Phosphorylation Patterns of Tumor Antigens in Cells Lytically

Vol. 40, No. 1JOURNAL OF VIROLOGY, Oct. 1981, p. 28-440022-538X/81/100028-17$02.00/0

Phosphorylation Patterns of Tumor Antigens in Cells LyticallyInfected or Transformed by Simian Virus 40

FRANS VAN ROY, LUCIE FRANSEN, AND WALTER FIERS*Laboratory ofMolecular Biology, State University of Ghent, B-9000 Ghent, Belgium

Received 19 January 1981/Accepted 4 June 1981

The phosphorylation sites of simian virus 40 (SV40) large tumor (T) antigenshave been analyzed by partial proteolysis peptide mapping and phosphoaminoacid analysis of the resulting products. At least four sites were found to bephosphorylated. An amino-terminal part of the molecule contained both phos-phoserine and phosphothreonine. One phosphothreonine residue was located inthe proline-rich carboxy-terminal end of the molecule, either at position 701 or atposition 708. The mutant dl 1265, which is defective in adenovirus helper function,lacked this phosphorylation site. In addition, the carboxy-terminal part of themolecule contained phosphoserine at a more central position. T-antigen-associ-ated proteins of SV40-transformed cells (nonviral T; 51,000 to 55,000 daltons) alsocontained multiple phosphorylation sites involving at least two serine residues inmouse antigens and an additional threonine residue in rat, human, and monkeyantigens. The latter residue and at least one phosphoserine residue were locatednear one terminus of the human NVT molecule. We did not find any evidence forphosphorylation of tyrosine residues in any of the multiple species of either largeT or nonviral T molecules. Several forms of large T antigens were extracted fromboth SV40-transformed and SV40-infected permissive and nonpermissive cells,and their phosphorylation patterns were compared. No evidence was found for adifferent phosphorylation pattern of T antigens in transformed cells.

The large tumor (T) antigen encoded by theearly A gene of simian virus 40 (SV40) is in-volved in a number of functions in both produc-tive infection and cell transformation (for a re-cent review, see reference 58). It regulates earlyviral message transcription, viral DNA replica-tion followed by late transcription in permissivecells, stimulation of cellular DNA synthesis, andthe initiation and maintenance of the trans-formed state. It is a phosphoprotein with anapparent molecular weight (Mr) of 90,000 to100,000. It has been reported that large T fromSV40-infected monkey cells contains phospho-serine (P-Ser) (55). Recently, this finding wasextended since both P-Ser and phosphothreo-nine (P-Thr) residues were detected in the largeT polypeptide (24, 39, 42, 62). By determiningthe specific activity of the large T antigen, 32Plabeled under steady-state conditions, it was es-timated that, on the average, each moleculecontains up to four phosphates, in a P-Ser/P-Thr ratio of 3 to 5:1 (62). In previous studies (39,42, 55, 62), tryptic peptide analysis of 32P-labeledlarge T antigen yielded only a single, well-sepa-rated phosphopeptide. This apparently con-tained a P-Thr residue, but much of the 32p labelstreaked or stayed at the origin in these peptidemaps, and only P-Ser was detected in this un-

28

resolved material. We have now tried to deter-mine the number ofphosphorylated sites oflargeT by performing phosphoamino acid analysis ofproteolytic intermediates resolved on polyacryl-amide gels. A minimum number of phosphory-lation sites was deduced by analyzing precursor-product relationships of the fragments obtained.

In view of the pleiotropic regulator role of thelarge T antigen, differential phosphorylation ofcertain sites may be physiologically very impor-tant. The activity of multiple enzymes and reg-ulatory proteins is known or presumed to beinfluenced by protein phosphorylation (32). Wehave searched also for evidence for regulation oftransformation by differential phosphorylationof SV40 large T in nonpermissive cells. Therationale for this investigation was the strikingassociation of tyrosine-specific protein kinaseactivity with several transforming gene products(1, 8, 20, 29, 30, 51, and multiple referencestherein), including the middle T antigen of pol-yoma virus (16).

In extracts of transformed cells, a prominentfraction of large T antigen is found specificallycomplexed with host-coded 48,000- to 55,000-dalton (48K to 55K) phosphoproteins, referredto as nonviral T (NVT) antigens (33, 36, 38, 44,52, 54, and references therein). Their role, if any,

PHOSPHORYLATION OF SV40 TUMOR ANTIGENS

in transformation is still completely hypotheti-cal, but similar or identical 50K phosphoproteinshave been detected also in chemically inducedtumor cells (12), Abelson murine leukemia virus-transformed cell lines (47), and several humantumor cell lines of widely different types (10).Recently, it has been found that this species-specific antigen is also present in uninfected,untransformed mouse and monkey cells, al-though in very low amounts. After infection withSV40, the synthesis and especially the phospho-rylation of these NVT antigens are much aug-

mented (19, 26, 28, 38, 44, 47, 52). Because of thesuggestive tumor-marker features of NVT anti-gens, we investigated the nature of their phos-phoamino acids. A minimum number of phos-phorylated sites was partly localized in the hu-man NVT molecule.

MATERIALS AND METHODSCell lines and virus. The different untransformed

and SV40-transformed cell lines in this study are listedin Table 1. All the cells were grown in Dulbeccomodified Eagle medium (DMEM), supplemented with10% newborn calf serum. Permissive CV-1 cells were

lytically infected with wild-type SV40 small-plaquestrain 776 as described previously (60). CV-1 cells werealso infected with "early" deletion mutants of SV40.

Mutant dllOO1 was grown in the presence of helpertsB4 mutant virus (35), and mutant d11265 is viable(7). Nondefective adenovirus type 2-SV40 hybrid virusAd2'ND2 (37, 58) was propagated in human A549cells.

Antibodies. The antitumor antiserum used in mostexperiments was an ammonium sulfate fraction ofpooled sera obtained from hamsters carrying SV40-induced tumors. It was generously provided by theResources and Logistics Branch, National Cancer In-stitute (lot no. 79X000128). Normal mouse serum (Ser-alab Ltd., Sussex, England) was used in control exper-iments. L21 monoclonal antibodies are directedagainst the mouse NVT antigen (27), and tissue cul-ture supernatant of this hybridoma was a gift of L. V.Crawford.

Labeling and extraction of cells. Before radio-active labeling, cells were washed twice with eithermethionine-free or phosphate-free DMEM supple-mented with 2% dialyzed newborn calf serum. After a

starvation time of about 0.5 h in the same media, cells

were labeled either with L-[3S]methionine (The Ra-diochemical Centre, Amersham, England; code SJ-204,>600 Ci/mmol, 200 ,uCi in 250 A/110 cm2 of cells) orwith 'Pi (The Radiochemical Centre; code PBS-43,carrier free, 0.5 to 1 mCi in 250 pl/10 cm2 of cells).Transformed cells were labeled for 4 h when they wereslightly subconfluent. Virus-infected permissive andnonpermissive cells were labeled from 44 to 48 hpostinfection.

