cell cultures (36), forms a stable complex with an activated form of cellular tyrosine kinase, pp6O-src (3, 9), and also with the related kinases pp62c-Yes (23) and p59&fy (4, 24). Associated *
MATERIALS AND METHODS Cell cultures. NIH 3T3 cells which were cloned for G418 sensitivity and high efficiency of transformation provided the basis for the cell lines presented in this communication. Cells were cotransfected with cloned middle T antigen DNA and the gene for neomycin resistance (Neor) and selected for G418 resistance as described previously (6). By metabolic labeling of G418-resistant clones with [35S]methionine, it was found that all of the cell lines used expressed roughly equivalent amounts of middle T antigen. NIH 3T3 cells
Corresponding author. 3895
3896
ULUG ET AL.
expressing the gene for Neor alone were used as controls. Cultures were maintained in Dulbecco modified Eagle medium containing 5% fetal bovine serum, 5% newborn calf serum, penicillin, and streptomycin. Metabolic labeling of cells with myo-[2-l3Hlinositol. To define steady-state inositol metabolite levels, at least two independently selected clones expressing each middle T antigen mutant were plated in duplicate at a density of 2 x 105 cells per 35-mm-diameter tissue culture dish. The culture medium was removed 4 to 8 h after passage and replaced with 1 ml of inositol-free Dulbecco modified Eagle medium containing 2.5% fetal bovine serum, 7.5% dialyzed fetal bovine serum, S ,ug of insulin per ml, 5 ,ug of transferrin per ml, and 10 to 25 ,uCi of myo-[2-3H]inositol (80 to 120 Ci/mmol; Amersham Corp.). Cultures were harvested 36 to 48 h later as described below. To monitor the effect of growth factor addition on phosphatidylinositol phosphate (PtdlnsP) levels, replicate cultures of normal and transformed cells were labeled with myo-[2-3H]inositol in inositolfree Dulbecco modified Eagle medium containing 0.5% serum for 36 h. The cultures were then treated with LiCl (20 mM) for 30 min and then incubated with or without human recombinant (B chain) or porcine PDGF (12.5 nglml) for an additional 10 min. Extraction of inositol metabolites from cells. Cultures labeled with myo-[3H]inositol were washed extensively with ice-cold phosphate-buffered saline and extracted by addition of 0.3 ml of ice-cold perchloric acid (4.5%). After 15 min at 4°C, the cells were scraped from the dishes and transferred to Eppendorf tubes. The culture dishes were washed with an additional 0.3 ml of perchloric acid, and the combined extracts were centrifuged (15,000 x g, 10 min). The inositol phosphate-containing supernatants were neutralized with 0.12 ml of EDTA (10 mM) and 0.5 ml of tri-N-octylamineFreon (1:1) (10) and stored at -20°C. The inositol lipidcontaining perchloric acid pellets were washed once with perchloric acid (0.5 ml) and once with EDTA (100 mM) at 4°C, and the final pellet was subjected to methylamine deacylation (18). The pellets were suspended in 50 ml of H20 to which was added 1.0 ml of methanol-40% methylaminen-butanol (4:4:1). After 45 min at 56°C, the samples were dried in a Speed Vac concentrator (Savant). The dried glycerophosphoinositide (GroPIns) products were suspended in H20, clarified by centrifugation, and extracted twice with butanol-petroleum ether-ethyl formate (20:4:1). The preparations were then dried and stored at -20°C. HPLC separation of GroPIns. Deacylated extracts of the inositol-containing lipids were analyzed by strong anionexchange high-pressure liquid chromatography (HPLC) with a Whatman Partisphere 5-SAX column as described by Stephens et al. (39). The HPLC apparatus consisted of a 1.0-ml injection loop, a digitally controlled mixing device, and two independent pumps [A, which pumped H20, and B, which drew 1.25 M (NH4)2HP04 [pH 3.8] with H3PO4]. With the unit running at 1 ml/min, samples (suspended in 0.5 ml of H20 and filtered) were injected and immediately subjected to the following linear elution gradients: 1 min to 2% B, 20 min to 4% B, 10 min to 100% B, 5 min to 100% B, 5 min to 0% B, and 30 min to 0% B. Alternatively, the same B buffer was used with the following elution program to optimize separation of GroPInsP2 isomers and the predicted GroPInsP3 product: 1 min to 2% B, 18 min to 4% B, 1 min to 11% B, 24 min to 11% B, 1 min to 23% B, 12 min to 24% B, 5 min to 100% B, 5 min to 100% B, 5 min to 0% B, and 30 min to 0% B. Fractions (0.4 ml) were collected directly in scintillation vials, mixed with 1.1 ml of methanol (50% in H20) and 3.0 ml
