Transformation by avian sarcoma viruses leads to phosphorylation of multiple cellular proteins on tyrosine residues.

0022-538X/82/050742-06$02.00/0

Transformation

by Avian Sarcoma Viruses Leads to

Phosphorylation

of

Multiple

Cellular Proteins on

Tyrosine

Residues

KARENBEEMON,t* THOMAS RYDEN,ANDE. ANN McNELLY TumorVirologyLaboratory, The Salk Institute, San Diego, California 92138

Received 8 October1981/Accepted 14 December 1981

Phosphoamino acidcompositionsweredetermined for 10 size classes ofcellular proteins, separated by electrophoresis through one-dimensional sodium dodecyl sulfate-polyacrylamide gels. Phosphotyrosine-containing proteins were observed in uninfected chicken embryo fibroblasts in every size class analyzed, ranging from approximately 20,000 to greater than 200,000 daltons. Transformation of chickenembryo fibroblasts by RoussarcomavirusorPRC II aviansarcomavirus led toincreases inphosphorylation of proteins attyrosine residues in all ofthese sizeclasses.A

large

fraction of the

phosphotyrosine-containing protein

molecules observedin Rous sarcomavirus-transformed cellswaslarger than 100,000daltons with a second broad peak in the 35,000- to

60,000-dalton

range. This study suggeststhatthereare anumberof substrates of viralorcellulartyrosine-specific protein kinases, which havenotyetbeen identified by other methods.

The src gene of Rous sarcoma virus

(RSV)

encodes a

protein

pp6Osrc

that is

responsible

for neoplastic transformation (4). Purified prepara-tions of pp6Osrc possess

protein

kinase activity specific fortyrosine residues (6, 7, 10,

15, 16, 20,

21). In uninfected chicken embryo fibroblasts labeled with

32p

under steady-state

conditions,

only about0.03% ofthe acid-stable

phosphory-lated amino acids in protein are phosphotyro-sines;however,this leveloftyrosine phosphor-ylation of cellular protein was observed to be 6- to10-foldhigher after transformation by RSV (15, 28). Studies with cells infected by tempera-ture-sensitive mutants of RSV show that this phosphorylation is readily reversibleupon

shift-ing

the temperature, suggesting that at least someof these additional proteinmolecules phos-phorylated at tyrosine residues are directly

phosphorylated

by

pp6Osrc

(28).Althoughnot all

transforming viruses have been shown to induce elevated levels of tyrosine phosphorylation of proteinsnor to encode proteins which either are themselvesor are associated with tyrosine-spe-cific protein kinases (28), several retroviruses appear to be very similar to RSV in having these two properties. This class of viruses includes Abelsonmurine leukemia virus, Snyder-Theilen and Gardner-Amstein feline sarcoma

viruses,

and the defective avian sarcoma viruses (ASV) whichhave oncogenes distinct from the RSV src gene: Fujinami sarcoma virus, PRC II, and Y73 (1, 2, 13, 18, 22, 23, 29-31).

t Present address: Department of Biology, The Johns Hop-kins University, Baltimore, MD 21218.

To further our understanding of the interac-tions between these viruses and their host cells resulting in transformed

phenotypes,

it is impor-tant to

identify

the cellular target protein or proteins which are

phosphorylated by

these

ty-rosine-specific

protein

kinases. Since

phospho-tyrosine

isaveryrare

protein

modificationeven

in transformed cells, it originally seemed that veryfewdifferent

proteins

might

be phosphory-lated at tyrosine residues,

making

it easy to

identify

potential

substrates of the

protein

ki-nase activity of pp6Osrc. Phosphotyrosine-con-taining proteins labeledin vivohavepreviously been identified

by

two-dimensional

gel

electro-phoresis (8, 9, 25, 26) and

by

analysis

of

specifi-callyimmunoprecipitated

proteins

(27). In addi-tion,severalproteins have beenphosphorylated at tyrosine residues in vitro in the presence of

pp6OSrc

(5, 7); however, in some cases these

proteins are not similarly modified in RSV-transformed cells (27).

