A single point mutation has pleiotropic effects on pp60v-src function.

Vol.62,No.6 JOURNALOFVIROLOGY,June1988, p. 1898-1906

0022-538X/88/061898-09$02.00/0

Copyright C)1988,AmericanSociety forMicrobiology

A

Single Point Mutation Has

Pleiotropic

Effects

on

pp60vsrc

Function

MELANIE J. WELHAM* ANDJOHN A. WYKEt

Imperial CancerResearch FundLaboratories, St. Bartholomew's Hospital, Dominion House,Bartholomew Close, LondonECIA 7BE, UnitedKingdom

Received 25 November1987/Accepted 22February1988

TheRoussarcoma virus mutant tsLA29 encodesapp6ov-src moleculethat istemperaturesensitiveforboth tyrosinekinase activityand itsabilitytolocateatthe cellperiphery.Thedefect inlocalizationappearsto bedue toaperturbationin eventsfollowing complexdissociation, since themutantenzymeshowsarapidlyreversible

association with the cytoskeleton when shifted between permissive and restrictive temperatures. Although tsLA29

pp60v-s'c

differsfrom the wild

type

atthree amino acid

residues,

studies withchimeric

proteins

show that only one of the mutations, an alanine-for-proline substitution at residue 507, accounts for all the temperature-sensitive characteristics. Moreover,asinglesecond sitemutation, atresidue427,canrestorethe

wildphenotype. Cells infectedwithachimeric virusencoding onlythealaninesubstitutionatposition507have

aconspicuouslyfusiformmorphology, suggestingthat this mutation also hassubtle effectsonpp6O-srcfunction thatare apparently compensatedforby theother mutations in native tsLA29.

The product of the v-src gene of Rous sarcoma virus (RSV) isa60-kilodalton phosphoprotein,

pp6Ov-sr,

whichis associated with the cytoplasmic face of the plasma mem-brane and with the cytoskeleton (5, 20, 21, 39). pp6Ov-src alone iscapableofinducing andmaintaining transformation of cells in culture, and, in common with many other viral oncogene products and growth factor receptors, its only well-documented function isthat ofatyrosine-specific pro-tein kinase.Anumberof potential substrates for thisactivity

are

phosphorylated

in RSV-transformed cells (14, 22, 38,

43), but in no case hastheirphosphorylationbeen shownto beasufficient precondition for cell

transformation,

suggest-ingthat crucial features ofpp6Ovsr" activity have yet to be identified.

Amajor contribution to the understanding ofv-src func-tion has been obtained from the physiological and genetic analysis ofa wide range ofRSV mutants (52). It has been possible as aresult tobuild up a consensus model defining domainsof thepp6Ov-srcprotein that affectdifferent aspects ofits biogenesis andfunction. Newly synthesized

pp6Ov-src

immediately enters acomplex with two cytosolic proteins, pp9OandppSO(2, 3).pp9Oisanalogoustothe90-kilodalton heat shock protein and the steroid hormone receptor-binding protein (41), whereas ppSO is a rare protein of no known function(1). This complex has been implicatedin the trans-portofpp6Ov-srctotheplasma membrane (1, 11), but it is not clearwhich regions ofpp6Ov-srcareimportantfor its forma-tion (52). Mutant proteins whose lesions lie within both C-terminal andN-terminal portions display abnormal kinet-ics ofcomplex formation or dissociation, whereas studies with antipeptide antibodies suggest that the carboxy-ter-minal region may be important in association with pp9O and ppSO (46, 52). Subsequent myristylation of the pp6Ov-sr N-terminal glycine residue is an absolute requirement for localizationattheinner face of the plasma membrane (7, 13, 18). Replacement of the N-terminal glycine with either

* Correspondingauthor.

t Present address: BeatsonInstitute for Cancer Research, Bears-den, Glasgow G611BD, Scotland.

alanine or glutamic acid results in proteins that are not myristylated, are unable to associate with the membrane (although retaining a fully functional kinase), and as a consequence are unable to transform cells (6, 25). Other mutants, which retain the N-terminal glycine residue but have extensive deletions in the amino-terminal region, also displayaninability to localize at the cellperiphery, suggest-ing that the amino-terminal 8- to 15-kilodalton region is important for attachment (18, 29, 31).

The catalytic or kinase domain, located in the carboxy-terminal half of the protein, wasinitially defined by proteo-lytic digestion (31) and more recently by the demonstration that many mutations mapping within this region affect the tyrosine kinase activity in vitro and in vivo (4, 52). The carboxy-terminal region may also play a role in amitogenic activity ofpp6vsrc, but this does not depend on a fully activetyrosine kinase(8, 23).

