Sequence and functional differences between Schmidt-Ruppin D and Schmidt-Ruppin A strains of pp60v-src.

Vol.64, No. 7 JOURNAL OF VIROLOGY,

JUlY

1990, P.3545-3550

0022-538X/90/073545-06$02.00/0

CopyrightX) 1990,American Society for Microbiology

Sequence and

Functional Differences

between Schmidt-Ruppin

D

and

Schmidt-Ruppin

A

Strains of

pp6Ov-src

SITA

REDDY,t DIANNE MAZZU,t

DENNIS MAHAN, ANDDAVID SHALLOWAY*

Department of Molecular and Cell Biology, The Pennsylvania State University, University Park, Pennsylvania 16802

Received5January 1990/Accepted18April1990

We show that

Schmidt-Ruppin

D pp6ov-src kinase activity is reduced byamutation previouslyshowntobe

associated with

Schmidt-Ruppin

A pp6Ov-srctemperaturesensitivity and that its reduced transformingactivity

is associated with

a

conformational

change in the SH3 region. The evolutionary relationship ofseven v-src

strains

was

studied

byusing parsimony analysis.

At

least

six

different v-src strains are commonly used,

often

with

the assumption

that

their biological effects are

equivalent. With

the

exception of two recently isolated

strains

(6), these

strains probably derive from the original

Rous tumor no. 1 isolate (19, 34). All v-src proteins

con-tain

the same

replacement for the carboxyl terminus of

pp60-csrc

but, because of rapid

retroviral mutation (3),

have diverged at multiple isolated residues during their

evolution

through

laboratory

passaging (2, 4, 15, 18, 28,

31-33).

Reduced transforming activities of SRD v-src relative to

SRA-SF v-src. We

discovered

that

chimeric

v-srclc-src

plas-mids

expressing Schmidt-Ruppin D (SRD) amino region

coding

sequences

combined

with c-src

carboxyl region

se-quences

had significantly lower focus-forming activity, when

tested by transfection into NIH 3T3 cells, than similar

constructs

containing Schmidt-Ruppin

A-San Francisco

(SRA-SF)

sequences

(Table

1

[26]). This

effect

was

evident

with

plasmids

in

which either Rous sarcoma

virus

(RSV)

long

terminal

repeats

(LTRs)

for low-level

expression

or

Moloney murine leukemia virus (MoMLV) LTRs

for

high-level

expression

were

used.

Nonchimeric SRD

v-src

had less

activity

than

SRA-SF v-src when tested with RSV LTRs but

not

when tested with

MoMLV LTRs

(Table

1).

(This

is

consistent with

our

earlier demonstration

that

transfection

assays

with weak

promoters

often

provide

more

sensitive

tests

for functional differences

than assays with strong

promoters

[26].) We

conclude

that

structural differences

between

SRD and SRA-SF

v-src

modulate

their

transform-ing

activities.

SRD

v-src

sequence.

To

identify

the

mutations

responsible

for

these

functional

differences,

we

sequenced

the SRD

v-src

gene

(Fig. 1).

We

found

that the SRD

and SRA-SF

(2,

15)

v-src

sequences

differ

in

nine

nucleotides, which

result in

five amino acids changes,

at

positions 62, 124, 318,

368,

and

461.

pp6ov(SRD)-src

kinase

activity

is reduced

primarily

by

its

Val-461-*Met

mutation.

The presence of Met-461 in SRD

v-src

rather than

Val-461

(present

in

SRA-SF

v-src)

was

of

*

Corresponding

author.

t

Presentaddress: Centerfor Cancer

Research, MIT,

Cambridge,

MA02139.

tPresent address:

University

of Nevada School of

Medicine,

Reno,NV89510.

particular interest since this mutation is required, but not

sufficient,

for the

induction of

temperature

sensitivity in

three mutants

of SRA-New

York

(SRA-NY) v-src (8, 21). To

study the effect of this mutation alone, we

constructed

plasmid

pMvsrcAD

expressing

a

SRA-SF mutant

having

a

Val-461-*Met

substitution

[pp6ov(sRA-SF)-src(M46l)]

and

plas-mid pMvsrcDA

expressing

a

Met-461-*Val SRD v-src

mu-tant

[pp6Ov(sRD)-src(V461)I

(Fig. 2). The relative

specific

activ-ities of

the

v-src

variants

were

measured with

the exogenous

substrate

(acid-denatured

enolase)

immune

complex

kinase

assay

(Table 1). Similar results

were

obtained in

experiments

in

which either monoclonal

antibody

EC10 (22) or 327 (16),

which binds different epitopes,

was

used.

pp6OV(sRD)-src

had

about

threefold

lower

specific activity

than

pp60vtSRASF)src;

analysis of

pp60v(SRA-SF)-src(M461)

and

pp60v-src(SRD)-src(V461)

showed

that this

reduction

was

associated with Met-461

alone. Garber

et

al.