J. VIROL.
of Quickszint 401 (Zinnser, Frankfurt am Main; Federal Republic of Germany), and counted in an LKB beta counter which was calibrated for quenching and background. Recovery with these HPLC protocols was over 90% of the input disintegrations per minute. To compensate for sample loss at late stages in GroPIns preparation, however, the levels of all deacylated inositol lipids are expressed as a ratio to the level of radioactivity present in the Ptdlns deacylation product GroPIns. HPLC separation of inositol phosphates. The perchloratesoluble inositol phosphate preparations were analyzed by HPLC with a Whatman Partisil 10-SAX (38) strong anionexchange column with ammonium formate-phosphate elution. Thawed samples were reextracted with tri-N-octylamine-Freon (1:1) and filtered before injection. At a flow rate of 1 ml/min, elution from the column was achieved by using linear gradients as follows, with pump B drawing 3.5 M ammonium formate (pH 3.7) with H3PO4: 10 min to 0% B, 55 min to 35% B, 20 min to 100% B, 5 min to 100% B, 5 min to 0% B, and 30 min to 0% B. With this protocol, all classes of inositol phosphates were readily identified. To analyze more precisely the levels of inositol bisphosphate and inositol trisphosphate isomers present, the following elution program was used: 10 min to 0% B, 1 min to 2% B, 10 min to 8% B, 30 min to 8% B, 1 min to 25% B, 15 min to 26% B, 10 min to 100% B, 5 min to 100% B, 5 min to 0% B, and 30 min to 0% B. Samples were generally collected beginning after the initial water wash (step 1) and then processed as described above. The contents of the individual inositol phosphate peaks were standardized to the total level of radioactivity present in the deacylated lipid preparation before butanol extraction. This compensated for any change in inositol uptake or pool size which may have occurred as a consequence of cell transformation. Immune complex Ptdlns kinase assays. For immune complex Ptdlns kinase assays, cells were rinsed with Trisbuffered saline (20 mM Tris [pH 8.0], 150 mM NaCl, 1 mM dithiothreitol, 0.1 mM orthovanadate) and lysed by addition of Nonidet P-40 containing LB buffer (150 mM NaCl, 20 mM Tris [pH 8.0], 1% Nonidet P-40, 1 mM dithiothreitol, 20 ,ug of leupeptin per ml, 1% aprotinin, 0.1 mM orthovanadate). These and all future steps were performed at 4°C. After 10 min, the lysates were clarified by centrifugation, protein contents were adjusted, and immune complexes were formed by addition of affinity-purified antibodies to the carboxy terminus of middle T antigen. Immune complexes were collected with Formalin-killed Staphylococcus aureus bacteria and washed three times with LB and twice with Tris-buffered saline. Kinase assays with the final pellets were performed in reaction volumes of 20 ,ul containing 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-NaOH (pH 7.4), 5 mM MgCl2, 1 mM dithiothreitol, 5 ,ug of lipid, 10 ,M unlabeled ATP, and 10 RCi of carrier-free [_y-32P]ATP. Stocks of lipid substrates (1 mg/ml) were prepared as follows: Ptdlns, PtdIns(4)P, PtdIns(4,5)P2, and phosphatidylserine (1:1:1:3 wt/wt) dissolved in CHC13 were dried under a stream of N2, suspended in 20 mM HEPES-1 mM dithiothreitol by sonication for 15 min at 4°C and frozen in small aliquots in liquid N2. After 10 min at 30°C, the kinase reactions were terminated by addition of 100 RI of HCI (1 N) and 250 Rl of CHCl3-MeOH (1:1). After centrifugation, the lower organic phase was washed with 125 RI of 1 N HCl-MeOH (1:1) and dried. Samples were suspended in a small volume of CHC13 and spotted onto Merck 60 silica gel chromatography plates. The plates were impregnated with 1% oxalate-1 mM EDTA in 40% methanol, air
VOL. 64, 1990
PHOSPHATIDYLINOSITOL TURNOVER AND MIDDLE T ANTIGEN
dried, and activated at 110°C immediately before use. Products were separated by using a solvent system containing
chloroform-methanol-acetone-glacial acetic acid-H20 (60: 20:23:18:11). Preparation of lipid standards. 32P-labeled PtdIns phosphate standards were prepared in vitro by using antiphosphotyrosine immune precipitates from PDGF-stimulated cells. NIH 3T3 cells rendered quiescent by incubation in 0.5% serum for 24 to 48 h were treated with 12.5 ng of PDGF per ml for 10 min. Immune precipitates prepared from
lysates
as
described above contained rabbit antiphosphoty-
