A novel oncogene, v-ryk, encoding a truncated receptor tyrosine kinase is transduced into the RPL30 virus without loss of viral sequences.

0022-538X/92/105975-13$02.00/0

Copyright© 1992,American Society forMicrobiology

A

Novel

Oncogene, v-ryk, Encoding a Truncated Receptor Tyrosine

Kinase

Is

Transduced

into the RPL30 Virus

without

Loss

of

Viral Sequences

RANJIA, BRUCE J. MAYER, TERUKOHANAFUSA,ANDHIDESABURO HANAFUSA* The Rockefeller University, 1230 York Avenue, New

York,

New York10021-6399

Received12 May 1992/Accepted 17 July 1992

The RPL viruses are acute oncogenic avian retroviruses isolated from chicken tumors. We carried out a genetic analysis of three of theviruses, RPL25, RPL28, and RPL3O. While RPL25 and RPL28 were shown to contain the erbB oncogene, RPL3O appeared to contain a novel protein tyrosine kinase oncogene. This gene,

v-ryk,wascloned and sequenced. The v-ryk oncogene contains a 1.39-kb nonretroviral sequence that includes

atyrosine kinasedomain which was inserted into the viral envelope protein gp37-coding region and fused in frame with upstream gp37 to generate a

p69m,37-y

fusion oncoprotein. Unlike that of other

acutely

transforming retroviruses, transduction of the v-ryk gene into RPL30 did not result in deletion of viral sequences. Sequence

analysis

suggested that v-Ryk is more homologous to receptor-type tyrosine kinases than

tononreceptor-typekinases. By reconstitution ofavirus fromits cDNA, the v-ryk oncogene has been shown to be

fully

responsible for the transforming activity of theRPL30virus.Antibodies specific to v-Ryk immuno-precipitated the v-Ryk oncoprotein from cells transformed by theRPL30 virus. The v-Ryk protein was shown

to be first synthesized as a 150-kDa precursor and then cleaved into the mature 69-kDa gp37-Ryk fusion protein, both parts of which were found to be localized to the membrane fraction. As expected from the sequence of v-Ryk, immunoprecipitates of v-Ryk from RPL3O-transformed cells were found to display a protein tyrosine kinase activity in vitro, and the levels oftyrosine-phosphorylated proteins are elevated in v-yk-transformed cells.

Acute oncogenic retroviruses have proven to be a rich

source of various categories of oncogenes. More than 25 viral oncogenes have beenmolecularly cloned and studied

(8). Sinceoncogenic retroviruses are the naturally selected

geneticsystemin whichimportantgenesinvolved in regula-tion ofproliferationanddifferentiation in normal cells were targeted, analysis of previously uncharacterized viral onco-geneswillbe valuable inidentifying novel cellular genes in these pathways that otherwise would be difficult to find.

Furthermore, identification ofnewoncogenesinretroviruses

notonly providesanimmediate indication that the genecan

be oncogenic, but it may also provide insights into the

mechanism of activation of the oncogenic potential of its

cellularhomolog.

The RPL25, RPL28, and RPL30 viruses are three acute

oncogenic avian retroviruses that were previously

unchar-acterizedgenetically. These viruseswerefirst isolated from chickentumors (21). Although theycause tumors in

chick-ens (21), the transforming genes present in these viruses havenotbeenidentified.Here,wereport thegenetic analy-sis of these three RPL viruses and themolecularcloningand

characterization of RPL30, which was found to contain a

novel viral oncogene. This viral oncogene isa newmember

of the protein tyrosine kinase gene family and appears to

encode a truncated receptor-type tyrosine kinase. It is

interestingthat unlike all oncogenicretroviruses studied so far, a cellular gene was transduced by the RPL30 virus without loss of any viral sequence.

*Correspondingauthor.

MATERIALS AND METHODS

Viruses, cells, and virus infection.TheRPL25,RPL28, and RPL30 viruses (21) were kindly provided by L. B. Crit-tenden of the Avian Disease andOncologyLaboratory,East

Lansing, Mich. The three viruseswere injectedinto newly

hatched chickens, and acutely transforming virus stocks

wererecovered from thesarcomasinduced in the chickens.

Preparation and maintenance of chicken embryo fibroblast

(CEF)

cultures and infection andpropagationof viruses have already been described (23). Briefly, CEF were prepared

from 11-day-old embryos and secondary cultures were

in-fected with the virus stock.

Oncogene blotscreenofoncogenicviruses.Probes

specific

for 19 known viral oncogeneswere preparedfrom

molecu-larly cloned DNA, and they contained no retroviral

se-quences. The viral oncogene probes usedwere as follows: felinefms, 1.4-kb PstI fragment from pSM3 (14); chicken ros,0.85-kbEcoRI-PvuII fragmentfrompROS (32);chicken

erbB, 0.56-kb BamHI fragment from pERB5

(51);

murine

abl, 1.6-kb SacI-HindIII fragment from pAB3sub3

(35);

feline fgr, 0.93-kb

SmaI-KpnI

fragment from pGR-FeSV (31);chicken src, 0.87-kbPvuII fragmentfrompTI107(44);

chicken

fps,

0.41-kb BamHIfragment frompBR-FO4 (20);

chicken yes, 0.43-kb BamHI

fragment

frompMR-YA

(42);

murine mos, 0.92-kb AvaI-HindIII fragment from pCMOS

(46); chicken mil, 0.58-kb BamHI-SphI fragment from

pMBS28(27);ratH-ras, 0.46-kb EcoRIfragmentfrompBS9

(17); rat K-ras, 0.38-kb SacII-XbaI fragment from

pHiHi3

(18); woollymonkey sis, 0.92-kbPstI-XbaI

fragment

from

pSSV (13); chicken

erbA-erbB,

1.2-kb SalI-BamHI

frag-mentfrompERBA(11); turkeyrel,0.97-kbEcoRIfragment

from

pEcoRI-rel

(6); chicken ski, 1.2-kb SacI

fragment

from

pSRski-1

(29); murine

fos,

1.0-kb PstI

fragment

from

pFOS1

(10);chicken myc, 0.8-kbClaI-EcoRI

fragment

from

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5976 JIA ET AL.

pCMYC1 (2); chicken myb, 0.35-kb EcoRI-SalI fragment

frompVM2 (28).A3.6-kb EcoRIfragmentof Roussarcoma

virus (RSV) (non-src region) from pSRA2was used as an

avian retrovirus positivecontrol(12).

To prepare the oncogene blot, 50 ng of each oncogene

fragment was denatured and blotted onto a nitrocellulose

filter by using a slot blot manifold. This blot was then

hybridizedwith 32P-labeled RPL virus cDNA generatedby

reversetranscription of viralgenomic RNAharvestedfrom infected cell culture media. Medium was collected at 4-h

intervals,prespunto removecells and

debris,

andthenspun for90 minat40,000rpmtopelletthevirus. Theviralpellets

weresuspendedin 10 mMTris-HCl

(pH

7.4)-5mMEDTA,

0.5%sodium

dodecyl

sulfate

(SDS)-1

mgofproteinaseK per ml

(1

ml of extraction buffer per25 mlof

original

medium

collected)andincubatedat37°Cfor 1 h. From 5to10 ,ug of viral RNA was usually recovered from 100 ml of culture

fluid.Theviral RNAswere thensubjectedtoreverse

tran-scription

in the presence of

[a-32P]dCTP

to generate

32P-labeled retroviruscDNAs(37).

cDNAlibraryandcloningof the viral oncogene.Total RNA

from RPL30 virus-transformed nonproducer cells (49) was

isolated, mRNAwas purified, andcDNAwas synthesized

by

standard procedures

(37).

Internal EcoRI sites of the

cDNA were methylated. The ends of the cDNA were

blunted with T4 DNApolymerase and then ligated to syn-thetic EcoRI linkers. The cDNAwas digested withEcoRI andrun on a0.8% agarosegel, andcDNAin thesizerange of 1to15 kbwasrecovered. This cDNAwasligatedtoAgtlO

vector arms to construct an RPL30 cDNA library. The

recombinant bacteriophage were packaged with Gigapack

Gold

Packaging

Extract

(Stratagene,

La Jolla, Calif.) in accordance with the manufacturer's protocol and used to infect

Escherichia

coliC600.

Three DNA probes derived from the gag,pol, and env

regions

of the genome of cloned helpervirus UR2AVwere

used to screen

105 plaques

from the RPL30cDNAlibrary

(see Fig. 2A).

Positive clones were

subjected

to Southern blot

analysis

withprobes derived from other

regions

of the UR2AV genome. The sizes of the cDNA inserts from the clones and the distancebetween the

hybridizing

probes on

the viral genome were compared. Those clones containing

cDNAinsertsthatwerelargerthan thedistancebetween the

hybridizing

probes were subcloned into the pBluescript phagemid

(Stratagene)

by usingstandardtechniques(37)and

sequenced.

DNA sequencing. Complete DNA sequences from both

strands of the clones identified above were determined by

the

dideoxy-chain

termination method

(38)

by using the

Sequenase sequencing

system

(United

States

Biochemical,

Cleveland,

Ohio). Overlapping fragments

for

sequencing

were generated

by

restriction

digestion

and

subcloning.

