JOURNAL OF VIROLOGY, Sept. 1990, p. 4454-4461 Vol. 64, No. 90022-538X/90/094454-08$02.00/0Copyright © 1990, American Society for Microbiology

A Completely Transformation-Defective Point Mutant ofPolyomavirus Middle T Antigen Which Retains Full Associated

Phosphatidylinositol Kinase ActivityBRIAN J. DRUKER,1* LEONA E. LING,' BRUCE COHEN,2 THOMAS M. ROBERTS,'

AND BRIAN S. SCHAFFHAUSEN2Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115,1 and

Department of Biochemistry, Tufts University Medical School, Boston, Massachusetts 021112

Received 16 February 1990/Accepted 15 June 1990

By using a random mutagenesis procedure combined with a recombinant retrovirus vector, mutants ofpolyomavirus middle T antigen (MTAg) were generated. Three new MTAg mutants with various degrees oftransformation competence were more thoroughly characterized. All of the mutants produced a stable MTAg,as assessed by metabolic labeling or immunoblotting, and each mutant possessed wild-type levels of associatedtyrosine kinase activity and associated phosphatidylinositol-3 (PI-3) kinase activity. One of these mutants, witha substitution of leucine for proline at amino acid 248 of MTAg (248m) was completely transformationdefective, as measured in a focus-forming assay. Furthermore, the pattern of phosphorylation of 248m in vivowas identical to that of wild-type MTAg, and the kinetics of association ofMTAg with an 85-kilodalton protein,the putative PI kinase, was not altered. Similarly, the pattern of PI derivatives obtained in an in vitro kinaseassay was not altered by the substitution at amino acid 248. Since the single base pair mutation at amino acid248 resulted in an MTAg that was completely transformation defective despite possessing wild-type levels ofkinase activities, this suggests that neither tyrosine kinase nor PI-3 kinase activity nor the combination of bothare sufficient for transformation by MTAg.

Polyomavirus middle T antigen (MTAg) is the principaltransforming protein of polyomavirus. Genetic analysis ofthe virus has shown this protein to be both necessary andsufficient for the transformation of established cell lines (2,18, 20, 28, 47). MTAg cDNA can efficiently induce pheno-typic transformation of immortalized cell lines and induceanchorage-independent growth and tumorigenicity when in-troduced into a variety of cell lines (9, 16, 23, 25, 33, 38, 39,47). Mutations which affect MTAg, but not large or small Tantigens, can severely impair the ability of polyomavirus totransform (5, 28, 46).Although MTAg has no known intrinsic biochemical ac-

tivities, at least three associated activities have been de-scribed previously. One of these is the association withcellular tyrosine kinases of the src family (src, fyn, and yes,but not lck) with an accompanying increase in the in vitrotyrosine kinase activity of complexed pp6Oc-srC (4, 8, 12, 14,15, 26, 29). A genetic analysis of the necessity of thisassociation for transformation has demonstrated that thisactivity is necessary but not sufficient for transformation byMTAg (for a review, see reference 32). To date, all MTAgmutants which lack this associated in vitro tyrosine kinaseactivity are also transformation negative. This raises thepossibility that MTAg transforms cells by allowing cellscontaining MTAg to mimic transformed cells containingactivated tyrosine kinases. However, there is ample evi-dence to suggest that the scenario may not be so simple.Mutants exist which are transformation negative yet are fullyactive in an in vitro tyrosine kinase assay (22, 31). Since onlya fraction of MTAg is complexed to cellular tyrosine kinases(3, 14), the remainder of the MTAg may perform otherequally as important functions.

* Corresponding author.

A second biochemical activity associated with MTAg isdue to an enzyme which catalyzes the phosphorylation ofphosphatidylinositol (PI) (22, 51). This PI kinase is novel inthat it phosphorylates position 3 of the inositol ring andbecomes tyrosine phosphorylated in response to cellularstimulation with growth factors or transformation by avariety of oncogenes (21, 49). The functions of the productsof this type 1 PI kinase are unknown; however, there is apossibility that they may have a role in mediating themitogenic effects of growth factor stimulation. It appearsthat MTAg, pp6O-src, and this PI kinase form a complex,since this activity can be immunoprecipitated from MTAg-transformed cells with either MTAg or src-specific antibod-ies and they fractionate together in a sucrose density gradi-ent (13, 21, 50).