TABLE 1. Cell lines used in this studyCell line Animal species and tissue Characteristics Source (reference)

CV-1 African green monkey Permissive for SV40 ATCC CCL 70kidney

BALB/c-3T3 clone A31 Mouse (BALB/c) em- Untransformed ATCC CCL 163bryo

SV-T2 Mouse (BALB/c) em- SV40-transformed BALB/c-3T3 ATCC CCL 163.1bryo clone A31

SV1o0 Mouse (BALB/c) em- SV40-transformed (56)bryo

Flll Rat (Fisher) embryo Untransformed (3)

Flll(WT648) Rat (Fisher) embryo SV40-transformed (3)

V15 Fl cll Rat (WAG) embryonic Transformed by BamHI-HpaII- (11)lung generated early fragment of

SV40 DNA (0.14 to 0.73 SV40map units)

A549 Human lung Carcinoma (22)

WI-38 Human fetal lung Diploid, untransformed ATCC CCL 75

WI-38 VA13 (2RA) Human lung SV40-transformed WI-38 ATCC CCL 75.1

WI-26 VA4 Human lung SV40-transformed WI-26 ATCC CCL 95.1

SV80 Human fibroblast SV40-transforned (57)

29VOL. 40, 1981

30 VAN ROY, FRANSEN, AND FIERS

At the end of the labeling period, the cells werewashed twice with ice-cold phosphate-buffered salineand then extracted in situ for 0.5 h at 00C with 200 plof extra^tion buffer per 10 cm2 of cells. In most exper-iments, extraction was carried out in a modified RIPAbuffer (50) consisting of 1% Nonidet P-40 (NP-40), 1%sodium deoxycholate, 0.1% sodium dodecyl sulfate, 150mM NaCl, 1 mM EDTA, and 10 mM Tris-hydrochlo-ride, pH 8.0. In some experiments, a lysis buffer (re-ferred to as NP-40 buffer) consisting of 1% NP-40, 137mM NaCl, 1 mM EDTA, 10% glycerol, and 50 mMTris-hydrochloride (pH 8.5) was used. To both lysisbuffers was added immediately before use either 500kallikrein inactivation units of aprotinin (SigmaChemical Co., St. Louis, Mo.) per ml or 300 ,tg ofphenylmethylsulfonylfluoride (Serva, Heidelberg,Federal Republic of Germany) per ml. The samebuffers were used throughout the immunoprecipita-tion procedures (see below). Lysates were scrapedfrom the plates, quick frozen, and stored at -30°C.

Immunoprecipitation. Cell extracts in NP-40buffer were clarified by low-speed centrifugation, andcell extracts in RIPA buffer were centrifuged eitherfor 15 min at top speed in a Beckman Airfuge or for 30min at 30,000 rpm in a Beckman R75 Ti rotor. Theseand all subsequent operations were carried out at 4°C.Generally, 15 pll of either hamster anti-SV40 tumor$erum or nonimmune serum was added to 200 Ml ofthe clarified lysate, and the mixture was incubated for2 to 3 h. Then 150 ,tl of a 50% (vol/vol, in RIPA orNP-40 buffer) protein A-Sepharose CL-4B beads(Pharmacia Fine Chemicals, Uppsala, Sweden) mix-ture was added, and the suspension was gently agitatedfor 1 h. The beads were pelleted and washed fourtimes with 1 ml of buffer (RIPA or NP-40). Finally,the beads were washed once with 62.5 mM Tris-hy-drochloride (pH 6.8) and then eluted twice by boilingfor 3 min, each time in 75 ,ul of elution buffer (62.5mMTris-hydrochloride [pH 6.8], 3% sodium dodecyl sul-fate, 7% /i-mercaptoethanol, 10% glycerol, and 0.01%bromophenol blue). The eluates were pooled and fro-zen at -30°C. Other lysate volumes were immunopre-cipitated under conditions adapted proportionally. Insome experiments, the eluted proteins were S-alkyl-ated with N-ethylmaleimide as described by Crawfordand O'Farrell (9).Polyacrylamide gel electrophoresis. Immuno-

precipitated proteins were analyzed by discontinuoussodium dodecyl sulfate gel electrophoresis as describedby Laemmli (34). All gels were made with a stocksolution containing 29.2% acrylamide and 0.8% bisac-rylamide. Separating slab gels were 1 mm thick andcontained various concentrations of acrylamide, asindicated in the figure legends. After electrophoresis,proteins were fixed with 50% trichloroacetic acid andstained with 0.1% Coomassie brilliant blue G in 7%acetic acid. Gels containing 3S-labeled proteins werefluorographed by means of EN3HANCE (New Eng-land Nuclear Corp., Boston, Mass.). Subsequently, thegels were dried and autoradiographed by conventionalmethods. In case proteins had to be recovered, thegels were dried immediately after electrophoresis with-out any washing or fixation steps. Proteins used asmolecular weight markers were phosphorylase b(94K), bovine serum albumin (67K), ovalbumin (43K),

carbonic anhydrase (30K), soybean trypsin inhibitor(20.1K), a-lactalbumin (14.2K; Pharmacia electropho-resis calibration kit), and, in addition, myosin (200K).

Partial proteolysis peptide mapping. Proteoly-sis mapping was performed essentially as described byCleveland et al. (5). Protein bands were excised fromthe dried gels. Almost all of the backing paper wasscraped off, and the gel pieces were then swollen insitu at the bottom of sample wells of a 3-cm-longstacking gel. For that purpose, 6-mm-wide samplewells were filled with 30 Il of buffer containing 20%glycerol, 0.5% sodium dodecyl sulfate, 2% ,B-mercap-toethanol, 1 mM EDTA, 125 mM Tris-hydrochloride(pH 6.8), and 200M,g of bovine serum albumin per ml.The swollen gel slices were then overlaid with 20 pl ofbuffer containing 10% glycerol, 0.5% sodium dodecylsulfate, 0.5mM EDTA, 0.001% bromophenol blue, 62.5mM Tris-hydrochloride (pH 6.8), awd appropriateamounts of Staphylococcus aureus V8 protease (MilesLaboratories, Slough, England), papain (Sigma Chem-ical Co.), or a-chymotrypsin (Sigma Chemical Co.).Electrophoresis was initially at 70 V until the dyemarker entered the separating gel. Then electropho-resis was completed at 25 mA per gel (16 by 16 by 0.1cm) or at 100 mA per gel (75 cm wide by 16 by 0.1cm). Besides the molecular weight markers mentionedabove, the following calibration proteins were alsoused: horse myoglobin (17.0K), lysozyme (14.3K), bo-vine a-chymotrypsin chain C (10.OK), trypsin inhibitorfrom lung (6.5K), insulin chain B (3.4K), and a-chy-motrypsin chain A (1.2K). In some experiments, awhole strip was cut out from dried first-dimensiongels, swollen to remove backing paper, loaded in itsentirety on top of the second-dimension gel, overlaidwith buffer containing proteases, and digested duringre-electrophoresis. In this two-dimensional analysis,the same buffers were used as for the peptide analysisof single protein bands, but all volumes and the di-mension of the sample well were adapted to the lengthof the first-dimension gel strips.Phosphoamino acid analysis. Proteins were

eluted from dried gel pieces essentially as described byBeemon and Hunter (2). The protocol included swell-ing, homogenization, elution, addition of bovine serumalbumin as carrier (50 fg/ml), and precipitation with20% trichloroacetic acid. The protein pellet waswashed successively with ethanol at -10°C, ethanol-ethyl ether (1:1, vol/vol) at -10°C, and acetone-trieth-ylamine-acetic acid-water (85:5:5:5, vol/vol/vol/vol) at0°C. The dried precipitates were dissolved in 100 pi of6 N HCI by heating at 100°C for 5 min. Acid hydrolysiswas performed by incubating for 2 h at 110°C in sealed100-1pl capillaries. The HCI was removed in vacuo, andthe samples were dissolved in 10,lM of water containingP-Ser (Sigma), P-Thr (Sigma), and 40-phosphotyro-sine (P-Tyr), each at 100 yg/ml. P-Tyr was synthesizedby the method of Eckhart et al. (16). The sampleswere spotted on cellulose thin-layer plates (E. MerckAG, Darmstadt, Federal Republic of Germany) andsubjected to two-dimensional analysis as described byHunter and Sefton (29). Electrophoresis was towardthe anode at pH 1.9 in the first dimension (until xylenecyanol FF reached the end of the plate), followed inthe second dimension by either electrophoresis towardthe anode at pH 3.5 (until orange-G reached the end

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of the plate) or ascending chromatography with iso-butyric acid-0.5 M NH40H (5:3, vol/vol). For selectedanalyses, only the first dimension was run. Internalmarkers were detected by staining with ninhydrin.The phosphoamino acids were eluted in water, andradioactivity was determined by addition ofAqualuma(Lumac, Basel, Switzerland) and scintillation count-ing.

