Polyoma Virus Middle T Antigen-pp6OC - The Journal of Biological ...

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Sep 19, 1991 - Purified Phosphatidylinositol 3-Kinase in Vitro*. (Received .... to1 3,4-bisphosphate; PtdIns-3,4,5-P3, phosphatidylinositol 3,4,5-tris- phosphate ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY Q 1992 by The American Society for Biochemistry and Molecular Biology,Inc.

Vol. 267, No. 8, Issue of March 15, pp. 5408-5415.1992 Printed in U.S.A.

Polyoma Virus Middle T Antigen-pp6OC'"" Complex Associates with Purified Phosphatidylinositol 3-Kinase in Vitro* (Received for publication, September 19, 1991)

Kurt R. Auger$, Christopher L. CarpenterQ1, Steven E. ShoelsonII, HelenPiwnica-Worms, and Lewis C. Cantley From the Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, the SHematologyOneolom Unit. Massachusetts General Hoswital.Boston. Massachusetts 02214, and the 11Joslin Diabetes Center, Haruard M e d i c i i S c h i , Boston, Massachusetts 02i15 '

Reconstitution of the polyoma virus middle T antigen (mT)-pp60" complex and phosphatidylinositol 3-kinase (PtdIns 3-kinase) has been accomplished in vitro with immunopurified baculovirus-expressed mTpp60""" and PtdIns 3-kinasepurified from rat liver. Both the 110- and 86-kDa subunits of the PtdIns 3kinase associated with the mT-pp60c-a" complex.The association of PtdIns 3-kinase with the mT-pp60""" complex was dependent on the protein-tyrosine kinase activity of pp6OC-"" as a kinase-inactivemutant (pp602g5c-ac) still complexed with mT, butthe mTPPf302g5c-s"complex was unable to bind PtdIns 3-kinase. The mT-pp60c-"" complex phosphorylated both subunits of PtdIns 3-kinase on tyrosine residues. The immunopurified mT-pp60C"" complex also associated with PtdIns 3-kinase activity from whole cell lysates, and this association was dependent upon the proteinof p~60"-~". Comparison of "Styrosine kinase activity labeled proteins from whole cell lysates which associatedwith immunopurified mT-pp6O"" and mTPP60ae5c-a"revealed proteinsof 110 and 85 kDa as the major peptides dependent on protein-tyrosine kinase activity for association with the complex. In addition, a synthetic phosphopeptide (13-mer)containing sequences conserved between the major tyrosine phosphorylation siteof murine polyoma virus mT, hamster polyoma virus mT, and the insulin receptor substrate (IRS-1) specifically blocked the association of the 85and 110-kDapolypeptides with the mT-pp60c-am complex. The ability toblock the association was dependent on the tyrosine phosphorylation of the peptide. Association of PtdIns 3-kinase activity was blocked concurrently. This is the first demonstration that the 110-kDa subunitof PtdIns 3-kinase can associate with mT-pp60""". This association in vitrois a step toward understanding protein-protein interactions important in the signal transduction pathway of oncogenic proteins.

Transformation by mouse polyomavirus requires middle T antigen, and thisprotein both regulates and is a substrate for protein kinases. A small fraction of the mT' molecules in fibroblasts is associated with the cellular proto-oncogenes pp6OC-"",pp62c-ye8, and p 5 P (1-4). In thecase of pp6OC"", this association results in theactivation of protein-tyrosine kinase activity (5-7). Transformation of fibroblasts by mT appears to require both activation of protein-tyrosine kinases and associationwith a phosphatidylinositolkinase (PtdIns kinase) (8-11). In addition, phosphorylation of tyrosine 315 of mT is critical for efficient transformation, PtdIns kinase association, and tumorigenesis (8, 9,12, 13). The PtdIns kinase activity that associates with mT/pp60""" has beenshown to associate with several activated protein-tyrosine kinases (8, 10, 14-19). The mechanism for association appears to require tyrosine phosphorylation and SH2 domains (reviewed in Refs. 20 and 21). PtdIns 3-kinase phosphorylates the 3-hydroxyl position of the inositol ring to produce phosphatidylinositol 3-phosphate (PtdIns-3-P) (16). In addition to phosphorylating phosphatidylinositol (PtdIns), the enzyme also phosphorylates phosphatidylinositol 4-phosphate(PtdIns-4-P)and phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) to produce two recently characterized phospholipids, phosphatidylinositol 3,4-bisphosphate (PtdIns-3.4-Pz) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3) (22-24), respectively. The production of these more highlyphosphorylated phospholipidsis dependent on the activation of protein-tyrosine kinases, and, more importantly, the temporal production of these lipids makesthem excellent candidates for important signaling molecules of cell division (25). Phosphatidylinositol 3-kinase was recently purified to homogeneity from rat liver cytosol (24) and was shown to be a heterodimer of 110- and 85-kDa subunits. The 85-kDasubunit is phosphorylated on tyrosine both in vivo in mT-transformed cells and upon co-precipitation with the mT-pp60'-s" complex in vitro (10). It has recently been demonstrated that tyrosinephosphorylated mT reassociates with pp85 in vitro (26). The

* This work wassupported in part by National Institutes of Health Grants GM 36624 and GM 41890 ( t o L. C. C . ) and CA 50767 ( t o H. P-W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Supported in part by Training Grant F32 HL07437 from the National Institutes of Health. To whom correspondence should be addressed Dept. of Physiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-956-6744;Fax: 617956-0445. 1Supported by an American Heart Association-Parke Davis clinician scientist award.

* The abbreviations used are: mT, polyoma virus middle T antigen; mT-pp60""", polyoma virus middle T antigen-pp6O""" complex; PtdIns 3-kinase, phosphatidylinositol 3-(hydroxy)kinase; PtdIns, phosphatidylinositol; PtdIns-3-P, phosphatidylinositol 3-phosphate; PtdIns-4-P, phosphatidylinositol 4-phosphate; PtdIns-4,5-P2, phosphatidylinositol 4,5-bisphosphate; PtdIns-3,4-P2, phosphatidylinoslto1 3,4-bisphosphate; PtdIns-3,4,5-P3, phosphatidylinositol 3,4,5-trisphosphate; HEPES, 4-(hydroxyethyl)-l-piperazineethanesulfonic acid; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; MES, 4-morpholineethanesulfonicacid; DME, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; Fmoc, N-(g-fluorenyl) methoxycarbonyl.