Genetic analysis of the necessity of this PI-3 kinaseactivity for transformation has revealed a relatively closeassociation between this activity and transformation (22).Mutants such as Py1178 (with a point mutation of Tyr-315 toPhe) and d123 (with a deletion of amino acids 302 to 326),which are transformation defective, are fully active in an invitro tyrosine kinase assay but are deficient in their ability toassociate with or activate PI-3 kinase activity (22). Thesedata suggest that this PI kinase activity may play a centralrole in transformation by MTAg. However, the mutantsd11015 (deletion of amino acids 338 to 347) and MG13(substitution of Arg for Ile at amino acid 178 and Cys for Trpat amino acid 180) are partially transformation defective andare reported to be as active in assays of PI kinase activity asthe wild-type MTAg (13, 22, 33).Two proteins with molecular masses of 36 and 63 kilodal-

tons (kDa) present in MTAg immunoprecipitates have beenrecently determined to be the catalytic and regulatory sub-units of protein phosphatase 2a (37). Again, genetic analysissuggests that the presence of these proteins is necessary but

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not sufficient for transformation by MTAg (19, 30, 35, 41,45). However, this conclusion is based on data regarding theassociation of the 36- and 63-kDa proteins with variousMTAg mutants, rather than measurements of phosphataseactivity.We have generated a large library of mutations in MTAg

by a random mutagenesis protocol combined with a pheno-typic screen for transformation-defective mutants by using aretrovirus vector (B. Druker and T. Roberts, submitted forpublication). Here we describe the biochemical properties ofseveral of these mutations. One of these mutations, whichresults in the substitution of leucine for proline at amino acid248, encodes an MTAg which is completely defective in itsability to form foci on a monolayer yet is fully active inassays of associated kinase activities. Thus, PI kinase activ-ity also appears to be necessary but not sufficient fortransformation by MTAg. This suggests that other activitiesas yet unidentified must be at least as important for trans-formation by MTAg as those described to date.

MATERIALS AND METHODS

Construction of mutant MTAg cDNAs. The vectors andrandom mutagenesis procedure used to generate the MTAgmutants have been described elsewhere (B. Druker and T.Roberts, submitted). An MTAg cDNA for a quadruplemutant containing base pair changes at nucleotides 740, 977,1244, and 1261, corresponding to base pair changes at aminoacids 190, 248, 337, and 343, respectively, as previouslygenerated is depicted in Fig. 1. A fragment obtained by SphIand XmnI restriction digests of this cDNA was placed into asimilarly cleaved wild-type MTAg cDNA backbone to gen-erate an MTAg cDNA with a single base pair change atnucleotide 740 (amino acid 190). Similarly, an XmnI- andPvuII-digested fragment was used to generate an MTAgcontaining a single base pair change at nucleotide 977 (aminoacid 248). Finally, a restriction digest of this cDNA withPvuII and NcoI was used for construction of an MTAgmutant with two changes at nucleotides 1244 and 1261(amino acids 337 and 343). Each of these cDNAs wasconstructed in pBD15 (B. Druker and T. Roberts, submit-ted), sequenced by dideoxy sequencing to confirm the pres-ence of the indicated change, and placed into the retrovirusvector pDOL- (24).

Cells and tissue culture. BALB/3T3 (clone A31) (fromC. D. Scher) and psi-2 cells (gift from C. Cepko) (6) weremaintained in Dulbecco modified Eagle medium supple-mented with 10% donor calf serum (GIBCO) at 37°C in 10%CO2.

Retrovirus infections and establishment of BALB/3T3clones. Transfection of psi-2 cells, infections with virussupernatants, drug selection, and isolation of G418-resistantclones were carried out as previously described (9).