Finally, a region in the amino-terminal half ofpp60vsrc, with minimum boundaries fromamino acids 81 and 169, is thought to play an ill-defined modulatory role in pp6Ov-src functioning. Many mutants with lesions in this region pos-sess kinase activity, but induce an aberrant morphology in transformed cells, possibly reflecting an impaired or altered substratespecificity (12, 24).

We havepreviously studied severaltemperature-sensitive (ts) mutants of RSV Prague A to elucidatethe relationships between structure and function in pp6Ovsrc. One such mu-tant, tsLA29, falls into the commonclass of mutants with a defective tyrosine kinase (44). Membrane association is ts for this mutant, whereas myristylation occurs normally at both permissive and nonpermissive temperatures (45). De-letion analysis has mapped a crucial mutation within the carboxy-terminalregion of the pp6Ov-src protein (16). In this study we have extended the physiological characterization of tsLA29 andprecisely defineditsmutations. Construction of chimeric v-src genes and analysis of the biochemical properties of the resultant mutant proteins have identified a critical tspoint mutation which alters the extreme carboxy terminus of pp60vsrc. This single amino acid substitution appearsto havepleiotropic effectsonv-src function. 1898

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MATERIALS AND METHODS

Cells, viruses, and antisera. Chicken embryo fibroblasts (CEF)wereprepared from fertileeggscultured and infected as previously described (47). Infected cells were grown at 35°C (permissive temperature) or41°C (nonpermissive tem-perature). Isolation of tsLA29 RSV and itsrevertants tothe wildtype were described previously (49-51).

tsLA29-transformed Rat-1 cellswere isolated by

coinfec-tion of Rat-1fibroblastsathigh multiplicity with tsLA29 and Carr Zilber-associatedvirus, followed by single-cell cloning (48a). tsLA29 Rat-1 cells were grownin Dulbecco modified Eagle medium supplemented with antibiotics and 5%

new-born calfserum at 35°C (permissive temperature) or39.5°C

(nonpermissive temperature). Tumor-bearing rabbit sera wereused as described previously (15, 44). The monoclonal

antibody to pp60vsrc, JB327, was kindly provided by J.

Brugge (32). The rabbit polyclonal anti-pp60vsrc antiserum

wasraised against the bacterial fusion protein by the method

of Gilmer and Erikson (19) with an expression plasmid

kindly provided by R. Erikson.

Biosynthetic labeling and cell fractionation.Plates (35 mm) of cellswereincubated for 30minto1 h withmethionine-free Dulbecco modified Eagle medium. For studies to localize pp60v-src, cells were labeled with [35S]methionine (100 ,uCi/ml) for 3 h. For pulse-chase analyses, cellswerepulsed for 15min with 200 ,uCi of[35S]methionine perml, washed twice with cold medium, and chased with cold complete medium forvarious time intervals atthe requiredincubation temperature.

Cells for fractionation were washed three times with ice-cold phosphate-buffered saline and incubated on ice.

Lysates were made in situ by adding 0.5 ml of modified cytoskeletal extractionbuffer(MCSK; 20 mM N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid [HEPES, pH 6.8], 3 mM MgCl2, 50 mM NaCl, 0.3 M sucrose, 0.5% Nonidet P-40 [NP-40], 1 mM CaCl2) to each dish. After 5 min, with occasional gentle rocking, the lysates were collected and a

further 0.5 ml of MCSK was addedto each dish, incubated for2 min, andremoved, andafinal 0.5-ml portion of buffer

was added and again incubated for2 min. All three lysates were pooled to give the soluble fraction. The residual insolublestructureswere solubilized with modified radioim-munoprecipitation assay buffer (RIPA; 1% NP-40, 1%

so-dium deoxycholate, 0.1% sodium dodecyl sulfate, 0.15 M NaCl, 0.01 M sodium phosphate [pH 7.0], 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride) or NP-40 buffer (1% NP-40, 150 mM NaCl, 20mM Tris [pH 6.8], 20 mM sodium PPi) and used asthe cytoskeletalfraction.

Immunoprecipitation and gel electrophoresis. Immunopre-cipitation with the monoclonal antibody JB327 and the polyclonal rabbit anti-pp60vsrc serum and sodium dodecyl-sulfate polyacrylamide gel electrophoresis were performed

aspreviously described(27, 30);in allcases 10% polyacryl-amide gelswere used. Gelswere impregnatedwithAmplify (Amersham Corp.), dried, and exposed on Kodak XAR-5 autoradiographic filmat -70°C.

Sedimentation analysis. Cells were labeled for 3 h with

[35S]methionine (100

,uCi/ml),

andlysateswere prepared by

usingMCSK and NP-40 buffers butin smaller volumes (0.5 ml total). Lysates were layered onto 10 to 30% glycerol gradientsin NP-40 buffer. Centrifugation wascarried out in

a Beckman SW50 rotor at 230,000 x g at 4°C for 17 h.