DNA sequences and deduced amino acid sequences were used to search the GenBank data base by the FASTA program

(33).

Viral plasmid construction. Clones 8-1 and 3-1 (see Fig.

2B)

weredigestedandligatedinto anonoverlappingRPL30 clone

(gp85-5'gp37-ryk-3'gp37-3'

long terminal repeat

[LTR]).

Thisconstruct was thendigestedwith

Sail

(within

gp85)

and

EagI (within

the 3'

polylinker)

andligatedto

SalI-and EagI-digested pSR-REP (9), which is an RSV-derived

retroviralvectorcontaining5'

LTR-gag-pol-gp85

(to the Sall

site).

The

resulting

plasmid construct, pRV-ryk,represents the

molecularly

cloned RPL30virus. Helper virus plasmid

pUR2AVhas been describedpreviously (49).

Transfection. Transfection of CEFwasperformed bythe

calcium phosphate method. Briefly, 4 to 10 ,ug of

plasmid

DNA per 60-mm-diameter dish was digested with EagI

(pRV-ryk)orSacI(pUR2AV), phenol-chloroformextracted,

precipitated with ethanol, and dissolved in 0.5 ml of 1 mM Tris-HCl(pH 8.0)-0.1 mM EDTA. A mixture of 10 ,ug of calf

thymus DNA and 55

p,l

of 2.5 M CaCl2was added to the

plasmid DNA. A mixture of 0.5 ml of 50 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 280 mM NaCl (pH 7.1), and 5 ,ul of 0.15 M Na2HPO4wasthen added dropwise with mild vortexing. This solution was

allowed to stand at roomtemperature for 30min, andthen thefineprecipitatewasaddedto7 x

105

secondary CEF per 60-mm-diameter dish and left for 8 hat37°C. After thattime,

the mediumwasreplaced with fresh medium.

Transformation, colony formation, and animal

tumorige-nicity

assays.Transfected, infected, and mock-treated(same

procedure except that no plasmid was added) cells were

maintained in medium containing 0.375% agar at 37°C. Transformationwasassessedby focus formation. To assay anchorage-independent growth, cells were trypsinized and replated in 10-cm-diameter dishes in 10 ml of medium containing 0.4% agaron alayer of 15 ml of the same medium containing 0.7% agar. Theplates were incubated at 40°C.

Tumorigenicity was assayed by injecting 0.1 ml of virus intothewing webs of newborn (7 days posthatching) White Leghorn chickens (SPAFAS, Inc.).

Generation of specific antiserum. The C-terminal unique region of v-Ryk was expressed in E. coli BL21 (DE3)pLysS

by using the T7 RNA polymerase expression system (36,41).

An 897-bp Sau3AI fragment containing the region from amino acid 463tothe C terminuswascloned intoa compat-ible BamHI site inthe linker region of the pET-3c vector. Plasmid-bearing bacteria were induced with

isopropyl-P-D-thiogalactopyranoside for 2 h, spun out, and lysed on ice by sonication in RIPA buffercontaining 1 mM phenylmethyl-sulfonyl fluoride. Proteins in the lysate were then separated by SDS-12% polyacrylamide gel electrophoresis (PAGE). The v-Ryk protein band was excised from the Coomassie blue-stained gel, washed with 10% methanol, dried, and homogenized in phosphate-buffered saline (PBS). Approxi-mately 100p,gofv-Ryk proteinwasemulsifiedwith Freund's complete adjuvant and usedtoimmunizerabbits,and 50 ,ug ofproteinwasused for boosterinjections. Preimmune serum

wascollected for control experiments.

Thespecificity of the antiserum was tested by an antigen

competition assay.v-ryk-transformed CEFwere labeled in

vivo with

32Pi

for 8 h(19) and thenlysed in RIPA buffer (see

below). Preimmune or immune serum(20 ,ul)was

preincu-bated in 120

pl

of RIPA buffer withorwithout 144p,gof the immune antigenorcontrolantigen(vector-bearingbacterial

lysates) for 3 hat 4°C and thenused to immunoprecipitate 14.4 p,g of32P-labeled cell lysate (see below).

Immunopre-cipitated proteinswere analyzed bySDS-7.5% PAGE.

Immunoprecipitation and in vitro kinase assay. Cell

cul-tures werewashed with ice-coldTris-glubuffer(25mM Tris, 137 mMNaCl,5 mMKCl, 0.7 mMNa2HPO4, 0.1%glucose, pH 7.4), and the cells were scraped off the plate, quickly

centrifuged, and frozen in a dry ice-ethanol bath. Frozen

pellets were suspended in RIPA buffer (10 mM Tris-HCl,

(pH 7.4), 5 mM EDTA, 150 mM NaCl, 10% glycerol, 1%

TritonX-100, 1%sodiumdeoxycholate, 1%Trasylol, 1 mM

phenylmethylsulfonyl fluoride,1 mMsodiumorthovanadate, 0.1 mMsodiummolybdate),incubatedonice for 20 min with

periodicshaking, and spun inamicrocentrifugeat4°C, and the supernatants were recovered. Protein concentrations

wereassayed witha ProteinAssay Kit(Bio-Rad Laborato-J. VIROL.

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THE v-ryk KINASE ONCOGENE

ries, Richmond, Calif.), and equal amounts of proteins were used in the subsequent assays. For immunoprecipitation, antibodies were added to the above-described cell lysates and incubated on a rotating wheel at 4°C for 1 h and then protein A-Sepharose (Pharmacia, Piscataway, N.J.) was added and the incubation was continued for another 30min. Theimmunoprecipitates were washed three times with RIPA buffercontaining 300 mM NaCl and twice with RIPA buffer containing 10 mM NaCl and then used directly either for SDS-PAGE analysis or for in vitro kinase assays.

For invitro kinase assays, the immunoprecipitates were washed twice with the kinase reaction buffer (see below) instead of RIPA buffer containing 10 mM NaCl, and then each was suspended in 30 ,ul of kinase reaction buffer (50 mMHEPESbuffer[pH7.5],10 mMMnCl2).Atotal of 5

pCi

of [-y-32P]ATPwas added to each sample, and it was

incu-batedat30°C for 10 min. Kinase reactions were terminated

by washingthe samplestwice with RIPA buffer containing

10mMNaCl and then subjected to SDS-10% PAGE. Rain-bow markers(14.3 to 200-kDa range; Amersham, Arlington

Heights,Ill.) were used as molecular weight standards in all protein gels. The gels were dried and exposed to X-ray film

at -70°Cfor 24 h withan intensifyingscreen.

Westernblotting(immunoblotting).Equalamountsof pro-tein from cell lysates (see above) were subjected to

SDS-10% PAGE and then electrophoretically transferred to a

polyvinylidene difluoride nylon filter (Millipore Corp.,

Bed-ford, Mass.)withtransfer buffer(2.5mMTris-HCl[pH 8.3],

9.2 mM glycine, 10% methanol). The filters were then probed withanti-Ryk serum orantiphosphotyrosine (PTyr) polyclonal antibodies, followed by incubation with

1"I-labeledproteinA(Amersham)asdescribedpreviously(22).

Autoradiographywasperformedasdescribed above. Partial protease mapping with Staphylococcus aureus V8 protease. 32P-labeled protein bands were excised from in vitro kinase assaygel andsubjectedtopartial digestion with S. aureusV8 protease asdescribedpreviously (7).

Pulse-chaseanalysis.CEFtransformed by the clonedv-ryk gene were labeled with

[35S]methionine

(110 ,uCi;

Amer-sham)for 1 h(pulse)in MEM(without methionine)medium

(GIBCO, Gaithersburg, Md.) after starvation in the same

medium for 2 h. The cellswerethenwashed,incubated with

completemedium,andchased for0, 15,or30 minor1, 2, 4, or 8 h. At those timepoints, cellswere lysed as described

above,

immunoprecipitated

with

anti-Ryk antibodies,

and

analyzed by SDS-10% PAGE.

Cell fractionation. Cells were swollen in hypotonic lysis

buffer (20 mM borate, 0.2 mM EDTA [pH

10.1]) (0.45

ml/10-cm-diameterdish) for 10min,collected,and

homoge-nized in a Dounce homogenizer (15 strokes), and then 8 volumes ofhypertonicsolution(0.5Mborate[pH

10.21)

was

added. Thelysatewas spunat500 xg and4°Cfor 10min,

the supernatant was collected and spun at

20,000

x gand

4°Cfor 30 min, and the supernatant

(soluble fraction)

was

collected. The

pellets

were dissolved in PBS and

layered

over sucrosegradients

(40

and20%

[wt/vol]

sucrosein

PBS)

and then spun at 100,000 xg and4°C for 1 h. The

plasma

membrane-enriched fractionwas

collected,

diluted in

PBS,

and spunat 100,000 xgand4°Cfor 30min, and the

pellets

werecollected asthe membrane fraction. Thetwofractions

werethensubjectedtobothWestern

blotting

and the in vitro kinase assay.

