Regulation of pp60c-src and its interaction with polyomavirus middle T antigen in insect cells.

0022-538X/90/010061-08$02.00/0

Copyright©D 1990, American Societyfor Microbiology

Regulation of pp60Ocsrc and Its Interaction with Polyomavirus

Middle T Antigen in Insect Cells

HELEN PIWNICA

WORMS,lt*

NIDHI G. WILLIAMS,' SENG H. CHENG,2ANDTHOMAS M. ROBERTS'

Dana-Farber CancerInstituteandDepartmentofPathology, HarvardMedical School, Boston, Massachusetts02115J, andLaboratory ofCellular Regulation, IntegratedGeneticsInc., Framingham,Massachusetts 017012

Received21June1989/Accepted15September 1989

High yields of soluble, biologically active pp60csrc and middle t antigen (MTAg) of polyomavirus were

produced in insect cells, using a baculovirus expression system. In mammalian cells, pp60csrc undergoes a

regulatory phosphorylationonTyr-527 in vivo and is autophosphorylatedonTyr-416in vitro. In insect cells, pp60C-srC was phosphorylated primarily on Tyr-416, although Tyr-527 was detectable at a low level. A

kinase-negativemutantofpp60C`c? was notphosphorylatedoneither Tyr-527or Tyr-416in insect cells and

thus isanexcellentbiochemicalreagent tosearchfor the regulatory kinase thatusuallyphosphorylates Tyr-527 inmammalian cells.MTAg synthesized in insectcells wasnotphosphorylatedontyrosine residues in vivoor

invitro, suggesting that it didnotassociate withany endogenoustyrosinekinases. However, MTAg isolated

fromcellscoinfected with viruses encoding both MTAg andpp60's'Cwasphosphorylatedontyrosine residues

both in vivo and in vitro.

pp60-csrc is the cellular homolog of the retroviral trans-forming protein

pp60v-src.

pp60c-src andpp60v srcare struc-turally andfunctionally related. Bothareassociatedwith the

plasmamembrane because ofan amino-terminal

myristyla-tion (3, 44). Bothpossessintrinsic tyrosine kinase activities

(12, 23, 32),although the kinase activity of pp60v-src has been estimated to be 10- to50-fold higher than that ofpp60c-src when measured in vitro (21, 24). Both proteins are

phos-phoproteins, although their sitesof tyrosine phosphorylation differ invivo. pp60`csrc is phosphorylated onSer-17 (11, 22)

and Tyr-527 (13) in vivo. Tyr-527 is located in the unique carboxyl terminus of pp60j-src and, therefore, is absent in pp6O -src. Data from a number of laboratories support the hypothesis that phosphorylation of Tyr-527 suppresses the

kinaseactivity and transforming potential of pp60-src in vivo (5, 6, 14, 16, 17, 24, 28, 29, 40). However, the kinase responsible for this negative regulation hasnotbeen identi-fied. Tyr-416 is the site of phosphorylation in vivo in (i) pp60v-src(37,46);(ii) transformingmutantsofpp60c`src (5, 9, 24, 28, 40), and (iii) that fraction of pp6Oc-src associated with the middle t antigen (MTAg) of polyomavirus (6). In each

case, phosphorylation of Tyr-416 correlates withenhanced kinaseactivity. Thus, full activation of pp6Oc-srcmayrequire

concomitantchanges inphosphorylationatbothTyr-527 and Tyr-416.

MTAg is the transforming protein of polyomavirus(47). It has no known intrinsic catalytic activities; instead, it is

thought to function by interacting with and altering the activitiesof cellularproteins. Three tyrosine kinases known tointeract withMTAgarepp60c-src (19), pp62c-Yes (30),and

pp59c-fyf

(7, 31). MTAg has been shown to activate the kinase activity of pp6Oc-src and pp62c-Yes (2, 30). Genetic studies have demonstratedthataninteraction withpp60csrc

is necessary for MTAg-mediated transformation (8, 20). How MTAg altersthe specifickinaseactivityofpp60f-src is unclear. The shift inphosphorylation fromTyr-527 to Tyr-416doesnot accountforthe entire activation(5),Complexed

*Correspondingauthor.

tPresent address: Department of Physiology, Tufts Medical

School, 136HarrisonAve., Boston,MA 02111.

pp60-src has also been showntobephosphorylatedon one or moreamino-terminal tyrosine residues (50). These

phos-phorylations have been detected in vivo only in thepresence

of sodium orthovanadate or in vitro in immune complex

kinase assays. Thus, their biological significance remains

unresolved. Inaddition, onlyasmallproportion of the total

amount ofMTAg and pp60csrc presentwithin acell atany

given time is found stably associated (1, 20). Levels of the complexcanbeincreasedonly marginally by overexpressing either pp6Ocsrc or MTAg (39, 41, 43) in mammalian cells.

Factors controlling the association of these two molecules

are presently not known. Unfortunately, the two proteins fail to interact when they are coproduced in bacteria (H.

Piwnica-Worms, D. Pallas, and T. M. Roberts, unpublished data) or in yeast cells (S. Kombluth and H. Hanafusa, personal communication) orwhen they are cotranslated in

vitro inarabbitreticulocytelysate (H. Piwnica-Worms, and

T. M. Roberts, unpublished data). A complete biochemical analysis awaits the development of a system in which

purified components can be added and theireffectscan be

assessedindividually in vitro.

Asa first step towards the development ofan assay, we

have overproduced MTAg and avian

pp60,csrc,

using a

baculovirusexpression system.We haveanalyzedthe post-translationalmodifications undergone by pp60csrc in insect cells, including myristylationandphosphorylation. In addi-tion, by coinfectingcells with recombinant virusesencoding

bothpp60"srcandMTAg,wehaveobtainedpp60c-src-MTAg

complexes.