Fractions(0.2 ml)werecollectedfrom thegradients, diluted

to 1 ml with NP-40 buffer, immunoprecipitated withJB327,

and

analyzed by

sodium

dodecyl sulfate-polyacrylamide gel

electrophoresis

on a 10%

polyacrylamide

gel.

Kinase assays.Cells seededon60-mm disheswerewashed three times with

phosphate-buffered

saline and

lysed

in NP-40 buffer. Kinase assays were carried out with

equal

amountsof

protein

foreach

sample

as

previously

described

(10, 44).

Tumor-bearing

rabbit serum no. 5which

displays

cross-reactivity

with c-src, was used for

immunoprecipita-tion.

Molecular

cloning

of

proviral

DNA.

High-molecular-weight

DNAwasisolated from tsLA29-transformed Rat-1cells and from CEF transformed

by

the revertant virus tsLA29 R2.

The

genomic

DNA was restricted with EcoRI and

electro-phoresed through

0.8%

agarose

gels,

and DNAbetween the sizes of2.2and 4.4kilobases

(kb)

was

electrophoresed

onto

DEAE81-cellulose paper

(previously

soaked

overnight

in 2.5 M NaCl and washed with distilled

water).

The DNA was

elutedfromthe paperat

37°C

with1.5 M

NaCl

in TE

(10

mM Tris

hydrochloride [pH 8.0],

0.01 mM

EDTA),

with exten-sive

vortexing,

for 1 to 2 h. The paper was removed

by

centrifugation,

and the DNAwas recovered from the super-natant

by

phenol-chloroform

extraction and ethanol

precip-itation. Size-selected DNA was

ligated

to agarose

gel-pu-rified

Agtwes.XB

EcoRI arms

(tsLA29)

or XNM1149 arms

(tsLA29

R2)

packaged

in vitro and recombinant

bacterio-phages

were

plated

onLE392

by using

standard

techniques

(33, 34).

Plaques

were

screened,

in

situ,

witha

32P-labeled

nick-translated

v-src-specific probe prepared

from the

612-base-pair

PstI

fragment

(nucleotides

8054 to 8666

[see

Fig.

5];

nucleotide

numbering

follows that of Schwartz et al.

[42]).

Two

positive

clones were identified for the tsLA29

library,

and four

positive

clones were identified for the

tsLA29 R2

library.

Sequencing

of tsLA29 and tsLA29 R2 DNA. The 3.1-kb

EcoRI

fragment

was

purified

fromone

positive

lambda clone foreach

virus,

subclonedintotheEcoRIsiteof

pUC13,

and

subsequently

digested

with a combination of

PstI, EcoRI,

and

BglII.

src-specific

sequences were subcloned into M13

vectors and used for

sequencing by

the chain termination

procedure (36,

40).

Construction of chimeras. Chimeric src genes were con-structed

by

using

standard

techniques.

Thevectorusedwas based on achimera between Rous-associated virus type 1

(RAV-1)

and avian

erythroblastosis

virus

(43a, 48),

whose salient features are

represented

in

Fig.

1. Retroviral

con-structs were made

by

ligation

of the 3.1-kbEcoRI B v-src

fragment

to EcoRI-cut and calf intestinal

phosphatase-treated

pRAVKpn3'-q.

Plasmids denoted

pRAV3'src

were

made which contained the v-src genes of the

Prague

A,

tsLA29,

and tsLA29 R2 strains. Each of the constructs

contained a

single

3'

long

terminal

repeat.

Isolation ofthe 5.5-kb

KpnI

fragment

from thesevectorsand

ligation

tothe 7.9-kb

KpnI

fragment

of

pRAVKpn5'

(Fig.

1),

containing

the 5'

long

terminal repeat, leads to formation of a

complete

retroviral construct. When such constructs are transfected

intoCEF

(26),

thecells

produce

replication-competent

virus which canbe collected in theculture medium and used for furtherrounds of infection.The

pRAV3'src

constructs

con-taining

the 3.1-kbEcoRI B

fragments

of tsLA29 and

Prague

A were

digested

with

SphI

and treated with calfintestinal

phosphatase,

which removed 584 base

pairs

of v-src

se-quence from nucleotides 8571 to 9155. The

SphI

584-base-pair

fragments

purified

from each vector were used to

generate chimeric v-src genes

containing

substitutions of

wild-type

and tsLA29v-srcsequences

(see

Fig.

5).

Complete

constructswere madefor the chimerasasdescribed

above,

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1900 WELHAM AND WYKE

KpnI

Kpnl So(I

WtEI

Cut

pRAVKpn3-V

withEcoRI. EcoRA

pRAVKpn3-Y KpnI _

PRAVA3bM

KpnI

Satl= 6.9kb 8-4kb

lco_RI-R

/ Ligate in 31kbEcoRlB fragment.