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reassociation was donein solution and by blotting after SDS finally 2 times with TNE (10 mM Tris-HC1 (pH 7.4), 100 mM NaC1, 1mM EDTA). The immunoprecipitates were used immediately for in denaturation. Tyrosine phosphorylation of mT appeared to vitro phosphorylation and association assays. regulate the subcellular distribution of the 85-kDa protein Purification of Phosphatidylinositol 3-Kinase-PtdIns 3-kinase was (26). The reassociatedpp85fromwhole cell lysates is the purified to homogeneity from rat liver as previously described (24). same 85-kDa protein found in the purified PtdIns 3-kinase The purified enzyme had a specific activity of approximately 500 complex (24). In addition, the 85-kDa subunit of PtdIns 3- nmol/mg/min when assayed as described below with PtdIns as the kinase hasbeencloned(27-29)andmay function as the substrate. The enzyme was stored at 4 "C in 50 mM MES (pH 6.7), 100 mM KCl, 0.5 mM dithiothreitol, 1pg/ml leupeptin and pepstatin mediator for the interaction betweenthe catalytic subunit of A and used within 2 weeks of purification. PtdIns 3-kinase and the activated platelet-derivedgrowth In Vitro Association of Phosphatidylinositol 3-Kinase with Immufactor (PDGF)receptor (27,28) and the mT-pp60c-"" complex nopurified mT-pp6P-""-Washed mT-pp60c-a" immune complexes were incubated with varying amounts of purified PtdIns 3-kinase in (27). 750 pl to 1ml of standard lysis buffer (see above) a t 4 "C on a platform Inan attempt to betterunderstand the importanceof PtdIns 3-kinase and mT-pp60""" complex formation in the rocker. The typical incubation was for 2-3 h after which the comwere collected by a brief centrifugation and washed twice with signal transduction pathway,we have reconstituted this com- plexes 1% Nonidet P-40 in PBS, twice with 0.5 M LiCl in 0.1 M Tris-HC1, plex in uitro using immunopurifiedmT-pp6O"""complex from and twice with TNE. overproducing insect cells and PtdIns 3-kinase purified from Mammalian whole cell lysates were prepared from confluent 10rat liver. When protein kinaseassays were performedin vitro cm cell culture plates. The monolayer was washed once with 4 "C both subunitsof PtdIns 3-kinase were phosphorylated on PBS and lysed in 1.0 ml of standard lysis buffer. After 10 min of tyrosine residues.The reconstituted complex utilized PtdIns, incubation on a rocker platform a t 4 'C, the cells were scrapeand lysates were cleared by centrifugation a t 12,000 X g PtdIns-4-P, and PtdIns-4,5-Pz as substrates to generate the harvested, for 5 min at 4 "C. Lysates were used immediately for association three newly described phospholipids. These results are con- experiments with the immunopurified mT-pp6O'"" complex. sistent with previous data obtained from in vivo studies (23). Peptides were mixed with whole cell lysates or the purified PtdIns Inaddition,immunopurified rnT-pp6Oc-'" associated with 3-kinase a t a final concentration of 50 p~ and incubated at 4 "C on PtdIns 3-kinase from a whole cell lysate after incubation in a rocker platform for 30 min. The mixture was then incubated with uitro, and this association was also dependent upon the pro- the immunoprecipitated mT-pp6O""" complexes as described above. Phosphatidylinositol Kinase Assays and Protein Kinase Assaystein-tyrosine kinase activity ofpp60""". The association of Phosphatidylinositol kinase assays were performed directly on the PtdIns 3-kinase and immunopurified mT/ppGO"-""could be immune complex as described (8, 22). Sonicated phospholipids were blocked by preincubatingthe whole cell lysate or the purified added to the washed beads, and the reaction was initiated with the enzyme with a synthetic peptidederivedfrom the mT se- addition of 50 p~ ATP, 5-25 pCi of [y-32P]ATP(3000 Ci/mmol), 5 quence. Tyrosine phosphorylation of the peptide was neces- mM MgClz in 20mM HEPES (pH 7.5). Reactions were incubated for The 50-p1 reactions were stopped by the sary to block association. The ability of the phosphorylated 5 min at room temperature. addition of 80 pl of 1 M HCl and 1 p1of 500 mM EDTA. The lipids peptide to block association of PtdIns 3-kinase with the mT- were extracted with 160 p1of methanol/chloroform (l:l),and the pp6OC-"" complex provides additional evidencefor the specific organic layer was collected for analysis. region of mT involved in binding PtdIns 3-kinase. This data Protein kinase assays were also performed directly on the immune complex with or without added PtdIns 3-kinase. The reaction was supports the model we have recently proposed (20). MATERIALS AND METHODS