MATERIALS AND METHODS

Celllines.Spodopterafrugiperda (Sf9)cellswereobtained from the AmericanTypeCulture Collection.Allprocedures relating to viral propagation, isolation, and plaquing were

performed assuggested by Summersand Smith (49). Generationofc-srcbaculoviruses. pSP68-4(RBS) (39) was

restrictedwithHindIII, andaBamHI linkerwasinsertedto generate pSP68-4RBS(Bam). pSP68-4RBS(Bam) was

re-stricted with NcoI, and a BclI linker (39) was inserted to generate pSP68-4RBS(Bcl-Bam). pSP68-4RBS(Bcl-Bam)

was restricted with BclI and BamHI, and the fragment 61

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encoding pp60csrc was isolated. This c-src fragment was cloned into BamHI-restricted and phosphatased pAC373 (49) to generate pAC373(c-src). A mixture of 2 jig of

pAC373(c-src)and 1jig of purifiedwild-type viral DNA was

transfectedintoSf9 cells.Four days later, medium

superna-tants wereremovedandcentrifugedat 1,000rpm for 10 min.

Clarifiedsupernatantscontainingwild-type and recombinant

viruseswere plaqued on amonolayer of Sf9 cells. Occlusion

negative plaques were picked and seeded onto 2.5 x 106

cells. Three days later, cells and mediumsupernatants were removed and centrifuged at 1,000 rpm for 10 min. The supernatant containing the virus was stored at 4°C. The

pelletedcells were lysedandimmunoprecipitated with EC10

serum (a monoclonal antibody specific for avian pp60csrc

[36]),andkinase assays wereperformedinvitro as described

previously (39). All lysates tested positive for the presence

of kinase-active pp6O-src. One supernatantwasselected and

taken through three rounds of plaque purification. A c-src

mutant encoding methionine rather than lysine at position 295 was constructed by site-directed mutagenesis as de-scribedpreviously (40). The Lys-295 to Met mutant was then

cloned intopAC373 togenerate pAC373(295) and

recombi-nantviruses were generated asdescribed above.

Generation of MTAg baculovirus. p890 (26)was restricted

withBclI andBamHI,and the fragmentencodingMTAg was

cloned intopAC373 which had been linearizedwith BamHI

and phosphatased. Therecombinantplasmidwasdesignated

pAC373(MTAg). A mixture of pAC373(MTAg) and

wild-type viral DNAwas transfected into Sf9 cells asdescribed

above for pp60-src.Twentyocclusion-negativeplaques were

picked and seeded onto cells as described above. After 6

days, cells and medium supernatants were removed and

clarified. Viral supematants were stored at4°C. Cellswere

lysedand normalized for protein content with the Bio-Rad

Laboratories protein assay kit, and proteinswere resolved

by 10% sodium dodecyl sulfate-polyacrylamide gel

electro-1

2

3

4

5

6

7

__

~~-pp60

phoresis (SDS-PAGE). Proteins were blotted onto nitrocel-lulose and probed with PY19 serum (polyclonal antibody specific for polyoma t antigens [34]) as described in the

protoblot immunoblottingsystem from Promega Biotec. One

viral stock was selected and taken through three rounds of plaquepurification.

Time course of protein production and accumulation.Cells, 3 x

106,

were seededinto each of 13 60-mm tissue culture

dishes. Oneplatewasmockinfected, sixwereinfectedwith

recombinant baculovirusencoding

pp6O-src,

and the

remain-ingsixwereinfected with recombinantbaculovirusencoding

MTAg. Cells were lysed at 15, 20, 24, 29, 40, and 52 h

postinfection (p.i.), and their protein concentration was

measured with the Bio-Rad protein assay kit. Total cell protein, 30 jug, was then resolved by SDS-PAGE,

trans-ferred electrophoretically to a 0.45-jim nitrocellulosefilter.

Thefilterwas thenblockedin 1% bovine serumalbumin in

TBST (10 mM Trishydrochloride [pH 8.0], 150 mM NaCl,

0.05% Tween 20) for 1 h at 37°C. The pp60c-src blot was

incubatedovernight with EC10serum (1: 2,500dilution),and

the MTAg blot was incubated for 4 h with PY19 serum (1:2,500 dilution). Both blots were then washed, incubated

with anti-mouse (pp60c-src blot) oranti-rabbit (MTAg blot)

alkaline phosphatase-conjugated immunoglobulin G for 45

min, and then developed by using the enzymatic color

reaction kit from PromegaBiotec.

Myristylation

ofpp6c-src.

Cells,

3 x 106,wereseededinto eachoftwo60-mmtissue culture dishes. Afterattachment,

the cellswereinfected with recombinantbaculovirus

encod-ing pp60c-srcatamultiplicity of infection of10. At 40hp.i.,

one plate was rinsed in methionine-free medium and then

labeledinmethionine-free mediumsupplementedwith 2 mM

glutamine and 0.67 mCi of [35S]methionine per ml. The

second plate was labeled with 2 mCi of[3H]myristic acid

(Dupont, NEN ResearchProducts) in afinal volume of1.5

ml. The[3H]myristic acidwaslyophilized and redissolvedin

12 34

5

6

7

FIG. 1. MTAg andpp6Oc-srcproduction and accumulation. Sf9 cells were either mock infected(lane 7, bothpanels) orinfected with recombinantbaculovirusesencodingpp6O-src(leftpanel)orMTAg(right panel)at amultiplicityof infection of10. Cellswereharvestedat various times after infection; cell lysates were prepared, and proteins were resolved by SDS-PAGE. The proteins were blotted onto nitrocellulose andprobed with EC10 (left panel)orPY19(right panel)serum. Lane 1,52 hp.i.;lane2,40hp.i.;lane3,29hp.i.;lane4,24 hp.i.; lane 5, 20hp.i.;lane6, 15hp.i.; lane 7, mock infected.

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15,ul of dimethyl sulfoxide prior to labeling. After 2 h, cells were lysed and proteins were analyzed directly by SDS-PAGE or immunoprecipitated with EC10 serum prior to SDS-PAGE.

Intrinsic and associated kinase activities. Cells, 9 x 106, were seeded intoeach of three 100-mm tissue culture dishes. After attachment, cells were mock infected or infected with recombinant baculovirus encoding MTAg or bothpp60-src and MTAg. Cell lysates were prepared at 40 h p.i. and normalized forprotein content. A 200-,ug amount of protein from each lysate was immunoprecipitated with eitherEC10 orPY19 serum, and kinase assays wereperformed in vitro. In vivo labeling and phosphopeptide mapping. Sf9 cells were infected with recombinant virus encoding

pp60c-src,

MTAg, or bothpp6Oc-src and MTAg each at a multiplicity of infection of 10. At 36 h after infection, cells were rinsed once

in phosphate-free medium and then incubated for 2 h in

phosphate-freemedium supplemented with 2 mM glutamine,

1.5% dialyzed calfserum, and 2mCiof32P-labeled P,per ml.