EccWil

StEI EcoRI

Koate 5.5kb KpnI

fragment

from

pRAV3s.

SacI

EcoRIs

Sac!

WEN

EcoRI... LTR

LTRE

EccRI EcoRI

IsoWte

719kb KpnI

fragment. pRAVKpn n

Complete pRA\tM Kpnl Kpnl

127kb

KpnL. 134kb Ligateto

5-5kb

KpnI Kpni

Sal R fragment from

pRAV3i

s

LTR53

mHI

BSt-

OstEl

sEI

EccoR

torn

FIG. 1. Construction of recombinant viruses.ThepRAVvectorsystembasedonRAV-1and avianerythroblastosis virus(43a, 48)was

used to reconstructreplication-competent virus. Theimportant featuresareshown in thefigure;detailsaregivenin thetext.

the mutations being checked in each construct by DNA sequencing.

RESULTS

pp6Ov-src is synthesized on cytosolic polyribosomes and entersacomplex withtwocellularproteins, pp9O andppSO. The half-life of the complex is 9 to 15 min, which is also approximately the time taken for newly synthesized pp6Ov-srctobecome localizedatthe innerface of theplasma membrane (2, 3, 11). This has led tothesuggestion that the complex plays a role in the transport of pp6Ov-src to the membrane (1). We previously found that pp6Ov-src of tsLA29 is tsin itsability tolocalize at the cell periphery (45). This apparent defect in membrane association is not due to a

failure of myristylation, which occurs normally at both temperatures. On initial analysis of thepp6Ov-src:pp9O:pp5O complex, someinstabilitytoRIPA bufferwasobserved, but

thiswasapparently independent oftemperature (45). These observations raise thepossibility thatadefect in theputative

transportmechanism is responsible for the reduced levels of pp60v-src associated with the plasma membrane in tsLA29-transformed cells.

Glycerol gradient sedimentation analysis of complex. To investigate the possibility of a transport defect, we first

examined the nature of the complex. Although we have

foundthecomplex formed between tsLA29 pp6Ov-src, pp9O,

andppSOtobe unstable in buffers containing ionic detergents (e.g., RIPA buffer), it is possible to demonstrate complex formation in buffers containing nonionic detergents (e.g., NP-40 and Triton X-100) by coimmunoprecipitation of pp60v-src and pp9O with JB327 (45; data not shown). There-fore, the behavior of the complexwasexamined by glycerol

gradient sedimentation analysis oftsLA29Rat-1cell lysates in bufferscontaining nonionic detergents.

Comparison of the sedimentation profilesatboth permis-sive(35°C) and nonpermissive (39.5°C)temperaturesfor the

soluble fractions shows both monomeric

(slower-sedi-menting) and complexed

(faster-sedimenting)

forms of

pp6O-vsrc

(Fig. 2).The

cytoskeletal

fractionatthe

permissive

temperature showsonly monomeric pp60-src. Thereare no bands

corresponding

to

pp9O

and

pp5O,

because these pro-teinshave along half-life and hence

incorporation

over3 his low. They can be identified, however, after

labeling

with [35S]methionine for 18 h (data not shown). The results indicatethat attherestrictive temperature tsLA29

pp60-src

isstill capable offorming acomplex with

pp9O

and

pp5O.

It isdifficult, however, tobe certain abouttherelative propor-tions ofmonomeric versuscomplexed forms ofthe

protein

because of variability ingradients and the absence of two distinctpeakscorresponding solelyto thesedifferent forms. There does, however, appear to be a shift toward the monomeric form at the restrictive temperature. Since an increased amount of

pp6Ov-src

is present in the soluble fraction at this temperature (see below), the increase in monomeric form isprobablyaneffectrather than a causeof

pp6Ov-src

temperature sensitivity. It seems unlikely,

there-fore,

that the

putative

defect in tsLA29

pp6O-src

transport

canbe totally explained bythebehavior of the complex. Pulse-chase analysis. Several reports have indicated that pp60 -src is associated with the plasma membrane and the cytoskeleton (5,20,29).Inthis setofexperiments, fractions correspondingtosolubleandcytoskeletalcomponentsofthe cell were prepared in situ on the tissue culture dish. This technique allows more rapid processing of pulse-chased samples. The preparationof crude membranes is somewhat slower and thus not so amenable in this study. We have found that both membranelocalization(45) and cytoskeletal association (seebelow) are ts for tsLA29

pp6Ov

src,although it is notpossibletoemphaticallyequate thetwo. To examine thekinetics oftranslocation, wetherefore decided to moni-tor the transport of

pp6Ov-src

to the cytoskeleton, since earlier events could be studied.