Transformation assays. Focus formation assays were per-formed as previously described (33). At the end of the assayperiod (3 to 8 weeks), cells were fixed with 3.7% formalde-hyde in phosphate-buffered saline and stained with methy-lene blue as previously described (9).

Soft-agar assays were carried out by seeding 2.5 x 104cells per 60-mm (diameter) dish in Dulbecco modified Eaglemedium containing 0.4% Bacto-Agar (Difco Laboratories)and 10% donor calf serum. Cells were fed every 3 days with1 ml of medium containing agar and allowed to grow for 3weeks before clones were scored for growth.

Radiolabeling of cells and immunoprecipitations. The pro-cedures for radiolabeling of cells and immunoprecipitations

have been published in detail (41, 42). In pulse-chase exper-iments, cells were labeled in Hanks salts (Flow Laborato-ries, Inc.) containing 50 to 500 puCi of [35S]methionine (DuPont, NEN Research Products) per ml for 90 min. Forlabeling with 32P04, cells were incubated in phosphate-freemedium containing 200 to 1,000 ,u.Ci of 32P04 per ml.Labeled cultures were lysed, incubated with antisera andprotein A-Sepharose, and washed as described previously(51). Washed immunoprecipitates were subjected to a sec-ond round of immunoprecipitation as described previously(10). Immunoprecipitates were boiled for 2 min in a sodiumdodecyl sulfate (SDS) dissociation buffer (0.0625 M Tris [pH6.8], 5% [wt/vol] SDS, 5% [wt/vol] 2-mercaptoethanol, 20%[vol/vol] glycerol, 0.015% [wt/vol] bromophenol blue) andanalyzed by SDS-polyacrylamide gel electrophoresis ondiscontinuous buffer SDS-polyacrylamide gels (27).Double immunoprecipitation protocol. Washed immuno-

precipitates were suspended in 200 ,ul of solubilization buffer(0.4% SDS, 50 mM triethanolamine [pH 7.4], 100 mM NaCl,2 mM EDTA [pH 7.4], 2 mM 2-mercaptoethanol [added justprior to boiling]) and boiled for 2 min. To the extract wereadded 0.5 M iodoacetamide to a final concentration of 10 mM(4 ,ul) and 1/4 volume of 10% Triton X-100 (50 ,ul), and themixture was placed on ice. Antibody and protein A-Sepha-rose were added to the mixture and incubated for 1 h.

Peptide mapping analysis. Peptide mapping analysis bylimited proteolysis on SDS-polyacrylamide gels was carriedout as previously described (42).

Isoelectric focusing. Isoelectric focusing of immunoprecip-itates for pp85 has been described previously in detail (10).Briefly, 60 ,ul of Garrels sample buffer (17) was added towashed immunoprecipitates, and the mixture was incubatedon ice for 30 min and loaded onto isoelectric focusingcylinders. First dimension 15-cm isoelectric focusing gelswere composed of 4% bisacrylamide (1.62/28.3), 8.0 M urea,4% Nonidet P-40, 2% ampholytes (LKB Instruments andPharmacia), 0.14% N,N,N',N'-tetramethylethylenediamine,and 0.14% ammonium persulfate. Samples were run at 400 to500 V/h for 9,000 V and then at 800 V for 1 h. Seconddimension electrophoresis of the focused proteins was on 5%acrylamide-SDS gels.