Cell CuEture-NIH/3T3 and Rat-1 fibroblasts were maintained by standard cell culture techniques in DME supplemented to contain 10% calf serum. Spodoptera frugiperda (Sf9)cells were cultured and used as described by Piwnica-Worms et al. (30). Sf9 cells were seeded into 60-mm plates (3 X lo6 cells), allowed to attach, and then were coinfected with baculovirus-mT and bacu1ovirus-pp60"'"", or baculo~irus-pp60"~'" as described (30). Cell pellets were prepared 40 h postinfection as described below. Preparation of Sf9 Cell Pellets, Lysates, and ImmunoprecipitatesSf9 cells were scrape-harvested from 60-mm cell culture plates in phosphate-buffered saline (PBS)and collected by centrifugation. The cell pellet was washed once with cold PBS and aliquoted to 1.5-ml microcentrifuge tubes and again separated by centrifugation. The supernatant was aspirated from the cells, and thepellets were quickfrozen in dry ice/ethanol and stored at -70 "C until the lysates were prepared. Lysates were generated from the frozen pellets by the addition of 1.0 ml of standard lysis buffer (137 mM NaC1, 20 mM Tris-HC1 (pH 7.4), 1 mMMgC12, 1 mM CaClz, 10% glycerol, 1%Nonidet P-40, 150 p M vanadate, 0.1 mM phenylmethylsulfonyl fluoride, 1 pg/ml aprotinin, and 1 pg/ml leupeptin) and incubated a t 4 'C with constant rocking for 15 min. Lysates were cleared by centrifugation a t 12,000 X g for 5 min at 4 "C. The mT-pp6OC-""complex was purified by immunoprecipitation essentially as described (31). Briefly, anti-mT (a-mT) antiserum (kindly provided byD. Pallas and T. Roberts, Dana-Farber Cancer Institute, Harvard Medical School) were added t o the lysates and incubated a t 4 'C for 2 h with constant agitation. The immune complexes were collected on protein A-Sepharose (CL4B, Sigma) that had been prewashed in 1%bovine serum albumin and then stored in a 50% suspension with PBS. Immune complexes were washed 2 times with 1% Nonidet P-40 in PBS, 2 times with radioimmune precipitation buffer (20 mM HEPES (pH7.5), 137 mM NaCl, 2 mM EDTA, 10% glycerol, 1%Nonidet P-40,0.1% SDS, 0.5% deoxycholate), twice with 0.5 M LiCl in 0.1 M Tris-HC1 (pH 7.4), and

done in 20 mM HEPES (pH 7.4), 10 mM MgClZ, 1-5 p~ ATP, and typically 10-50 pCi of [T-~'P]ATP(3000 Ci/mmol) per assay. Experiments for the time course of phosphorylation of PtdIns 3-kinase by the mT-pp60""" complex were done with 100 p~ ATP. To analyze the phosphorylation of purified PtdIns 3-kinase, the purified enzyme was added directly to the washed immune complex prior to theprotein kinase reaction. The reaction was stopped by the addition of 2 X SDS gel loading buffer, and samples were boiled for 5 min prior to SDSPAGE. Phosphoamino Acid Analysis-Phosphoamino acid analysis of the 3ZP-labeledproteins from the SDS gel was performed as described (32). Briefly, radioactive gel slices were excised after autoradiography, fixed in 30% methanol for several hours, and dried by rotoevaporation. The gel slices were treated with tosylphenylalanyl chloromethyl ketone-trypsin (Worthington) in 50 mM ammonium bicarbonate at 37 "C overnight. The supernatants were collected and dried and washed with successively decreasing volumes of H20 (eg. 1 ml, 500 pl, 300 pl, 200 pl, 100 pl). The tryptic fragments were hydrolyzed with 6 M HCl a t 100 "C for 90 min. Samples were then dried and washed with HzO. The samples were dissolved and analyzed by electrophoresis in pyridine/acetic acid/water (1:10189, v/v/v) for 90 min at 800 V with nonradioactive standards. The standards were visualized by spraying with 0.2% ninhydrin in acetone, and radiolabeled samples were visualized by autoradiography. Two-dimensional analysis was also performed as described (32). Electrophoretic separation in the first dimension was done in formic acid/acetic acid/HzO (25:78:897, v/v/v). Plates were thoroughly dried and then developed by ascending chromatography for the second dimension in a 2-propanol/HC1/HzO (7015:15, v/v/v) solvent system. Standards andsamples were visualized as described above. Tran3'S-label of NIH/3T3 Cells, Rat-1 Cells, and Sf9 Cells-Rodent cells were grownto approximately 80-90% confluence in 10-cmplates in DME supplemented to contain 10% calf serum. The culture medium was removed and replaced with 5 ml of DME minus methionine (GIBCO) that contained 500 pCi of Tran%-label (ICN, >lo00 Ci/ mmol) supplemented with one-third of the amount of unlabeled methionine normally used. The cells were cultured for 12-48 h and