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[image:3.612.140.476.70.330.2]

350A _{350B 3950A}

1 3 5 7 9 11 13 15 17 19 21 1 3 5 7 9 11 13 15 17 19 21 23 1 3 5 7 9 11 1 115 17 1921 23

FIG. 2. Glycerolgradient sedimentation analysis oftsLA29cellfractions and immunoprecipitationofpp604-src. tsLA29Rat-1cellswere

labeled with [35S]methionine for 3 h at the permissive (35°C) and nonpermissive (39.5°C) temperatures. Cell fractions were made and

sedimentedthrough 10to30%glycerol gradients.pp6v-srcwasimmunoprecipitated fromalternatefractions withmonoclonal antibody JB327.

The direction ofsedimentation is from lefttoright in eachcase. (A) Soluble cell fraction; (B)cytoskeletal cell fraction. Molecularmass

markersareindicated and expressed in kilodaltons.

The results (Fig. 3) demonstrate that at the permissive temperature there is a gradual increase in the amount of pp60v-srcappearing in the cytoskeletal fraction, correspond-ing to transport ofpp60v-src from the cytosol to the cyto-skeleton. We could identify pp6Ov-src in the cytoskeletal fraction aftera15-min chase. When pulse-chase analysesare

carriedoutwithPrA-transformed Rat-1 cells,somepp6Ov-src isapparentinthecytoskeletal fraction afternochase period at both permissive and nonpermissive temperatures (data notshown). It isnotclear whetherthis is duetomore rapid transportortothefact thatagreaterproportion of pp6Ov-src islocalized atthe cytoskeleton in these cells. At the

nonper-missive temperature, little or no tsLA29 pp6Ov-src became associated with the cytoskeletal fraction. However, upon shiftfrom thenonpermissivetothepermissive temperature, the soluble material accumulated under restrictive condi-tionsefficiently shiftedtothecytoskeletal fraction (Fig. 4A). Itthereforeappearsthat pp6Ovsrcis transported normally at 35°C, but thatat39.5°C (nonpermissive temperature) there is

areversible defect,independent of complex formation, that

either prevents the protein from associating at the cell periphery orrenders this association extremely labile. This raises the question of whether conformational changes within pp6Ov-src itself are responsible for its behavior or

whether it is unableto interact with other, as yet unidenti-fied, proteins which may be important for anchorage of

35C

pp6fi-vsr to the cytoskeleton and plasma membrane. To address these questions further, we analyzed prelabeled

pp60-src in tsLA29 Rat-1 cells by shifting them from the permissive tothenonpermissive temperature. The distribu-tion ofpp6O-src altered dramatically after this shift, alarge

proportion being found in the soluble fraction after 1 hatthe nonpermissive temperature(Fig. 4B). This shows that

asso-ciation with the cytoskeleton is a reversible phenomenon

and suggests that conformational changes occurring within the pp6Ov-src protein itselfare responsible for the tempera-turelability. Theapparentbiastoward monomericpp6Ov-src in the soluble fraction at the restrictive temperature in the glycerol gradients (Fig. 2) also suggests that many of these

defective moleculesarenotcomplexed with ppSO and pp9O. Weare currently investigating whether pp6Ov-src prelabeled at 35°C and shifted to 39.5°C becomes associated with the complex after the shift.

Genetic lesion in tsLA29. With the physiological data accumulated fortsLA29, itwasofgreatinteresttosequence the mutant src gene and to correlate the functional abnor-malities observed with structural alterations inv-src itself.

The nucleotide sequence of the entire v-src gene of tsLA29wasderived fromcodingsequencescontained within the3.1-kb EcoRI B fragment cloned into Agtwes.AB.

The nucleotide sequence revealed three pointmutations which lead to alterations in the predicted amino acid

se-395C

LENGTH OFCHASE

(minutes) 0 15 , 30 , ,60

A B A B A B A B A B

S

t

4 3---- 45

A B A B A B A B A B

pp6O-FIG. 3. Pulse-chase analysisofpp6O-vrctransport. tsLA29Rat-1cells weregrownat thepermissive(35°C)andnonpermissive(39.5°C)

temperatures,pulse-labeledwith[35S]methioninefor15min,and chased withcompletemedium for the timeintervalsindicated; cellfractions

werethenmade,andpp6ov-srcwasimmunoprecipitatedwith the monoclonalantibodyJB327. Lanes:A, soluble cell fraction; B, cytoskeletal

cellfraction.

92-5-

69-pp6O

45-

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1902 WELHAM AND WYKE

A tB

S z S C S C S C' S S

C._ _ _ _s ' . ..