In vitro kinase reactions. Confluent cultures of MTAg-expressing cells were lysed, immunoprecipitated, andwashed as previously described (51), except that lysis buff-ers contained 1 mM sodium orthovanadate. Protein amountswere determined by using the Bradford assay (Bio-RadLaboratories), and immunoprecipitations were performed onextracts containing equal amounts of total cell protein byusing a rabbit polyclonal antiserum (36). In vitro proteinkinase assays were performed as described previously (22).Phospholipid kinase activity was determined by incubatingwashed immunoprecipitates for 5 min at 25°C in a buffercontaining 40 jig each of PI (Avanti Polar Lipids), PIP, andPIP2 (Sigma) per ml, 80 ,ug of phosphatidylserine per ml, 10puM ATP (0.5 mCi/ml), 50 mM HEPES (N-2-hydroxyeth-ylpiperazine-N'-2-ethanesulfonic acid), 10 mM MgCl2, and 1mM EGTA (pH 7.5). Labeled phospholipids were extractedwith CHC13, methanol, and HCI, as previously described(49) and separated by thin-layer chromatography on silica gel60 plates (EM Science) in n-propanol-glacial acetic acid-water (65:2:33). Phospholipids were visualized by iodinevapor staining with radioactivity in PI, PIP2, and PIP3, asdetermined on a Betascope radiation detector (Betagen).Counts were normalized to the amount of MTAg, as deter-mined by immunoblotting of cell lysates. Analysis of thestructure of phospholipid products was carried out by high-

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BamH II 1

73 173

746 809

SphxI

710

Amino acids

740

977

A to G

C to T

1244 C to T

1261 G to A

Nco I

740 977 1 L2741244 1261

190 248 337 343

190

248

337

343

Hind III

EcoR I (Bgl II)

1561 1656

421

Asn to Asp

Pro to Leu

Pro to Leu

Ala to ThrFIG. 1. Schematic of MTAg mutations. Shown is the MTAg cDNA used for mutagenesis. It was cloned into pBD15 as a BamHI-Bglll

fragment, as described elsewhere (B. Druker and T. Roberts, submitted). The four base pair changes introduced into MTAg are indicated byvertical lines, and the corresponding amino acid changes are indicated below. The quadruple mutant, MTm, was fragmented, as describedin Materials and Methods, to produce an individual or double mutant, referred to by the amino acids containing the following substitutions:190m, 248m, and 337/343m.

performance liquid chromatography separation of the deacy-lated 32P-labeled phospholipids.

Immunoblotting. Immunoblotting was performed as previ-ously described (34), with the exception that 2% gelatin(Bio-Rad) was used to block excess binding sites on thenitrocellulose. Rabbit antipolyomavirus T antigen serum wasprovided by D. Pallas (36) and was diluted 1:1,200 forincubation with blots.

RESULTSTransformation assays of mutant MTAgs. MTAg mutants

were generated by using a random mutagenesis procedureand constructed into a recombinant retrovirus vector. Trans-formation-defective, MTAg-expressing cell lines were ob-tained by retrovirus infection of murine fibroblasts withtransiently expressed retrovirus. The retrovirus from one ofthese lines was rescued, and the entire MTAg coding regionwas sequenced (B. Druker and T. Roberts, submitted). Thebase changes and corresponding amino acid changes areshown in Fig. 1. From this MTAg mutant, containing fourbase changes compared to the wild type, three new MTAgmutants were constructed, as described in Materials andMethods. The individual mutants will be referred to by theamino acid corresponding to the base pair carrying a muta-tion (i.e., 190m, 248m, 337/343m). Each of the MTAg mu-tants was placed into the retrovirus pDOL-, transfectedinto psi-2 cells with transiently expressed virus used to infectBALB/3T3 cells, as previously described (9).

Results of a typical focus-forming experiment are pre-sented in Fig. 2. These results demonstrated that the doublemutant 337/343m was comparable to the wild type in itsfocus-forming ability, whereas 190m had slightly diminishedfocus-forming capability (smaller foci and fewer in number)and 248m was completely deficient in its ability to producefoci on a monolayer. By microscopic examination, cells

expressing 248m have a more-rounded morphology and growto a higher saturation density compared with control fibro-blasts but are not as rounded as cells expressing wild-typeMTAg (data not shown). This point can be seen by compar-ing the density of G418 colonies resulting from infection withcontrol, 248m, or MTAg-containing retrovirus (Fig. 2). How-ever, cells expressing the 248m mutant do not form foci on amonolayer. In fact, cells expressing the mutant 248m weremaintained for 7 weeks as a monolayer without any fociresulting, even though 73 G418 colonies were obtained inparallel cultures at 3 weeks postinfection. Soft-agar assays ofpooled G418 colonies demonstrate that both wild-typeMTAg- and 337/343m double mutant-expressing cells arecapable of growth in soft agar. However, cells containingretrovirus alone or retrovirus with the 190m or 248m pointmutations were incapable of growth in soft agar (data notshown).MTAg protein production. In order to determine whether