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T Antigen-pp6WsKComplex

then harvested as follows. Radioactive culture medium was aspirated, c-src 2YSc-sre and the cell monolayer was washed 3 times with ice-cold PBS. The m T /m p pT6/ 0p p 6 0 cells were lysed by the addition of 1 ml of standard lysis buffer (see above) and incubated on a rocker platform for 15-20 min a t 4 "C. The lysate was harvested with the aid of a cell scraper and cleared of + + insoluble material by centrifugation a t 12,000 X g for 5 min a t 4 "C. P t d I n s 3 - k i n a s e : The lysates were used immediately for association assays. Sf9 cells were labeled a t 38-40 h postinfection with Tran=S-label Ptd n s P as described (30). Peptide Synthesis-The phosphopeptide RENEpYMPMAPQIH corresponding to the hamster mT sequence (33) was synthesized Ptd n s q essentially as described (34). Ne-Fmoc-L-phosphotyrosine(FmocTyr(P))was prepared by treating L-phosphotyrosine (Sigma) with NPtd I n s q (9-fluorenylmethoxycarbony1)succinimide(Aldrich) (35). Solid-phase peptide synthesis was conducted on a Milligen/Biosearch 9600 synthesizer using His(Trt)-PAC resin (Milligen/Biosearch) a t a substitution level of 0.37 mmol/g. Acylation reactions with standard side chain-protected amino acids (Arg(Pmc), Glu(t-butyl))were conducted with 4 eq each of Na-Fmoc amino acid, l-hydroxybenzotriazole, and diisopropylcarbodiimide in dimethyl formamide for 1 h a t 22 "C;the peptide bond-forming reaction with Fmoc-Tyr(P) was conducted manually for 2 h with 3 eq of each reagent. Asn and Gln were incorporated as the respective pentafluorophenyl esters. N"-Fmoc deprotections were carried out with a mixture of piperidine, toluene, and dimethyl formamide (30:35:35, v/v/v) for 7 min. The peptide was cleaved from the resin, and side chain-protecting groups were removed by treatment with amixture of trifluoroacetic acid, thioanisole, ethanedithiol, and anisole (905:3:2, by volume) for 2 h a t 4 "C. The cleaved product was precipitated in diethylether, desalted on a column (2.6 X 100 cm) of Bio-Gel P-2, equilibrated in 3M acetic acid, and lyophilized. The major product was purified by preparative HPLC (Waters Prep 4000) on a Dynamax-3OOA 12-pm C8 column (41.4 X 250 mm)equipped with a matched guard column; peptides were eluted witha mobile phase composed of acetonitrilein 0.05% aqueous trifluoroacetic acid (80 ml/min). Amino acid analysis (N/D, 1.0; Q/ 1 2 3 4 E, 3.0; P, 2.0;A,1.0; M, 2.0; I, 1.0; Y,1.0; H, 0.9; R, 0.9) and fastFIG. 1. Analysis of a phosphatidylinositol kinase reaction atom bombardment mass spectrometry (molecular ion a t m/z 1699.3) after association of purified rat liver PtdIns 3-kinase with were as predicted. Thin Layer Chromatography, Deacylatwn of Phospholipids, and baculovirus-expressed, immunopurified mT-pp6Pm and The mT complexes were immunopurified as deHPLC Anulysis-Intact phospholipids were analyzed on oxalate- mT-pp60296'~"". treated silica gel 60 plates (E. Merck) in a solvent system of 1- scribed and incubated with or without purified PtdIns 3-kinase a t propanol, 2 M acetic acid (6535, v/v). For experiments inwhich only 4 "C for 2 h in standard lysis buffer. Phosphatidylinositol kinase PtdInsP and/or PtdInsP, were analyzed, the faster chloroform, meth- assays were done with a mixture of phosphatidylserine, PtdIns, anol, 2.2 M NHzOH (9:7:2, v/v/v) solvent system was utilized. Phos- PtdIns-4-P, andPtdIns-4,5-P2 as lipid substrates (1:1:1:2). Immunopholipids were deacylated and analyzed by HPLCas previously purified, baculovirus-expressed mT-pp6O""" (lanes 1 and 2) and mTdescribed (31). 3H-Labeled phosphoinositides and inositol standards pp60295E.""(lanes 3 and 4 ) were used. Purified PtdIns 3-kinase was were obtained from Du Pont-New England Nuclear, and the lipids added (lunes 2 and 4 ) or was omitted (lanes 1 and 3).The migration were deacylated by the same method used for the 32P-labeledsamples. of PtdInsP, PMInsP2, and PtdInsPsstandards is indicated. The standards and samples were co-injected with the nonradioactive nucleotides ADP and ATP for each HPLC analysis. In order to testfor in vitro association between the mT-pp-

60'"" complex and PtdIns 3-kinase, purified rat liver PtdIns 3-kinase (24) was incubated with the baculovirus-expressed Associationof Immunopurified mT-pp6OC-'" with Purified mT-ppGO'~". After incubation at 4 "C, the beads containing Phosphutidylinositol3-Kinase Is Dependent on Protein-Tyro- the complex were washed and then assayed for lipid kinase sine Kinase Activity-PtdIns 3-kinase associateswith the mT- activity. As shown in Fig. 1, PtdIns, PtdIns-4-P, and PtdInspp60'-"" complex in vivo and can be immunoprecipitated from 4,5-P2kinase activities associated with the mT-pp60'-3m commT-transformed cells using antibodies against mT or pp60'- plex whenpurified PtdIns 3-kinase was added. However, very (8,9, 16). The mT-pp6O"" complex has been generated in little PtdIns kinase activity associated with the protein kiinsect cells by co-infection with recombinant baculoviruses nase-deficient mT-pp6029k-"" complex (Fig. 1, lane 4 ) . This is encoding mT and pp60""" genes (30). This complex has been in spite of there being equivalent amounts of complex in each shown to have protein-tyrosine kinase activity. Both mT and case (data not shown). Thus, thein vitro association of PtdIns pp60'.'" are phosphorylated on tyrosine in the co-infected 3-kinase with the mT-pp6O"" complex appears to be dependcells, and the purified complex has tyrosine kinase activity ent o n the protein-tyrosine kinase activity of pp60""". These toward exogenous substrates. A kinase-defectivecomplex, results are consistent with previous in vivo findings that mT mT-pp60zgk.3m, has also been characterized.' The pp60295c~s" must associate with and activate pp60"'"" in order for PtdIns mutant protein associates with mT butdoes not have protein tyrosine kinase activity. The mT-pp6O""" and mT/pp60'95c~"mkinase association (9, 13). The results in Fig. 1 demonstrated that immunopurified complexes were purified from infected Sf3 cells using a-mT mT-pp60c-"" and purified PtdIns 3-kinase associate in vitro. antibodies conjugated to protein A-Sepharosebeads. The purified complexes lacked PtdIns 3-kinase activity, although Association wasalso demonstrated by adding purified PtdIns a small amount of PtdInsP kinase activity was detected (Figs. 3-kinase directly to the insect cell lysate and incubating the mixture at 4 "C.After incubation, a-mT antiserum was added, 1 and 3). andthe immunecomplexeswerecollectedon protein A* H. Piwnica-Worms, unpublished observations. Sepharose andthen washed.Lipid kinase assays demonRESULTS