Nucleotide sequence accession number. The sequences

re-portedhere have been

deposited

in theGenBank data base

underaccessionno. M92847.

RESULTS

Genetic analysis of threeRPLoncogenicretroviruses.Three ofthe RPL series ofacuteoncogenic retroviruses, RPL25,

RPL28,and RPL30,werefirst isolated from chickentumors

in farmflocksbyBurmester's groupattheRegionalPoultry Research Laboratory (now named the Avian Disease and

OncologyLaboratory)in EastLansing, Mich.,in the 1950's and 1960's(21).These RPL viruseswereshowntobe ableto

induce chickenerythroblastosis, fibrosarcomas, endothelio-mas, viscerallymphomatosis, nephroblastoma,

osteopetro-sis, and hemorrhages. The nature of the lesionswas influ-encedbythe virus dose inoculated, the ageat inoculation,

and therouteof inoculation(21).Since these viruses hadnot

previously been genetically characterized, we decided to

analyzethe transforminggenes presentin these three RPL

viruses.Injectionof the viruses into newly hatched chickens resulted in induction ofsarcomasfrom whichacutely

trans-forming avianviruseswere recovered. These RPL viruses

werealso showntobeabletotransform CEF in culture and

wereused for furtheranalysis. First,wedetermined whether

theyencode oncogenes that havealreadybeen identifiedin other oncogenic retroviruses. A screening method which allowsrapid comparison ofnewretroviral genes toknown viral oncogenes was developed. Oncogene-specific DNA

fragmentswereisolated fromapanelof 19plasmids contain-ingknownviral oncogenes. Each DNAprobewascarefully

chosensothatit containednovirus-derived sequences that could hybridize nonspecifically with the retroviruses to be tested. These probes were bound to nitrocellulose

filters,

and this oncogeneblotwasthen

hybridized

under conditions

of moderate

stringency

with 32P-labeled cDNA made

by

reversetranscriptionof different RPL virus

genomic

RNAs. These viral RNAs were obtained from virion

particles

re-leased from CEF transformed with the RPL viruses. If any of these RPL viruses contained a known oncogene, its cDNAwould beexpectedto

hybridize

tothe

corresponding

probeonthe oncogeneblot. The results of the

screening

are shown in

Fig.

1. Allthree RPLvirus cDNAs,as

expected,

hybridized to thepositive control, a DNAfragment of the

avian retrovirus structural gene. RPL25 and RPL28 also

hybridizedtothe slotscorrespondingtotheerbBand

erbA-erbB probes. Southern

blotting

of the

restriction-digested

original oncogene-containing

plasmids

with the RPL25 and

RPL28 cDNAs demonstrated that the

hybridization

was

specific

toerbB sequences

(data

not

shown).

This kind of Southern

blotting analysis

also indicated that the low-level

hybridization to the src and

myb

probes observed with

RPL25 and RPL30was due tocontamination of the probe

fragmentwith non-oncogene-derived viral sequences

(data

not

shown).

Furthermore, Northern

(RNA)

blots of RNAs from cells infected with RPL25 and RPL28hybridizedwith

an erbB probe,

showing

specific hybridization

bands not

present in RNA from uninfected CEF

(data

not

shown).

These results demonstrated that the RPL25 and RPL28 viruses contain the erbB oncogene,similartoavian

erythro-blastosis virus

(AEV),

whose cellular counterpart is the

epidermal growth factor receptor

(15, 51).

More

interest-ingly,

no

specific hybridization

of RPL30 to any of the oncogene

probes

was

detected,

suggesting

that the RPL30

virus containsanovel oncogene.Likealmost all of theacute

oncogenic retroviruses,RPL30 is alsoa

replication-defective

virus and needs a helper virus to produce infectious viral progeny

(data

not

shown).

The size of the RPL30 viral genome, 9

kb,

is

unusually

largeamongaviansarcomaviruses and is exceeded

only by

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5978 JIA ET AL.

LTR gag pol env LTR

I a I I I _UR2AV

gag pol env

B

LTR gag pol

clones:

RPL30

*: i

FIG. 1. Hybridization of three RPL virus cDNAs to oncogene

probes.DNAprobeswereisolatedfrom 19 known viraloncogenes

and boundtonitrocellulosefiltersasdiagrammedin the lowerright panel (seethetextforprecise probeidentities).32P-labeled cDNAs preparedfromRPL25, RPL28,andRPL30virionswerehybridized

tofiltersasdescribed in thetext. ALV,avian leukosis virus.

that of RSV(9.31 kb),which contains thesrc oncogene in additionto all of the viralgenes(48). To determine what,if

any, viral genetic information had been deleted from the

RPL30 virusduringtheprocessofoncogenetransduction,as seen in almost all of the oncogenic retroviruses character-ized so far, nine probes spanning the whole retroviral

genomewerepreparedfrom cloned UR2AVDNA and then hybridizedtothe 9-kbRPL30genome. The resultsshowed, surprisingly, that all nine probes hybridized to the RPL30

genome(datanotshown).Thissuggestedthat eithernoviral sequencedeletion occurredduringtransduction of the

onco-geneoronlyasmall deletion occurred.Inotherexperiments, immunoprecipitation of lysates from RPL30-infected CEF with tumor-bearing rabbit serum showed elevated invitro

tyrosine kinase activity relative tolysates from uninfected CEF (data not shown). Tumor-bearing rabbit serum was

raised in rabbits bearing RSV-induced tumors and

recog-nizes avian retroviral structural proteins in addition to

pp60-s'c (4).Thispreliminarycharacterization of theRPL30

virus strongly suggested that RPL30 contains a novel ty-rosine kinaseoncogene.Therefore,wedecidedtoclone this

oncogenemolecularly.

CloningofthetransforminggeneintheRPL3O virus.Since

wedidnothaveaspecific probefor thetransforminggenein theRPL30virusand didnotknow thepreciselocation of the

geneinthe RPL30genome,wedecidedtousethreeprobes

derived from UR2AVgag,pol, andenvtoscreen acDNA

libraryconstructed from RPL30-infected nonproducer CEF (Fig. 2A).The rationale of this strategywastoisolateRPL30

env env LTR

iI

-+

onc

1.7kb

2.2 kb

2.5kb

RPL30

3-1

8-1

[image:4.612.62.299.72.356.2]

2-4

FIG. 2. Cloningof thetransforminggenein theRPL30virus.(A) Diagram of the UR2AVgenome,ahelpervirusrequiredforRPL30 replication.gag, pol, and env denote the viral structural genes.

Three DNA probes usedintheinitialscreeningfor theoncogenein RPL30arerepresented bythick bars beneath the positionsin the viral genome fromwhich theywere derived. (B) Cloning of the oncogenein the RPL30virus. The three thick bars represent the threeoncogene-containingclones thatwereisolated from thelibrary andsequenced.Their sizesareindicated beneath thebars,and their

names areindicatedto theright of thebars. Above the bars isa

diagram showingthe location of thetransforminggenein theRPL30 viralgenome; this informationwas deduced from thecloningand sequencinganalyses.

viralgene-oncogenefusionfragments. The fusionfragments

could then be identified asclones containinginserts larger

than thoseexpectedfromprototypic retrovirusse uences.

Six positive clones were isolated from 3 x 10 plaques

from the RPL30 cDNA library with the env probe, eight

were isolated with thepol probe, and none were isolated

from 4 x

104

plaques with thegag probe. The inserts in

positive clones were excised from the phage vectors and

subjectedtoSouthern blotanalysis. TheSouthern blotswere

firsthybridizedwiththeprobesused in thelibrary screening toconfirm theauthenticityof theclones and then

rehybrid-ized withprobesfromregionsin theUR2AVgenome

adja-cent tothose from which thescreening probeswerederived.

The insert sizewascompared withthe distance betweenthe

hybridizing probes on the UR2AV genome to determine whether the size was consistent with or larger than that

distance. Three clones originally detected with the 3' env

probe caught our attention. These three clones were later

foundtobe identical to each other and arerepresented by

clone 3-1 inFig.2B.The inserts fromthese cloneswere 1.7

kblong,andtheyalsohybridizedwithanLTRprobe butnot

witha5'envprobe(datanotshown). However, the distance

between the5'end ofthe3'envprobe (0.8 kb) and the 3' end

of the LTRprobe (0.6 kb)isonly1.4kb. Thenegative results

obtained with the 5' env probe suggested that these 1.7-kb

clones containsequencesin additiontotheviralgenes;the

extrasequences werepotentiallythose of the RPL30

onco-gene.Theremainingcloneswerenotfoundtocontainextra sequences. By subcloning and sequencing, clone 3-1 was

foundtocontaina1,665-bp insert. Atthe3' end of thisinsert

is the viral3'envgp37gene,followedby the LTRsequence.