the transformation defect in these mutants was simply a lackof MTAg protein production, pooled G418 colonies and 6 to10 individual G418 colonies of each of the above clones wereselected for further analysis. By immunoblot analysis with apolyclonal rabbit anti-T serum, a protein of 56 to 58 kDawhich is presumed to represent MTAg was seen in allMTAg-expressing clones (Fig. 3). Steady-state levels ofMTAg expression in the individual clones vary over a three-to fivefold range (data not shown), with no clear differencesin levels of MTAg expression between any of the mutantsand the wild type. In order to more specifically address thestability of MTAg, [35S]methionine labeling of individualcolonies was performed. Each of the mutants was pulse-labeled in a manner similar to that of the wild type (Fig. 4,lane a). Examination of the results of a 60-min chase (Fig. 4,lane b) gives no indication that any of the mutants, including248m, is particularly unstable. Also from the chase experi-

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TRANSFORMATION-DEFECTIVE MIDDLE T ANTIGEN MUTANT 4457

A-G418

/\

pDOL-II

190m

248mn

///

337.343m/n

B

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**.. .1/.0

337,3T3 DOL WT 190 248 343

200-

97 -

68-

,. . 0

.'

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FIG. 2. Focus-forming assay ofMTAg mutants. Focus formationon monolayers of BALB/3T3 cells after infection with mutant andwild-type MTAg retroviruses. Cells were split between two 100-mm(diameter) plates after infection. Cultures were incubated in Dul-becco modified Eagle medium plus 10% donor calf serum with (B)and without (A) G418 for 3 weeks and then fixed and stained withmethylene blue. pDOL-, Retrovirus without insert; MT-WT, ret-rovirus with wild-type MTAg.

ment, it is possible to see the 58,000-molecular-weight (58K)form of MTAg that represents a modified species, withphosphorylations differing from those of the 56K form ofMTAg. Again, each of the mutants appears normal. Asassessed by one-dimensional analysis, each of the mutantsappears to have a similar complement of associated proteinin comparison with that of the wild-type MTAg (data notshown). In particular, associated proteins of 63 and 36 kDacan be identified in one-dimensional gels of this mutant. Dataregarding an 85-kDa-associated protein will be presentedbelow.

Tyrosine kinase activities and phosphorylation of MTAgmutants. G418 colonies obtained from control retrovirus orthe various MTAg constructs were analyzed for the ability ofMTAg immunoprecipitates to phosphorylate tyrosine resi-dues in vitro. Colonies were selected for approximatelyequal levels of MTAg expression, as judged from immuno-blots normalized for total protein (Fig. 3). All of the MTAgmutants, including transformation-defective 248m, demon-strated wild-type levels of tyrosine kinase activity in vitro(Fig. SA). This experiment was repeated with several indi-vidual colonies of 248m-expressing cells with similar results(data not shown).Although transformation-defective 248m clearly has asso-

4 5 -

2 9 -

FIG. 3. Immunoblot analysis of MTAgs expressed in BALB/3T3fibroblasts. Nonidet P-40 lysates of individual G418 colonies ex-pressing control retrovirus, wild-type MTAg, or the various mutantswere separated on a 10o SDS-polyacrylamide gel and electro-phoretically transferred to nitrocellulose. Blots were probed withrabbit polyclonal antipolyomavirus T-antigen serum (36) and devel-oped with an alkaline phosphatase-conjugated anti-rabbit secondantibody. Lanes: 3T3, control fibroblasts; DOL, fibroblasts contain-ing retrovirus without an insert; WT, wild-type MTAg; 190, 190m;248, 248m; 337,343, 337/343m. Numbers to the left of the gel indicatemolecular masses (in kilodaltons). *, 56- to 58-kDa protein pre-sumed to represent MTAg.