Cell lysates were prepared in a phosphate-RIPA buffer (10

mMsodiumphosphate [pH 7.2], 150 mM NaCl, 1% Nonidet P-40,0.1%sodium deoxycholate, 0.1% SDS, 2 mM EDTA)

supplemented with 2 mM phenyl-methylsulfonyl fluoride,

0.1% Aprotinin, and 2 mM sodium orthovanadate, and

pp60c-srcwasimmunoprecipitated with eitherEC10 orPY19

serum. Staphylococcus V8 and tryptic protease mapping was performed as described previously (14, 30), and

cyano-genbromidemappingwasperformedasdescribed by Jove et

al. (25). Phosphoamino acid analysis was performed as

described previously (35).

RESULTS

cDNAsencoding normal pp60c-src,a c-src mutant

contain-ing methionine rather than lysine at position 295, and the

MTAgofpolyomavirus werecloned intothe BamHI site of

pAC373 (49) to generate pAC373(c-src), pAC373(295), and

pAC373(MTAg), respectively. Sf9 cells were then

trans-fected with a mixture of wild-type baculoviral DNA and

pAC373(c-src), pAC373(295),orpAC373(MTAg).Fivedays

after transfection, medium supernatants containing both

wild-type and recombinant viruses were removed and

plaqued on a monolayer of Sf9 cells. Recombinant virus

plaqueswereidentified visually by theirocclusion-negative

phenotype (49). Recombinant virus was

purified

from

con-taminating

wild-type virus

by

three rounds ofplaque

purifi-cation.

Protein production and accumulation. The time course of

protein production

and accumulation in insect cells was

examined asfollows. Sf9 cells were infected with

recombi-nant viruses encodingeither

pp6Oc-src (Fig.

1,left

panel)

or

MTAg(Fig. 1,right panel)at amultiplicity of infection of10.

Atvarious times afterinfection, cell lysates were

prepared

and protein production was examined

by

Western

blotting

(immunoblotting).

pp6Jc

srcandMTAgwerefirst detected20

hp.i. (lane 5), increased

linearly

untilabout 40 h

(lane

2),and

continued to accumulate

gradually

from 52 to72 h

(lane

1

and data not shown). Breakdownproductswere detectable

by about 30 h p.i. (lane 3),

though

the bulk ofthe

protein

remained stable for at least 3

days (data

not

shown). By

Coomassie blue

staining,

we estimated that

pp6c-src

and

MTAgcomprise

approximately

1% oftotal cell

protein.

pp60CSrCismyristylatedininsectcells.

pp({-csrc

is

myristy-latedin avianandmammalian cells

(3,

44)

andinyeast cells (29). To determine whether it was

myristylated

in insect

cells, we infected cells with recombinant baculoviruses

encoding pp6Oc-src and thenlabeled the infected cells with

either [3H]myristic acid(Fig. 2, right panel) or

[35S]methio-nine (Fig. 2,left panel).Total cell lysates or

immunoprecip-itates prepared by using antiserum specific for pp6Oc-src were then resolved by SDS-PAGE. Although pp6f4-src was the major protein labeled with

[35S]methionine,

several

addi-tional proteins were also labeled during this time period.

Since the labeling period was started at 40 h p.i. when host protein synthesis is shut off, these proteins represent late virus-specific proteins.pp6Oc-srcwas the onlyprotein labeled with [3H]myristic acid. We do not know the fraction of

pp60-)srcthatismyristylated.

Intrinsic and associated kinase activities of pp60cs1c and MTAg in insect cells. To determine whether (i) pp6Oc-src

producedin insect cells was enzymatically active, (ii) MTAg

could associate with any endogenous insect tyrosine ki-nases, and (iii) MTAg and pp60-csrc would associate when

coproduced in insect cells, we performed the following

experiments: insectcells were mock infected (Fig. 3, lane 1)

orinfected with recombinant baculovirusesencoding MTAg

(lane 2) or MTAg and

pp6fc-src

(Fig. 3, lanes 3 and 5). Cell

lysates were prepared and immunoprecipitated with either

EC10serum (34)specific for avianpp6Oc-src(lane 5) or PY19 serum (36) which recognizes MTAg (lanes 1, 2, 3, and 4). Kinase assays were performed in vitro. MTAg was

phos-phorylatedin immunecomplex kinase assays in the presence

35s

T E

3T

T

E

FIG. 2. Myristylation ofpp6csrc. Sf9 cells were infectedwith recombinant virus encoding pp6Ocsrcandat 40hp.i. labeledwith either[35S]methionine(leftpanel)or[3H]myristicacid(rightpanel). Cell lysateswere preparedafter2hoflabeling, andproteins were

resolved directly by SDS-PAGE (T) or immunoprecipitated with EC10serumpriortoSDS-PAGE(E). Exposuretimeswere16 h for 35Gand 96 hfor'H.Arrowheads denoteppcswre.

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1

2

3

4

5

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SRCP--4MTe

30

FIG. 4. Association betweenpp60csrc and MTAg in vivo. (Top) Cellswereinfected with virusesencoding MTAg (lane 1), pp6c-src (lane 4), or bothMTAg and pp60c-src (lanes 2 and 3). At 36 hp.i., cellswerelabeled with

32pi.

Celllysateswereprepared and immu-noprecipitated with either PY19 (lanes1and2)orEC10(lanes 3 and 4) serum.Immunoprecipitateswereresolvedby SDS-PAGE. (Bot-tom) Lanes1, 2, and 3 are the results ofphosphoamino acid analysis performedonMTAg isolated from lanes 1, 2, and 3, respectively, of thegel shown in the top panel. Lane 4 is the result of phosphoamino acidanalysis performedonpp6OcsrC isolated from lane 4 of the gel shown in thetop panel.