At the5'end,however,theinsert contains 818 bp ofnonviral

sequenceswhicharehomologous tothecatalyticdomain of protein tyrosine kinases(Fig. 2B). To isolatethefull-length

RPL25 RPL28 A

probes:

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cDNA for this putative transforminggene, the kinase frag-ment from clone 3-1 was used as a probe to rescreen the

RPL30 cDNA library. Numerous positive clones were

iso-lated, and twoof them (clones 8-1 and 2-4), containing the

longest inserts (2.2 and 2.5 kb, respectively), were chosen

forsubcloning and sequencing (Fig. 2B). Clone8-1contains 234 bp of the viralenvgp85 gene atthe 5' end, followed by 538 bpofthegp37gene,1,390 bp of nonviralsequences,and

10 bp of the gp37gene. Clone2-4 started with 378bp of the envgp37geneatits5' end, followed by1,390 bp of nonviral

sequencesand77 bp of 3' gp37, andended with 298 bp of the

LTRsequence.Therefore, both clones 8-1 and 2-4appeared

to contain the full-length putative oncogene. Since this

1.39-kb nonviral sequence has a protein tyrosine kinase

domain,wenamed thisputative viraloncogenev-ryk, for the

RPL30virustyrosine kinase.

Sequenceanalysisofv-ryk.Thesequenceofthe3'region of theRPL30 virus, including the v-ryk oncogene, is shown in

Fig. 3A. The v-rykgenewasinserted into the viral envelope

protein gp37 coding region within theenvgene. Itcontained an open reading frame of 442 amino acids, followed by a

62-bp3' untranslated region containinganAATAAA

polya-denylation signal. This open reading frame was fused in

framewith theupstreamviral gp37gene, which contains a

transmembrane domain. Thus, the putativeoncogenein the

RPL30 virus encodes a 621-amino-acid gp37-Ryk fusion

proteinwith a predicted molecular mass of 69 kDa termed

P69gp37-,yk. Interestingly, in contrast to almost all acute oncogenic retroviruses, v-rykwasinserted into the 3' region

of theviral envelope protein gp37 genewithout deletion of

anyviralsequence. The DNA sequences flanking the v-ryk oncogene in the RPL30 viral genome match perfectly the

continuous sequenceintheenvgene of other similar avian retroviruses, likeRSV(39) and UR2 (32).

InthesequenceC terminaltothetransmembrane domain ofgp37,p69WI37-ik has four amino acids fromgp37, followed

by thev-Ryk sequence, whichconsists of a42-amino-acid

N-terminal domain,a277-amino-acid protein tyrosinekinase

catalytic domain, anda 123-amino-acid C-terminal domain. The structure of v-Ryk is diagrammed in Fig. 3B. The boundaries ofthe kinase catalytic domain were defined as

describedby Hanks etal. (25). Itcontains the ATP-binding

siteconsensus sequenceG-X-G-X-X-G andalysine residue

atposition 334 (Fig. 3A), which is invariantamongall protein

kinases (24). v-Ryk contains all 11 of the subdomains

con-served in all of the known protein kinases (24). Further analysis revealed that the sequenceofv-Ryk more closely

resemblesthatoftyrosine kinases rather thanthat of serine-threoninekinases.Namely, 38 of40 highly conserved amino acidsoverthewholecatalytic domainof 42 tyrosine kinases

(24) were present in v-Ryk. In the regions thought to be

importantindetermining the substratespecificity ofakinase

(20, 25), D-L-A-A-R-N in subdomainVIb ofreceptor-type tyrosinekinaseswasconserved in v-Ryk and

P-I/V-KIR-W-T/M-A-P-Einsubdomain VIII ofmosttyrosine kinaseswas

P-V-K-W-i-A-i-Einv-Ryk; both regionswerequitedifferent

from those characteristic of serine-threonine kinases. This

stronglysuggested that v-Ryk isa tyrosinekinase. Tyr-469

corresponds to the conserved autophosphorylation site in otherkinases.Furthermore, intheposition correspondingto

the kinase insert of the platelet-derived growth factor and

colony-stimulating factor 1receptors (25),v-Ryk contains a

seven-amino-acid insert withthesequenceLEMAPQF. The

functional significance ofthis insert is not known. Beside this short kinase insert, v-Ryk contains an additional

six-amino-acidinsert,LSSQQI, betweenkinasedomainsIVand

V. The full-length v-Ryk sequence was used to search for

protein sequence homology in GenBank. The region of

v-Rykhomologous to otherproteins is limited to the kinase catalytic domain(Fig. 4). The highest homologies found in the kinase domain were to theaxl-UFO oncogene product (67.3% amino acididentity), the ark gene product (67.3%),

thev-sea oncogene product (47.4%), the humanmet

onco-geneproduct (43.2%), the human insulin receptor (41.4%),

the bovineinsulin-like growthfactor 1 receptor(41.1%),and the humanretoncogeneproduct(40.0%).axl(32a)andUFO (26a) are the same gene, isolated from human myeloid

leukemia cells, which encodes a putative tyrosine kinase receptor. ark isa murine putative tyrosinekinase receptor gene(35a). The v-sea oncogene is the transforminggene of avian erythroblastosis retrovirus S13, and its cellular ho-molog is a receptor-type tyrosine kinase (40). Since these

highlyhomologous kinasesarereceptor-typemolecules and

v-Ryk is only 36.1% homologous to c-Src in the kinase

catalyticregion, c-Rykismorelikelyareceptor-typethana

non-receptor-type tyrosine kinase. Another interesting

fea-tureofv-Ryk is that the 123 amino acids C terminal to the kinase domain show no significant homology to any other

protein in GenBank. Since this domain contains three

ty-rosine residues, theymaybepotential autophosphorylation

sites and thus may havesomeregulatoryrole(s),asproposed

fortyrosine residues in theC-terminal region ofthe

epider-malgrowth factor receptor (45)orthe Neuoncoprotein (1).

Biologicalactivityofv-ryk.The RPL30 virus caused trans-formation of CEF 10 to 14days after infection. The

trans-formed CEFexhibited an elongated and fusiform

morphol-ogyand grewto ahighercell density than normal CEF(Fig.

5D).

These transformed CEF formed colonies, but control cells did not (Fig. 6D), reflecting anchorage-independent

growth. The RPL30 virus induced chicken sarcomas 5 to 6 weeks after injection into the wing webs of 1-week-old chickens(datanotshown).Todetermine whether thecloned v-ryk gene is fully responsible for thetransformingability of the RPL30 virus, we tested the biological activity of this

putative oncogene.To dothis, v-rykwascloned into

retro-virus vector pSR-REP to construct retroretro-virus pRV-ryk, which contains the 5' LTR, gag, pol, and gp85 from RSV

andgp37-rykand the 3' LTR from RPL30. When pRV-ryk

wascotransfected into CEF with pUR2AV, the CEF became transformed and formed focimorphologically indistinguish-able from those ofRPL30-transformed cells after 10 to 14

days (Fig.

SC),

while neither pUR2AV itself nor mock

transfection caused any transformation (Fig. 5A and B). Whenthesetransformed cellswereplatedon agar,colonies verysimilar to those induced by the original RPL30 virus formed (Fig. 6C). Virus collected from these pRV-ryk-transformed cells induced sarcomas in chickens within 6 weeks, and histological examination of the sarcomas re-vealed that theywere fibrosarcomas and

rhabdomyosarco-mas(datanotshown).Therefore, the cloned v-ryk gene can fully reproduce the transforming capacity of the RPL30 virus.

Biochemical analysis of the v-Ryk oncoprotein. To avoid cross-reaction with othertyrosine kinases, the unique region C terminal to the kinase catalytic domain of v-Ryk was

chosen as an immunogen to generate specific anti-v-Ryk antibodies in rabbits (see Materials and Methods). The

specificityof theserum wastested inanantigen competition assay. Figure 7 shows that in lanes 3, 5, and 7, cold

immunogencompeted successfullyto remove aband witha

molecularmassof 70kDaimmunoprecipitated from in vivo

phosphate-labeled RPL30-transformed cells by sera from

VOL. 66,1992

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5980 JIA ET AL.