ciated tyrosine kinase activity, it is important to compare itsphosphorylation to that of the wild type. For this purpose,proteolytic digestion with Staphylococcus aureus V8 prote-ase was performed on wild-type MTAg and 248m labeled invitro with [32P]ATP (Fig. 6, lanes 1 and 2). The 24K and 18Kfragments contain the tyrosine phosphorylation sites at 315and 322; the third, lower fragment, which is labeled in doublemutants lacking tyrosines 315 and 322 (40), is presumablyderived from tyrosine 250. The wild type and mutant areidentical. In vivo labeling with 32P04 was predominantly onserine residues, although tyrosine phosphorylation was de-tected (40, 44). This in vivo phosphorylation is predomi-

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A B C D E a b c d e A DOL WI '190

97-

68-

FIG. 4. Pulse-chase analysis of MTAg mutants. MTAg-BALB-expressing BALB 3T3 cells were pulse-labeled (lanes A to E) with[355]methionine (1 mCi/ml) for 60 min and either extracted or chased(a to e) for 60 min by using a 10-fold excess of unlabeled methionine.Extracts were double immunoprecipitated by using anti-T serum, asdescribed in Materials and Methods. Samples were resolved on a7.5% SDS discontinuous polyacrylamide gel. Lanes: A and a,d11015; B and b, 190m; C and c, 248m; D and d, 337/343m; E and e,wild type. The large arrowhead represents the 56K and the smallerarrowhead represents the 58K form of MTAg.

nantly in the same region of the molecule as Tyr-250 (43; B.Schaffhausen, unpublished data). While the possibility existsthat a mutation at amino acid 248 affects the patterns ofmiddle T phosphorylation in vivo, lanes 3 and 4 (Fig. 6) showthat this did not seem to be the case. The V8 mappingpatterns of mutant and wild-type MTAg labeled in vivo with32P04were indistinguishable.PI kinase activity-pp85 association. Since there was no

obvious defect in associated tyrosine kinase activity or thepattern of phosphorylation of 248m compared with those ofthe wild type, a second, associated biochemical activity wasinvestigated. This associated activity, PI kinase activity,appears to correlate as well or better with transformationthan does associated tyrosine kinase activity in existingMTAg mutants (22). The levels of PI kinase activity inMTAg immunoprecipitates from control cells, wild-typeMTAg-expressing cells, and cells expressing the variousMTAg mutants are shown in Fig. SB. Results demonstratethat all mutants, including the transformation-defective mu-tant 248m possess wild-type levels of the associated PIkinase activity.The PI-3 kinase immunoprecipitable from MTAg-trans-

formed cells is capable of phosphorylating PIP and PIP2 aswell as PI, as opposed to PI-4 kinase, which has a distinctsubstrate preference for PI (1). As it is unclear which ofthese multiple PI substrates are the physiologically relevantsubstrates, MTAg immunoprecipitates from control, wild-type, or 248m-expressing cells were incubated with PI, PIP,and PIP2 in a kinase reaction (Fig. 7). Again, the mutant248m possessed wild-type levels of PI kinase activity in thisassay. In order to confirm that the products of this reactionwere PI-3 kinase derivatives, these products were deacy-lated and analyzed by high-performance liquid chromatogra-

45-

B

PIP-

337,DOL WT 190 248 343

* 9

0-

FIG. 5. Kinase activities associated with mutant MTAgs. Immu-noprecipitates were prepared from extracts containing equalamounts of MTAg, as determined by densitometric analysis of theimmunoblot in Fig. 3. Immune complex kinase reactions with[32P]ATP were performed as previously described (22). Lanes arelabeled as described in the legend to Fig. 3. (A) MTAg phosphory-lation in an in vitro immune complex kinase assay. Arrowheads tothe left of the figure denote the migration of MTAg and an 85-kDaassociated protein. (B) Phosphorylation of PI. Products of reactionsperformed in vitro were analyzed by thin-layer chromatography andautoradiography as previously described (22). 0, Origin; PIP, PIphosphate.

phy. These results demonstrated that the products producedin this reaction comigrated with PI-3 standards (data notshown). Thus, the mutant 248m does not appear to have adefect in the generation of PI-3 derivatives, as assessed invitro.