(5) have

previously reported

that

Val-461-*Met mutation

does

not

decrease the kinase

activity

of SRA-NY

pp60v-src.

The

difference

from

our

results

might

be due

to

the

use

of different types of kinase assays

or

due

to

the

additional amino acid differences (at residues 124

and

301) between the SRA-NY and SRA-SF

pp6Ov-src

strains.

We suspect that the

equal focus-forming

activities

of

pMvsrcAD

and

pMvsrcDA

reflect lack of

sensitivity of

the

focus-forming

assay

with MoMLV

LTR

plasmids (26)

and

that differences between the

pp6ov(SRA-SF)-src(M46l)

and

pp6ov(SRD)-src(V461)

focus-forming activities

would be

ob-served if RSV LTR

expression plasmids

were

used

(cf.

the

focus-forming

activities of

the MoMLV and RSV LTR

v-src

plasmids pMvsrcD, pMvsrc, psrcll,

and

pRvsrcA).

Altered

pp6Ov(SRD)-src

V-8 protease

cleavage

pattern.

Partial

digestion of

either 32P-labeled

immunoprecipitated pp6Oc-src

pp6Ov(sRA-sF)-c

.or

pp60v(5RA

SF)-src(M461)

with

Staphylo-coccus aureus

V8

protease

yielded

34-kilodalton

(kDa)

ami-no-terminal

Vi

and

26-kDa

carboxyl-terminal

V2

fragments

and

18-

and 16-kDa V3 and

V4

amino-terminal

subfragments

(Fig.

3).

However,

an

altered pattern

was

observed

for

pp60_v(SRD)-src

and

pp6Ov(sRD)-src(v46l):

this

contained

re-duced

amounts

of the V3 and V4

fragments

and

a

novel

9.5-kDa V5

fragment

that

was

only

weakly

present

in the

pp6Ov(sRA-sF)-src

digests.

The

V3,

V4,

and

V5

fragments

of

both

pp6Ov(SRD)-src

and

pp60v(SRA-SF)-src

contained only

phosphoserine,

while all the

V2

fragments

contained

only

phosphotyrosine

(data

not

shown).

The

pp6Ov(SRD)-src

and

3545

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3546 NOTES J. VIROL.

D ACTCTGCTGGTGGCCTCGCGTACCACTGTGGCCAAGCGGTAGCTGGAACGTGCAGCCGACCACC

A G G

D 1 MET GlySer SerLys Ser Lys Pro LysAsp Pro Ser GlnArg Arg Arg Ser Leu GLuPro ProAsp Ser Thr His His Gly Gly Phe Pro D 1 ATG GGG AGT AGC MG AGC MG CCT MG GAC CCC AGC CAG CGC CGG CGC AGC CTG GAG CCA CCC GAC AGC ACC CAC CAC GGG GGA TTC CCA

D 31 Ala Ser GLn ThrProAsnLysThr AlaAla ProAspThr HisArg Thr Pro SerArgSer PheGlyThrVal Ala Thr GluPro Lys Leu D 91 GCC TCGCAGACC CCC AAC MG ACA GCA GCC CCC GAC ACG CAC CGC ACC CCC AGC CGC TCC TTC GGG ACC GTG GCC ACC GAG CCCAAG CTC

c GlyGLy

D 61 Phe Glu AspPheAsnThr SerAspThrVal Thr Ser ProGlnArg Ala GLyAla LeuALa GLy GlyVat Thr Thr PheVal Ala LeuTyr D 181 TTC GAG GAC TTC AAC ACT TCT GAC ACC GTT ACG TCG CCG CAG CGT GCC GGG GCA CTG GCT GGC GGC GTC ACC ACT TTC GTG GCT CTC TAC

A G

A Gly

c Arg Thr Asp

D 91 Asp Tyr Glu Ser Trp lieGtu ThrAspLeuSer Phe Lys Lys Gly Glu ArgLeuGln Ile Val Asn Asn ThrGlu Gly Asn Trp Trp Leu D 271 GAC TAC GAG TCCTGG ATT GAA ACG GAC TTG TCC TTCMG AMGGA GAACGC CTG CAG ATT GTC AACAAC ACG GM GGT AAC TGG TGG CTG