rosine antibodies. Antisera were collected following multiple injections with O-phosphotyramine-conjugated keyhole limpet hemocyanin (prepared by the method of Pang et al. [31]), and phosphotyrosine-specific antibodies were purified as already described (32). Ptdlns kinase assays were performed as described above, and after chromatography, PtdInsP, PtdInsP2, and the predicted PtdInsP3 were identified by autoradiography, scraped from the plates, and directly subjected to methylamine deacylation. When subjected to
deglyceration (detailed below), 32P04-labeled GroPInsP2 and GroPInsP3 prepared in vitro yielded products chromatographically indistinguishable from Ins(1,3,4)P3 and Ins(1,3,4,5)P4, as reported by Auger et al. (1). 3H- and 14C-labeled PtdIns(4)P and PtdIns(4,5)P2 standards were prepared from metabolically labeled middle T antigen-transformed cells. Cultures (approximately 5 x 106 cells) were incubated in 5 ml of inositol-free Dulbecco modified Eagle medium containing 10% dialyzed fetal bovine serum and 5 ,uCi of myo-[14C]inositol or 50 ,uCi of myo[3H]inositol for 24 to 30 h. Labeled cultures were treated with 10 mM LiCl and 50 mM sodium orthovanadate for 2 h, rinsed with phosphate-buffered saline, and harvested with perchloric acid as described above. The supematant was neutralized and reserved for preparation of inositol phosphate standards (see below). The perchloric acid precipitate was suspended in a small volume of H20, and inositol lipids were extracted by addition of CHCl3-MeOH-HCl (100:200: 1). After 1 h at 4°C, phases were separated by the addition of 1/3 of a volume of CHC13 and 1/3 of a volume of H20. After centrifugation, the lower organic phase was reextracted with 1 volume of MeOH-1 N HCI (1:1) and dried. Samples were spotted onto oxalate-EDTA-impregnated silica plates and subjected to chromatography by using a solvent system containing CHCl3-MeOH-4 M NH3 (9:7:2). Compounds were visualized by autoradiography, scraped from the plates, and deacylated directly. Preparation of inositol phosphate standards. 3H- or 14Clabeled Ins(1,4)P2, Ins(1,3)P2, and Ins(1,4,5)P3 were prepared by periodate-catalyzed deglyceration of GroPIns(4)P, GroPIns(3)P, and GroPIns(4,5)P2, respectively, as previously described (17). 3H-labeled Ins(1,3,4,5)P4, InsP5, and InsP6 were prepared as previously described (37; P. T. Hawkins, D. J. M. Reynolds, D. R. Poyner, and M. R. Hanley, Biochem. Biophys. Res. Commun., in press). Ins(1,3,4)P3 was purified by HPLC from extracts of myo-
["4C]inositol-labeled cells treated with sodium orthovanadate in the presence of LiCl as detailed above. Ins(1,3,4)P3 prepared in this manner comigrated in HPLC analysis with 3H-labeled Ins(1,3,4)P3, whose level was elevated after PDGF treatment of quiescent fibroblasts (1). 3H- or 14Clabeled Ins(3,4)P2 and Ins(4,5)P2 were prepared from Ins(1,3,4)P3 and Ins(1,4,5)P3, respectively, by alkaline phosphatase treatment (17). InsP3 was incubated with 3 U of bovine alkaline phosphatase (Sigma Chemical Co., St. Louis, Mo.) in the presence of 5 mM MgCl2-20 mM etha-
3897
TABLE 1. Middle T antigen mutants used in this study Mutant Prototype class
Mutass
Wild type d18
I
II III
d11015 d123
NG59 Neor
a
Transforming phenotype phenotype
Binding of of Binding Ptdins pptcdnrc
~~~~~~~~~3-kinase
+
+
+
+
+
+
a a
+
+
-
-
-
_
_
_
+
Cells expressing dl23 and d11015 middle T antigens occasionally gave rise
to small foci.
nolamine (pH 9.5) for 60 min at 20°C. Samples were diluted 10-fold with water, and InsP2 was purified by chromatography on AG-1 (formate) columns as described below. 32plabeled Ins(1,2)P2 was prepared by boiling Ins(1,3)P2 in the presence of 1 N HCI for 10 min as described by Stephens et al. (39). The samples were cooled, neutralized with ammonia, and lyophilized. All inositol phosphate standards purified by HPLC were desalted as follows. Peaks of radioactivity were pooled, diluted 10-fold with water, and bound to small 1-ml columns of anion-exchange resin AG-1 (X8; 200/400 mesh), formate form (Bio-Rad Laboratories, Richmond, Calif.). After extensive washing with 0.15 M ammonium formate-0.1 M formic acid, the columns were treated with 0.4 M ammonium formate-0.1 M formic acid to elute InsP2 or 0.75 M ammonium formate-0.1 M formic acid to elute InsP3. The eluates were neutralized with ammonia and lyophilized. Statistical analysis. Statistical significance was determined by t test with the StatView statistics program (Brainpower, Inc., Calabasas, Calif.).