RESULTSDifferent species of large T and NVT an-

tigens used in this study. Both SV40-trans-formed and SV40-infected permissive and non-permissive cell cultures were labeled with either[3S]methionine or 32Pi. Proteins were extracted,immunoprecipitated with antitumor antiserum,and separated on polyacrylamide gels. Figures 1and 2 show autoradiographs of representativeprotein profiles, 3S labeled and 32p labeled, re-spectively. Two distinct regions are obvious. Thefirst region contains large T antigens with an Mrranging between 78K and 125K, all of which arephosphorylated. A summary of the protein spe-cies analyzed is given in Table 2. It was reportedpreviously that large T has an anomalously highapparent Mr in sodium dodecyl sulfate-poly-acrylamide gels (13, 25, 48, 53). Although thecalculated Mr of large T, based on the nucleotidesequence of SV40 DNA, is 81,632 (reference 58),we will refer here to undegraded large T inproductive infection as an 88K species. Faster-migrating forms of large T with apparent Mr of83K and 78K are believed to be proteolyticdegradation products (49, 53), generated espe-cially during extraction of productively infectedcells (Fig. 1, lanes b and c, and Fig. 2, lanes a toc). Apparently larger forms are present too. Mostextracts of both infected and transformed cellscontained minor but significant amounts of aphosphorylated 91K species, which was barelydetectable by 3S labeling. In addition, severalcells contained a 95K large T antigen. The latterwas the major species in SV-T2 cells (Fig. 1, laned, and Fig. 2, lanes d and h). SV-T2 cells alsocontained minor amounts of even larger T anti-gens with Mr of 105K and 125K, respectively.Such super T forms have been detected previ-ously in transformed mouse and rat cells (33, 42,54). Monkey cells infected with mutant dl;001specifically synthesized a 33K phosphoproteinfragment of large T (48) (Fig. 1, lane b, and Fig.2, lane b). Monkey cells infected with mutantdl 1265 yielded an apparently normal set of largeT phosphoprotein species (6, 13) (Fig. 2, lane c).Human cells infected with the hybrid virusAd2+ND2 synthesized 42K and 56K proteinswhich are related to SV40 large T antigen (40)but are rather poorly phosphorylated (62) (Fig.1, lane g, and Fig. 2, lane f). The SV40 small t

a b c d e f g h

1 2Z5 .05 -

83--78 _

51 -

45 -

.56qu~11 . ~~~~~~53

.42

33.

FIG. 1. Immunoprecipitates of 3S-labeled large Tand NVT antigens from different SV40-infected andSV40-transformed cell lines. Cells were labeled andextracted with RIPA buffer as described in the text.Immunoprecipitation wasperformed with either con-

trol serum (lane a), hamster antitumor serum (lanesb to e and g) or L21 anti-NVT monoclonal antibodies(lanes f and h). Proteins were analyzed in a 10%polyacrylamide gel. The numbers in this and thefollowing figures refer to the apparent Mr (x 103) ofclearly distinguishable protein bands. The followingcells were used: SV40 dl1001-tsB4-infected CV-1(lanes a and b); SV40 776-infected CV-1 (lane c); SV-T2 (lane d); SV40 776-infected A549 (lanes e and f);and Ad2+ND2-infected A549 (lanes g and h). The45K band in lane c and the 120K band in lane g are

presumably SV40 VPI and adenovirus 2 hexon, re-

spectively.

antigen has not been found to be phosphory-lated.The second region of the gel contains the

phosphorylated NVT antigens, with Mr of ap-

proximately 50K to 55K. In our hands, NVTantigens of infected monkey cells and infected or

transformed mouse cells migrated as 51K spe-cies. Infected or transformed rat and humancells contained slightly larger NVT antigens(53K) (Fig. 2, and see Fig. 6). SV80 cells con-

tained a 53K-55K NVT doublet as reported pre-viously (10, 25, 26, 44). Strikingly, more NVTantigens could be (co-) immunoprecipitated withantitumor antiserum after extraction of monkeyand human cells with NP-40 buffer instead ofRIPA buffer (compare lanes i and j of Fig. 2).RIPA buffer is known to disrupt (nonspecific)

120

VOL. 40, 1981 31


g

.125tQ5- gSs88

_ 856

.42

i.

.. 4.

88

a .55

FIG. 2. Immunoprecipitates of 32P-labeled large T and NVT antigens from different SV40-infected andSV40-transformed cell lines. Cells were labeled and extracted with either RIPA buffer (lanes a to i) or NP-40buffer (lane j). Proteins were immunoprecipitated with antitumor serum and analyzed on 10% (lanes a to fand j) or 8% (lanes g to i) polyacrylamide gels. Parts of a preparative gel are shown in lanes a to f. Thefollowing cells were used: SV40 776-infected CV-1 (lane a); SV40 dllO01-tsB4-infected CV-1 (lane b); SV40d11265-infected CV-1 (lane c; this preparation was alkylated before analysis); SV-T2 (lanes d and h); SV40776-infected A549 (lane e); Ad2+ND2-infected A549 (lane f); Flll(WT648) (lane g); and SV80 (lanes i and j).

molecular interactions much better than NP-40buffer does (50) and apparently also dissociatesprimate NVT-large T complexes. A lower sta-bility of primate NVT-large T complexes hasbeen noticed recently by others, too (10, 14, 28).L21 monoclonal antibodies, directed againstmouse NVT antigens, precipitated only very lowamounts of a 53K NVT antigen from humanA549 cells, unless these cells were infected withSV40 (Fig. 1, lane f; the higher mobility of thesame antigen in Fig. 1, lane e, is due to largeramounts of immunoglobulin G in the antitumorantiserum). Extracts of Ad2+ND2-infected A549cells yielded a 53K phosphoprotein upon im-munoprecipitation with antitumor antiserum(Fig. 1, lane g, and Fig. 2, lane f). However, thisspecies was not precipitable with L21 antibodies(Fig. 1, lane h) and yielded phosphopeptidesatypical for human NVT antigens (see Fig. 6,lane g). Thus, in agreement with others (14), wefound that Ad2+ND2 hybrid virus seems to beunable to induce NVT antigen efficiently.Phosphoamino acids of large T and NVT

antigens. Preparative amounts of large T andNVT proteins were labeled with 32p; in appro-priate cell cultures, immunoprecipitated, andseparated by gel electrophoresis. Protein bandswere excised, eluted, and hydrolyzed in 6 N HCI.The resulting radioactive phosphoamino acidswere miixed with unlabeled amino acids andquantitated by two-dimensional analysis. Figure3 shows profiles of typical hydrolysates, and the

results are summarized in Tables 2 and 3. Wefound, by two different methods of analysis, thatneither large T nor NVT from SV40-infected orSV40-transformed nonpermissive cells con-tained any detectable amounts of P-Tyr. In ad-dition, the P-Ser/P-Thr ratio of large T wasremarkably constant in both infected and trans-formed cells. The NVT antigens of monkey,human, and rat cells infected or transformed bySV40 also contained both P-Ser and P-Thr, butthe latter was present only in low amounts.However, the absence of P-Thr in NVT antigensof infected and transformed mouse cells wasconfirmed several times and illustrates the hostcell species specificity of NVT.At least four phosphorylated sites in

large T antigen. Undegraded 32P-labeled 88Klarge T of productively infected monkey cellswas analyzed by partial proteolysis with increas-ing amounts ofStaphylococcus V8 protease (Fig.4A). Digestion at low enzyme concentrationsyielded 58K and 31K phosphorylated interme-diates. These same molecules were obtained bydigestion of 35S-labeled large T, suggesting thatthey were derived, probably by a single cleavage,from different and complementary parts of thelarge T protein. The use ofhigher concentrationsof protease generated the following prominentphosphopeptides: a group with Mr ranging be-tween 31K and 27K, a doublet with Mr of 19Kand 18K, respectively, and several smaller mol-ecules. The locations of the 58K and 31K inter-