Polyoma Virus MiddleT Antigen-ppGO"-""Complex strated thatpurified PtdIns 3-kinase associated with the mTpp60"'"" complex in the crude insect cell lysate (data not shown). A monoclonal antibody (EC10) directed against pp60"'" also immunoprecipitated the mT-pp6O""" complex and associated lipid kinase activity (data not shown). Phosphorylation and Phosphoamino Acid Analysis of the Phosphatidylinositol 3-Kinase-Purified PtdIns 3-kinase is a heterodimer of 85- and 110-kDa proteins (24). The 85-kDa subunit has been shown to be phosphorylated on tyrosine in uiuo in mT-transformed cells (10, 24). In order to determine if one or bothof the subunits were phosphorylated on tyrosine residues in uitro bymT-ppGO"'", protein kinase reactions were performed directly with the immunoprecipitated complex. PAGE of the proteinsafter the phosphorylation reaction established that both the 110- and 85-kDapolypeptides were phosphorylated (see Fig. 2A). As seen in Fig. 2A, more label was incorporated into the 85-kDa subunit than into the110kDa subunit. This result reflects preferential phosphorylation of the 85-kDa subunit as PtdIns 3-kinase purified from rat liver has equimolar amounts of the 85- and 110-kDa subunits (24). The purified enzyme has two distinct but related 110kDasubunits with slightly different mobilities on SDSPAGE. 35S-Labelingstudies of cultured cells (discussedbelow) indicated that thedifference in phosphorylation between the 85- and 110-kDa subunits is not due to differential immunoprecipitation or release from the immune complex, as similar amounts of labeled protein remained in thecomplex. For both the 110- and 85-kDa polypeptides the phosphorylation was saturable andreached a plateau after30 min (data not shown). The stoichiometry of phosphorylation was calculated to be 1.2 mol of phosphate/mol of 85-kDa protein added to the assay. The stoichiometry was 0.38 mol of phosphate per mol of 110-kDa protein. Phosphoamino acid analysis demonstrated that both the 85- and 110-kDa subunits were phosphorylated on tyrosine residues, as expected for phosphorylation by mT-ppGO""" (Fig. 2, B and C). Complex Formation of [email protected]" with Phosphatidylinosito1 3-Kinase from Whole Cell Lysates-The immunopurified mT-pp60""" also associated with PtdIns 3-kinase from fibroblast lysates. NIH/3T3 or Rat-1 lysates were prepared from confluent cells in standard lysis buffer as described under "Materials and Methods." Immunopurified mT-pp60""" complexes were added to the whole cell lysates and incubated at 4 'C.The protein A-Sepharose-conjugated mT-pp60c-""complex was then washed with detergent and salt andassayed for associated phospholipid kinase activity. The resultant samples were extracted and deacylated for analysis by HPLC anion exchange chromatography. As depicted in Fig.3, PtdIns, PtdIns-4-P, and PtdIns-4,5-P2 kinase activities were present. The HPLCmigration positions of the threeproducts were consistent with the structure PtdIns-3-P, PtdIns-3,4-P2, and PtdIns-3,4,5-P3. Immunopurified mT-pp60295e-""(the kinase-inactive mutant) was unable to associate with the endogenous PtdIns 3kinase activity of fibroblast whole cell lysates as would be expected from the results with purified PtdIns 3-kinase (data not shown). A small amount of PtdIns-4,5-P2 was generated in the immunoprecipitates from both mT-pp6O""" and mTpp60206c~"" complexes (Fig. 3B). This is consistent with the results in Fig. 1 in which some PtdInsP kinase activity was found associated with both of these complexes in theabsence of added PtdIns 3-kinase. Thus, the ability of the mT-pp60""" to associate with PtdIns 3-kinase from whole cell lysates was also dependent upon the protein-tyrosine kinase activity of pp60""".

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The amount of PtdIns 3-kinase activity that associated with the mT-pp6O"-"" complex was dependent on the amount of lysate in theincubation. Titration of Rat-1 lysates against a fixed amount of mT-pp6O""" complex revealed the immune complex was in excess, and lysate from approximately 2.5 X IO6cells represented 50% saturation (data notshown). Proteins Associated with Baculouirus Expressed [email protected]""from Whole Cell Lysates-To define the specific proteins which associated with the protein-kinase active complex NIH/3T3 cells were labeled with Tran3'S-label, and whole cell lysates were prepared. Immunopurified protein A-conjugated mT-pp60c-""and mT-pp60295e-"" were added to separate radiolabeled lysates and incubated at 4 "C for 3 h. The complexes were washed with Nonidet P-40, LiC1, and TNE. The associated proteins were analyzed by SDS-PAGE, and the results areshown in Fig. 4. Although a number of radiolabeled peptides associated with both the protein-kinase active (lane I ) and -kinase inactive (lane 2) complexes, the most striking difference was observed in the 85- and the 110-kDa regions as indicated by the arrows (Fig. 4). A band at 173 kDa also preferentially associated with the active mT-ppGO"'" complex. These results are analogous to the results obtained with the purified PtdIns 3-kinase, in that both the 110-kDa doublet and the 85-kDa polypeptide associated with the mT-pp60c-a" complex in uitro. Peptides That Block the Association of PtdIns 3-Kinase with the [email protected]'" Complex-Data presented demonstrate that tyrosine kinase activity in the mT-pp60c-""complex is required for association of PtdIns 3-kinase. The major site of tyrosine phosphorylation on murine mT by pp60"-""is tyrosine 315 (36). Amino acid sequence in this region of mT is conserved in various protein-tyrosine kinases that havebeen observed to associate with PtdIns 3-kinase (20). The in uitro association was further characterized, and the importance of tyrosine phosphorylation was directly tested. Competition experiments were done with a phosphorylated and unphosphorylated version of a synthetic 13-amino acid peptide based on the region of hamster mT. This sequence is related to murine mT at tyrosine 315 (see Table I). Preincubation of the phosphorylated peptide with whole cell lysates blocked association of PtdIns 3-kinase with the immunopurified mT-pp6O""" complex (Fig. 5, lanes 5 and 6). Preincubation of the unphosphorylated version of the peptide with cell lysates did not affect the association (Fig. 5, lanes 3 and 4 ) . The phosphorylated version of the peptide could also block the association of PtdIns 3-kinase activity with the immunopurified mT-ppGO"-"" when purified PtdIns 3-kinase was used for the association assay (data not shown). Proteins that were prevented from associating with the mT-pp6O"-""complex by the phosphorylated peptide were also determined. Whole cell lysates from Tran35S-labeled Rat-1 cells were preincubated in the absence of peptide, with phosphorylated peptide or unphosphorylated peptide, and then association with exogenously added mT/pp60c-"" was monitored. Fig. 6 shows that proteins of similar molecular weight to thesubunits of purified PtdIns 3-kinase (a doublet around 110 kDa and an 85-kDa band) associated with the kinase active complex (lane 2) butnot with the protein kinasedeficient complex (lane 1). The unphosphorylated peptide did not affect this association (compare lanes 3 and 4 with lanes 1 and 2). However, the phosphorylated peptide specifically blocked three proteins (lane 6, see arrows) from associating with the kinase active mT-pp6O"-"" complex. Other, higher molecular weight proteins that associated in a protein kinasedependent manner (compare lanes 1 and 2 or lanes 3 and 4 )

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B

A

1

2

C

.... .. ...; .