Previous work has closely linked the PI-3 kinase activityto a phosphoprotein with an approximate molecular mass of85 kDa. As with previously reported experiments, an 85-kDaprotein was phosphorylated in the kinase reaction in vitroand correlates with the presence of the associated PI kinase

248 343

mob, .1jam 14WAPAA. -a

-.4- ~~~~~~~~~~~~~~~~~~~~~~~~..- ~ ~~~~~~~~~~~~~~~~~~~~~

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1 2 3 4

PULSE

WILD TYPE

, 4,

248

.* 'a

CHASE

18 '.- 4_

FIG. 6. Peptide mapping of phosphorylated 248m. Digestion was

carried out on wild type (lanes 1 and 3) or 248m (lanes 2 and 4).MTAg was labeled in vitro (lanes 1 and 2) with [32P]ATP or in vivo(lanes 3 and 4) with [32P]orthophosphate. After resolution on 7.5%acrylamide SDS gels, digestion on a 15% acrylamide second dimen-sion gel was carried out with 40 ,ug of S. aureus V8 protease per ml.Middle T-specific peptides, including the 24K and 18K peptidesrepresenting the major tyrosine phosphorylation sites, are indicatedby arrowheads.

activity (Fig. 5). Figure 8 shows isoelectric-focusing patternsof pp85 from anti-T immunoprecipitates from either wild-type or 248m-expressing cells labeled with [35S]methionine.The multiple forms represent phosphorylated pp85 species(10). As expected from the PI kinase results, 248m associateswith pp85. Further, the pp85 associated with 248m is modi-fied similarly to that associated with the wild type. It hasrecently been shown that pp85 undergoes a rapid associationwith MTAg, after which it can then dissociate (10). Todetermine whether pp85 might be impaired in its dissociation

4000 -

3000 -

C.)2000 -

1000

0

248m WT-MT

FIG. 7. Phospholipid kinase assay of MTAg 248m. Immunopre-cipitates of MTAg and associated proteins from cells expressingwild-type MTAg and 248m were assayed for the ability to phosphor-ylate PI, PIP, and PIP2. Activity is shown as counts per minute(CPM) incorporated into PIP (-), PIP2 (U), and PIP3 (U) productsnormalized to MTAg protein, as described in Materials and Meth-ods.

FIG. 8. Pulse-chase analysis of pp85 association with MTAgmutants by isoelectric focusing. Wild-type MTAg or 248m-express-ing 3T3 cells were labeled with [35S]methionine (1 mCi/ml) for 60 minand either immediately extracted or chased for 60 min by using a10-fold excess of unlabeled methionine. Double immunoprecipitatesmade with anti-T antiserum were analyzed by 2-dimensional isoelec-tric focusing gel electrophoresis.

from MTAg, which would result in the transformation de-fect, a chase experiment was performed (Fig. 8). As can beseen, release of pp85 from 248m appeared to be similar tothat with the wild type.

DISCUSSIONSeveral new mutants of polyomavirus MTAg obtained

from our library of MTAg mutants (B. Druker and T.Roberts, submitted) have been more thoroughly character-ized. The mutant 337/343m contains two changes in the sameregion deleted in the mutant d11015 (deletion of residues 338to 347) (30). Both d11015 and the mutant 337/343m are fullyactive in assays of associated kinase activities; however, asopposed to d11015, the mutant 337/343m retains full trans-forming capabilities. The mutant 190m is of interest, as it isincapable of growth in soft agar yet is nearly wild type in itsability to form foci on a monolayer. Thus, this mutant mayhelp to define a region and/or functions which are necessaryfor anchorage-independent growth. The mutant 248m isparticularly interesting, as it appears to be as active aswild-type MTAg when assayed for associated tyrosine andphosphatidylinositol kinase activities yet is completelytransformation defective. Thus, this mutant demonstratesthat neither activity nor the presence of both activities issufficient for transformation by MTAg. While several com-pletely transformation-defective mutants with wild-type lev-els of associated tyrosine kinase activity exist, the onlypreviously reported mutants with wild-type levels of associ-ated PI kinase activity retain detectable levels of transform-ing ability (13, 33).The PI-3 kinase activity has been shown to be important