In this study the phosphotyrosine-containing proteinspresent in cells were labeled with

32p;

in vivo and then quantitatively analyzed after frac-tionation into size classes by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophore-sis. Although this technique affords much less resolution of individual proteins than does two-dimensionalgel analysis, it is more quantitative andcircumventsproblems due to specific losses of proteins or to lack of detection of some proteins. Specific issues which are addressed include: (i)the minimumnumberof phosphoty-rosine-containing proteins in uninfected

ASV-742

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VOL.42,1982

transformed cells, (ii) the approximate size of the most abundant phosphotyrosine-containing proteins in transformed cells, (iii) the relative abundance of previously identified potential substrates of ASV protein kinases, and (iv) a comparison ofsubstrate specificities in vivo of tyrosine-specificprotein kinases present in unin-fected cells with those in cells transformed by RSVand byPRC II ASV.

Since phosphotyrosine represents a very small fractionof thephosphorylatedamino acids inprotein, it was first necessarytooptimize the amountof

32Pi

resultinginphosphotyrosine after in vivo labeling. Thiswasaccomplishedby two modifications of published procedures (15). It hadpreviouslybeenshown that altering the time of radiolabeling of the cells from 18 to 2 h resultedinafourfoldgreater fraction of the total label in phosphoamino acids derived from pro-teinpresent asphosphotyrosine(17),apparently owingto afasterturnoverrateforphosphate on tyrosine than for thatonphosphoserineor phos-phothreonine.Wefound that labelingcellsfor 4 hledtosignificantly moreincorporationof

32p,

while maintainingthe higher relativeabundance

of

label in phosphotyrosine. The second

modifi-cation

was to change the time of incubation of

proteins with 6 N

HCl

at 110°C. Since the tyrosine-phosphatebond is somewhat more la-bile in acid than that of serine-phosphate or threonine-phosphate (24), prolonged acid treat-mentleadstopoorerrelativerecoveries of

phos-photyrosine.

Wefound that1 hof acid

hydroly-sis led to a higher relative abundance of

phosphotyrosine

recovered, although

1hof

hy-drolysis

ledto poorertotal levels of

phospho-aminoacidrecoverythandid2hof

hydrolysis.

Phosphoproteins

present in chicken embryo

fibroblasts infected with

Schmidt-Ruppin,

sub-group A

(SR-A)

RSV were

initially

compared

with those inuninfected cells by

SDS-polyacryl-amide

gel

electrophoresis.

Both cultures were

labeled with

32p;,

solubilized

in radioimmune

precipitation buffer

(RIPA) (2),

clarified

by

cen-trifugation, and the

supernatants were

electro-phoresed

on

discontinuous

SDS-polyacrylamide

gels.

The

insoluble

pellet

was not

analyzed,

although it

has

previously

been shown that the

RIPAsupernatant contains about thesame frac-tion of

phosphotyrosine

as does the whole cell

(28).

Therewas nodifference in the

phosphopro-teins present in the two cell types that was

discernible

by

this type of

analysis (data

not

shown);

thus,

RSV didnotappearto

perturb

the

gross

pattern

of cellular

phosphorylation.

This is not too

surprising,

since the RSV-coded

protein

kinase

activity

is

specific

for

tyrosine,

and this is a very rare modification even in RSV-trans-formed cells.

Toobserve

specific

effects of

pp60Src

andany

NOTES 743

other tyrosine-specifickinasesactivatedby RSV transformation,

32P-labeled

lysates were first electrophoresed through SDS-polyacrylamide gels, and the gels were then sectioned into 10 size classes. Proteins were eluted and their phosphorylatedamino acids were analyzed after partial acid hydrolysis by a two-dimensional electrophoretic separation as previously de-scribed (15, 28). The acid-stable phosphoamino acids were identifiedby theircomigration with

internal

markers. Afterquantitation of

radioac-tivity recovered comigrating with each of the phosphoamino acids, the percentage of phos-photyrosine recovered in each size class of proteinwas determined and is plotted in Fig. 1 for uninfected cells and for cells infected with SR-A RSVorwith PRCIIASV. Differences in the efficiency ofrecovery of proteins from one size class to the next would not affect these results since each gel fraction was analyzed independently.

Intheuninfected

cells,

phosphotyrosine-con-taining proteins were observed in every size class analyzed, ranging from approximately 20,000 to greater than 200,000 daltons. The greatestrelative abundance ofphosphotyrosine was present in proteins having sizes ranging from about

35,000

to60,000 daltons (greater than 0.15% perfraction). The lowest relative abun-dance of phosphotyrosine was in proteins of 20,000to35,000 daltons (< 0.10%perfraction). Becauseof the

small

amountofphosphotyrosine in the uninfected cells, quantitation was most difficult here.