RESULTS Ptdlns, PtdlnsP, and PtdInsP2 are phosphorylated in middle T immune precipitates. Middle T antigen mutants have been previously classified on the basis of the ability to bind pp60c-src (Table 1). All transforming (class I) and nontransforming (class II) middle T antigen mutants bind pp6Oc-src, but nontransforming class III mutants have lost the ability to bind pp6Oc-src. According to this classification, only class I and certain class II mutants retain p81 binding and have associated PtdIns 3-kinase activity (6, 8, 20, 30). It has been recently reported by Auger et al. (1) that PtdIns 3-kinase activity associated with the PDGF receptor was capable of utilizing Ptdlns(4)P and PtdIns(4,5)P2 as substrates. To test whether the middle T antigen associated PtdIns 3-kinase also recognized these substrates, lysates from cells harboring prototypes of middle T antigen mutant classes were precipitated by using middle T antibodies and immune complex kinase assays were performed by using a mixture of PtdIns, PtdInsP, and PtdInsP2 as a substrate. PtdIns 3-kinase activity associated with wild-type and d18 middle T antigen utilized all three substrates with roughly equal efficiencies to produce products with predicted PtdInsP, PtdInsP2, and PtdInsP3 structures (Fig. 1). Formation of these products in vitro was dependent upon the presence of the respective lipid substrate, Mg2+ and micromolar ATP in the reaction mixture (data not shown). Among class II mutants, d11015 but not d123, middle T antigen exhibited Ptdlns 3-kinase activity with all three substrates. Under identical conditions,
3898
ULUG ET AL.
J. VIROL.
_
_
* 9 9 * 0 *
* *
1
9
2
3
*
4
6
5
7
8
* 9 * *
9
PtdinsP PtdInSP2
PtdInsP3
10
FIG. 1. PtdIns kinase activity associated with middle T antigen mutants. Nonidet P-40 extracts (250 jig) of cells expressing NG59 (lanes 1 and 2), dl23 (lanes 3 and 4), d11015 (lanes 5 and 6), d18 (lanes 7 and 8), or wild-type (lanes 9 and 10) middle T antigen were precipitated by using antibodies to the carboxy terminus of middle T antigen (mt.c), and kinase assays were performed by using PtdlnsPtdlnsP-PtdInsP2-phosphatidylserine (1:1:1:3) as the substrate. In odd-numbered lanes, an excess of mt.c peptide was included during precipitation. Positions of products with predicted Ptdlns(3)P, Ptdlns(3,4)P2, and Ptdlns(3,4,5)P3 structures are indicated. This plate was exposed for 4 h by using Fuji RX film with an enhancing screen at -70°C.
NG59 (class III) middle T antigen and Neor controls (data not shown) exhibited no appreciable PtdIns 3-kinase activity. We conclude that PtdlnsP and PtdInsP2 substrates are also recognized by the middle T antigen-associated PtdIns 3-kinase. HPLC separation of polyphosphoinositides in middle T antigen-transformed cells. Although PtdIns 3-kinase activity was specifically precipitated from polyomavirus middle T antigen-transformed cells by antisera directed to either middle T antigen or pp6Ocsrc, it remained unclear whether formation of this complex affected inositol metabolite levels in vivo. To address this question, the levels of polyphosphoinositides were monitored in cultures metabolically labeled with myo-[3H]inositol. Steady-state inositol metabolite levels were defined by incubation of cultures with myo[3H]inositol in medium with reduced inositol content. In addition to added insulin, transferrin, and dialyzed serum, the labeling medium contained 2.5% undialyzed serum which contained sufficient growth factors (and inositol) for proliferation of nontransformed cells. Under these conditions, uptake of inositol and subsequent incorporation into PtdIns were not grossly affected by middle T antigen transformation. Lowering the inositol content of the culture medium further resulted in a very reduced pool of free labeled inositol in the transformed cells, complicating direct comparison of metabolite levels in the various cell lines (data not shown). Cultures were labeled for 36 to 48 h with myo-[3H]inositol and harvested with perchloric acid. The lipid-containing fractions were deacylated and analyzed by -.
10000
1000
A '. 5000I 50
Qj o
° 0
0
-~~~
50
100
C~~~
500
N n
C4
0
C
150
500
na.
+~ ~ ~ ~L
0
Fraction Number FIG. 2. HPLC separation of the deacylation products of inositol-containing compounds from cells expressing d11015 middle T antigen. Perchloric acid-insoluble extracts (5 x 106 dpm) from [3H]inositol-labeled cells were deacylated and analyzed by HPLC as described in the text (open symbols). 32P-labeled lipids generated in vitro by using antiphosphotyrosine immune precipitates were deacylated for use as standards (closed symbols). Elution of the predicted GroPIns(3,4)P2 (B) and GroPIns(3,4,5)P3 (C) products is compared with the elution pattern of deacylated cell lipids.