I

. .

FIG. 2. Phosphorylation of purified PtdIns 3-kinase by the immunopurified mT-pp60"'" complex. A protein kinase reaction was performed using immunopurified mT-pp60""" complex in the presence or ahsence

of purified PtdIns 3-kinase. The protein kinase reaction was initiated with the addition of [y-""I]ATP and stopped after 10 min with the addition of 2 X gel loading buffer (see "Materials and Methods"). The sampleswere hoiled for 5 min and analyzed on a 6% polyacrylamide gel. A, autoradiography of gel-separated proteins. L a n p 1 is m7" pp60""" in the absence of PtdIns 3-kinase, and lane 2 is in the presence of PtdIns %kinase. Arrows indicate the position of the 110-kDa, 85-kDa,pp60"" (BO-kDa), and mT (58-kDa) proteins. H , two-dimensional phosphoamino acid analysis of the 85-kDa hand from the in vitro kinase reaction was performed as descrihed under "Materials and Methods." P-SER, phosphoserine; P-THR, phosphothreonine; P-TYR, phosphotyrosine. C, one-dimensional phosphoamino acid analysis of the 110-kDa proteins from the in vitro kinase reaction.

As pointed out previously, receptor-type tyrosine kinases that bind PtdIns 3-kinase have short stretches of sequences with similarity to the Tyr-315 region of mT (20).In the PDGF DISCUSSION obey receptor, the sequences around Tyr-740 and Tyr-751 theserules(Table I), andthelattersite is known to be We have reconstituted purified PtdIns 3-kinaserecomwith Both Tyrbinant, baculovirus-expressed, immunopurified mT-pp6O""" phosphorylated in response to PDGF binding (37). in vitro. The PtdIns 3-kinase associated with protein-tyrosine751 and Tyr-740 have been implicated in binding PtdIns 9kinase (37-40), and peptides phosphorylated at Tyr-740 or kinaseactive mT-ppGO'."" but failed to associatewithan inactive mutant complex. ATP was not required during the Tyr-751 have been shown to block association of purified PDGF receptor with PtdIns %kinase from cell lysates (40). precipitation, indicating that phosphorylation of PtdIns 3mT-pp6O""" are analogous tothe kinase itselfwas not neededfor tight complex formation. Thus,ourresultswith Previous work has shown that mT is phosphorylated on Tyr results with the PDGF receptor and extend these studies to when co-expressed with pp6W."" ininsect cells(30). The show that purified PtdIns 3-kinase will complex with mTpp6OC.""without the need of other cellularfactors. ability of the wild-type mT-pp6O""" complex but not the Inadditiontoreceptor-typetyrosinekinases,the major mutant to associate with PtdIns 3-kinase (Fig. 1) thus suggests that tyrosine phosphorylationof the mT-pp6O"'" com- substrate of the insulin receptor tyrosine kinase (IRS-1) contains several repeats of sequencessimilartotheTyr-915 plex is needed for the association. This result is consistent with the observation that mT must be phosphorylated on Tyr region of polyoma m T (41) (Table I). This protein is not a .?-kinase in insulinin order to blot the 85-kDa subunitof PtdIns 3-kinase using tyrosine kinase but associates with PtdIns stimulated (but not unstimulated) cells (41). IRS-1 has two the Western blot procedure(24, 26). Consistent with this idea, a phosphotyrosine-containing regions of sequence that are quite similar to the hamster mT peptide based on a region highly conserved between hamster phosphopeptide used for competitionstudies in this paper and murine polyoma m T (Table I) blocked association of (Table I). Further work is needed to determine the sequence PtdIns 3-kinase activity with mT-pp60c-"". The non-phos- specificity of the tyrosine-phosphorylated peptide domains that bind PtdIns 3-kinase and what proteins utilize these phorylatedpeptidehadno effect on the association. The phosphotyrosine of this sequence is analogous to Tyr-315 of domains for recruitmentof this enzyme. The results presented here provide the first evidence that murine mT, themajor site of tyrosine phosphorylationin vivo the 110-kDa subunit of PtdIns %kinase associates with the (36).A mutation of Tyr-315 to Phe reduced (but did not eliminate) association with PtdIns 3-kinase i n vivo (8), and mT-ppGO'."" complex. The 110-kDa subunit was not phosa n antibody against thisregion preferentially immunoprecip- phorylated as well as the 85-kDa subunitin the reconstituted itates the fractionof mT thatdoes not associate with PtdIns complex (Fig. 2) and was not previously detected in "P(7:-3-kinase (13). Mutations in the region of Tyr-315 also com- labeled proteins associated withm T immunoprecipitates from promise the transforming ability of polyoma virus without polyoma-infected cells (10). However, using [,""S]methionineaffecting the tyrosine kinase activity of the mT-pp60""" com- labeled cells, the major proteins that associated with baculoplex, indicatingthat thisregion is criticalfor in viuo targeting virus-expressed mT-pp6O""" complex and failed to associate of the protein-tyrosine kinase(20). with the kinase-defective mutant complexmigrated at the were not blocked by the phosphorylated peptide(lanes 5 and 6).