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PHOSPHORYLATION OF SV40 TUMOR ANTIGENS 33

TABLE 2. Phosphoamino acid analysis of large T antigen species in different cell lines and their majorphosphorylated Staphylococcus V8 proteolytic fragments

Relative amount ofCell linea Large T speciesb phosphoamino acidc V8 phosphopeptidesd

P-Ser P-Thr

CV-1 (SV40) 88K 74 26 58K + 31KCV-1 (dl-1001) 33K' 72 28 31KCV-1 (dl-1265) 88K 82 18 58K + 31KBALB/c-3T3 (SV40) 88K 72 28 58K + 31KSV-T2 125K NIY ND 59K + 48K + 42K + 37K

105K ND ND 59K + 48K + 42K + 37K95K 71 29 59K + 37K88K ND ND 59K + 31K

SV101 95K ND ND 59K + 37K88K 71 29 59K + 31K

Flll (SV40) 88K ND ND 58K + 31KFlll (WT648) 88K 72 28 58K + 31KV15 F1 Cll 88K 72 28 58K + 31KA549 (SV40) 88K 72 28 58K + 31KA549 (Ad2+ND2) 56K 50 50

42K 46 54WI-38 (SV40) 88K ND ND 58K + 31KWI-38 VA13 88K ND ND 58K + 31KWI-38 VA4 88K ND ND 58K + 31KSV80 88K 70 30 58K + 31Ka Cells infected by wild-type SV40 776 are referred to by their name followed by SV40 in parentheses. Names

of other viruses used are indicated in a similar way. All cells were labeled with 32Pi for 4 h before extraction andimmunoprecipitation (Fig. 2).

b These data are shown partially in Fig. 1 and 2.c These data are shown partially in Fig. 3 and 5. The amount of each phosphoamino acid is expressed as the

percentage of the total counts in the three phosphoamino acids examined. Typical counts per minute in theradioactive spots were 2,000 for P-Ser, 700 for P-Thr, 32 to 38 for P-Tyr, and 30 to 39 for control regions in thesame chromatograph. P-Tyr was never detectable in significant amounts.

d These data are shown partially in Fig. 5 and 7.'Besides this deletion-specific protein, the normal set of SV40 large T proteins was present because of the

presence of helper tsB4 virus.fND, Not done.

mediates in the large T protein was furtherinvestigated by the use of SV40 mutants.Mutant dllOOl is generated by enzymatic ex-

cision of HindII/III fragments H and I (35) fromSV40 DNA. Assuming no other alterations, theknown nucleotide sequence predicts a stop co-don TAA immediately beyond the new HindII/III A-B fragment junction (21, 61). This mutantDNA would then code for a protein fragmentcontaining the first 272 amino acids of large Tonly (see Fig. 10). Such 33K protein has indeedbeen detected in immunoprecipitates (48; seeabove). It is phosphorylated and yielded, uponmoderate digestion with V8 protease, a 31Kphosphopeptide (Fig. 5, lane b). The latter isapparently identical to the 31K intermediate tonormal-sized large T, since both were cleavedfurther by V8 protease to the 19K-18K doublet,whereas smaller phosphopeptides were hardlydetectable in either case (Figs. 4B and 6, lane b).On the other hand, phosphorylated 56K and42K proteins, coded for by the hybrid virus

Ad2+ND2, were also analyzed. These proteinsshare a common carboxy terminus with the nor-mal large T antigen (40, and references therein)(see Fig. 10). Upon partial V8 proteolysis, theyyielded neither a prominent 31K fragment nora 19K-18K doublet (Fig. 5, lane d, and Fig. 6,lanes f and h). Instead, their phosphopeptidescomigrated largely with those generated fromthe 58K phosphopeptide of large T by extendedV8 hydrolysis (Fig. 4B). We can conclude fromthis and other evidence (see below) that the 31Kand 58K proteolytic intermediates originatefrom the amino-terminal and carboxy-terminalends of large T, respectively. McCormick et al.(42) obtained similar results by analysis ofamino-terminal peptides.The 31K and 58K phosphopeptides were sub-

jected to acid hydrolysis and phosphoamino acidanalysis (Fig. 5, lanes e to g). Both fragmentscontained P-Ser as well as P-Thr residues, theformer always being the major phosphoaminoacid (Table 4). Identical results were obtained

VOL. 40, 1981


T- Fi11 (WT)Ser - P

*Thr-P *

p

a ! ' ST911.1

NVT-Fll1(WT)

Se. -P

Ihr-P

B

P,1i' VT 2er-F

hr-P

I.; yr-P

Thr-P l

- Ser-PN!.i'

C --

NVT-SO80

'ivyr-P

D E - F

FIG. 3. Phosphoamino acid analysis of large T (A, B, and C) and NVT antigens (D, E, and F). Phospho-proteins were labeled with 'Pi and immunoprecipitated as described in the text and in the legend to Fig. 2.The following cells were used: Flll(WT648) (A and D); SV40 776-infected CV-1 (B); SV80 (C and F); and SV-T2 (E). Theproteins were isolated by gel electrophoresis, eluted, and subjected to acid hydrolysis. Radioactivephosphoamino acids were analyzed by two-dimensional separation and identified as described in the text.The first dimension was electrophoresis atpH 1.9 in all cases. The second dimension was electrophoresis atpH 3.5, except for (C), where chromatography was carried out. (A) was overexposed to show that not eventrace amounts of P-Tyr were present. For additional data, see Tables 2 and 3.

TABLE 3. Phosphoamino acid analysis ofNVTantigensa

Relativeamount of

Cell line NVT species phosphoaminoacid

P-Ser P-ThrCV-1 (SV40) 51K 95 5BALB/c-3T3 (SV40) 51K 100 0SV-T2 51K 100 0SV101 51K 100 0Flll (WT648) 53K 92 8V15 Fl cll 53K 94 6SV80 53K + 55K 90 10

a Data shown partially in Fig. 3. See Table 2 fordetails.

for 32P-labeled T antigens from SV80 and WI-26VA4 cells. The 33K protein, coded for by dl 1001,and its V8-generated 31K fragment also con-tained both P-Ser and P-Thr residues (Table 4).Finally, the 42K as well as the 56K proteinscoded for by Ad2+ND2 contained both phos-phoamino acids. However, the degree of phos-phorylation of the latter proteins was low, andtheir P-Ser/P-Thr ratio differed from that ofthe58K intermediate of normal large T (Table 2).These deviations could be due to altered tertiary

structure, altered subcellular compartmentali-zation, or diminished metabolic stability of thehybrid-encoded polypeptides (15). We may ten-tatively conclude that at least four phosphory-lated sites are present in the large T molecule.However, for validation, this conclusion requires(i) that both electrophoretic bands (31K and58K) are chemically homogeneous and (ii) thatthese fragments contain no overlapping phos-phorylated sequences. This was investigated asfollows.