PHOSPHATIDYLINOSITOL TURNOVER AND MIDDLE T ANTIGEN
VOL. 64, 1990
TABLE 3. Levels of putative Ptdlns(3,4)P2 and PtdInsP3 products in cells expressing middle T antigen mutants
TABLE 2. Levels of Ptdlns(3)P and Ptdlns(4)P in cells expressing middle T antigen mutants Cell type
(no.) Wild type (5) d18 (2) dllO15 (5) d123 (6) NG59 (4) Neor (5)
Mean + SEM % of radioactivity' PtdInsP2 Ptdlns(4)P PtdIns(3)P
0.205 0.203 0.200 0.175 0.177 0.169
+ 0.017b ± ± ± ± ±
0.015b
0.006b 0.008 0.004 0.008
2.46 2.35 2.49 3.41 3.80 3.39
± ±
0.07b 0.04b 0.14b
± 0.22 ± 0.23 ± 0.36
5.44 3.94 5.45 5.52 4.93 6.46
3899
± ±
0.03b 0.32b
±
0.30b
± 0.26 ± 0.14b ± 0.48
a Mean levels of PtdIns(3)P, Ptdlns(4)P, and PtdInsP2 are expressed as percentages of the radioactivity associated with Ptdlns after deacylation and HPLC separation. Total levels of Ptdlns ranged from 0.5 x 106 to 2.0 x 106 dpm. b Significantly different from Neor (P - 0.05).
strong anion-exchange HPLC (39). A representative example of the HPLC analysis of polyphosphoinositides from [3H]inositol-labeled cells expressing d11015 middle T antigen is illustrated in Fig. 2. 32P04-labeled standards in this experiment, corresponding to the deacylation products of PtdIns(3)P and the putative PtdIns(3,4)P2 and PtdIns (3,4,5)P3 compounds, were generated in vitro by using anti-phosphotyrosine immune complexes prepared from quiescent NIH 3T3 cells treated with PDGF. As described previously, Ptdlns(3)P represents approximately 0.2% of the total inositol-containing lipid and its deacylation product eluted immediately before GroPIns(4)P on strong anionexchange HPLC (peak at fraction 32). The putative PtdIns(3,4)P2 product represents approximately 0.01% of the total inositol-containing lipid fraction in these cells, and its deacylation product eluted immediately before GroPIns (1,4)P2 in this HPLC system (peak at fractions 85 and 86 in Fig. 2B). Finally, the putative PtdIns(3,4,5)P2 product represents approximately 0.02% of the total inositol-containing lipid in these cells and its deacylation product (peak at fraction 140 in Fig. 2C) eluted between Ins(1,4,5)P3 and InsP4 (data not shown). We found that deglyceration of the 32P04-labeled putative PtdIns(3,4)P2 and Ptdlns(3,4,5)P3 products from in vitro kinase assays yields compounds which comigrated with Ins(1,3,4)P3 and Ins(1,3,4,5)P4 standards (data not shown), confirming the tentative structure assignments of Auger et al. (1). Levels of Ptdlns(3)P in middle T antigen-transformed cells. A summary of the results from two experiments designed to define Ptdlns(3)P levels in cells expressing transforming and nontransforming middle T antigen mutants is presented in Table 2. When normalized to the level of PtdIns, the PtdIns(3)P content was modestly but consistently (P < 0.05) elevated (15 to 25%) in cell lines expressing d11015, d18, or wild-type middle T antigen relative to the level in cells expressing Neor alone. By contrast, the levels of PtdIns(4)P were reduced in the cell lines which express middle T antigen with associated Ptdlns 3-kinase activity. PtdInsP2 levels were reduced in all cell lines expressing middle T antigen, and no correlation could be established between either cell transformation or the presence of Ptdlns 3-kinase activity in vitro and PtdInsP2 levels. From these results, we conclude that middle T antigen which has associated Ptdlns 3-kinase activity in vitro also activates PtdIns 3-kinase in vivo. The levels of Ptdlns(3)P in polyomavirus-transformed cells, while low, could not be further stimulated by addition of PDGF, and they approximated the levels seen after PDGF stimulation of quiescent normal fibroblasts (E.T.U., unpublished data).
Mean + SEM % of radioactivity' PtdInsP3
Cell type (no.)
Ptdlns(3,4)P
Wild type (2) dlOl15 (3) d123 (2) Neor (2)
0.0181 + 0.0037 0.0091 ± 0.0028 0.0037 ± 0.0007 0.0095 ± 0.0055
0.0348 ± 0.0083 0.0219 ± 0.0073 0.0111 ± 0.0017 0.0117 ± 0.0009
a Mean levels of Ptdlns(3,4)P2 and PtdInsP3 are expressed as percentages of the radioactivity associated with Ptdlns after deacylation and HPLC separation. Levels of Ptdlns in these samples ranged from 4 x 106 to 10 x 106 dpm.