Acomparisonbetween these results and those ofa parallel experiment using chicken embryo

fibroblasts

transformed by SR-A RSV is also

displayed in Fig. 1. Again, every gel fraction analyzed contained protein

phosphorylated

at

tyrosine;

however, such

proteins

were present

in greater relative abundance here than in the

corresponding

protein

size class derived from

uninfected cells. More than 0.5% ofthe total

phosphoamino

acids recovered were

phospho-tyrosines

in fractions

containing

proteins

of

25,000to30,000 and35,000to

80,000

daltons. In

iis

experiment,

the

highest

relative abundance

of

phosphotyrosine

was found in

proteins

of

40,000to

50,000

daltons.

However,

some

varia-tion was

observed

from one

experiment

to

an-other. Three

different

experiments

are

com-pared, all

using

SR-A

RSV-transformed

chicken embryo fibroblasts

(Table 1).

In the first two

experiments,

labeling

wasfor4handtreatment

withHCIwasfor 1 h.However,in

experiment 3,

cells

were labeled for 16 h and

proteins

were

treatedwith HCI for 2 h.Differencesinboththe absolute and relativelevels of

phosphotyrosine

recovered per fraction were

observed

in these

experiments.

This may bedue eitherto

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TABLE 1. Relative abundanceofphosphotyrosine

in individualsize classesofprotein from RSV-transformedcells

Protein size %Phosphotyrosine per gel fraction (xlO-3daltons) Expt 1 Expt 2 Expt 3

>150 0.28 0.48 NDa

100-150 0.45 0.26 0.19

80-100 0.46 0.43 0.17

60-80 0.51 0.41 0.37

50-60 0.60 0.40 0.39

40-50 0.82 0.67 0.65

35-40 0.51 0.55 0.65

30-35 0.42 0.26 0.35

25-30 0.60 0.84 0.47

20-25 0.50 0.22 0.10

aND, Not

determined.

mental variability, for example, differences in theslicing of fractions from geltogel,oritmay representactualheterogeneity existingbetween

differentpopulations of RSV-transformed cells. Inall of theseexperiments, the highestrelative amounts of phosphotyrosine per gel fraction

appeared in the middle of the gel region

ana-lyzed, encompassing proteins with sizes of

ap-proximately 35,000 to 60,000 daltons.

Interest-ingly, the uninfected chicken embryo fibroblasts also had the highest levels ofphosphotyrosine

perfraction in these size classes, although the

overall profiles are not identical for uninfected and RSV-infected cells.

Inchicken embryofibroblasts transformed by PRCIIASV,therelativephosphotyrosinelevels

were highestin the samefractions asfor RSV,

with theexceptionthat the 100,000-to 150,000-dalton fraction from PRC II-transformed cells contained amuch greater amount of phospho-tyrosine (Fig. 1). It is likely that at least a

largefractionof thephosphotyrosine in this size class is duetothe PRC II-encoded polyprotein P105gagfps, which has been showntobe heavily phosphorylated in vivo attyrosine residues (2,

22; unpublisheddata). P105 was also themajor

phosphotyrosine-containing protein detected in

PRC II-infected cells by two-dimensional gel

electrophoresis (9).

Analysis of Snyder-Theilen feline sarcoma

virus-transformedmink lungcellsbythis

proce-duregaveresults ratherlike those with PRCII,

despite the difference incelltypes.Thegreatest

percentage of phosphotyrosine-containing

pro-teinswasfound inafractionwith sizesof83,000

to 100,000 daltons, which includes the virus-coded presumptive transforming protein P85

that has several phosphotyrosine residues in

vivo (Beemonand McNelly,unpublished data).

In addition, relatively large levels of

phospho-tyrosine-containing proteins were observed in

the

37,000-

to

68,000-dalton

size classes (data

not

shown).