A

TSCCCO AnCSCCOGCGA TOI OTC GAC LC ACACMaAARTTTCAIC AMA CCA AAM 60

phe pro11l loU proglyva1 trpv-1*ap a.rthrginglymenph. thrlyeprolye 20

gps

O00 CTA CCI CCCOCA ATTTTCCTC ATrTOC OATCOC WATOGC> BOAATTCCTAOT120

&laleu pro pro ciaIleph. ileeye giy*aprg &IatrpginglyIlepro*sr 40

COT CCA 0TA 0C GMOC CCCTOC TATCTA WOA AAACT? ACCATO TTAOA CCC AAC CATACA 160

argproval glyglyprocya tyrloU giylye leo thrmetleu*lapro&&n his thr60

CATAT? CAC AAM ATACTTOCTAT TCC TCO CAA MCA OC AMA CAM TT CIUA COT? 240

aspilehis lys ileleu atlasnner serg1n thrglyva1 erghi. ph. ergserv-1 60

qP37->

TCA CATCT? GAT CA? ACA TOC TCA GAC GAA CAGCT? TG OCTCCTACA BCA AA ATC 300

*-S hi. loU asp aspthreye *apglU va1 ginloU tapglyprothraia argile 100 TT? BATCTATC TTACC CCA GOO OTAWCAOCCCC CAAOC CTA AX GM ATCCM AMA 360

ph.&lamer ileleoU al proglyva1 &ia*- &lagin &laloU erggln Ileglo erg 120

CTACC TOTTOOTCTC?AAA CAM OCTAAC TT?ACA MATCACTC CT?Go CGMTTATT 420

loU clacys trpserv-1 lys gin cla can loU thr thr merleo leo gly asploU loU 140

CAT CGMCC MA ACT AT? CGA CAT0CC OT? CTC CAM AAC CCA C00 AT? GMTT? TT? 480

cap cap vclthr serile crghis lac vcl loU gin canarg &ia ciaile*apph. leu 160 CT? CTA OCTCAM CM? TOTAACCACT? GC AT? TOTTOOC TTC AAM CT? AMT540

leu leou hi. glyhis glycye lys capile&lagSlyntCy. eyeph. can lou *er180

GAM CACAOCGMCT ATA CAM AAC TTC?C CAM CTAAT? AMU GMCAM OTC AAM AMGATC .00

caphi. oar glU c&ailegin lys lys ph. gin loU netlye glu his vl can lysllo 200

0GC GMCAGCCAC CCA ATCCG ACTTOO CTCQGAGMA TTA TC CB WAATS C0T GM660

giyval cap ser capproileglysertrp1iU argglyleo ph. glyglyil* glyglU 220

TOGGC ATA CAC TTACTAAAMCGA CT CTT TTS CT? OTACT? ATC TTATT CTA 0TA 720

tap ilehisloU ioU lyeglyloU leo iougiyleo va1 vai ile lou loU loU vcl240 GTC TOC CT? CCT TOC CTT TT? CAM TTS OTATCT A WCCGA AAC ATCacTACT ACA760

vaicys ioU proCye ioU leo ginph. vai serser aerthrSrg lye netthr thrthr260

...A... Iv-ryk-2,

OTe OTAAAM TacACA CCM AA AAM TCCTAC TOC COOAMA 0CCOTCGMCT?ACATTO 640 vcl val&an tyrthr cialys lye eertyr eye crgarg*a1 v-1 giu loU thr leo gly 280

AMCC? GGACTC AGC AGC GAM CTC CAM CAM AAC CT? C GM SCTOT AT? AMA AAM 00

earlou glyvcl aermerglU loU gin gin lys loU gin capv.1 va1 ileaeparg cen 300

kinasoI -->

GCCCT?AGO CT? AAC GTC CT?CGA GAM CGM TTC TCA ATC GAM GA C0T960

alaloU aer loUglylye vcllouglygluglygluph. glyser va1met glo giyarg320 CTCACC CMGCOCAA GGC ACCCCA CaM AAC GT OCTC? AAC ACCAT? AAM TTG OATAAC 1020 loU ner gin proglu glythrprogin lys v.1 &lav.1 lys thrsetlys lOU cap can 341

TT? TCCCAT AMA GAM ATACMCM TTC CTC AMTGMWcA BATOC ATAAAC Gac TT GM1060

ph.*erhi. argglo ileglugluph.ahleo glo cilac&a eys1il lya capph.*ap360

Cac COC AATCST ATC AAM CTC CTAGOT GT? TOC ATCCM CT?AOCTCTCAM CAM ATCCCC 1140

his pro &anva1 ilelye loU ioU glyvc& cys ileglu loU aer aergin ginilepro360

AAM CCCAT?CT CTT CTCCCATTC AT? AAM TATCT CMCTC CACACCT CT? CT? COC1200 lys pro netvclvcl loU proph. met lys tyrgly cap1lU hi. aerph. loU loU crg400

TCCCOGCTCCA0 AT? oCC CCC CAM TTC OT CCCCT? CAM ATC CT? CT? AM TTCAT? GT? 1260

aararg loUglU met*iaproginph. vclproloU gin matleu l-u lyeph. met vcl420 GAM AT? 0CCCT? Oa ATC CGM TAC CTC AOC AM CO0 CA TmCTT CAC AO OAT TT? GOG 1320

cap 1ie&aloU glynet glu tyr loU serser crggin ph. loU hi. crg caploU &la440

OCTC00 AAC TOC AT? TTACC0 GA? CACAT? ACG GTGTOT GT? WCAGM TT? c0 CT? TCC1360 ilaarg cancys met leo crg capaspmetthr vcl eye va1&ca capph. glylou ser460

AM AM ATC TA AOC GO GMATM TACTCOT CM CO ATACCAA AT CCAOT? AAM 1440 lys lye il tyr aergly captyr tyr crg gingly arg1ie cal lysmetprovcl lye 460

TOO ATT ATAGMTCCCTC CT MAC COTGT TAC accACCAM ATOATT TOO ¢cA1500

trp ile cal ili glu serlou cla cspargv1ltyrthrthrlyeser cspvaltrp cla 500

TT 00c an ACCAT? TOOGMa ATA 0CO ACC AMA GO AT? ACTCCC TMAC cCA 0o OTCCAM 1560 ph. glyvalthrmettapglu iliea thr erggly met thrprotyrproglyvalgin 620

AACCACGMAT? TATGMTATCTATTC Cac GM CAM COOCTCAAA AACCCT AmAACTOC 1620

canhi. glu iletyrglutyr leu ph. hi. glygln cap leulye lyeproglu can eye 540

T?ACATGAM CT? TAC OATATCAT? TCC TCCTOC TOOAo OGCTGM OCT OC GACCOAC00 1660

lou capglu l-o tyr capi1e met aer eer cye trparg cal glupro ila cap crgpro560 ACC TT TCA CAGCT? aAAOT CATCT? GMA AacCT? TT? GA aCc CTTCCT 0CCC00 AG 1740

thrph. ,erginleu lye vcl his leu glu lysil- lou glunor leuproalaproarg560

IC-tere ->

TCCAAC CACG? ATc TA GTC AACACC MCCCTOCeaGM GA aocCCC GACTCCAce 1600

gly merlye capval iletyrvcl canthr*ar leuproglu gluser pro cap eorthr 600 CMCGMTT? 0C0 TT? CAT TCOG GT ATCCCC CAA GMOG TCTGMTT? CGCCCCGMG GM 1660

gln cap l-ugly louasp serval11. pro gln*1- capsor caplou*approgly cap620

ATT OCTGMCCCTOCTOC ?CC CAC acM AAM GCAGCGCT? OT?COAOT? GMAT? CACGAM 1920

1lo cal gluproeys cye serhisthr lys c,laal leuv.1 cal v.l aspilohis cap640

T MGTAC GT? CTTCAAAI GMA 0CaO CCCaAGMCAT CTTACVT? COA1660

glyglyser crgtyrvcl leugluserglu glyser prothrglu cap cal tyrvclpro660

CAGCT? CCC CAC GM 0O0 TCO TooACC GM 0CCaMC ACC TTO CCTOT? Aac TCG2040

gln leuprohisglugly ser cal trp thrglu cia aerthr leuprovcl gly*erser680

CTTocA OCT Ca CTC CCATOT CT CAT TOC CTC cMcM TCC GA CTGCTOTOA 2100 lou cia cal gln leupro eys cla cspglycysleuglu cap*-r glu alaleu lou '" 699 AT? CM CCT C CAM aoCAOC GT? TOAATC AMC ACA CM aA TTTTATTT AA AAaCAM 2160

TCT TATTA TTCACTCG CTA TCO COAGOA ATACAM GMATT Aea AGA TAM TAM GCA2220

^3'env-->

CCC TAAAM aMC acW ATAGOCCBO TTCCTCTAM TCO TOTCAT ACCTO TT? CGM C0 TAM 2280

TCO A?CGOTTAM CATACM GCMTST AAM AGOTCO CT? AWT AWTOCCGACA AMTT? AM2340

CTACAM TAM GCC AAC CT TGACCO OCA T?S GOA TCA AQ AAM CT? CT? MG T?T AMCC0 2400

TT? TT?.CT? COC OAT CTA C GOTAGo TTTACM COT AAC TOG 0o CCaTQA TAT2460

OTATAM CCA ACMG 0o CT? CO TTSS TAC CC ATMaGCeC CTC A" AMTAM TOG2520

AM COc TTT TOC ATA AGO AATOT AWT CTT An CAM aM TA AMTOT ATA2580

^ U3 -->

TTaCOA MAT AAM COAATC 0CC TOATOCACC aAA AA COTATTATATOATCC CATTooTOO 2640 TOAAGOACC CACCT? AMG OCA TAT 00c CCT TMA TMA AACTCT CTG TCCTSGC0TCAT TCC2700

T?C CTCAT?C BATCA TCT acM COG CAM AMT ATCAT? OGATa CAM GA BOC aMc ATT CAT2760

COT 0GCCOATOC TCA TTG CT? TCA CTAACG aWT TAT 0TA ACC CAT GMTOTACT TaA OCT 2620

TOT AW TOC TAM CAA TAA AWI 0CCATT TTA CCT CCC ACC ACaTTC OT? TOCACC TOO OTT 2660

R--> A US-->

CAT 0O CG0 aCCOTC OATTCCTCAC0G CTACQAOACCT GA TOA aoCaMA aGo CTT CAC 2940

COa 0C0 CAT?CT AT?ACW CCA TOATCAAAA CT? AAA acc TACACTAT TT CCCAACCOO3000 cell->

CMOCAAT?