Levels of novel PtdInsP2 and PtdInsP3 species in middle T antigen-transformed cells. It has been recently demonstrated that elevated levels of products with putative Ptdlns(3,4)P2 and Ptdlns(3,4,5)P3 structures accumulated upon treatment of quiescent smooth muscle cells with PDGF (1). To define whether polyomavirus-transformed cells also exhibit levels of these novel products, we compared the HPLC elution patterns of 32P-labeled GroPInsP2 and GroPInsP3 standards generated in vitro with those of deacylated lipid preparations derived from middle T-expressing cells (as in Fig. 2B and C). Because of the exceedingly low levels of these compounds in normal fibroblasts (0.005 to 0.01% of the radioactivity associated with PtdIns), duplicate or triplicate samples of deacylated 3H-labeled lipid preparations were pooled to yield enough of a sample to permit detection of these compounds. The levels of these products were elevated two- to threefold in transformed cells (Table 3). Furthermore, at least PtdInsP3 levels appeared to correlate with the presence of PtdIns 3-kinase activity in cells expressing middle T antigen mutants. While the very low levels of these compounds precluded the derivation of statistically significant values, we tentatively conclude that a correlation appears to exist between the accumulation of these novel products in vivo and the detection of middle T-associated PtdIns 3-kinase activity in vitro. Inositol phosphate levels in middle T antigen-transformed cells. The very low levels of the Ptdlns 3-kinase products in cells with active Ptdlns 3-kinase may reflect rapid processing of these products to other compounds. We therefore also screened our panel of middle T antigen mutants for changes in the levels of soluble inositol phosphates. Perchloric acidsoluble inositol phosphates were neutralized with tri-Noctylamine and subjected to strong anion-exchange HPLC using a Partisil 10 cartridge and ammonium formate-phosphate elution. Our initial experiments focused on separation of the major classes of inositol phosphates with the elution programs detailed in Materials and Methods. A summary of the results of two experiments using various clones of each mutant class is presented in Table 4. All cells expressing middle T antigen mutants exhibited significantly elevated total levels of inositol phosphates compared with cells expressing Neor alone, but no pattern could be established between elevated inositol phosphate levels and cell transformation. For example, d18 was fully transforming but exhibited inositol phosphate levels comparable to those in NG59, which fails to associate with src family tyrosine kinases or transform cells. This was most apparent when the levels of InsP1, the most abundant acid-soluble inositol metabolite, were examined in the various cell lines. Furthermore, when the levels of InsP1 in cell lines expressing d123 or d18 middle T antigen were compared, no change which correlated with Ptdlns 3-kinase activation was observed. Likewise, these
3900
ULUG ET AL.
Cell type (no.)
J. VIROL. TABLE 4. Phosphoinositide levels in cells expressing middle T antigen mutants Mean ± SEM % of radioactivitya
InsP, Wild type (8) d18 (10) d11015 (8) d123 (8) NG59 (4) Neor (8)
13.18 7.64 12.37 9.80 8.61 6.95
± 0.59b ± 1.16b ± 0.74b ± 1.18b ± 1.01b ± 0.42
InsP2
InsP3
0.322 ± 0.018b
0.1215 ± 0.0131
InsP4
0.0735 ± 0.0060b 0.0686 ± 0.0090" 0.0683 ± 0.0064b 0.0375 ± 0.0036 0.0297 ± 0.0037 0.010 0.0269 ± 0.0039 a Mean phosphoinositide levels are expressed as percentages of the radioactivity associated with the lipid-containing fraction (ranging from 0.5 x 106 to 2.0 x 106 dpm) after perchlorate extraction. b
Significantly different from neor (P
_
0.228 0.294 0.207 0.196 0.175
± ± ± ± ±
0.014b 0.017b 0.015 0.023
0.0925 0.1223 0.1043 0.0937 0.0927
± ± ± ± ±
0.0057 0.0072 0.0075 0.0093 0.0102
0.05).
initial studies also revealed that the total levels of InsP3 were statistically invariant among cell lines expressing transforming and nontransforming middle T antigen mutants. We conclude that gross changes in the levels of inositol phosphates do not correlate with the presence of activated Ptdlns 3-kinase or cell transformation. A correlation was established between the overall levels of InsP4 in cell lines which express activated PtdIns 3-kinase. Cells expressing wild-type, d18, or d11015 middle T antigen exhibited a twofold increase in the level of InsP4 compared with cell lines which express either d123 or NG59 middle T antigen. Because of restrictions imposed by the HPLC method used, however, we were unable to define whether this represents accumulation of a particular InsP4 isomer. InsP2 levels were also elevated in cells with activated Ptdlns 3-kinase. Significantly elevated levels of InsP2 were detected in wild type-, d18-, and d11015-transfected cell lines relative to cell lines expressing d123, NG59, or