32P-labeled large T antigens were extractedfrom dl 1265-infected CV-1 cells. This mutant isknown to lack only the nine carboxy-terminalamino acids of large T, which are replaced byfour new residues (59) (see Fig. 10). The normal-sized large T of dl 1265 (see above) was degradedby limited amounts ofV8 protease to apparentlynormal 31K and 58K intermediates (Fig. 5, lanec). However, phosphoamino acid analysis ofthese peptides showed that the 58K fragmentlacks P-Thr (Fig. 5, lane i; Table 4). Thus, the58K phosphopeptide contains the extreme car-boxy-terminal end of large T, and, in addition,the 58K region in the gels did not contain de-tectable contaminating material encompassingphosphorylated sequences of the 31K fragment.In a further experiment, 31K and 58K phospho-peptides of wild-type large T protein were iso-

T -CV I ISV)..........._

hr-P 0

T-SV 800 Ser-P

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A a b c d e

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11

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58* W9

__-* ~~~~~~~31

27*

23.

1.7= _ * 191 J3. * * * : _ * 1B

11 *

FIG. 4. Partial proteolysis peptide mapping ofnP-labeled large T antigen of SV40 776-infected CV-1cells with Staphylococcus V8 protease. (A) Large Tantigen (88K) was labeled and purified by immuno-precipitation and electrophoresis on a 8% polyacryl-amide gel as described in the legend to Fig. 2. Theexcised protein band was subjected to partial prote-olysis with increasing amounts of V8protease duringreelectrophoresis on a 12.5% polyacrylamide gel asdescribed in the text. The following amounts of V8were used: 1 ng (lane a), 10 ng (lane b), 100 ng (lanec), 1 pg (lane d), and 10 pg (lane e). (B) The phospho-peptides of large T were excised from gel lanes simi-lar to that depicted in lane a of (A). Then they weresubjected to extended proteolysis with increasingamounts of V8 protease during re-electrophoresis ona 12.5% polyacrylamide gel. Either 58K (lanes a to e)or 31K (lanes a' to e') fragments were analyzed. The

TABLE 4. Phosphoamino acid analysis of V8proteolytic fragments of large T antigena

Relative amount ofCell line phosphopep- phosphoamino acid

tide species P-Ser P-Thr

CV-1 (SV40) 88K 74 2658K 70 3031K 75 25

CV-1 (dl-1001) 33K 72 2831K 75 25

CV-1 (dl-1265) 88K 82 1858K 100 031K 73 27

a These data are shown partially in Fig. 5. See Table2 for details.

lated from the gel and each was subjected sep-arately to extended V8 proteolysis (Fig. 4B).Preliminary experiments had shown that bothfragments yielded an 18K phosphopeptide. Butit can be seen that the 58K-derived 18K frag-ment moved slightly faster than the 31K-derivedfragment, and the former was also much moreeasily degradable to typical smaller molecules.Moreover, since the 31K and 58K intermediatescontain the amino terminal and carboxy termi-nal, respectively, it is very unlikely that theycould contain an overlapping segment as largeas 18K. The following findings further argueagainst the existence of a smaller overlappingphosphorylated fragment: (i) phosphopeptides(30K, 28K, and 27K) slightly smaller than the31K intermediates were generated by V8 prote-olysis, but these were formed at V8 concentra-tions higher than those necessary to obtain 58Kintermediates (Fig. 4A); (ii) V8 protease cleavedoff a 2K fragment from the dl 1001-encoded 33Kprotein, which is apparently not phosphorylated;and (iii) V8 proteolysis yielded an apparentlynormal set of 58K-related phosphopeptides fromthe shorter Ad2+ND2-encoded 56K protein.Conclusive evidence on this topic came also fromthe analysis of other large T antigen species(manuscript in preparation). Finally, Schwyzeret al. (49) have shown that a 40K fragment ofSV40 large T, located centrally with the amino-terminal at residue 131, is not phosphorylated invivo (see Fig. 10).Phosphorylation patterns of large T in

permissive and nonpermissive cells. Thephosphoamino acid analysis of large T antigen(see above) suggested that there were no majordifferences between the phosphorylation pat-

following amounts of V8 were used: Ong (lanes a anda'), 10 ng (lanes b and b'), 100 ng (lanes c and c'), Ipg (lanes d and d'), and 10 pg (lanes e and e').

VOL. 40, 1981 35

....0~ ~ .0 4 F) S e

r ~ ~ ~ ~~~0. P - 't,

.I j n

FIG. 5. Phosphoamino acid analysis of proteolytic fragments of 32P-labeled large T species. Large Tantigen or related molecules were labeled and purified by immunoprecipitation and electrophoresis on a 10%polyacrylamide gel as described in the legend to Fig. 2. The excised protein bands were subjected to partialproteolysis with V8protease (25 ngper 3-cm sample well) during electrophoresis on a 12%polyacrylamide gel(lanes a to d). The following proteins were analyzed: 88K of SV40 776-infected CV-1 cells (lane a); 33K ofSV40dl1l01-infected CV-1 cells (lane b); 88KofSV40dl1265-infected CV-1 cells (lane c); and 56KofAd2+ND2-infected A549 cells (lane d). Selected bands of these gels were excised and subjected to acid hydrolysis, andtheir phosphoamino acids were separated by one-dimensional electrophoresis at pH 1.9 (lanes e to j; foradditional data, see Table 4). The following analyses are shown: the SV40 776-encoded 88K large Tprotein(lane e) and its 58K (lane f) and 31K (lane g) fragments; and the SV40 d11265-encoded 88K large Tprotein(lane h) and its 58K (lane i) and 31K (lane j) fragments.

d et f g h

qo -.. ;a=1~~~~~~~~I 5348

i" 3 al

..

_

*i.

.....,.

.*-. gm 1 3

aW

FIG. 6. Partial proteolysis peptide mapping of selected 32P-labeled large T and NVT forms. Proteins were

labeled and purified by immunoprecipitation and electrophoresis on a 10% polyacrylamide gel as describedin the legend to Fig. 2. The excised protein bands were subjected to partial proteolysis with V8 proteaseduring reelectrophoresis on a 10 to 17.5% polyacrylamide gradient gel. The three panels each group lanes ofthe same gel run. In 6-mm sample wells, either 1 pg (lanes a to i) or 1 ng (lanesj to 1) of V8protease was used.The following proteins were analyzed: 88K of SV40 776-infected CV-1 (lanes a and c); 33K of SV40 dl-1001-infected CV-1 (lane b); 53K NVT ofSV40-infected A549 (lanes d andj); 53K NVT of WI-26 VA4 (lane e); 42K(lane f), 53K (lane g), and 56K (lane h) phosphoproteins ofAd2+ND2-infected A549; 51K NVT of SV40 776-infected CV-1 (lanes i and 1) and 51KNVT ofSV-T2 (lane k).

36

88 _ _

,,8_a

?-. *_ -..