FIG. 4. Temperature shift analyses of pp6O-src localization in labeled tsLA29 Rat-1 cells. In each case cells were pulse-labeled for 15 min with [35S]methionine and chased for1 hat the original incubation temperature, after which the samples were temperature shifted and incubated forafurther 1or2 h. Identical samples weremaintained at the original temperature. At the appro-priate time points fractions were made corresponding to soluble (lanesS) and cytoskeletal (lanes C) cell fractions, andpp6Ov-srcwas immunoprecipitated with polyclonal rabbit anti-pp6O antisera. (A) Temperature shift from the nonpermissive to the permissive tem-perature; (B) shift from the permissive to the nonpermissive tem-perature. Lanes: 1, cells maintained at theoriginal temperature for a1-h chaseperiod; 2, cellsmaintainedattheoriginal temperature for a3-h chase period; 3, cells maintained attheoriginal temperaturefor a1-hchase and then shifted and incubated forafurther1-hchase; 4, cells maintained at the original temperature fora 1-hchase and then shifted and incubated for a further 2-h chase. Molecular mass markersareexpressed inkilodaltons.

quence; all three were within the kinase domain. These changesaresummarized in Fig. 5. Thealterationatposition 8180, which changes the coding of amino acid 351 from lysine to arginine, is probably conservative. The tyrosine kinases v-erbB,v-abl,and v-ros all have an arginine instead oflysineat thecomparableposition,andit islikely that this mutation does not play a crucial role in determining the functional abnormalities of tsLA29. The alteration at nucle-otide 8251 (amino acid 375), which changes alanine to threonine,lies in the centerof the kinasedomain.Thethird

Plasmid

pRAVPrA

pRAV29

Virus

6I

alteration, at nucleotide 8647, which leadstosubstitution of alaninefor proline at amino acid 507, lies in the regionwhere genetic complementation studies predicted a crucial muta-tion (16). To test the relative contribumuta-tion of each ofthese mutations to the phenotype of tsLA29, we constructed chimeric v-src genes by usinganRAV-1-avian erythroblast-osis virus (AEV10) vector system (Fig. 1).

Construction of chimeras. The SphI 584-base-pair frag-ment, containing the coding region for amino acids 482 to 526, was exchanged between tsLA29 sequences and PrA wild-type sequences (Fig. 5). The resultant chimeras were checked by DNA sequence analysis. The parental and chimeric pRAV derivatives were transfected into CEF, transformed foci were picked and expanded, and recombi-nant viruses were recovered from the culture fluid of the infected CEF. These recombinant viruses were used in further studiesonmorphology,localizationofpp6Ov-src,and invitro kinase activity.

Morphology. The titers of the recombinant viruses ob-tained were all comparable. The wild-type RAVPrA pro-ducedfociatboth temperatures, whereas RAV29 was ts for focusformation(Fig. 6).ThepRAV29:PrAconstruct, which contains the coding sequences for the mutant amino acids 351 and 375, was able to induce focus formation at both temperatures, thefocitendingtobeveryroundin morphol-ogy. The pRAVPrA:29 construct, containing the coding sequencefor the mutant amino acid at position 507 alone, inducedfocus formationat thepermissivetemperatureonly. Thesefociwereratherfusiformin theirmorphology, as were thecells whentransformed in mass culture. Thus, notonly did it appear that the mutation at amino acid 507 was responsible for the ts phenotype, but it also had subtle effects on morphology. A region in the amino-terminal portion ofpp6O-src has previously beenconsidered impor-tant in determining morphology, so the observation that a carboxy-terminal mutation also has effects on morphology raises the possibility that interactions between domains of pp6Ov-src arerequiredfor normal functioning.

Myristic N KINASE C v-SrC

Acid I I C

pp60-K Y

295 416

EcoRI Pstl PstI Pstt Pstt Sphlt PstlSphl EcoRI

I a yy lyr~~v N I

144 6489

Mutat ions

RAV PrA

RAV29

351 Lysine _ Arginine 375 Alanine_ Threonine 507 Proline -_ Alanine

pRAV29:PrA RAV29: PrA 351 Lysine - Arginine

375 Alanine- Threonine

[image:5.612.57.291.76.181.2]

pRAVPrA:29 RAVPrA:29 C V'/ZZZ 3 507 Proline Alanine

FIG. 5. RSV variants used in this study. The nucleotide numbering follows that of Schwartz et al. (42). Chimeric v-src genes were

constructedbetween PrAwild-typeand tsLA29sequences. The SphIsitesusedaredenoted by the lines bisectingthe chimericgenes.The

positions ofthe nucleotidemutationsaredenoted (V). The 584-base-pairSphI fragmentwas used inamix-and-match approachto generate

pRAV29:PrAand pRAVPrA:29. The parental and chimeric genes wereconstructed into thesamereplication-competentplasmid and then transfectedinto CEF. Viruseswere recoveredas described in thetext.