Neor. We optimized the separation of InsP2 isomers by using isocratic ammonium formate-phosphate elution. Illustrated in Fig. 3 are the elution positions of Ins(1,2)P2, Ins(1,3)P2, Ins(1,4)P2, Ins(3,4)P2, and Ins(4,5)P2 standards (panel A) in comparison with the 3H-labeled inositol phosphates present in d123 (panel B) and d11015 extracts (panel C). The increase in radioactivity recovered in InsP2 in d11015-transformed cells could be largely accounted for by the increase in radioactivity comigrating with the Ins(3,4)P2 and Ins(1,2)P2 standards. When this analysis was extended to include the other cell lines expressing middle T mutants, the level of radioactivity which comigrated with the Ins(1,2)P2 standard was found to be elevated approximately twofold in all of the cell lines expressing middle T antigen which is capable of binding to pp6c-src (Table 5). By contrast, an approximately twofold increase in the Ins(3,4)P2 fraction was consistently and significantly observed only in cell lines with activated Ptdlns 3-kinase activity. The levels of Ins(1,4)P2 were invariant among the cell lines analyzed, with the exception of d18, which exhibited reduced levels of this compound. The finding of elevated levels of a compound comigrating with Ins(3,4)P2 prompted us to reexamine the levels of Ins(1, 3,4)P3 in middle T antigen-transformed cells. Ins(1,3,4)P3 is a known precursor of Ins(3,4)P2 (35). By using a shallow gradient of ammonium formate-phosphate during HPLC elution, we obtained clear separation of the Ins(1,3,4)P3 and Ins(1,4,5)P3 isomers (Fig. 3). We found significantly elevated levels of a compound which comigrated with Ins(1,3,4)P3 in cells transformed with wild-type middle T antigen (Table 6). When extended to include the middle T antigen mutants, this analysis revealed increased levels of this compound in all cell lines which exhibited Ptdlns 3-kinase activity in vitro, although the low levels of radioactivity in this analysis
prevented derivation of significance values. By contrast, no differences in Ins(1,4,5)P3 levels which correlated with either cell transformation or associated Ptdlns 3-kinase activity in vitro were found. We conclude that association of Ptdlns 3-kinase activity with middle T antigen appears to correlate with elevated Ins(1,3,4)P3, but not Ins(1,4,5)P3, levels. DISCUSSION Middle T antigen-transformed cells have elevated levels of PtdIns(3)P. Binding of middle T antigen to cellular tyrosine kinase pp60csrc and related kinases is an essential step in cell transformation by polyomavirus, yet little is known about the consequences of this interaction. Insight into this question may be gained by the finding of a third member of this complex, an 81- to 85-kilodalton protein whose presence correlates with Ptdlns 3-kinase activity. p81 is phosphorylated on tyrosine and therefore represents a likely substrate for pp60c-src kinase activity in vivo. In this study, the levels of Ptdlns(3)P were significantly elevated in cells expressing wild-type and certain mutant middle T antigens. We conclude, therefore, that association of Ptdlns 3-kinase activity with middle T antigen in vitro correlates with activation of Ptdlns 3-kinase activity in vivo. Association of middle T antigen with activated pp6Ocsrc and related kinases is required, but not sufficient, for changes in the level of Ptdlns(3)P. Cell lines expressing d123 middle T antigen, for example, bind pp6Ocsrc and partially elevate its kinase activity but do not exhibit changes in cellular Ptdlns(3)P levels. Efficient binding of p81 and, hence, activation of PtdIns 3-kinase activity may be dependent upon the phosphorylation state of middle T antigen. Talmage et al. (42) have demonstrated that middle T antigen lacking the major site of tyrosine phosphorylation does not have associated Ptdlns 3-kinase activity. We note that this site is missing in d123 middle T antigen but present in another middle T antigen mutant (82JF3); both fail to bind p81 (8). PtdIns 3-kinase activity associated with the middle T antigen phosphorylates the D-3 position of the inositol ring to form Ptdlns(3)P (35, 43) and products with proposed Ptdlns(3,4)P2 and PtdIns(3,4,5)P3 structures (1). We have extended these findings to show that PtdIns 3-kinase activity associated with both wild-type and mutant middle T antigens also recognizes PtdIns(4)P and Ptdlns(4,5)P2 substrates. Phosphorylation of these substrates was dependent upon the presence of Mg2+ and high ATP concentrations, whereas formation of PtdIns(3)P by Ptdlns 3-kinase was efficient at low ATP concentrations in the presence of Mn2+ (data not shown). The reason why the different substrates are phosphorylated under different reaction conditions is not clear. The modest extent to which Ptdlns(3)P levels are elevated
VOL. 64, 1990
PHOSPHATIDYLINOSITOL TURNOVER AND MIDDLE T ANTIGEN
3901
400 300 Q
200 100
0
2000 1500 0.