FIG. 3. Intrinsic and associated kinase activities ofp60c-srcand MTAg. Sf9 cells were mock infected (lane 1) or infected with recombinantviruses encoding either MTAg(lane 2) orMTAg and pp6Oc-src (lanes3 and5). Celllysateswerepreparedat40hp.i. and immunoprecipitatedwithEC10 (lane 5)or PY19(lanes 1,2, 3,and4) serum, and kinase assays were performed in vitro. Lysates were also prepared from NIH 3T3 cells transformed by MTAg(lane 4 [10])andimmunoprecipitated withPY19serum, andkinase assays wereperformed invitro. The protein with the greatest electropho-reticmobility in lane4is a MTAg breakdownproduct (39).Forlanes 1, 2, 3,and 5, 200 ,ug of total cellproteinwasimmunoprecipitated, whereas 1.5 mg of totalcell proteinwasimmunoprecipitated in lane4.

(lanes3and5)butnotin the absence(lane 2) of

pp6Oc-src.

In

immunoprecipitates prepared with EC10 serum (lane 5),

bothpp60csrc and MTAgphosphorylation isevident. EC10

serum immunoprecipitates both complexed and free pp60

c-src,

both ofwhichcanbephosphorylatedinvitro.This accounts for the significant phosphorylation ofpp6Oc-src in

thisexperiment. Inimmunoprecipitates preparedwith PY19

serum(lane 3), onlythepp6Ocsrc complexed toMTAg was

immunoprecipitated and, since MTAg is thepreferred

sub-strate, phosphorylation ofpp6Ocsrc is less evident. MTAg was also phosphorylated in vivo by pp6Ocsrc when both

proteinswerecoproduced in insect cells (Fig. 4). Cells were

infected with viruses encoding

pp6fc-src

(Fig. 4, lane 4) or

MTAg (lane 1) or with both recombinant viruses (lanes 2 and 3)andwerelabeledwith

32Pi.

Cell lysates were prepared and

immunoprecipitated with either PY19 (lanes 1 and 2) or

EC10 (lanes3and 4) serum. pp6Ocsrccoimmunoprecipitated

with MTAg, using antisera specific for MTAg (lane 2), and

MTAg coimmunoprecipitated with

pp6Ocsrc,

using antisera

specific forpp6Oc-src (lane 3). pp6Oc-src wasphosphorylated

primarily on tyrosine and serine residues (Fig. 4, bottom

panel). MTAg was phosphorylated on serine when

synthe-sized aloneininsect cells (lane 1)andwasphosphorylatedon

tyrosine as well as serine when coproduced with pp6oc-src (lanes 2 and 3). We also found that MTAg reacted with an

anti-phosphotyrosine antibodyinimmunoblots fromlysates

prepared from cells coinfected with viruses encoding both MTAg andpp6Oc-src butnotfromlysatesprepared from cells infected with virus encoding MTAg only (datanot shown). Thus, MTAgwas phosphorylatedon tyrosine both in vitro and in vivo only when coproduced with pp6oc-src in insect cells.

pp60j-srcisphosphorylatedprimarilyonTyr-416 and Ser-17

ininsectcells.To determine where thepp6Oc-src produced in insect cells was phosphorylated in vitro and in vivo, two-dimensional tryptic phosphopeptide mapping was per-formed.Tyr-416wasthe majorsite ofautophosphorylation

(Fig. 5A, B, andC).Tryptic digests of pp6O-src synthesized

and labeled in insect cells gave rise to two major tryptic

phosphopeptides (Fig. SD). Species2migratedat aposition

similartothetryptic peptide containingSer-17,and species

1 migrated at a position similar to the tryptic peptide

containing Tyr-416.Toverify this, wemixedtryptic digests

ofpp6Oc-srcsynthesizedininsect cells withtryptic digestsof

a c-src mutant thatwehave previously shown to be

phos-phorylatedonSer-17and Tyr-416 (40) (Fig. SE).Wedonot

know what fraction of pp6ocsrc produced in insect cells is

phosphorylatedonSer-17,but phosphotyrosine was present in greaterquantities thanphosphoserine by phosphoamino

acid analysis (Fig. 4, bottom panel) and more label was

incorporated into the peptide containing Tyr-416 than into

that containing Ser-17 (assuming equal recovery of both

peptides; Fig. 5D). Thus, it is unlikely that there is a

stoichiometricphosphorylation of Ser-17.

Since the phosphopeptide containing Tyr-527 is often

difficultto recoverduring trypticphosphopeptidemapping,

wealsoperformedone-dimensional cyanogen bromide

map-ping (data not shown). Using this method, we found that

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A

A

D

B

E

C

A

F

@1

.2

*1

.2

;

_A

A

FIG. 5. Tryptic phosphopeptide mapping ofpp6Oc-srclabeled in vitroand in vivo. pp6Oc-srcwasimmunoprecipitated fromlysates of (A) infectedSf9cells or (B) NIH 3T3 cells overproducingpp6O-src,andautokinase assays were performed in vitro.Alternatively,pp6Oc-scwas immunoprecipitatedfrom 32P-labeledlysates of (D)infected Sf9 cells or (E) NIH 3T3 cells expressing a c-src mutant in which Tyr-527 is replaced by Phe. Immunoprecipitated proteins were digested with trypsin, separated by electrophoresis and chromatography in two dimensions, and autoradiographed. (C) Mix of phosphopeptides from panels A andB;(F) mix ofphosphopeptides from panels D and E. Spots 1and 2 designate the phosphopeptides containing Tyr-416 and Ser-17, respectively. The origins are marked witharrowheads. Electrophoresis atpH 8.9 was performed in thehorizontal dimension with the anode at the right.

pp6&c-src

was also phosphorylated to a small extent on Tyr-527(-10%of the level seen at Tyr-416). To distinguish

betweenthe possibility thatTyr-527 was being

phosphory-lated by a kinase present in Sf9 cells other than pp6c-src

itself, weexpressed akinase-negativemutantofpp60csrcin

insect cells and compared its phosphorylation pattern with

thatofnormal

pp60>src,

usingone-dimensional staphylococ-calV8protease mapping(aprotocol in whichphosphate on

Tyr-527 is stable). The kinase-negative mutant was

con-structed by replacing lysine residue 295 in pp60csrc with

methionine. Lys-295 is in the ATP-binding domain of

pp60c-src

and is essential for kinase activity (25, 48). The

Lys-295toMet mutant hasbeenshown to be phosphorylated

onTyr-527 in aviancells (25). Ifanendogenous insect kinase

wasresponsible for phosphorylatingTyr-527, then this

res-idue would beexpected tobephosphorylated in theLys-295

mutant;if,onthe otherhand,pp60csrcwasitselfresponsible

for thisphosphorylation,then theLys-295mutantwouldnot

bepredictedtobephosphorylatedonTyr-527.The presence

ofV8

fragments

1, 3, and 4(Fig. 6) indicated that the Lys-295 mutant wasphosphorylated within its amino terminus,

pre-sumably on Ser-17. However, the carboxy-terminal V8

fragment (V2) containing Tyr-416 and Tyr-527 was not

present in the mapofthe Lys-295 mutant (Fig. 6, lane 3),

indicating

thatthismutant wasnot

phosphorylated

oneither

Tyr-527orTyr-416.