k

0 4 5 148464237

< .*mmi SI


terns of this protein in different cells, includinginfected permissive and nonpermissive cells andtransformed nonpermissive cells. We have triedto further substantiate this conclusion by partialproteolysis peptide mapping of 32P-labeled largeT molecules from different sources (Fig. 7 and8). Digestion with limited amounts of V8 pro-tease yielded phosphorylated 31K and 58K frag-ments of all 88K large T molecules examined(Fig. 7A; Table 2). The constant P-Ser/P-Thrratio in these large T molecules (Table 2), to-gether with the fairly constant relative amountsof radioactivity in the 58K and 31K fragments,suggests that the same sites are phosphorylatedsimilarly in all cases. Other digestion patterns,obtained with higher concentrations of V8 pro-tease (Fig. 7B; other data not shown), were inagreement with this idea. Phosphopeptides, typ-ical for each part of the large T molecule (forexample, 19K-18K and 13K-ilK molecules, re-spectively), were indeed conserved fairly well inthese digests. Since it could be difficult to detectthat two neighboring phosphorylation sites onthe same V8 proteolysis intermediates were dif-ferentially influenced by the transformed state,digestion was performed also with several con-centrations of other proteolytic enzymes knownto generate multiple intermediates. Typical pa-pain and chymotrypsin digestion patterns of 32p_labeled 88K large T proteins are shown in mostlanes of Fig. 8. The overall correspondence ofthese patterns is striking, making it unlikely thatdifferential phosphorylation of large T occursduring transfornation.The V8 digestion patterns of the 95K, 105K,

and 125K large T and super T molecules of SV-T2 and SV101 cells were more complicated.They all yielded slightly larger 58K fragments,but the increase of Mr was reflected mostly inthe 31K parts of large T (Fig. 7A, lane c; Table2). Upon further digestion with V8 protease,these striking differences largely disappeared.Peptides patterns of 3S-labeled large T speciesin mouse cells became very similar if not iden-tical to those of large T in other cells (Fig. 7B,lanes j to m). However, all 32P-labeled large Tmolecules of SV-T2 or SV101 cells, including the88K molecule, generated an atypical 26K phos-phopeptide, and the 13K-ilK doublet was alsomodified (Fig. 7B, lane c). So far, we are notcertain whether these molecules are changed inmethionine-poor regions or whether the phos-phorylation patterns is modified. Recently, itwas reported that certain forms of super T an-tigen contain duplications of some sequences,which can be situated either in the amino-ter-minal or in the carboxy-terminal half ofthe largeT protein (41, 42, 54).

Additional minor differences could be repro-

ducibly detected upon careful examination ofpartial proteolytic digests of several 32P-labeledlarge T antigens. Some of these are indicated inFig. 7 and 8 by arrowheads beside individuallanes. Generally, these changes did not correlatewith the permissive or nonpermissive state ofthe cells. Rather, the SV40-infected state and,on the other hand, the SV40-transformed statedetermined the cleavage pattem, although largeT of F111(WT648) behaved somewhat interme-diately. These minor changes were due presum-ably either to variability of the infecting virus orto alteration of integrated genomes and did notresult from modified phosphorylation reactionsduring established transfornation. Indeed,though most proteolysis patterns of 3S-labeled88K large T antigens were hardly distinguish-able, minor differences were detected here alsobetween the patterns of infected and trans-formed cells, respectively. Moreover, on a fewoccasions, changes in minor 3S-labeled inter-mediates could be correlated with changes in the3P-labeled antigen profile (for example, Fig. 8,lanes m to r).Phosphorylation sites in NVT antigens.

Partial digestion of both 3S-labeled and 32P-labeled NVT antigens with proteolytic enzymesgenerated peptide profiles definitely differentfrom those of large T antigens (Fig. 6, lanes d, e,and i to 1; Fig. 8, lanes g and h). In addition, hostcell species specificity of these NVT antigenswas reflected in these cleavage patterns. Wehave analyzed the partial proteolysis profiles ofa single species of 3P-labeled NVT antigen inmore detail to determine a minimal number ofphosphorylation sites (Fig. 9). Hydrolysis of the53K NVT antigen of SV80 with minimalamounts of V8 protease generated two largephosphorylated intermediates with Mr of about48K and 39K and, in addition, a set of phospho-peptides with Mr lower than 15K (Fig. 9A). Thelatter includes a heavily labeled broad bandconsisting of at least three peptides, 14K, 12K,and 9K. Phosphopeptides with apparent Mrlower than 5K are referred to by letters. Undersimilar proteolytic conditions, 48K and 39K frag-ments were found also to be major fragments of3S-labeled NVT antigens of SV80. It was likelythat the large and small fragments, respectively,were derived from different complementaryparts of the protein, which implies that at leasttwo phosphorylated sites are present on thehuman NVT molecule (Fig. 9D). Limited V8digestion of other NVT antigen species alsoyielded similar large and small phosphorylatedintermediates (Fig. 6, lanes j to 1; Fig. 9A, laned).

Further evidence came from two-dimensionalproteolysis profiles of 32P-labeled NVT antigen

37VOL. 40, 1981

38 VAN ROY,

AFRANSEN, AND FIERS

V 8 10 ng

a b c d e f

S

*. i11I

88

t * S

a

37

9e 3.

g h

I a

, f58

9* 31

19

p p p p p p p p p

Ba b c d e f

S.'ff

V 8 1 pg

g h i J

88

58

3 1

e~~ ~ ~~~~~~#WJX 28

. 2

_.,..D- .

_. ... .E ..

* 4

p p p p p p

_

FSrsa,~~~~~~~~~~~~~~~~~~~~~~~.

p..

p p p s

FIG. 7. Partial proteolysis peptide mapping of different species of large T antigens with staphylococcus V8protease. Large T antigens, labeled either with 'Pi or with ['SJmethionine (indicated in the figure asp ands, respectively) were extracted from both infected and transformed cells and isolated on 8% polyacrylamidegels as described in the legends to Fig. 1 and 2. The protein bands were excised and partially digested asdescribed in the text. V8protease was used at concentrations of 10 ng (A) and I pg (B) per 6-mm sample well,and the gels were either 12.5 to 17.5% (both left panels) or 10 to 17.5% (both right panels) polyacrylamidegradients. Major differences between different lanes are indicated by the Mr (x 103) of the fragments; minordifferences are indicated by arrowheads (see text). The following polypeptide species were analyzed: 88K ofSV40-infected CV-1 (lane a in A and lanes a and k in B); 88K ofF111(WT648) (lanes b and g in A and laneb in B); 95K of SV-T2 (lane c in A and lanes c and m in B); 88K of SV80 (lane d in A and B); 88K ofSV40-infected A549 (lane e in A and B); 88K of V15 Fl cli (lane f in A and B); 88K ofSV40-infected WI-38 (lane hin A and lane g in B); 88K of WI-38 VA13 (lane i in A and lane h in B); and 88K of WI-26 VA4 (lanes i andi in B).

J. VIROL.

k I m

*: 27

a 24-22

1 8A.SUaa-W

S S S


A B Cchym

2 0 ,ug

abc d e f g h ~~~~~MM i jkIm67 ^

4 *

*4 3 6 7.

*8~~~~~~~~~~3- 4 s

eii 0 30* t-}'~~~~-2!|afi h' *u.~~~2~~~~~1

1 4

3 0 pg

n o p q r

I

Jl p *a

1W.=*f g [ :_~~~~~~~V

P P P P P p p p p p p P S s s P p P

FIG. 8. Partialproteolysis peptide mapping of different species of large T and NVT antigens. 32p_ and 35S-labeled large T antigens (indicated as P and S, respectively) were isolated andpartially digested as describedin the legend to Fig. 7, but here, 10 ng ofpapain (pap) (A), 20 pg of a-chymotrypsin (chym) (B), or 30 pg of a-

chymotrypsin (C) were used per 6-mm sample well. Either 12.5 to 17.5% (A and B) or 10 to 17.5% (C)polyacrylamide gradient gels were used. Only the Mr (x 103) ofmarkerpolypeptides (M) is indicated. Regionsof identical Mr are indicated by connecting lines between (B) and (C). Arrowheads point out minor differencesbetween the peptide patterns (see text). The following peptides species were analyzed: 88K of SV40-infectedCV-I (lanes a and n); 88K ofFl11(WT648) (lanes b and 1); 95K ofSV-T2 (lane c); 88K ofSV80 (lanes d, k andm); 88K of SV40-infected A549 (lanes e and j); 88K of V15 P1 cli (lanes f and i); 51K NVT ofSV-T2 (lane g);53K NVT ofF111(W648) (lane h); 88K of WI-26 VA4 (lanes o and p); 88K of WI-38 VA13 (lane q); and 88K ofSV40-infected WI-38 (lane r).