A G

c Leu

D 121 Ala HisSer ValThr Thr Gly Gln Thr Gly Tyr Ile Pro Ser AsnTyr Val Ala Pro SerAspSer Ile GinAla Glu GluTrp Tyr Phe

b 361 GCT CAT TCC GTG ACT ACA GGACAG ACG GGC TAC ATC CCC AGT AAC TAT GTC GCG CCC TCA GAC TCC ATC CAG GCT GAA GAG TGG TAC TTT

A C C

A Leu

D 151 Gly Lys Ile ThrArg Arg Glu SerGluArg Leu Leu Leu Asn Pro GluAsn ProArg Gly Thr Phe LeuValArg Glu Ser GluThr Thr D 451 GGGMGATC ACT CGT CGG GAG TCC GAG CGG CTG CTG CTCMC CCC GAA MC CCC CGG GGA ACC TTC TTG GTC CGG GAG AGC GAG ACG ACA D 181 LysGly Ala Tyr Cys Leu Ser VaL Ser AspPhe Asp AsnAla Lys GlyLeu AsnVal Lys His Tyr Lys Ile Arg Lys LeuAspSerGly

D 541 MA GGT GCC TAT TGC CTC TCC GTT TCT GAC TTT GACMC GCCMG GGGCTC MT GTG MG CAC TAC AAG ATC CGC AAG CTG GAC AGC GGC D 211 Gly PheTyr Ile ThrSer ArgThr GlnPhe Ser Ser LeuGln Gln Leu Val Ala Tyr Tyr Ser LysHisAla AspGly LeuCys HisArg

D 631 GGC TTC TAC ATC ACC TCA CGC ACA CAG TTC AGC AGC CTGCAG CAG CTG GTG GCC TAC TAC TCC AAA CAT GCT GAT GGC TTG TGC CAC CGC D 241 Leu ThrAsnVal Cys ProThrSer Lys Pro GlnThr GlnGly LeuAla Lys Asp Ala Trp Glu Ile Pro Arg Glu Ser Leu Arg Leu Glu D 721 CTG ACCMC GTC TGC CCC ACG TCCMG CCC CAG ACC CAG GGA CTC GCCMG GAC GCG TGG GMATC CCC CGG GAG TCG CTG CGG CTG GAG D 271 Vat Lys LeuGly GlnGlyCysPhe GlyGlu Val Trp Met Gly Thr Trp AsnGlyThr ThrArgVal Ala Ile Lys Thr LeuLys Pro Gly D 811 GTG MGCTG GGG CAG GGC TGC TTT GGA GAG GTC TGG ATG GGG ACC TGGMC GGC ACC ACC AGA GTG GCC ATA AAG ACT CTG MG CCC GGC

c Asn Arg

D 301 Thr Met Ser ProGluAla Phe LeuGln GluAla Gln Val Met Lys Lys Leu GlnHis Glu Lys Leu Val Gln Leu TyrAla Val Val Ser D 901 ACC ATG TCC CCG GAG GCC TTC CTG CAGGMGCC CAA GTG ATGMG MGCTC CAG CAT GAGMG CTG GTT CAA CTG TAC GCA GTC GTG TCG

A G G

A Arg

c Thr

D 331 GluGlu Pro lieTyr lieVal lieGlu Tyr Met Ser Lys GlySer Leu Leu AspPhe Leu Lys Gly GluMet GlyLys Tyr Leu Arg Leu D 991 GMGAG CCC ATC TAC ATC GTC ATT GAG TAC ATG AGCMG GGG AGC CTC CTG GAT TTC CTGMG GGA GAG ATG GGCMG TAC CTG CGG CTG

c Ala

D 361 Pro GlnLeuVal Asp MetAlaAspGin lieAla SerGlyMetAla TyrVal Glu Arg Met Asn Tyr Vat HisArg Asp Leu ArgAla Ala

D 1081 CCACAG CTC GTT GAT ATG GCT GAT CAG ATT GCA TCC GGC ATG GCC TAT GTG GAG AGG ATGMC TAC GTG CAC CGA GAC CTG CGG GCG GCC

A C

A Ala

D 391 Asn IleLeuVal GlyGlu Asn Leu Val Cys Lys Val Ala AspPheGly LeuAlaArg Leu lieGlu Asp Asn Glu Tyr Thr Ala Arg Gln

D 1171 MCATC CTG GTG GGGGAG MC CTGGTG TGCMG GTG GCT GAC TTT GGG CTG GCA CGC CTC ATC GAG GACMC GAG TAC ACA GCA CGG CM

D 421 Gly Ala LysPhe Pro IleLys TrpThrAla ProGluAla Ala Leu Tyr GlyArg Phe Thr Ile Lys Ser AspVat Trp Ser Phe Gly Ile