70S2 7454 8054 8571 8666 9155 9238

I

-j

Izz--- , 1- -III I ZZ-J..

rzzzzz-z ZZZ,/'i P71

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350

RAVPrA 0A

iz\

RAV29:PrA

RAV29

..

4.4-'X''.

42 4... q. ..; ~

[image:6.612.118.513.77.551.2]

RAVPrA:29

FIG. 6. Morphologies ofvirus-infected CEFatthe permissive(35°C)andnonpermissive(41°C)temperatures.Fociwerephotographed6

to8dayspostinfection.

Subcallular localization. Having shown tsLA29tobetsfor membrane and cytoskeletal association (45; see above), it

was importanttodetermine the localization of the chimeric pp6jv-src proteins. Infected CEF were plated onto 35-mm dishes and metabolically labeled with [35S]methionine, and MCSK and NP-40 buffers were used to make lysates as

described above. TheJB327monoclonalantibodyandrabbit anti-pp6Ovsrc antiserum were used to immune precipitate pp60v-srcfrom thelysates. Both RAV29 and RAVPrA:29are

tsforassociation with the cytoskeleton, but RAVPrA and RAV29:PrA behave in atemperature-independent manner

(Table 1). Hence, it appearsthat the change atamino acid 507 is sufficient toconfer temperature sensitivity on cyto-skeletalassociation.

Kinaseactivityofchimericproteins.All three mutations lie withinthepredicted region for the kinase domain, andhence

we needtoevaluatetheireffectonpp6Ov-src kinaseactivity.

In vitro kinaseassayswereperformedonNP-40lysates from CEF infected with the chimeric recombinant viruses.Equal amounts of protein were assayed in each case, and the resultsarerepresented in Fig. 7. Thelevel of

immunoglob-ulinheavy chainphosphorylationobserved in the uninfected CEF track is due to cross-reactivity of the tumor-bearing rabbit serumused withc-srcand assuchispresent in each sample assayed. The pp6Ov-src proteins of RAVPrA (wild type) and RAV29:PrA both display a high level of kinase activitythatisindependentoftemperature.The RAV29and

RAVPrA:29 recombinant pp6Ov-src proteins, on the other

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1904 WELHAM AND WYKE

TABLE 1. Subcellularlocalization ofpp6o-src by cellfractionation

% ofpp6vOSrcatfollowing

Infectingvirus Cellfractiona growth tempb

35°C 410C

RAVPrA S 5 24

C 95 76

RAV29 S 10 60

C 90 40

RAV29:PrA S 5 15

C 95 85

RAVPrA:29 S 30 70

C 70 30

aS,Soluble cellfraction; C, cytoskeletalfraction.

b Each value represents theproportion ofpp60v-sr,determinedby densi-tometricscanning,in each cell fractionexpressedas apercentageofthetotal solubleplus cytoskeletal fractions.Theresultspresentedarethe average of twoindependent experiments.

hand, have ahighactivityatthepermissivetemperature,but thekinaseactivity is reducedatthe nonpermissive temper-ature.Thisreductionis dueto adecrease in

specific

activity at restrictive temperature rather than to adifference in the half-life of the pp6Ov-src protein, which is the same at the permissive and nonpermissive temperatures (data not shown).Itthereforeappears thatthe mutationatresidue 507 is also the crucial lesion in determining the temperature sensitivity ofthe tyrosine-specificprotein kinase.

Nucleotide sequence of revertant tsLA29 virus-src gene. Previous studies have shown that the reversion of otherts mutants to the wild phenotype resulted from second-site lesionsthat wereeither close to, or remotefrom,theforward mutation (17). The nucleotide sequence of the complete v-src gene encoded by tsLA29 R2 was derived from se-quencescontainedwithinthe3.1-kbEcoRIfragment cloned into XNM1149. The revertant contained all the forward mutations of tsLA29, but a single compensating mutation was located at nucleotide 8409. This distant second-site mutation changes lysine to arginine in the center of the kinasedomainataminoacid427andeffectivelyrestoresthe behavior ofpp60v-srctothatofwild-type PrA RSV (data not shown).

DISCUSSION

The mutant tsLA29 is ts for tyrosine kinase activity and also in itsability to locate at the cellperiphery (44, 45).We

§* * : *,: *

*

FIG. 7. Invitro kinase assaysperformed on lysates from CEF infected withthe RSV variants used in this study. The products of thereactionswere separated on a sodium dodecyl sulfate-polyacryl-amidegelelectrophoresisgel(10%

pojyacrylamide)

and autoradio-graphed. Abbreviation: IgG, 42P-labeled immunoglobulin heavy chains.

showherethat itsaberrantlocalizationisnotattributableto

defective

complex

formation with

pp90

and

ppSO

or to aberrantdissociation from thesetwo

proteins, although

the

complex

does show evidence of

slight abnormality

(45).