DISCUSSION

Inthisreport, wedescribetheapplication ofabaculovirus

expressionsystem to(i) produce large

quantities

of

soluble,

biologically

active

pp60csrc

and MTAg;

(ii) produce large

quantities ofakinase-negativemutantof

pp60-src

tobe used

as substrate to identify and purify kinases that

regulate

pp60c-src

inmammalian

cells;

and

(iii) develop

asystemfor

the study ofMTAg-pp60c-src interactions invitro. The

bac-ulovirus expression system utilizes a helper-independent

virus (Autographa californica nuclear polyhedrosis virus)

whichcanbe grown tohigh titers incells adapted to Spinner

culture(49).Thus,highlevelsofrecombinantproteincanbe

obtained with relative ease.We haveobtained large

quanti-ties of soluble, biologically active pp6Oc-src and MTAg,

putting intherealm ofpossibilityathree-dimensional

struc-turalanalysis of both proteins.Inaddition, baculovirusesare

noninfectious to vertebrates andtheirpromoters have been

showntobe inactiveinmammalian cells(4). This isaclear

advantage overother eucaryotic viralvector systems when

expressing oncogenes or other potentially dangerous

pro-teins. Further, unlike bacterial expression systems, the

baculovirus systemis a

eucaryotic expression

system and,

therefore, utilizesmanyoftheprotein

modification,

process-ing, and transport systemswhichoccurinhighereucaryotic cells.

Two of the three posttranslational modifications of

pp60c-src

which

usually

occurin

higher

eucaryotic

cells also

take place in insect cells. These are

myristylation

and

phosphorylation on Ser-17.

Myristylation

has also been

shown to occur in yeast cells (29). In mammalian

cells,

Ser-17 is

phosphorylated by

a

cyclic AMP-dependent protein

kinase(11, 38). Ahomologous enzymewiththe

capacity

to

recognize avian

pp6Oc-src

must therefore exist in Sf9cells.

Theonlyinvertebratehomolog ofc-srcthat hasbeencloned

andsequencedis from

Drosophila

melanogaster.

The amino

terminus of the

Drosophila

gene

diverges

greatly

from its vertebrate homolog,but it does encode aserineat

position

26which appearstobe

homologous

toserine17 in vertebrate

pp60c-src

(45).

Phosphorylation

of

Tyr-527,

the third modifi-cation of

pp60c-src

that

usually

occurs in mammalian

cells,

was also detectable at low levels in Sf9 cells. That the

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4V1

:V2

4V3

* 4s

*V4

FIG. 6. Staphylococcol V8protease mapping. pp60csrc(lanes 1 and2)and theLys-295to Metc-srcmutant(lane 3)were

immuno-precipitated from 32P-labeled lysates of infected Sf9 cells and resolvedbySDS-PAGE. Gel bandscontainingtherespective

pro-teins wereexcised and digestedwith staphylococcalV8 protease, and the proteolytic digestion products were resolved on a 15% polyacrylamide-SDSgel.V8fragments1, 3,and 4 are derivedfrom the amino terminusofpp6Oc-src,and V8fragment2 isderived from thecarboxylterminus ofpp6Oc-src.

Lys-295mutantwasnotphosphorylatedonTyr-527suggests that this low level ofphosphorylation was due to auto- or

transphosphorylation by pp6Ocsrcitself.

Drosophila c-src has been shown to encode a tyrosine residue ata position homologous to Tyr-527 in vertebrate c-src (45). Thus, the invertebrate src homolog may be

regulatedby amechanism similar to that used in vertebrate cells. The findingthat the Lys-295 mutantofpp6Oc-src was not phosphorylated on Tyr-527 in insect cells can be

ex-plainedinseveralways. (i)A527 kinasehomologmaynot be

presentininsect cells. (ii)The 527kinasemaybepresentin insect cells but its activity may be saturated by the large quantitiesof the c-srcmutantbeing produced. However,the

Ser-17 kinasewas not saturated under theseconditions.(iii)

The Tyr-527 kinase may be more labile than the Ser-17

kinase.

pp60csrc

is synthesizedlate in the viral replication cycle, long after host protein synthesis has been shut off. Thus, the Tyr-527 kinase may have been present prior to

viralinfection butmayhaveturnedoverbythetimepp6Oc-src synthesis began. (iv) The determinants that make up the recognition site for the 527 kinase may not be shared betweenavianpp6Oc-srcanditsDrosophila homolog. Thus,it

is possible that an insect 527 kinase exists but does not recognize avian pp60csrc. (v) Finally, it is still formally possible that

pp60c-src

is itself the 527 kinase, but that it

functions poorlyin yeastand insect cells(15, 16, 29).

Recent studies strongly suggest that Tyr-527 is a major

regulatory site inpp6Ocsrc. Phosphorylation ofthis residue has been shown to suppress the kinase activity and trans-forming potential of pp6Ocsrc Other members of the src

familyoftyrosinekinasespossessaTyr-527 homolog. Thus,

suppression by phosphorylation at this residue may be a

general mechanism for regulating thebiochemical and

bio-logicalactivities of the entiresrcfamilyoftyrosinekinases.

Thefinding that theLys-295mutantisnotphosphorylatedon Tyr-527 in insect cells makes the baculovirus system ideal

for preparinglarge quantities of this mutant to be used for

identifying and isolating the 527 kinase from mammalian

cells. In addition, other regulatory kinases, besides that which phosphorylates Tyr-527, may also be identified

by

using the approach.