B

v-ryk

gag pol gp85

I l M

LTR

gpif r

~~~~~~-

xx 0 #4

TMso,

17

LTR

catalytic

C-term

FIG. 3. cDNAsequenceandstructureof the 3' portionof theRPL30virus. (A)cDNAsequence and translatedprotein sequence ofthe

3'portionofRPL30,includingthe v-tykoncogene. The starting site foreachgene regionisindicated byasinglecaretunderneath thefirst

nucleotidefollowedby the name anddirection of thatgene. Aseries ofcaretsunderlinesthetransmembrane domainof gp37, an asterisk

marks theATP-bindingsiteatLys-334in thekinase catalytic domain of v-Ryk, andc-termdenotes theC-terminaldomain of v-Ryk (seepanel

B).Aseries of threeasterisks indicatesthestopcodon for theproteinencoded bythisgene,andthe AATAAA polyadenylation signalsite isunderlined.The numbersfor nucleotidesand amino acidsaretothe right of the corresponding lines. (B) Genomicstructureof theRPL30

virus. Theviralgene names are indicatedabove their locations, and the coding regions for thetransmembrane domain, kinase catalytic domain,and C-terminal domain(C-term)of v-Ryk,aswellasthestopcodon,arelabeledunderneath. Thetrianglesdenote the kinaseinserts. Thisdiagramis drawn approximatelytoscale.

J.VIROL.

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[image:6.612.167.467.576.629.2]

v-RYK NALSLGKVLGEGEFGSVMUGRLSQPEG--TPQKVAVETMKLDNFSHRIIEEFLSEAACIKDrDHPN

AXL/UFO HKVALGKTLGEGEFGAVKEGQLNQDDS---ILKVAVTMKIAICTRSELEDFLSEAVCMKEFDBPN

ARK HKVALGKTLGEGEFGAVKEGQLNQDDS---ILKVAVKTKIVIC;TRSELEDFLSEAVCMXEFDHPN

v-SEA LITHRSRVIGRGHFGSVYHGTYMDPLL--GNLHCAVESLHRIT-DLEEVEEFLREGILMKGFHHPQ

HIR EKITLLRELGQGSFGMVYZGNARDIIKGEAETRVAVXTVNESA-SLRBRIEFLNEASVMKGFTCHH

MET LIVHFNEVIGRGHFGCVYHGTLLDNDG--KRIHCAVKSLNRIT-DIGBVSQFLTEGIIIMKDFSHPN

IGF1R EKITMSRELGQGSFGMVYEGVAKGVVKDEPETRVAIKTVNEAA-SMRERIEFLNEASVMKEFNCHH

RET KNLVLGKTLGZGZFGKVVEATAFHLKGRAGYTTVAVKMLKENA-SPSELRDLLSEFNVLKQVNHPH

c-SRC ESLRLEVKLGQGCFQEVWMGTWNG---TTRVAIETLEPGTM--SPEAFLQEAQVMEKLRHEK

v-RYK VIELLGVCIELSSQQI-PKPMVVLPFXKYGDLHSFLLRSRLEMAPQF---VP AXL/UFOVMRLIGVCFQGSERESFPAPVVILPFKHGDLEESFLLYSRLGDQPVYL---PT ARE VMRLIGVCFQGSDREGFPEPVVILPFKKHGDLSLHLYSRLGDQPVFL---PT

v-SEA VLSLLGVCLPRH----GLPLVVLPYNRHGDLRHFVRAQERS---PT HIR VVRLGVVSEG---QPTLVVMELKAHGDLKSYLRSLRPEAENNPGRPP---PT MET VLSLLGICLRSE----GSPLVVLPYKHODLRNFIRNETHN---PT IGF1R VVRLLGWSQG---QPTLVIMELKTRGDLKSYLRSLRPEMENNPVLAP---PS RET VIELYGACSQD---GPLLLIVEYAKYGSLRGFLRESRKVGPGYLGSGGSRNSSSLDHPDERALT

c-SRC LVQLYAVVSE---EPIYIVTEYKSKGSLLDFLKGETGKY---LR

v-RYE LQMLLKFKVDIALQEYLSSRQFLHRDLAARNCHLRDDTVCVADFGLSKIKYSGDYYRQG--RIA

AXL/UFO -QILVEFKADIASQIZYLSTKRFIHRDLEAARNCLNENKSVCVADFGLSEKIYNGDYYRQG--RIA

ARE -QMLVKFADIASQTLSTKRFITR tDLAA.RNC)LNENMSVCVADFGLSEEIYNGDYYAKG--RIA

v-SEA VKELIGFGLQVALGKEYLAQKKFVERDLAARNCNLDETLTVKVADFGLARDVFGKEYYSIRQHRHA

HIR LQEMIQMAAEIADQIAYLNAEKFVHRDLAARNCXVAHDFTVKIGDFGMTRDIYETDYYRKG--GKG

MET VKDLIGFGLQVAKANKYLASKKFVHRDLAARNCMLDEKFTVKVADFGLARDMYDKEYYSVHNKTGA

IGFlR LSEMIQMAGEIAD(MAYLNANKFVSREAARNCNVAEDFTVKIGDFGMTRDIYETDYYRKG--GKG

RET MGDLISFAWQISQQ4QYLAEMKLVHRDLAAREILVAEGRKMRISDFGLSRDVYEEDSYVKR--SQG

c-SRC LPQLVDMAAQIASGKArVERMNYVHRDLRAANILVGENLVCKVADFGLARLIEDNEYTAR ---QGA v-RYK IKPVENlARIESLADRVYTTKSDWVAFGVTf NATRGMTPYPGVQNHEITEYLFHGQRLKKPENCL

AXL/UFOIOIVEWIAIESLADRVTTSKSDVSFGVTRNNIATRGQTP!PGVENSEIYDYLRQGNRLKQPADCL

ARK IKPVKWIAIESLADRVTTSKSDVNLFGVTD4NKIATRGQPYPGVENSEIYDYLRKGNRLKQPVDCL

v-SEA KLPVRNMALESLQTQEFTTKSDNSFGVIJNILLTRGASPYPEVDPYDMARYLLRGRRLPQPQPCP

HIR LLPVRHMAPESLEDGVFTTSSDWSFGVVLWITSLAEQPYQGLSNEQVLKFVMDGGYLDQPDNCP

MET KLPVEWMALZSLQTQEFTTSDVWSFGVVLNZEMTRGAPPYPDVNTFDITVYLLQGRRLLQPEYCP

IGF1R LLPVRHMSPZSLKDGVFTTYSDVSFGVVLWEIATLAEQPYQGLSEEQVLRFVMEGGLLDKPDNCP

RET RIPVEWMAIUSLFDHIY!TQSDVWSFGVLLWKIVTLGGNPYPGIPPERLFNLLKTGHRMERPDNCS

c-SRC KFPIEWTAPKAALYGRFTIKSDWSFGILLTULTTEGRVPYPGMVERBVLDQVERGYRMPCPPECP

v-RYE DELTDIMSSCNRAEPADRPTFSQELVHLUELLESL

AXL/UFO DGLYALSRCNELNPQDRPSFTELREDLZNTLKAL

ARE DGLYALKSRCNELNPRDRPSFAELREDLENTLKLC

v-SEA DTLYGVKLSCWAPTPEERPSFSGLVCELERVLASL

HIR ERVTDLRRMCNQFNPNMRPTFLEIVNLLEDDLHPS

MET DPL!EVNLKCWHPEAEMRPSFSELVSRISAIFSTF

IGF1R DMLFELXIMCNQYNPEMRPSFLEIISSIKEEMEPG

RET EEMYRLMLQCWKQEPDERPVFADISEDLEEMMVER

[image:7.612.58.299.75.335.2]

c-SRC ESLHDLNCQCVREEPEERPTFEYLQAFI.DYFTST

FIG. 4. Homology of v-Ryktoother tyrosine kinases.The v-Ryk protein sequence is aligned with those of other tyrosine kinases (sequencesarefromGenBank). Thenameof each protein isonthe left, dashes denotegaps in thesequences introduced for maximal

alignment,and theresidues inv-Ryk thatarealsopresentinatleast twoother kinasesarein boldletters.

Control

three immunized rabbits. This competition was specific,

becausethe controlantigen (vector withnoinsert) couldnot compete successfully for binding of the antibodies to the

70-kDa band (lane 1). In addition, preimmune serumcould not precipitate the 70-kDa protein from 32P-labeled cells

(lanes 8and9).

This70-kDaphosphoproteinwasalsoimmunoprecipitated

frompRV-ryk-transformed CEF (Fig. 8, lane 4) butnotfrom

pUR2AV-transfected cells (lane 2). The size of this phos-phoprotein,70kDa, is consistent with thepredicted molec-ular mass of v-Ryk, as determined from its sequence.

Therefore, this 70-kDa protein is believed to be thev-Ryk oncoprotein.

Since the v-ryk geneencodes aputative tyrosine kinase, weperformedin vitrokinaseassays onimmunoprecipitates

ofv-Ryk. Following incubation of anti-v-Ryk immunopre-cipitates fromv-ryk-transformed CEF and nontransformed

CEF with

[.y-32PjATP,

two proteinswithmolecularmasses

of70 and 150 kDa becamephosphorylatedinboth pRV-ryk (Fig. 9, lanes 2 and 4)- and RPL30 (data not

shown)-transformed cells. These two phosphoproteins were not

detected in mock-transfected (data notshown) or

UR2AV-transfected(lane 6)CEF.Phosphoaminoacidanalysisof the

two proteins showed that the phosphorylation was on

ty-rosineresidues(datanotshown).We concluded from these experiments that v-Ryk is indeed a tyrosine kinase. The

70-kDaproteinis believedtobeautophosphorylated v-Ryk,

and the 150-kDa band is either a protein substrate which coimmunoprecipitatedwithv-Rykor av-Ryk-related protein

whichcanalso berecognized

by

theserum weused.

v-Ryk antibodies were also used in Western blotting analysis. These antibodies detected a prominent 150-kDa

UR2AV

-c Ak

..:5leBS

pRV-ryk+UR2AV RPL30+UR2AV

FIG. 5. Transformation of CEFby

v-iyk.

CEFweremock transfected(A),transfected with thehelpervirusplasmid pUR2AV (B)orthe cloned

v-ryk

construct

pRV-ryk plus

pUR2AV

(C),

orinfected with theRPL30virusplus helpervirus UR2AV(D).Cellmorphologyat20

days

aftertransfectionorinfection is shown.

Magnification,

x100.

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[image:7.612.137.474.418.694.2]

5982 JIA ET AL.

Control UR2AV

[image:8.612.142.482.77.354.2]

pRV-ryk+UR2AV RPL30+UR2AV

FIG. 6. Colonyformation ofv-ryk-transformedcells. As forFig. 5,cellswereputonagar 20days after transfectionorinfection, theplates wereculturedat40°C, coloniesappeared2weekslater,and thesepicturesweretaken 3 weeks later. Magnification, x30.

Construct_Contrut UR2AV_U2AV+UR2AVpRV-ryk

RPL30

_UR2AV+

SeruLm111 303

Ao ;-- +

304 305 Prc-lmm1ni

+

± - ± - ± KD Serum P I P I P I

PP1P

aWWSk4WI'm

t g #_ ~~200

6974

TX4xEWXW:

~

69

- 46

1 2 3 4 5 6 7 8 9

FIG. 7. Antigen (Ag)competitionassay.v-ryk-transformedCEF