Polyoma Middle Virus

TAntigen-pp6O""" Complex

A

1

gPldlns-3-P

5413 295c-src

c-src

m T / p pm6T0 / p p 6 0

1.200 lo4

- 210 kDa

1.000 10'

g

so00

d

6000

173 kDa

4000

+

110 k D a 3

- 97 kDa

2000

85 kDa 4

0

- 68 kDa

Elutlon t h e (rnln)

B 5000 4000

IgPldlns-3.4-P2

T

f

1000

62

60

0

3000

*$

58

68

66 64 Elutlon tlme (rnln)

70

C

T

2

FIG. 4. "S-Labeled proteins from whole cell lysates of NIH/ 3T3 fibroblasts that associatewith immunopurified, baculovirus-expressed mT-pp60"'"" andmT-pp60286"~"~ after in vitro association. NIH/3T3 cells were labeled with Tran35S-label,and

2ooo

whole cell lysates were prepared as described. Immunopurified, pro(lane 2 ) was tein A-conjugated mT-pp60""" ( l a n e 1 ) or mT-pp60z9k-sm incubated with the radiolabeled lysates, and then the immune complexes were washed and analyzed by SDS-polyacrylamidegel electrophoresis. The arrows denote the location of the 110- and 85-kDa polypeptides. Molecular mass markers are indicated on the right.

1500 1000 500 0

1

lns-l.3.4,S-P4

8'2

86

90 94 9 8 106 102 Elution tlrne (mln)

FIG. 3. HPLC analysis ofthedeacylatedproducts from PtdIns, PtdIns-4-P, and PtdI11s-4,Fi-p~ kinase reactions on immunopurified mT-pp60c-"" proteinA complexes after association with NIH/3T3 wholecell lysates. A, elution profile of the deacylated [32P]PtdInsP(solid line) and conjected [3H]gPtdIns4-P standard (broken line) (where g is glycero). The immunopurification of mT-pp6O""" and association with the NIH/3T3 lysate was performed as described in the text. The lipid kinase reaction was done with equal amounts of phosphatidylinositol and phosphatidylserine (by weight) and deacylated directly after organic extraction of the reaction mix. E, elution profile of the deacylated lipids from the PtdIns-4-P kinase reaction (solid line) coinjected with [3H]gPtdIns4,5-Pz (broken line) standard. The reaction was as described in A, except that phosphatidylinositol 4-phosphate and phosphatidylserine were used as the lipid substrates (50% of each by weight). C, elution profile of the deacylated reaction product from the PtdIns-4,5-Pz kinase reaction (solid line) coinjected with [3H]inositol standards (broken line). The reaction was performed as described above, except that phosphatidylinositol 4,5-bisphosphate and phosphatidylserine (50% of each by weight) were used as substrates. In A, B, and C, the arrows indicate the migration positions of gPtdIns-3-P, gPtdIns-3,4Pz, and gPtdIns-3,4,5-P3produced by deacylating standards produced with purified PtdIns 3-kinase.

Pep.

P-pep.

"

7 -PIP

b 1

2

3

4

5

6

- PIP, - Origin

7

FIG.5. Association of PtdIns3-kinase activity from Rat-1 cells with immunopurified mT-pp60c-"" complex.Whole cell lysates were generated as described under "Materials and Methods" from Rat-1 cells and thenincubated or not with the indicated peptide. The mT-pp6F" complex was purified, incubated with the lysates, washed, and assayed for PtdIns kinase activity (as in Fig. 3A). The lipids were extracted and analyzed by TLC. Each sample was done in duplicate for this experiment except lane 7 which is immunopurified mT-pp60c'smwithout addition of cell lysate. Lanes I and 2, mT-pp60""" and cell lysate; lanes 3 and 4 included the unphosphorylated peptide (50 p ~ in) the lysate; and lanes 5 and 6 were preincubated with the phosphorylated peptide (50 p ~ ) Standards . visualized by iodine vapor are indicated by circles;PIP, phosphatidylinositol phosphate; PIP, phosphatidylinositol bisphosphate; pep., unphosphorylated peptide; P-pep., phosphorylated peptide.

positions of the 110- and 85-kDa subunits of the purified PtdIns 3-kinase. A third, unidentified protein of higher mo- kinase associate with the mT-pp6O"'" complex in polyoma lecular weight was also selectively immunoprecipitated with infected cells and that these are the major cellular proteins wild-type mT-pp6O"'"". The phosphopeptide specifically that associate with the complex in a tyrosine kinase-dependblocked association of the 85- and 110-kDa proteins and ent manner. PtdIns 3-kinase activity. These results are consistent with On the basis of previous results it is likely that the85-kDa the idea that both the 85- and 110-kDa subunits of PtdIns 3- subunit of PtdIns 3-kinase is regulatory and provides the link

Polyoma Virus Middle T Antigen-ppGO"-"' Complex

5414 1nT/pp66"~' 295c-src mT/pp60

Pep. P-pep.

- + - + + - + - - + + - . -

-

-

- + + + +

"

173 kDa 4

110 kDa

E.

85 kDa

+ 1

2

3

4

5

6

FIG. 6. Associationof"S-labeled proteins fromRat-1 whole cell lysates with mT-pp6Wam or mT/pp60296c"mwith or without preincubationwith peptide. Rat-1cells were labeled with Tran3'S-label, and lysates were prepared as described. The lysates were incubated with phosphorylated peptide (lanes 5 and 6), unphosphorylated peptide (lanes 3 and 4 ) , or no peptide (lanes 1 and 2). The samples were then incubated with immunopurified mT-pp6029b8m (lanes I, 3, and 5) or mT-pp60c-am(lanes 2, 4, and 6). The immune complexes were collected and analyzed by SDS-PAGE on a 6% gel. The upper double arrows denote the location of the 110-kDa proteins, and the lower arrow indicates the location of the 85-kDa protein.