We have previously reported a region in the

carboxyl

terminus of

pp6Ocsrc,

borderedby Asp-516andPro-525,that

is required for MTAg association in mammalian cells

(9).

Since thisregion is not conserved in theDrosophilapp6Oc-src homolog (45), we did not predict an interaction to occur betweenMTAg and the insecthomolog of pp6Ocsrc. Indeed, MTAg synthesized in Sf9 cells was not detectably associated with any endogenous insect kinases when assayed in vivoor in immunecomplex kinase reactionsassayed in vitro. How-ever,MTAg did form a complex with avianpp6Oc-src in cells coinfected with viruses encoding both proteins. These re-sults make the baculovirus expression systempreferable to other expression systems for studying complex formation because: (i) preparations ofMTAg from higher

eucaryotic

cells would be expected to be contaminated with endoge-nous pp6Oc-src and (ii) pp6Oc-src and MTAg do not interact when coproduced in yeast cells (Kornbluth and Hanafusa, personal communication) or bacteria (Piwnica-Worms etal.,

unpublished data) or when cotranslated in vitro, using a

rabbit reticulocytelysate (Piwnica-Worms and Roberts, un-published data). In addition, overproduction of either com-ponent in mammalian cells does not significantly increase levels of complex formation (39, 41, 43). These results suggest that there may be aninterplaybetween both positive

andnegativefactors which act inconjunctiontoregulate the

interactionof pp6Ocsrc and MTAg in mammalian cells. For

example, the posttranslational modifications undergone by

MTAg (i.e., phosphorylations) may be a positive factor

regulating complexformation in mammalian cells (42). We

caneasily test this hypothesis by comparing the

posttrans-lational modications undergone byMTAg ininsect cells and

thosewhichoccurinmammalian andyeast systems. Factors that inhibit complex formation in mammalian cells may include regulatory proteins, such as the 527 kinase, that compete with MTAgfor pp6Ocsrc binding. The baculoviral

expressionsystem may be the systemof choice for

identify-ing and purifying these various factors from mammalian cells.

Complex formation between MTAg and pp6Oc-src was

assayed both in vivo and in vitro. We found that MTAg

coimmunoprecipitatedwithpp6Ocsrc using antisera specific

for pp6Oc-src, and thatpp6Oc-srccoimmunoprecipitated with

MTAg, using MTAg-specific sera. In addition, MTAg was

phosphorylated on tyrosine in vivo only when coproduced

withpp6Oc-src and in vitro only when pp6Ocsrc was present in

the immunoprecipitates. Because the majority ofpp6O-src

producedin insect cells is not phosphorylated on Tyr-527, its

kinaseactivityisconstitutively high. It has, therefore, been difficult to determine whether the pp6Ocsrc associated with

MTAgisactivatedcompared with its unassociated

counter-part. Todate, we have been unable to measure any substan-tial enhancement in kinase activity when pp6csrc is

com-plexed to MTAg in insect cells. We are currently mapping

the complexed pp6Oc-src and MTAg to identify any novel

posttranslational modifications that occur as a result of

complex formation.

There remains uncertainty as to whether the complex formedbetween MTAgandpp6Ocsrc in insectcells is

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[image:6.612.108.248.77.304.2]

tional. Because of the large quantities of protein

being

produced, the complexformationweobserved could be due

to a nonspecific aggregation of the two proteins. Using

antisera specific for MTAg, we have observed

coimmuno-precipitation ofpp6fvsrc and the v-fmsgene product when

eitheris coproduced withMTAg ininsectcells (althoughnot

to the same extent as with pp60-src). Neither of these

proteins has beendemonstrated to associate with MTAg in

mammalian cells. However, we do not observe

MTAg-pp6&c-src

complexes sedimenting to the bottom ofsucrose

gradients, aswouldbeexpected forlarge nonspecific

aggre-gates. We also donot observea major shift inthe

sedimen-tation of eitherMTAgofpp60csrc from coinfectedcells when

compared with their sedimentation from singly infected

cells. When isolated from mammalian cells, the complex

sediments in the region of the gradient expected for a

220-kilodaltoncomplex (19, 20). These resultsmight suggest

that we have no complex formation inside insect cells, but

ratherthetwoproteinsassociateafterthecellsarelysedand

incubatedfor extended periods, as isthe case during

immu-noprecipitations. Alternatively, the complex isolated from

mammalian cells may contain proteins in addition to

pp60c-src and MTAgwhich contribute toitsgreater

sedimen-tation velocity, and thecomplex formedin insectcells may

be deficient in these other proteins. Several proteins have

been shown to associate with MTAg in mammalian cells

(33), and a

potential

phosphatidylinositol

kinaseof

approxi-mately 85 kilodaltonshas been shown tobe associatedwith

MTAg-pp60csrc

complexes

isolated from mammalian cells

(18, 27). Further studies will

be required

to determine

whether MTAg and pp60csrc form afunctional

complex

in

insect cells.

Regardless of whether a functional

complex

is formed

betweenpp60c-srcandMTAgwithininsect cellsatthe timeof

theirsynthesis, thissystem

provides

a

powerful

tool forthe

study ofpp60csrc and MTAg interactions in vitro. We are

currently purifying large

quantities

of MTAg, free of

con-taminating tyrosine kinases, andlarge

quantities

of

pp6&-src

as afirststeptowardsthe

development

ofasystem in which

purifiedcomponents canbe added and their

ability

todrive

complex formation canbe assessed

individually.

ACKNOWLEDGMENTS

Wethank R. Jovefor adviceonthecyanogenbromide mapping, Debbie Morrison for the baculovirus encoding the v-fms gene product, and A. Desai for secretarial assistance.

This work was supported by Public Health Service grants CA30002 and CA43803 to T.M.R. and R29 CA50767 to H.P.-W. from the National Cancer Institute.

LITERATURE CITED

1. Bolen, J. B., V. DeSeau, J. O'Shaughnessy, and A. Shokreh. 1987. Analysis of middle tumor antigen and pp60csrc interac-tions in polyomavirus-transformed ratcells. J. Virol. 61:3299-3305.