werelabeled in vivo with32Pi,lysed,immunoprecipitatedwith either preimmune(Pre-Imm)serum(lanes8and9)or serum303(lanes 1, 2,and3),304(lanes4and5),or305(lanes6 and7)intheabsence ofantigen(lanes 2, 4, 6,and8)orin thepresenceofimmuneantigen (lanes 3, 5, 7, and9) orcontrol antigen (lane 1), and analyzedon

SDS-7.5% PAGE. The arrowhead shows the bandthatwas

specif-icallyeliminatedbycompetitionwith the immuneantigen.

1 2 3 4 5 6

FIG. 8. Immunoprecipitation of the v-Ryk oncoprotein. CEF transfected withpRV-ryk plus pUR2AV (lanes3 and4)orpUR2AV alone(lanes1and2)orinfectedwith theoriginal RPL30virusmixed withUR2AV(lanes5 and6)werelabeled in vivo with32pi,lysed, immunoprecipitated with either preimmune serum (P) or immune

serum (I), and analyzed by SDS-10% PAGE. The arrowhead indicates the protein that was present only in v-ryk-transformed cells.

- 200

KD

97,4

- 69

- 46

- 30

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[image:8.612.344.536.409.650.2] [image:8.612.87.275.423.648.2]

THEv-ryk

-- +

KD 200 200

_ 97.4 97.4

_ 69 69

_ 46

46

- 30

30

1 2 3 4 5 6

FIG. 9. In vitro kinaseassay.CelllysatesfromCEFtransfected witheitherpRV-ryk plus pUR2AV (lanes1to4)orpUR2AV alone (lanes 5 and 6) were immunoprecipitated with either preimmune

serum(P)orimmuneserum(I), subjectedtoanin vitrokinaseassay

in thekinasereactionbuffer, and analyzed bySDS-10% PAGE. The arrowheads indicate the two proteins that were present only in v-ryk-transformedcells.

protein in v-ryk-transformed CEF (Fig. 10). Upon careful

examinationof lanes3 and4 inFig. 10, another specific band

withamolecularmass of 70kDacanbeseen (indicated by

thearrowatthe lowerposition); thisbecameobvious when the filmwasexposed longer (datanotshown). These results

were obtained with serum 303; the same results were

ob-tained withtwootherantisera(datanotshown).These data showed thatpRV-ryk-transformed cells have the same

bio-chemical properties as RPL30-transformed cells, further

provingthattheclonedv-rykgenecanfully recapitulatethe

transforming activityof theRPL30virus.

Biosynthesis of thev-Rykoncoprotein. On Westernblots (Fig. 10),theanti-Rykantibodiespredominately recognized

a150-kDaprotein, suggesting thatthe150-kDa protein is the

precursor ofthe 70-kDa protein. To studythe relationship

betweenthesetwoproteins, the 70-and150-kDa bandswere

excised fromthe invitro kinaseassaygelandsubjectedtoS.

aureus V8 protease mapping. The V8 protease digestion

patternsofthe twoproteinswere similar(Fig. 11), suggest-ing that the 70-kDa protein is derived from the 150-kDa protein.Toruleoutthepossibilitythatthe150-kDaprotein

arosefromdimerization of the 70-kDaprotein,

v-ryk-trans-formed cells were pulse-labeled with [35S]methionine and thenchasedfor various periodswith cold methionine. The results (Fig. 12) demonstrate that the 70-kDa protein is

derived from the 150-kDa protein. The 150-kDa protein

peaked 15 min after the pulse-labeling step and then was

1 2 3 4

FIG. 10. Western blot of thev-Ryk oncoprotein. Cell lysates

from CEF transfected with pRV-ryk plus pUR2AV (lane 3) or

pUR2AValone(lane 2),mocktransfected(lane 1),orinfected with the original RPI30 virus (lane 4) were run on SDS-10% PAGE, transferred to a polyvinylidene difluoride nylon filter, andprobed witha 1:200dilution ofv-Ryk-specificantiserum. Thearrowheads indicate the two proteins that were present only in v-ryk-trans-formed cells.

gradually chasedintothe 70-kDaprotein,whichpeakedat2 h. Because anti-Rykantibodiesweregenerated against the

mature form of v-Ryk, other proteins derived from the

precursorwouldnotberecognized bythis antiserum.

Enve-lope proteins ofretroviruses are known to be first

synthe-sized as theenv polyprotein precursorPr95, which is then cleaved intotwo matureenv proteins, gp85 andgp37, by a

cellular protease(5).Webelieve thatanalogoustothis,inthe

RPL30 virus, an Env-Ryk precursorfusion protein is first

synthesizedand then cellular protease cleaves thisprecursor

intogp85andP69W-37ryk(Fig. 13).Theglycoprotein complex

of RSV lacking the cytoplasmic domain ofgp37 has been

showntobestably expressedonthe cell surface inamanner

similar to thatofwild-type RSV (34). Therefore, the cyto-plasmic tail of gp37 is not required for assembly of the

envelopeglycoproteins intovirions. In Fig. 12,the labeling

of the150-kDaproteinwasstrongerthan that of the70-kDa

protein,consistent with resultsobtained from Western

blot-ting experiments (Fig. 10), which show that the 150-kDa

protein is more abundant than the 70-kDa protein. The in vitro kinaseassaydata(Fig. 9)show that the70-kDamature oncoproteinseemstoexhibithigherkinaseactivitythan the 150-kDaproteinorthatthe150-kDaprotein

autophosphory-lates poorly. V8 mapping revealed that all of the phos-phopeptideswerederived from the 70-kDaprotein (Fig. 11), asexpectedifthe otherprotein (gp85) isextracellular.

Cellular localizationof thev-Ryk oncoprotein. The

recep-torlikestructureof

P69337ryk

suggeststhatthisoncoprotein

is localizedonthe cell membrane. Cellfractionation

exper-iments with sucrose gradients were performed to test this pRV-ryk p

+UR2AV 4

P I

_t

_4r,

pRV-ryk UR2AV

PUR2AV

p I p I

KD

pRV-ryk RPL30 UR2AV VOL.66,1992

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5984 JIA ET AL.

pRV-ryk

RPL30 KD

I

p

-

200

pol gp85 gp37

-

97.4

-

69

-

46

v-ryk gp37

stop LTR

*

150 kD Envprecursor-Ryk

*

Cellularprotease

AGP37

v-Ryk

MKSN=~~~

69kD

[image:10.612.318.556.72.251.2]

GP85 GP37-Ryk

FIG. 13. Biosynthesisof the v-Ryk oncoprotein. This diagram shows thattheRPL30 virus (top line)first synthesizes a 150-kDa Env-Ryk precursorprotein (middle line)and then has it cleaved into twomatureproteins,gp85and

p69j37-yk

(bottom line). TM,

trans-membrane domain.

150kD70kD 150kD70kD

FIG. 11. V8 mapping of two proteins fromv-ryk-transformed cells. The 150- and 70-kDa proteins from either pRV-ryk- or

RPL30-transformedcellswereexcisedfromthe in vitro kinaseassay

gel, subjected toV8mappinganalysisasdescribedinthe text, and

run onSDS-12.5% PAGE.

prediction.Theresults(Fig. 14)showed thatonthebasis of the data from both the in vitro kinase assay and Western

blotting analysis, v-Ryk is indeed located primarily in the

membrane-enrichedfraction rather than in thecytosol frac-tion. This is consistent with the idea that P69gP37-ryk is a

membrane-boundreceptorlikemolecule.

Phosphotyrosine-containingproteinsareincreased in

v-ryk-transformed cells. Since thev-rykgene is atyrosine kinase

oncogeneonthe basis ofsequenceanalysis and the in vitro kinaseassay, weexamined whether therewas anincrease in

phosphotyrosinelevels inv-ryk-transformedcells. Antiphos-photyrosine antibodieswereusedinaWesternblotanalysis

0'

15'

30'

1h

2h

of whole-cell lysates. The results in Fig. 15 show that in v-iyk-transformed CEF, there were multiple

phosphoty-rosine-containing proteins which were not detected in

un-transformed cells; among them were the 150- and 70-kDa

proteins, whichpresumably correspond tov-Ryk

oncopro-teins.Thissuggested that v-Ryk tyrosine kinase activity is required for the transforming phenotype. The identities of those multiple tyrosine-phosphorylated proteins are not known, but they are presumably substrates for the v-Ryk