To quantitatively analyze the fraction of the total phosphotyrosine recovered in proteins of varioussizes present in cells transformedby SR-A

RSV,

the recovery ofradioactivity from the gel was monitored. When electroelution was

performedfor 20h,itwasfoundthat radioactiv-itywasobtained from all sizeclasseswith rela-tively equal efficiency. The proportion of the totalamountofphosphoprotein inacell

present

in each size class was determined by summing recoveriesofphosphoaminoacids from uniform samples ofeluted proteins. The percentage of totalphosphotyrosinerecoveredwasthen calcu-lated for each size class. Figure 2 shows the resultof suchananalysisofphosphoproteinsin RSV-transformedcells. These dataarefrom the

same experiment as in Fig. 1 and in Table 1, experiment 1. Although most of the total phos-photyrosine recovered was present in proteins of35,000to60,000 daltons,there was a surpris-ingly large amountof the totalphosphotyrosine presentinproteinsof100,000to150,000daltons and even larger. If there were a bias in the efficiency ofelution,it should favor the smaller proteins, making this result even more striking. These data suggest that a relatively large number ofdifferent cellular proteins are

phos-phorylated at tyrosine residues as a direct result oftransformation by RSVorPRC II and estab-lish a minimum number of 10 potential

sub-strates of the various tyrosine-specific protein kinases in a cell. The actual number of such proteins isprobably muchlarger,since the sepa-ration method used here provided no resolution between proteins of similar size.Thus, the sub-strate specificity oftyrosine-specific protein ki-nases appears to bebroader than was suggested by experiments in which potential substrates werefractionated by two-dimensional gel elec-trophoresis. Such studies detected only a few phosphotyrosine-containing proteins in trans-formed cells having sizes of 36,000, 28,000, 43,000and46,000 daltons(8, 9, 25,26).

Themostsurprisingfinding of this study was thelarge number ofphosphotyrosine-containing protein molecules having sizes greater than 100,000 daltons in RSV-transformed cells. The identity or absolutenumberofthese proteins is notknown. Suchproteins accountedfor approx-imately 30% of the phosphotyrosine in RSV-transformed cells, as determined in this

assay,

but had notbeen detected by two-dimensional gel analyses. Thisdiscrepancy could be due to the presence of a large number of different

phosphotyrosine-containing

proteins (each of

low abundance), to chargeheterogeneity, or to the inability of these large proteins to be re-solved by isoelectric focusing. Vinculin, a

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0.8r

0.71-0.6

0.5

0.41-

0.31-

0.2-

0.1-L

100 80 60 5040 35 302520

MWx

io-3

PRCII

I

150 100 80 60 50 40 35 30 25 20

MW x 10-3

FIG. 1. Relative abundance of phosphotyrosineinindividual size classes of cellularproteins.Uninfected, SR-ARSV-transformed, and PRC II-transformedchicken embryofibroblastswere grown on35-mmculture dishes

andlabeled with 2to6mCi32P1 (ICN) for4h (3). Cellswerelysedat4°Cin radioimmune precipitationbuffer, clarifiedbycentrifugation (2), and the supernatants werebroughtto afinalconcentration of 2% SDS-10%

2-mercaptoethanol-50 mM Tris-hydrochloride(pH 6.8)-10%glycerol. Afterbeing boiled for 2min,sampleswere

electrophoresedondiscontinuous SDS-polyacrylamidegels,usingthebuffers of Laemmli (19). The resolvinggel

wasapproximately 13cmhigh, 14cmwide, and 2mmthick and contained 0.1% SDS, 15%acrylamide, and 0.09%obisacrylamide. After electrophoresis, together with molecular weight markers (Bio-RadLaboratories), wetgelswereautoradiographed for 5to20min. Subsequently,the gelswereslicedhorizontally into10fractions, each approximately 1 cm high. Both the autoradiograms and the molecular weight markers were used to standardize fraction boundaries from geltogel. Proteinswereelectroeluted from each gel slice in 5to10ml of buffercontaining 0.1% SDS, 0.05 MNH4HCO3, and 5% ,B-mercaptoethanol for 6to20 h at100to 200 mA. Recovery was monitored by themeasurement of Cerenkov radiation. Proteins wereprecipitated with 20%o trichloroacetic acid, and the proteinpelletwaswashed withchloroform-methanol(2:1). Proteinswerepartially hydrolyzed with 200 ,ld of 6 N HCI for 1 hat100°C.Phosphoamino acidswereresolvedby two-dimensional electrophoresisoncellulosethin-layer plates (E. Merck Labs), firstatpH 1.9andsubsequentlyatpH3.5bythe procedure of Hunter and Sefton (15); 0.5 Fg of each phosphoamino acid marker (phosphoserine and phosphothreoninewere obtained fromSigma ChemicalCo.; phosphotyrosine was agiftfrom T.Hunter) was coelectrophoresedwith the radioactivesamplesand visualizedby stainingwithninhydrin.Afterautoradiography of thethin-layer plates, phosphoaminoacidscomigratingwiththemarkerswereeluted withpH1.9buffer,and theirradioactivitywasquantitated byscintillationcounting.The relativeabundance ofphosphotyrosineineach gel fraction is plottedas afunctionof the molecularweight (MW) of theproteinsin that sizeclass.