2. Bolen, J. B., C.J. Thiele, M. A. Israel, W. Yonemoto, L. A. Lipsich, and J. S. Brugge. 1984. Enhancement of cellular src

geneproduct associatedtyrosylkinaseactivity following

poly-omavirusinfection and transformation. Cell38:767-777. 3. Buss, J. E.,and B. M.Sefton. 1985.Myristic acid, a rarefatty

acid, is thelipid attachedtothe transformingprotein ofRous sarcomavirus and itscellularhomolog.J.Virol.53:7-12. 4. Carbonell, L. F., M. J. Klowden, and J. K. Miller. 1985.

Baculovirus-mediatedexpressionofbacterial genesin

dipteran

andmammalian cells. J. Virol. 56:153-160.

5. Cartwright, C. A., W. Eckhart, S. Simon, and P. L. Kaplan.

1987. Cell transformationby

pp60csrc

mutatedin the carboxy-terminalregulatory domain. Cell49:83-91.

6. Cartwright,C. A.,P. L. Kaplan, J.A.Cooper,T.Hunter,and W. Eckhart. 1986. Altered sites of

tyrosine

phosphorylation

in

pp60c-src

associated with

polyomavirus

middle tumor

antigen.

Mol. Cell. Biol.6:1562-1570.

7. Cheng,S.H.,R.Harvey,P.C.Espino,K.Semba,T.Yamamoto,

K.Toyoshima,and A. E. Smith. 1988.Peptideantibodiestothe human c-fyngene product demonstratepp59fyn is

capable

of

complexformation with the middlet

antigen

of

polyoma

virus.

EMBOJ. 7:3845-3856.

8. Cheng,S.H., W.Markland,A. F.Markham,and A. E.Smith. 1986. Mutations around the NG59 lesion indicate an active association ofpolyomavirus middle-T

antigen

with

pp60c-src

is

requiredfor cell transformation. EMBOJ. 5:325-334.

9. Cheng,S.H.,H.Piwnica-Worms,R.W.Harvey,T.M.Roberts,

and A. E. Smith. 1988. The carboxy terminus of

pp6('-src

is involved in complex formation with the middle-T

antigen

of

polyomavirus. Mol. Cell. Biol. 8:1736-1747.

10. Cherington, V., B. Morgan, B. M.

Spiegehnan,

and T. M. Roberts.1986.Recombinant retroviruses that transduce

individ-ualpolyoma tumor

antigens:

effectson

growth

and

differentia-tion. Proc. Natl. Acad. Sci. USA 83:4307-4311.

11. Collett,M.S.,E.Erickson,and R. L. Erikson.1979.Structural

analysis oftheavian sarcomavirustransforming

protein:

sites

of

phosphorylation.

J. Virol.29:770-781.

12. Collett, M. S., A. F. Purchio, and R. L. Erikson. 1980. Avian

sarcomavirustransforming protein p60srcshows

protein

kinase

activity specificfortyrosine. Nature(London)285:167-169.

13. Cooper,J. A.,K. L. Gould, C. A. Cartwright, andT. Hunter. 1986.

Tyr5"

is

phosphorylated

in

pp60csrc:

implications

for

regulation.

Science 231:1431-1434.

14. Cooper, J. A., and C. S. King. 1986.

Dephosphorylation

or

antibodybindingto thecarboxyterminus stimulates

pp6Oc-src.

Mol. Cell. Biol.6:4467-4477.

15. Cooper, J. A.,and A. MacAuley. 1988. Potential

positive

and

negative

autoregulation

of

pp60C-src

by

intermolecular

autophos-phorylation.Proc. Natl.Acad. Sci. USA 85:4232-4236.

16. Cooper, J. A., and K. Runge. 1987. Avian pp60-src is more

active when

expressed

in yeastthan in vertebrate fibroblasts.

Oncogene 1:297-310.

17. Courtneidge, S. A. 1985. Activation of the

pp60csrc

kinase

by

middle T

antigen binding

or

by

dephosphorylation.

EMBOJ. 4:1471-1477.

18. Courtneidge, S. A., and A. Heber. 1987. An 81kd

protein

complexed

with middle t

antigen

and

pp60-src:

a

possible

phosphatidylinositol

kinase. Cell 50:1031-1037.

19. Courtneidge, S. A., and A. E. Smith. 1983.

Polyoma

virus

transforming protein

associates with the

product

ofthe c-src

cellular gene.Nature

(London)

303:435-439.

20. Courtneidge, S. A., and A. E. Smith. 1984. The

complex

of

polyoma

virus middle-T

antigen

and

pp60c-src.

EMBO J. 3: 585-591.

21. Coussens, P. M., J. A.

Cooper,

T. Hunter, and D.

Shalloway.

1985. Restriction ofthe in vitro and in vivo

tyrosine protein

kinaseactivities of

pp60c-sc

relativeto

pp6o-src.

Mol. Cell. Biol. 5:2753-2763.

22. Cross,F.R.,and H.Hanafusa.1983.Local

mutagenesis

ofRous

sarcomavirus: the

major

sites of

tyrosine

phosphorylation

of

pp6Oc-src

are

dispensible

fortransformation. Cell34:597-607.

23. Hunter, T.,and B. W.Sefton. 1980.

Transforming

gene

product

of Rous sarcoma virus

phosphorylated tyrosine.

Proc. Natl. Acad. Sci. USA 77:1311-1315.

24. Iba,H.,F. R.Cross,E.A.Garber,and H.Hanafusa.1985.Low level of cellular

protein

phosphorylation by nontransforming

overproduced p60c-sc.

Mol.Cell. Biol. 5:1058-1066.

25. Jove,R., S. Kornbluth,and H. Hanafusa. 1987.

Enzymatically

inactive

pp60csrc

with altered

ATP-binding

site is

fully

phos-phorylated

on its

carboxy-terminal regulatory region.

Cell 50: 937-943.

26. Kaplan,D. R., B.Bockus, T.M. Roberts, J. Bolen,M. Israel,

and B. S. Schaflhausen. 1985.

Large-scale

production

of

poly-omamiddle T

antigen by using

genetically

engineered

tumors.

Mol.Cell. Biol.5:1795-1799.