for cellular growth and transformation. This activity iselevated in a variety of transformed cells and in cellsstimulated with growth factors (21). In the case of theplatelet-derived growth factor receptor, deletion of an inter-vening sequence in the cytoplasmic portion of the tyrosinekinase domain results in a receptor which retains platelet-derived growth factor-stimulated increase in tyrosine kinaseactivity and is capable of stimulating PI-4 turnover. How-ever, this receptor does not associate with the PI-3 kinaseand does not activate DNA synthesis (11). In contrast to theplatelet-derived growth factor receptor, stimulation of thecolony-stimulating factor type 1 (CSF-1) receptor does notresult in an increase in PI-4 turnover or activation ofphospholipase C. However, there is an accumulation of PI-3derivatives in CSF-1-stimulated cells (48). As noted above,this PI kinase activity correlates well with the transformationcompetence of MTAg mutants. Although this data arguesstrongly for an important role for the derivatives of the PI-3

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kinase in mediating cellular growth and transformation, the248m mutant of MTAg suggests that other activities are alsonecessary. However, the results reported here only demon-strated that PI-3 kinase activity is present in MTAg immu-noprecipitates assayed in vitro. We are currently comparingthe pattern of PI-3 kinase products from wild-type and248m-expressing cells in vivo.The mutant 248m appears to associate with a similar set of

proteins, compared with wild-type MTAg, as assessed byMTAg immunoprecipitates from metabolically labeled cells.In particular, proteins of 36 and 63 kDa, which have recentlybeen identified as the catalytic and regulatory subunits ofprotein phosphatase 2a (37), are present at wild-type levels.This mutant has no obvious difference in its pattern of invivo phosphorylation, in pulse-chase experiments or byanalysis of V8 maps. Similarly, there is no alteration in thekinetics of association of the mutant MTAg with an 85-kDaprotein, the putative PI kinase.At this point, we can only speculate as to the defect

present in the mutant 248m. First, the mutant MTAg couldfail to interact properly with an as yet unidentified proteinnecessary for transformation. In this case, it is possible thatthe mutant might associate with this protein but fail toappropriately alter the activity of the protein or simply fail toassociate with this protein. Similarly, the kinetics of associ-ation with a critical substrate necessary for transformationother than the 85-kDa protein could be altered in the mutant.Second, it is possible that this mutant does something morethan wild-type MTAg, that is, it might associate and activatea growth-inhibiting protein or inactivate a growth-stimulat-ing protein. Third, the subcellular localization of the mutantMTAg might differ from the wild-type protein. In support ofthis notion is the finding that the tetrameric sequence NPXYof the low-density lipoprotein is necessary for coated pit-mediated internalization (7). This sequence occurs in MTAgat amino acids 247 to 250. It has been previously reportedthat tyrosine 250 is important for MTAg-mediated transfor-mation (32); here we show that proline 248 is also essential.Current experiments are directed at determining whetherthis sequence affects the localization of MTAg.

ACKNOWLEDGMENTS

We thank W. Morgan, D. Pallas, C. Cepko, and R. Myers forproviding reagents and A. West, H. Mamon, W. Morgan, M.Brown, M. Corbley, H. Piwnica-Worms, D. Morrison, D. Kaplan,and D. Pallas for assistance and advice regarding a variety oftechniques used in these studies.

This work was supported by the American Cancer Society PRTF75 (B.J.D.) and Public Health Service grants CA30002 (T.M.R.) andCA34722 (B.S.S.) from the National Institutes of Health.

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