kinase.

DISCUSSION

Inthis study,we analyzed three previously

uncharacter-ized RPLoncogenic retroviruses; twoof them, RPL25and RPL28,were shown to contain the erbB oncogene in their

genomes. Wethenfurtheridentified,and subsequent

molec-S M S M

4h

8h

--

150kD

.... ..:

.-m

4-

70

kD

1

2

3

4

5

6

7

FIG. 12. Pulse-chase analysisof thev-Ryk oncoproteins. CEF transfected withpRV-ryk plus pUR2AVweremetabolically labeled invivo with[35S]methioninefor 2h,chased for the times indicated at the tops of the lanes, immunoprecipitated by v-Ryk-specific antiserum,andanalyzed by SDS-10% PAGE.

MW

-

200

kD

- 97.4

.4

-

69

-

46

1 2 3 4

FIG. 14. The v-Rykoncoprotein is localized onthe cell

mem-brane.CEF transfected withpRV-ryk plus pUR2AVwere fraction-atedintosoluble (S) and membrane(M) fractions as described in

Materialsand Methods. Thesetwofractionswerethensubjectedto either Western blotting analysis with v-Ryk-specific antiserum (lanes 1 and 2) or an in vitro kinase assay (lanes 3 and 4) and analyzed bySDS-10% PAGE. Thearrowsindicatethetwoformsof thev-Rykprotein. MW,molecularmass.

RPL30

-

30

- 21.5

- 14.3

I I . r..

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pRV-ryk RPL30

UR2AV - + + +

4r .1:

.,F 7t .-*,: :,- o

200

- 97.4

-- 69

- 46

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1 2 3 4

FIG. 15. Phosphotyrosine-containing proteins are increased in

v-ryk-transformedcells.AWesternfilterwaspreparedasdescribed

inthe legendtoFig. 10 and blotted with polyclonal antiphosphoty-rosine antibodies.The arrowheads indicate the positionsof thetwo

v-Rykproteins.

ularlycloned, anoveltransforminggenefrom thegenomeof

the third virus, RPL30. This viral oncogene, v-ryk, is a member of the protein tyrosine kinase gene family. It

in-serted itself into the RPL30 virus envelope gene without

deletionofanyof the viralsequencesand is fused inframe

with thegp37gene upstream toform a69-kDa gp37-v-Ryk

transmembrane protein, P69gp37-yk. v-ryk can transform CEF, form colonies, and induce tumors in chickens, fully recapitulating the transformingactivity of theoriginalRPL30

virus. The v-Ryk oncoprotein is first synthesized as a

150-kDaprecursorproteinand then cleavedinto themature

p69gp37-ryk protein. v-Ryk has a tyrosine kinase activity in

vitro and is phosphorylated in vivo in v-ryk-transformed cells. Furthermore, elevated phosphotyrosine-containing protein levelsarefound in thesev-ryk-transformed cells.

Viral oncogenes are transduced from cellular genes by nononcogenic retroviruses. The v-erbB oncogene was first identified in the AEVgenome (strainsAEV-ES4 and AEV-R), together with another oncogene, erbA (47). The erbB

gene,however,isnotalways cotransduced into retroviruses

with theerbA gene, since erbB transducingviruses lacking

erbA have also been isolated (3, 30, 50). The finding that RPL25 and RPL28both contain only the erbB oncogene is

consistent withthe notion that the transforming activity of

erbBis stronger.

All acute oncogenic retroviruses lose part of their viral

genes during the oncogene transduction process, thus be-comingreplication deficientand relyingonhelpervirusesto

replicate and assemble infectious viral particles (43). Al-though the Schmidt-Ruppin and Prague strains of RSV

contain thev-srcgeneyetpreserveallof the viral structural

genes andarereplication competent, there isevidence that

the original RSV was a replication-defective transforming virus (16). Thecurrent replication-competent strains of RSV wereprobably produced after a second recombination event. TheRPL30virus is the first example of a virus that did not lose any of the viral sequences in transducing a proto-oncogene, although the envelope protein gp37 coding region was disrupted by insertion of the v-ryk gene, rendering the virusdeficient in replication. The reason that no viral genes were deleted in RPL30 is probably that these are homolo-goussequences between retroviral sequences and the c-ryk

gene at the region of the insertion. This will become clear when the sequence for the c-rykproto-oncogene is known.

Some viral tyrosine kinase oncogenes were transduced from cellular receptors for peptide growth factors. For example, v-erbB in AEV was transduced from the epidermal growth factor receptor (15), and v-sea was presumably transduced from a cellular receptor (8, 26). Since v-Ryk more closelyresembles receptor-type tyrosine kinases than nonreceptor tyrosine kinases, c-rykmay encode another cell surfacereceptor. We are currently cloning and characteriz-ing thec-ryk gene, and results from preliminary experiments have confirmed thatc-ryk isindeed a receptor-type tyrosine kinase (26b). Therefore, it is very likely that transduction of the c-ryk gene by the RPL30 virus rendered this kinase active in the absence of its ligand. The two oncogenes erbB and sea are particularly interesting because of their pro-cesses of viral transduction and the structures of their final oncogene products. These two oncogenic proteins are simi-lar to v-Ryk in that their basic mechanism of activation is truncation ofextracellular domains and the final products still maintaintransmembrane domains, albeit the one of v-ErbB is derived from theepidermal growth factor receptor and those of v-Sea and v-Ryk came from viral env product gp37. Althoughrelatively high amino acid identities with Axl-UFO and Ark(67.3%) in the kinasecatalytic domains were found, thepreliminary data on the cloning ofc-rykindicate that they areencoded by different genes (26b). In addition, the catalytic domain of v-Ryk contains sequences similar to polymerase chain reaction fragments JTKll (32b), tyro-12, tyro-7, and tyro-3 (28a). Particularly, tyro-12 has 53 of the total of 54 amino acids identical to those of v-Ryk. However, the rela-tionships between v-Ryk and thesepolymerase chain reaction fragments are difficult to judge because these polymerase chain reaction fragments are only 50 to 60 amino acids long and are derived from the most conserved region within the tyrosine kinase catalytic domain.

The results of Western blot analysis (Fig. 10) and the pulse-chase experiment (Fig. 12) indicate that the 150-kDa precursor protein is moreabundant thanp69w37-ryk in

v-ryk-transformed cells. This raises the interesting question of whether the Env-Ryk precursor protein has transforming ability or whether cleavage is required for transformation. Other unanswered questions about the v-ryk oncogene in-clude the function of the C-terminal sequences ofv-rykand the small inserts in the kinase domain. We are currently addressing these questions by mutagenesis of thev-ryk onco-gene.

ACKNOWLEDGMENTS

We are particularly indebted to L. B. Crittenden of the Avian Disease andOncologyLaboratory,EastLansing, Mich., for gener-ouslyproviding us with theRPL30virus. Wesincerely thank K. A. Sokol of the Laboratory Animal Research Center for excellent assistance inpathology studies and many members of our laboratory fortechnical help. We are grateful to G. Scholz for critical reading ofthemanuscript.

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5986 JIA ET AL.

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