27. Kaplan,D.R.,M.Whitman,B.

Schaffhausen,

D.C.Pallas,M.

on November 10, 2019 by guest

http://jvi.asm.org/

White,L. Cantley, and T. M. Roberts.1987. Common elements in growth factor stimulationandoncogenic transformation: 85kd phosphoprotein and phosphatidylinositol kinase activity. Cell 50:1021-1029.

28. Kmiecik, T. E.,andD.Shafloway. 1987.Activation and

suppres-sion of pp60c-src transformingability bymutation ofitsprimary sites of tyrosinephosphorylation. Cell49:65-73.

29. Kornbluth, S.,R. Jove, andH. Hanafusa.1987.Characterization of avian and viral pp60src proteins expressed in yeast. Proc. Natl. Acad. Sci. USA 84:4455-4459.

30. Kornbluth, S., M. Sudol, and H. Hanafusa.1987.Association of polyomavirus middle-T antigen with c-yes protein. Nature

(London)325:171-173.

31. Kypta, R. M., A. Hemming, and S. A. Courtneidge. 1988. Identificationandcharacterization of pp59fyn(asrclike protein tyrosine kinase) innormaland polyomavirustransformedcells. EMBOJ.7:3837-3844.

32. Levinson, A. D., H. Oppermann, H. E. Varmus, and J. M. Bishop. 1980. Thepurified product of the transforminggeneof

avian sarcoma virus phosphorylates tyrosine. J. Biol. Chem. 255:11973-11980.

33. Pallas, D. C., V. Cherington, W. Morgan, J. DeAnda, D. Kaplan, B. Schaffhausen, and T. M. Roberts.1988.Cellularproteinsthat associate with themiddle and smalltantigensofpolyomavirus. J. Virol.62:3934-3940.

34. Pallas, D. C., C. Scley, M. Mahoney, E. Harlow, B. S. Schaff-hausen, and T. M. Roberts. 1986. Polyomavirus smalltantigen: overproduction inbacteria andutilization formonoclonal and polyclonal antibodyproduction.J. Virol.60:1075-1084. 35. Pallas,D. C., and F.Soloman. 1982.Cytoplasmic microtubule

associated proteins: phosphorylationatnovel sites is correlated with their incorporationinto assembled microtubules. Cell 30: 407-414.

36. Parsons, S. J., D. J. McCarley, C. M. Ely, D. C.Benjamin,and T.Parsons. 1984. MonoclonalantibodiestoRoussarcomavirus

pp6O-srcreactwithenzymatically activecellular pp6Osrcof avian andmammalian origin.J.Virol. 51:272-282.

37. Patschinsky, T., T. Hunter, F. S. Esch, J. A. Cooper, andB. M.

Sefton.1982.Analysis of thesequenceof amino acids surround-ingsites oftyrosine phosphorylation. Proc. Natl. Acad. Sci. USA 79:973-977.

38. Patschinsky, T., T. Hunter, and B. M. Sefton. 1986. Phosphor-ylationof thetransforming protein ofRoussarcomavirus:direct

demonstration of the phosphorylation ofserine17and identifi-cation of an additional site of tyrosine phosphorylation in

pp60v-srcofPragueRous sarcomavirus. J. Virol. 59:73-81. 39. Piwnica-Worms, H., D. R. Kaplan, M. Whitman, and T. M.

Roberts. 1986. Retrovirus shuttle vector for study of kinase activities ofpp6Oc-src synthesized in vitro and overproduced in vivo. Mol.Cell. Biol. 6:2033-2040.

40. Piwnica-Worms, H., K. B. Saunders, T. M. Roberts, A. E. Smith,andS. H.Cheng. 1987.Tyrosinephosphorylation regu-lates thebiochemical and biologicalproperties of pp6Ocsrc. Cell 49:75-82.

41. Raptis, L.,H.Lamfrom,and T. L.Benjamin.1985. Regulation of cellularphenotype andexpression of polyomavirus middlet antigenin ratfibroblasts. Mol. Cell. Biol. 5:2476-2485. 42. Schaffhausen, B. S., and J. Benjamin. 1981. Comparison of

phosphorylation oftwopolyomavirus middleTantigens in vivo and in vitro. J. Virol. 40:184-196.

43. Schaffhausen,B.S.,B.J. Bockus,K. L.Berkner,D.Kaplan,and T. M. Roberts. 1987. Characterization of middle T antigen expressed by usinganadenovirusexpression system.J. Virol. 61:1221-1225.

44. Schultz,A.M.,L. E.Henderson,S.Oroszlan,E. A.Garber,and H. Hanafusa. 1985. Aminoterminalmyristylation of the protein kinase p60srC, aretroviral transforming protein. Science 227: 427-429.

45. Simon, A.M.,B.Dress,T.Kornberg, andJ.M. Bishop. 1985. The nucleotide sequence and the tissuespecific expression of Drosophilac-src.Cell 42:831-840.

46. Smart, J. E.,H.Oppermann,A. P.Czervlofsky,A. F.Purchio, R.L.Erikson, and G. M.Bishop. 1981.Characterization of sites fortyrosinephosphorylation in the transformed protein of Rous sarcoma virus (pp6ov-src) and its normal cellular homologue (pp6Oc-src). Proc.Natl.Acad. Sci. USA 78:6013-6017. 47. Smith, A. E., and B. K. Ely. 1983. The biochemical basis of

transformationby polyoma virus. Adv. Viral Oncol. 3:3-30. 48. Snyder,M.A., J.M.Bishop, J.P.McGrath, and A. D.Levinson.

1985.AmutationattheATPbinding site of pp6Ov-src abolishes kinaseactivity, transformation, and tumorigenicity. Mol. Cell. Biol. 5:1772-1779.

49. Summers,M.D.,and G. E. Smith. 1987.Amanual of methods for baculovirus vectors and cloned insect cell culture proce-dures. TexasAgricultural Experiment Station, College Station. 50. Yonemoto, W., M.Javis-Morar, J. S.Brugge, J. B. Bolen, and M. A. Israel.1985.Tyrosine phosphorylation within the amino-terminal domain ofpp60c-src molecules associated with polyoma virus middle-sizedtumorantigen. Proc. Natl. Acad. Sci. USA 82:4568-4572.

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