130,000-dalton cytoskeletal protein, has also

beenidentifiedasapotentialsubstrate ofpp60(rc

(27). However, the number of molecules of

vinculinphosphorylated ontyrosine residues is

muchtoolow (B. Sefton, personal

communica-tion) to account for any significant fraction of

the phosphotyrosine-containing proteins larger than 100,000 daltons detected in this study. In

PRC II-transformed cells, the large amount of

phosphorylation of the viral protein P105 at

tyrosine residues (Fig. 1; 2, 9, 22) obscures

detectionbythis method ofphosphorylated

cel-lular proteins in this sizerange, making it diffi-culttocomparetoRSV-transformed cells.

The 36,000-dalton protein, which appearsby

two-dimensional gel analysis to be the major substrate of RSV transformation-induced phos-phorylation (also of transformation by several

otherretrovirusesorof activation ofthe epider-mal growth factor receptor-associated kinase

activity [8, 9, 11, 12, 14, 25, 26]),accounted for

no morethan15%of the totalphosphotyrosine

on proteins in this analysis (Fig. 2). It is not

knownwhether there areadditional

phosphoty-c

0

._

C) 0

I.-

L-8

7

6

C

I-S

-4

0-R 3

2

NOTES 745

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746 NOTES

20

I-.

-J

F--J

al

1O0

0'

L

150 10080 60 50 40 35 30 25 20 MWx103

FIG. 2. The relative abundance of phosphotyro-sine-containing proteins of different sizes in SR-ARSV-transformed cells. The data from RSV-trans-formed cellsshown in thelegendtoFig.1 and Table1, experiment 1,has beenreplottedhere todemonstrate theproportionof the totalphosphotyrosine-containing proteinsrepresentedin eachsize class.

rosine-containing proteins presentin the

35,000-to40,000-dalton size classwhich would diminish

further the fraction of totalcellular phosphotyro-sine present in the 36,000-dalton protein. Both

RSV- and PRC II-transformed cells contained

several substrates of tyrosine-specific protein kinases in the30,000-to60,000-dalton size class.

Theseproteinsarelikelytoincludethose previ-ously identified by other methods (8, 9, 15).An

exception to the apparent similarity between

RSV- andPRCII-transformed cellsin phospho-tyrosine-containing proteins has been observed invinculin, which isnotphosphorylated in PRC II-transformed cells (27).

Since uninfected cells are likely to contain several tyrosine-specific protein kinases, in ad-ditiontopp60c-src (the cellular homolog of viral pp60V-src),it isnotpossibletocomparesubstrates

ofkinases encoded byv-srcandc-srcsimply by

comparison of phosphotyrosine-containing

pro-teins in transformed and nontransformed cells. Itisnotknown, furthermore,whether kinases in

additiontopp60-src itselfareresponsiblefor the

elevated level of tyrosine phosphorylation of

proteins in RSV-transformed cells. However,

the profiles of phosphotyrosine-containing

pro-teins, although not identical, are not grossly

dissimilar between uninfected and

RSV-trans-formed cells. These results are consistent with

thehypothesis that the various viraland cellular

tyrosine-specific kinases sharemanyof thesame

protein substrates with perhaps only

quantita-tive differences in preference for different sub-strates.

Inconclusion,itappearsthat there are several potential substrates of ASV-codedor-activated protein kinases which needtobe further charac-terized,particularly those in the high-molecular-weight region. The apparently large number of substrates ofprotein kinases makes it easier to explainthe

pleiotropic

natureof the transformed phenotype. However, it makes it much more difficult to ascertain which of these substrates are essential for transformation.

This workwas supportedbyPublic Health Service grant CA-23896 from the National Cancer Institute.

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