TABLE I Sequence of the hamster mT peptide and similar sequences from proteins known to associate withPtdIns 3-kinase The hamster mT peptide sequence was synthesized and used for experiments as described in the text. The analogous sequence in the PDGF receptor has been shown to be involved in the binding of PtdIns 3-kinase after receptor phosphorylation. The specific sites of tyrosine phosphorylation in hamstermT and rat IRS-1 have not been mapped (indicated by *). Y,phosphorylated tyrosine; the number in parentheses indicates the location of the Tyr in the protein sequence. Seauence

Protein

Hamster mT Murine mT Rat IRS-1 Rat IRS-1 Rat IRS-1 Human PDGF Rec.j3 Human PDGF Rec.6

(298*) (315) (608*) (628*) (lolo*) (751) (740)

RENEYMPMAPQIH EEEEYMPMEDLYL TDDGYMPMSPGVA GNGDYMPMSPKSV APVSYADmTGIA ESVDWPMLDMKG SDGGYMDMSKDES

between mT and the110-kDa subunit. The 110-kDa subunit is probably the catalytic subunit of P a n s 3-kinase (42). The 85-kDaprotein has two -80-amino acidstretches of homology to the amino-terminal non-catalytic domain of pp60'"" (SH2 domain) (27-29). SH-2 domains have been shown to bind to tyrosine-phosphorylatedregions of target proteins to form 'tight complexes both in vivo and in vitro (43) (reviewed in Refs. 20 and 21). Tyrosine-phosphorylated mT specifically blots the 85-kDa (but not the 110-kDa) subunit of PtdIns 3kinase using a Western blot procedure (24, 26). The regions of the 85-kDa subunit required for the association with mT and with the 110-kDa subunit are under investigation. Finally, reconstitution of purified PtdIns 3-kinase with mTpp60""" did not have a major effect on substrate specificity. The purified enzymeutilizes three substrates in vitro (PtdIns, PtdIns-4-P, and PtdIns-4,5-Pz), and the relative activities

toward these substrates are very sensitive to assay conditions (24). However,association of PtdIns 3-kinase from whole cell lysates with the mT-pp6OC*'"did change the substrate specificity from using onlyPtdIns toutilizing PtdIns, PtdIns-4-P, and PtdIns-4,5-Pz (24): Cells transformed with polyoma mT or stimulated with PDGF have increased levels of PtdIns-3,4Pzand PtdIns-3,4,5-P3 but little change in PtdIns-3-P suggesting that transformation by polyomavirus or by activation with PDGF enhances phosphorylation of PtdIns-4-P and/or PtdIns-4,5-Pz by PtdIns 3-kinase (22,23,44).Usingour standard assay conditions the ratio of products formed when a mixture of all three substrates was presented was similar whether pure enzyme or reconstituted mT-pp6O""-PtdIns 3kinase complex was used. The ratio was also similar to that found in mT immunoprecipitatesfrom mT-transformed cells (23). The factors that affect in vivo specificity of this enzyme remain to be determined. Acknowledgments-We thank B.C. Duckworth and R. Kapeller for help in purifying PtdIns 3-kinase and J. Falk and M. S. Atkisson for their excellence in culture of the Sf9 cells. We also thank Brian S. Schaffhausen for helpful discussions and critical reading of the manuscript. REFERENCES 1. Cheng, S. H., Harvey, R., Espino, P. C., Semba, K., Yamamoto, T., Toyoshima, K., and Smith, A. E. (1988) EMBO J. 7,38453855 2. Courtneidge, S. A., and Smith, A. E. (1983) Nature 303, 435439 3. Kornbluth, S., Sudol, M., and Hanafusa, H. (1987) Nature 325, 171-173 4. Kypta, R. M., Hemming, A., and Courtneidge, S. A. (1988) EMBO J. 7,3837-3844 5. Bolen, J. B., Thiele, C. J., Israel, M. A., Yonemoto, W., Lipsich, L. A., and Brugge, J. S. (1984) Cell 38,767-777 6. Courtneidge, S. A. (1985) EMBO J. 4,1471-1477 7. Raptis, L., Lamfrom, H., and Benjamin, T. L. (1985) Mol. Cell. BWl. 5,2476-2485 8. Whitman, M., Kaplan, D. R., Schaffhausen, B., Cantley, L., and Roberts, T. M. (1985) Nature 315,239-242 9. Kaplan, D. R., Whitman, M., Schaffhausen, B., Raptis, L., Garcea, R. L., Pallas, D., Roberts, T. M., and Cantley, L. (1986) Proc. Natl. Acud. Sci. U.S. A. 83,3624-3628 10. Kaplan, D.R., Whitman, M., Schaffhausen, B., Pallas, D.C., White, M., Cantley, L., and Roberts, T. M. (1987)Cell 50, 1021-1029 11. Courtneidge, S. A., and Heber, A. (1987) Cell 50,1031-1037 12. Carmichael, G., Schaffhausen, B. S., Mandel, G., Liang, T. J., and Benjamin, T. L. (1984) Proc. Natl. Acud. Sci. U.S. A. 81, 679-683 13. Talmage, D. A., Freund, R., Young, A. T., Dahl, J., Dawe, C. J., and Benjamin, T. L. (1989) Cell 59,55-65 14. Sugimoto, Y., Whitman, M., Cantley, L.C., and Erikson, R. L. (1984) Proc. Natl. Acud. Sci. U.S. A. 81, 2117-2121 15. Macara, I. G., Marinetti, G. V., and Balduzzi, P. C. (1984) Proc. Natl. Acud. Sci. U.S. A. 81,2728-2732 16. Whitman, M., Downes, C. P., Keeler, M., Keller, T., and Cantley, L. (1988) Nature 332,644-646 17. Varticovski, L., Drucker, B., Morrison, D., Cantley, L., and Roberts, T. (1989) Nature 342,699-702 18. Fukui, Y., Kornbluth, S., Jong, S.-m., Wang, L.-h., andHanafusa, H. (1989) Oncogene Res. 4,283-292 19. Ruderman, N. B., Kapeller, R., White, M. F., and Cantley, L. C. (1990) Proc. Natl. Acud. Sci. U.S. A. 87,1411-1415 20. Cantley, L. C., Auger,K.R., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R., and Soltoff, S. (1991) Cell 64, 281302 21. Koch, C. A., Anderson, D., Moran, M. F., Ellis, C., and Pawson, T. (1991) Science 252,668-674 22. Auger, K.R., Serunian, L. A., Soltoff, S. P., Libby, P., and Cantley, L. C. (1989) Cell 57, 167-175

K. R. Auger, unpublished data.

Middle Polyoma Virus

T Antigen-pp6PsmComplex

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5415

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