of SV80 cells. The phosphoprotein was digestedin the first dimension with low amounts of V8protease and in the second dimension with var-ious increasing amounts of this same protease.Figure 9B shows representative gel patterns.Careful examination of several gels revealed thefollowing precursor-product relationships (seealso the scheme in Fig. 9D). The 48K fragmentcontained the whole 39K fragment and part ofthe peptides with Mr below 5K (a, b, and e).Assuming that the 48K fragment lacks the ex-treme right end of the protein, then the 39Kfragment lacks still more of this same end. In-deed, the latter fragment yielded a 35K inter-mediate, a 25K-21K-12K fragment family, anda 22K-18K-12K fragment family, but no detect-able peptides at all with Mr below 5K. Additional34K, 30K, and 28K fragments were all related toeach other and must be located in the right endof the protein, since they contain the complete

set of very small phosphopeptides (a to e; notfully visible in Fig. 9B), but not much more thatis detectable by 32P labeling. Another family ofintermediates (44K, 32K, and 22-20K) is a minorone and should be located in the so-called leftpart of the molecule. The smaller intermediatesof the right end fall into two classes, comprisingpeptides 14K, 9K, a, c, and e, and 12K, b, and d,respectively.Phosphoamino acid analysis of several inter-

mediates was performed (Fig. 9C). Uncleaved53K protein and the 34K intermediates bothwere found to contain minor amounts of P-Thr.However, each of the internediates 48K, 39K,25K, and 22K contained P-Ser only, whereas the14K-9K aggregate yielded both P-Ser and P-Thr. From these results, we conclude that atleast three phosphorylated sites are present inhuman NVT antigen, as summarized in Fig. 9D.A P-Thr residue is located within a 5K segment

pap1 0 ng

39VOL. 40, 1981


Pk-r -

d(: ::: ::.~

ba

*0

53 t .,

B

53~~~~3

0 5:$ 4 844_&4

0~~~~~~~~^ ',;

A e ie s: 0 r, g

I -5 48

- 39-- 34

312

21 2-2

Ia-

1 4t 2o 44 -%x39 0 i 2- -$,

5 448

4 439

3 432

2825

22.2 120

1 8

_0 _

ii:.

0----a.8

b

_....IL.e410 I "

Pemr0

P *s.,

P Ser P T r

s .w ox ..

A 5 3

48 K

39 K14 F

34 ix

FIG. 9. Phosphorylation sites ofhuman NVT antigens. (A) 32P-labeled NVT antigens ofSV40-transformedcells were purified by immunoprecipitation and electrophoresis on a 8% polyacrylamide gel as described inthe legend to Fig. 2. Excised protein bands were subjected to partial proteolysis with Staphylococcus V8protease during re-electrophoresis on a 12.5% polyacrylamide gel. The top of the gels is at the left, and thefollowing NVTproteins were hydrolyzed: 53K ofSV80 with 200pg of V8 protease (lane a); 53K ofSV80 with2 ng of V8 (lanes b and c); 53K of WI-26 VA4 with 2 ng of V8 (lane d). In this and the other panels, thenumbers refer to the Mr (x 103) ofmajor fragments, and letters alongside the lanes identify fragments with Mrbelow 5K. (B) Gel strips similar to lanes b and c of (A), together with undegradedpolypeptide bands ofSV80NVT (marked 53 at the periphery of the panels), were subjected in the second dimension to extended partialhydrolysis with V8protease as described in the text. One gel strip was digested with 33 ng of V8protease, i.e.,2 ngper 6-mm length ofsample well (a). Another gel strip was digested with 3.3,ug of V8, i.e., 200 ngper 6-mmlength of sample well (b). The second-dimension gel was 15% polyacrylamide. (C) Major V8-generatedintermediates of 32P-labeled SV80 NVT molecules were excised from the gels in (A) and subjected to acidhydrolysis. Their phosphoamino acids were separated by one-dimensional electrophoresis at pH 1.9. Thepeptide aggregate with Mr between 9K and 14K was analyzed as a whole. (D) Schematic representation ofmajor proteolytic fragments ofSV80 NVT and their presumptive phosphorylation sites. Arrowheads point atmajor V8 cleavage sites.

at one end of the molecule. A second phos-phorylated residue is a P-Ser located within a9K segment situated between the 5K segmentmentioned above and the center of the molecule.The latter residue is present in the 48K inter-mediate but absent from the 39K fragment. Thesmall peptides a and b, but not c and d, shouldcontain this residue. At least one more P-Serresidue is located in the rest of the molecule.Our data do not allow us to localize this remain-ing phosphorylation site(s) more precisely.

DISCUSSION

In earlier reports (39, 42, 55, 62) large T fromSV40-infected monkey cells was found to con-tain both P-Ser and P-Thr residues. We haveanalyzed the phosphoamino acid content oflargeT, extracted from both SV40-infected and SV40-transformed cells, with special emphasis placedon the detection of potential P-Tyr residues. Inaddition, it was of interest to look for P-Tyrresidues in NVT antigens, since these proteins

53

b

14' 12V% ,9

u

f

J. VIROL.

F ^. r


can be considered as tumor markers and espe-cially because a cellular 50K protein, coprecipi-tating with pp6fjrc in immunoprecipitates-aproperty reminiscent ofNVT antigens coprecip-itating with SV40 large T-was found to containP-Tyr as well as P-Ser residues in vivo (29). Ourresults show that P-Tyr was completely absentfrom different species of both large T and NVTantigens labeled in vivo in several different cells.The methodology used was sensitive enough todetect this phosphoamino acid unless only avery tiny fraction of molecules was phosphory-lated on tyrosine, for example, due to high turn-over. Also, very recently Greenspan and Carroll(24) mentioned the absence of P-Tyr residues inlarge T antigen of transformed mouse cells.

Furthermore, we tried to determine the num-ber of phosphorylated sites of large T by analyz-ing the phosphoamino acids of proteolytic inter-mediates resolved on polyacrylamide gels. Thelarge T protein is cleaved by low amounts ofStaphylococcus V8 protease into 58K and 31K

U. 7 6.S S.S U.4

phosphorylated intermediates. We have foundthat these fragments are derived from comple-mentary and apparently nonoverlapping partsof the large T molecule. The 31K fragment con-tains the normal amino-terminus of the proteinand a major phosphorylation site (Fig. 10). Oth-ers have assigned a major phosphorylation siteto a similar fragment of large T or even a shorterone (within the first 130 amino-terminal aminoacids) (42, 48, 49, 62). Now we find that thisamino-terminal fragment contains at least twophosphorylation sites, since both P-Ser and P-Thr are detectable upon hydrolysis. It shouldalso be mentioned here that the first 82 aminoacid residues, which large T antigen shares withsmall t antigen, are apparently not phosphory-lated in small t. The 58K fragment of large Twas rather weakly phosphorylated under ourexperimental conditions and was shown to con-tain the carboxy-terminus of the protein (Fig.10). This phosphopeptide also contains both P-Ser and P-Thr residues. Thus, at least four phos-

1.3 6.2 6.1 N.V.776

di 1265

d1 1001

Ad2*N02

776

I~ ~ ~ ~ C

_ ____ I

1 32 83 733NH2 -4........... A COOH

1 272i -1....... i

?

331K-P ...........

V- Ser + V- Thr I

766

di 1265

di 1001

AS2*ND2

3 K - 776

*S K -11265

33 K- l 1001

:2 K IAd2No2

40KFIG. 10. Schematic representation of the presumptive sites ofphosphorylation in SV40 large T antigen.