1000
500
0
2000 1500 1000
500
0 0
50
1 50
1 00
Fraction Number FIG. 3. HPLC separation of perchloric acid-soluble inositol-labeled compounds from cells expressing d11015 and d123 middle T antigen mutants. The elution of standards using the HPLC separation program described in Materials and Methods is illustrated in panel A. The standards eluted sequentially (from left to right) as follows: [32P]Ins(1,3)P2, [14C]Ins(1,4)P2, [32P]Ins(1,2)P2, [14C]Ins(3,4)P2, [14C]Ins(4,5)P2, [14C]Ins(1,3,4)P3, [3H]Ins(1,4,5)P3, [3H]InsP4, [3H]InsP., and [3H]InsP6. The elution profiles of d123 (B) and dlOl15 (C) phosphoinositides determined by the same program are presented for comparison. Note a reduction in the level of radioactivity associated with Ins(1,2)P2, Ins(3,4)P2, Ins(1,3,4)P3, and InsP4 peaks in d123 relative to d11015. Elutions of equivalent amounts of phosphoinositides (based on 5 x 106 dpm of the respective inositol lipid fraction) were subjected to separation in panels B and C.
in middle T antigen-transformed cells is comparable to the level of PtdIns(3)P induced in cells after stimulation with PDGF, when Ptdlns 3-kinase is maximally activated (E.T.U., unpublished data). The level of Ptdlns(3)P was only marginally elevated in serum-starved middle T antigentransformed cells after PDGF treatment, yet the transformed cells could still respond to PDGF as measured by Li+sensitive inositol phosphate accumulation (7) or immune precipitation of PtdIns 3-kinase activity with antiphosphotyrosine antibodies (E.T.U., unpublished data). This suggests that although the inositol-signalling pathway is intact in middle T antigen-transformed cells, there is a maximum level of PtdIns(3)P that accumulates in cells. Our data suggest that middle T antigen-transformed cells have attained this level.
TABLE 5. Levels of inositol bisphosphate isomers in cells expressing middle T antigen mutants Mean ± SEM % of radioactivitya
Cell type
(n
=
4)
Ins(1,4)P2
Wild type 0.0696 0.0472 dl8 0.0885 dllO15 d123 0.0992 Neor 0.0754
± 0.0056
Ins(1,2)P2
0.0089b 0.0150b 0.0771 ± 0.0105b 0.0641 0.0054b
0.1659 0.1092 0.1236 0.0586 0.0310 ± 0.0012 0.0507 0.0714
0.0018b,c 0.0849 + 0.0071 + 0.0192 ± 0.0058
Ins(3,4)P2
+ 0.0237b,c ±0.0112 ±0.0133 ± 0.0065 ± 0.0043
a Mean inositol bisphosphate levels are expressed as percentages of the radioactivity associated with the lipid-containing fraction (0.5 x 106 to 2.0 x 106 dpm) after perchlorate extraction. b Significantly different from Neor (P s 0.025). c Significantly different from d123 (P - 0.005).
ULUG ET AL.
3902
J. VIROL.
TABLE 6. Levels of inositol trisphosphate isomers in cells expressing middle T antigens mutants Mean ± SEM % of radioactivitya
Cell type (no.)
Ins(1,3,4)P3
Wild type (6)
0.0200 0.0147 0.0148 0.0108 0.0114
d18 (4) d11015 (6) d123 (6) Neor (8)
Ins(1,4,5)P3
± 0.0040b ± 0.0021 ± 0.0027 ± 0.0028
0.0933 0.0639 0.0985 0.0919 0.0796
± 0.0010
± ± ± ±
0.0043 0.0046 0.0100 0.0053 ± 0.0109
Mean inositol bisphosphate levels are expressed as percentages of the radioactivity associated with the lipid-containing fraction (0.5 x 106 to 2.0 x 106 dpm) after perchlorate extraction. b Significantly different Neor (P s 0.05). a
Ptdlns(4)P levels were significantly reduced in cells expressing activated Ptdlns 3-kinase. It is unclear why our data on PtdIns(4)P levels differ from those previously described. Kaplan et al. (21) reported a 50% increase in Ptdlns(4)P levels in polyomavirus-infected cells relative to those of mock-infected controls. Such a discrepancy may arise from differences in the lipid extraction protocol or from the use of virus-infected cells. We feel that the reduction in Ptdlns(4)P levels we observed is not a consequence of phospholipase C activation during extraction, because a concomitant increase in the levels of immediate phospholipase C products, e.g., Ins(1,4)P2, was not observed. One possibility is that reduced Ptdlns(4)P levels in cells which have activated PtdIns 3kinase arise from a limitation in the availability of substrate Ptdlns to the kinase which produces PtdIns(4)P. By using a dual [3H]- and [14C]inositol-labeling procedure, Stephens et al. (39) demonstrated that turnover of the inositol moiety of Ptdlns(3)P was similar to that of Ptdlns(4)P, suggesting that these compounds arise from a common pool of Ptdlns. Alternatively, reduced PtdIns(4)P levels may arise from an effect on the other kinases, phosphatases, or phospholipases which maintain PtdIns(4)P and Ptdlns(4,5)P2 levels in equilibrium. A further effect of middle T antigen expression on inositol lipid metabolism involves the appearance of elevated GroPIns levels in cells which express middle T antigens which associate with Ptdlns 3-kinase. Elevated GroPIns levels in these cells most likely arise from concerted phospholipase A2 and A1 activity on Ptdlns. These results will be presented elsewhere (E.T.U., unpublished data). Ptdlns(3,4)P2
Ptdlns(3)P
Pdldns