These

findings

areincontrast tothose foranumber of other RSV mutants, in which defects in subcellular localization canbe attributedalmost

entirely

toabnormal behavior of the

complex (23,

35).

Furtherexamination

by using

pulse-chase

experiments

demonstratesareversibletsdefect in the

asso-ciation of tsLA29

pp60v-src

with the

cytoskeletal

cell frac-tion. This

presumably

reflects a

perturbation

in events

following complex

dissociation. A defect in amino-terminal

myristylation

might

produce

this

phenotype,

but

myristyla-tion oftsLA29

pp60v-src

is not ts

(45).

Wetherefore suspect that the mutant

protein

displays

a

temperature-dependent

conformational

change

that reduces its kinase

activity

and

precludes

either an inherent

ability

to

persist

at the

cyto-skeleton or its

capacity

to interactwith unidentified mole-culesatthatlocation. Inany event, it appears that

myristy-lation of

tsLA29

pp60v-src

alone is not

sufficietit

to ensure

stable associationwith the

plasma

membrane and

cytoskel-eton.

The defects observed in

physiology

canbe attributedtoa

single-point

mutation resulting

in thesubstitution of alanine

for

proline

at amino acid 507. The coordinate effect of this

single

mutation on both kinase

activity

and

localization

is

demonstrated

by

studies with chimeric

pp60v-src

molecules

and

emphasized by

the factthat a

single

second-site

muta-tionat

position

427, close totheconserved

alanine,

proline,

and

glutamic

acid motif

(residues

430to

432),

fully

restores

thefeatures

of

wild-type

pp60v-sr.

The crucialtslesionis at

the

carboxy-terminal

end ofthe conserved kinase domain

adjacent to a region which has been suggested to have a

regulatory

role in

pp60v-src

function

(37).

The observation

thatthe mutated

residue,

proline 507,

is

highly

conservedin a wide range of

tyrosine

kinases also suggests that it may itself

play

a role in the

regulation

of the kinase or other activities of

pp60v-src.

The Chou and Fasman

(9)

rules

predict

a coil/turn structure in the

region

ofthe mutation.

Substitution of alanine for

proline

may decrease the

flexi-bility

ofthe molecule andso

disrupt

the

relationships

ofthe

regulatory C terminus to the

kinase

or other domains.

Alternatively,

such a decrease in

flexibility

could affect

interactions withother

proteins

which may be

important

in

pp6fiv-src

function and

biogenesis.

Itcan be

envisaged

that this might

coordinately

affect both kinase

activity

and

pp60V-srC localization,

but thefact thata

distant

second-site

mutation,

also at a

highly

conserved residue

(lysine

427)

within thekinasedomain,compensatesforthis defect shows that such an

explanation

is

inevitably

facile.

Itis

interesting

that the chimericprotein containing only

the mutationat amino acid 507 has subtle effects uponcell morphology. Infected cells are markedly

fusiformn,

in con-trast to the rounded cell morphology seen when CEF are infected with either the parental ts virus, the chimera RAV29:PrAcontaining justthe 351and 375mutations,or the wild-type revertantRAV29R2. It appears that the two mu-tations at amino acids 351 and

375,

although having

no detectable effecton

pp6Ovsrc

physiology, are in some way

compensating

in theparentaltsLA29virus for the effect the

lesionatamino acid 507 has on

cell

morphology. Alterations

in

the amino-terminal region are more usually associated withdefects in cellmorphology.CEFtransformedby mutant

viruses

which have amino-terminal deletions, as well as

those infected

by

many recovered avian sarcoma viruses, exhibit fusiform

morphologies

(13, 27, 28). However, other J.VIROL.

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[image:7.612.93.255.597.672.2]

mutations mapping to the carboxyterminus also appear to influence cellmorphology. Our

wild-type

Prague A clone has a different residue at amino acid 502 compared with other cloned v-src genes (17), and this clone tends to transform cells with a slightly fusiform morphology (Fig. 6). Other reports have also mentioned such phenomena, although the effect issubtle (35). This suggests that the C-terminal amino acids spanning residues 500 to 510 may play a modulatory role in determiningcell morphology, perhaps byinfluencing the access of

the

pp6Ov-src tocertain targets. Moredetailed studies on the location and molecular interactions of the mutant pp60-src should be informative.

ACKNOWLEDGMENTS

We are grateful to all members ofthe laboratory for technical advice, to S. Kellie, M.Owen, and G. Petersforcriticalcomments; andtoNikkiPinchen for her endless patience duringthe preparation of the manuscript.

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