Part of the genome of wild-type SV40 (strain 776) is indicated by segmented blocks (each segment represents0.1 map unit). SV40-specific RNA is shown by uninterrupted blocks. Deletions of mutants d11265 and dllO01are indicated by dots. Solid lines in the DNA and RNA ofthe Ad2+ND2 hybrid viruses represent adenovirus-specific sequences (31). Intervening sequences in the large TmRNA are also indicated by dots (map unit 0.60to 0.53). Arrowheads point at the major V8 cleavage site in the large T protein. It is not known whether theAd2+ND2-encoded 56K and 42K proteins have an SV40- or adenovirus type 2-specific amino-terminal(question marks). The 40K fragment, located at the center of the large T protein, is a tryptic intermediatewhich lacks phosphate, according to Schwyzer et al. (49).

DNA

RNA

Protein

5 I K 1

Vs fragments

P-ser P-ThrI 7117-76

-T- I I I I

I I I --I 1: I -r---

::: I I .

1 z::=z3- -

I -r

I I

VOL. 40, 1981 41


phorylated sites are present in large T antigen.Our present data do not allow us to localize theP-Ser residue of the carboxy-terminal part oflarge T in more detail. On the other hand, the58K fragment obtained from the large T antigenof mutant d11265 was found to be phosphory-lated on serine residues only. This mutant lacksthe last nine amino acids of large T, and theseinclude two potentially phosphorylated residues,Thr (701) and Thr (708). It is noteworthy thatmutant dl1265 is viable in monkey cells butretains only 4% of the wild-type helper capacityfor growth of human adenovirus in monkey cells(6). Besides other explanations for this finding,including the loss of a peculiar proline-rich car-boxy-terminal segment (6, 59), the disappear-ance of a phosphorylation site could also beresponsible for this particular functional defect.From the data in Table 4, it can be calculated

that under our experimental conditions the fourdifferent (classes of) sites in large T are phos-phorylated with the following approximate mo-lar efficiency: NH2. [P-Ser (0.45).P-Thr (0.15)]* P-Ser (0.30) * P-Thr (0.10) *COOH, if the phos-phorylation efficiency ofcomplete large T equals1.00. However, it is difficult to draw any firmconclusions from these data about the existenceof differentially phosphorylated species of largeT, because of the following arguments. It hasnot been established that all four sites wereradioactively labeled to metabolic equilibrium.The rate of decomposition during acid hydroly-sis differs for P-Ser and P-Thr, and furthermore,their yield is very much dependent on the com-position of the phosphopeptides from whichthey are derived (4). Finally, we do not knownow whether, for instance, the 31K phosphopep-tide contains one or more phosphorylation sitesinvolving serine residues. In principle, the latterproblem can be solved easily by isoelectric fo-cusing of 32P-labeled large T molecules. How-ever, using published methodology (9, 23), wewere unable to obtain a limited number of dis-crete focused spots of either integral S-alkylatedlarge T molecules or S-alkylated 58K and 31Kphosphopeptides. This may be due to tenaciousand heterogeneous aggregation of large T mole-cules during analysis (23, 46).

It is of considerable interest that up to fourphosphorylation sites have so far been recog-nized in the large T molecule, since differentiallyphosphorylated molecules may correlate withdifferent biological functions of the large T pro-tein. Phosphorylation of the large T protein oftsA mutants has been shown to be modified atthe nonpermissive temperature (17, 62). Poly-mers of large T and complexes of this proteinwith host-specific NVT antigens turned out tobe highly phosphorylated, whereas monomers

were under-phosphorylated (19, 24, 43). Large Tantigen in SV40 nucleoprotein complexes maybe differentially phosphorylated (39), and exten-sive phosphorylation of large T is correlatedwith high (nonspecific) binding affinity for hostDNA and chromatin (24, 45). It remains to beseen whether these different physiological formsof large T have different and specific sites phos-phorylated or whether increased phosphoryla-tion is due to a concerted modification of allsites. Improved localization and quantitation ofthe different phosphorylated residues in large Tcould provide an answer to this question.

Protein phosphorylation is enhanced andmodified in SV40-transformed cells (18, andother references therein; unpublished data).Thus, it cannot be excluded a priori that thefunction of large T protein is influenced by dif-ferential phosphorylation in infected permissivecells on one side and transformed cells on theother. However, we found no evidence at all forthis hypothesis within the sensitivity limits ofour detection methods. Both the phospho-amino acid content and the pattern of phos-phorylated proteolysis intermediates turned outto be very similar if not identical for the majorlarge T species isolated from several differentsources. Minor differences were not consistentlyfound in transformed cells or infected nonper-missive cells and could generally be related tochanges in the primary structure of the protein.Thus, it is suggested that no minor form of largeT antigen with a peculiar phosphorylation pat-tern is selected during establishment of transfor-mation and that nonpermissive cells do notphosphorylate large T antigen strikingly differ-ently than do permissive cells.

Finally, the 48K to 55K nonviral T antigensseem to be gaining wide-spread interest becauseof their suggestive tumor-marker features. Two-dimensional analysis of 32P-labeled NVT anti-gens gives several somewhat streaking spotswith similar Mr but different charge (10; unpub-lished data). Thus, it can be suggested thatdifferent phosphorylated states of NVT exist,possibly also related to different states of aggre-gation. SV40 infection of monkey cells enhancesnot merely synthesis but also phosphorylationof NVT antigen (28). But the question remainswhether this increase is merely quantitative orwhether it is also qualitative. We present here apreliminary analysis ofthe phosphorylation sitesof NVT antigens. Indeed, we found evidence formultiple phosphoamino acids: at least two P-Serresidues and one P-Thr residue in human NVTantigens. The P-Thr residue was found in sub-molar amounts, was derived from a region neara terminus of the molecule, and was apparentlyabsent from mouse NVT antigens. Further stud-

J. VIROL.

PHOSPHORYLATION OF SV40 TUMOR ANTIGENS 43

ies on phosphorylation sites of both large T andthese NVT antigens could reveal interestingstructural and regulatory relationships withtheir respective functions.

ACKNOWLEDGMENTSWe are grateful to N. Bouck, E. Fanning, J. Feunteun, and

M.-L. Hammaskjold for providing us with various cell lines.Viruses were kindly provided by P. Berg, C. Cole, G. Walter,and D. Nathans. L21 hybridoma supernatant was a generousgift of L. V. Crawford. We thank J. Van der Heyden for growthand titration of virus stocks and W. Kuziel for assistanceduring the production of the manuscript.

This investigation was supported by grants from the Fondsvoor Geneeskundig Wetenschappelijk Onderzoek and fromthe Kankerfonds of the Algemene Spaar- en Lijfrentekas ofBelgium. One of us (LF.) thanks the Belgian Institute voor

Wetenschappelijk Onderzoek in Nijverheid en Landbouw fora fellowship.

LITERATURE CITED

1. Barbacid, M., K. Beemon, and S. G. Devare. 1980.Origin and functional properties of the major geneproduct of the Snyder-Theilen strain of the feline sar-coma virus. Proc. Natl. Acad. Sci. U.S.A. 77:5158-5162.

Z. Beemon, K., and T. Hunter. 1978. Characterization ofRous sarcoma virus src gene products synthesized invitro. J. Virol. 28:551-566.

3. Bouck, N., N. Beales, T. Shenk, P. Berg, and G. DiMayorca. 1978. New region of the simian virus 40genome required for efficient viral transformation. Proc.Natl. Acad. Sci. U.S.A. 75:2473-2477.

4. Bylund, D. B., and T.-S. Huang. 1976. Decompositionof phosphoserine and phosphothreonine during acidhydrolysis. Anal. Biochem. 73:477-485.

5. Cleveland, D. W., S. G. Fischer, M. W. Kirschner,and U. K. Laemmli. 1977. Peptide mapping by limitedproteolysis in sodium dodecyl sulfate and analysis bygel electrophoresis. J. Biol. Chem. 252:1102-1106.

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Phosphorylation Patterns of Tumor Antigens in Cells Lytically