D 1261 GGT GCC AAG TTC CCC ATCMG TGG ACAGCC CCC GAG GCA GCC CTC TAT GGC CGG TTC ACC ATC MG TCG GAT GTC TGG TCC TTC GGC ATC

c Val Val Gln

D 451 Leu LeuThr Glu LeuThr Thr Lys GlyArg Met Pro Tyr ProGlyMet GlyAsnGlyGlu Val Leu Asp ArgVal GluArg Gly Tyr Arg D 1351 CTG CTG ACT GAG CTG ACC ACC MG GGCCGG ATG CCA TAC CCA GGG ATG GGC MC GGG GAG GTG CTGGAC CGG GTG GAG AGG GGC TAC CGC

A G

A Val

c Lys

D 481 Met Pro Cys Pro Pro Glu Cys Pro Glu Ser Leu His Asp Leu Met CysGln Cys Trp Arg Arg Asp ProGlu GluArg Pro Thr PheGlu

D 1441 ATG CCC TGC CCG CCC GAG TGC CCC GAG TCG CTG CAT GAC CTT ATG TGC CAG TGC TGGCGGAGG GAC CCT GAG GAG CGG CCC ACT TTT GAG

A C

c Phe Glu Asp Tyr Phe Thr Ser Thr Glu Pro GlnTyrGlnProGlyGluAsnLeu

D 511 Tyr Leu GlnAla Gln Leu Leu ProAla CysVal Leu Glu Val Ala Glu

D 1531 TAC CTG CAG GCC CAG CTG CTC CCT GCT TGT GTG TTG GAG GTC GCT GAGTAGTGCGCGAGCAAAATTTAAGCTACAACMGGCAAGGCTTGGCCGACAATTGCA

A T A

FIG. 1. SRDv-srcsequence. Thenucleotide sequence reported here will appear in the GenBank and EMBL data bases under accession number M33292. The coding sequence begins at position 1. Nucleotides and amino acids that are different in SRA-SF v-src are shown in lines marked A. Amino acids that are different in

pp60-src

are shown in lines marked c. The sequence was determined by Maxam-Gilbert sequencing (17) of fragments fromasubcloneof SRDv-srcphageXSR-RSV-D-5 (29). Allregions were sequenced at least twice and, in most regions, onbothDNAstrands.

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[image:2.612.96.533.94.645.2]

NOTES 3547

non-chimeric

pIrr1

psrcl11 A= L pRvsrcA

pRcsrc

v-src/c-src

_

chimeras

-*

pRS21 Lt--=

RSV LTR vector

pA

pA

codon 515

pA

pA

pRPB5

non-chimeric

pMvsrcD r

pMvsrc

MoMLV

vector

I _H__

_\_NN~, 4,

pMcsrc \'r* I"

v-src/c-src chimeras

pMvcsrcD *

pMBB4

v-src/v-src chimeras

pMvsrcDA E _

codon 431

_'

codon 431

.,.,________________________

pMvsrcAD F-EX 7\'

FIG. 2. src expression plasmids. Only eucaryotic regions are shown. LTRs and coding regions are boxed. Coding sequences are from chickenc-src, SRD v-src, orSRA-SF v-src and are indicated by hatching. Dots above the psrcll coding sequence indicate the positions of amino acid differences between SRD and SRA-SF v-src. pA marks the positions of simian virus 40 early region polyadenylation sites. Recombinations were made at eitherBglI (codon 431) orPstI (codon 515) sites. psrcll (35), pMvsrc, and pMcsrc (9), pRvsrcA, pRcsrc, pRS21, pRPB5, and pMBB4 (26) have been described. Fragments from these plasmids were cross-ligated to create the other plasmids. The pMvsrc, pRvsrcA, pRPB5, and pMBB4 coding sequences were derived from plasmids donated by H. Hanafusa.

pp6oV(SRA-SF)-src

had

identical tryptic

phosphopeptide

maps

(data

not

shown).

The

release

of

a

small

amount

of V5 fragment from

pp60v(SRA-SF)-src

and

comparison of the SRD and SRA-SF

sequences

show

that

the SRD

V5 cleavage site does

not

result from the

introduction of

an

additional

glutamic acid

residue.

Thus,

it is

likely that

accelerated V5 cleavage

results

from

a

mutation-induced conformational change

in the

pp60v(SRD)-src

amino region. This

conformational

change

could

explain

the

reduced

transforming activity of

pMvcsrcD,

the v/c-src chimera

containing

SRD-specific

amino acids at

positions 62, 124, 318, and 368, relative

to

pMBB4,

the related SRA-SF

chimera

(Fig.

2 and Table

1).

This reduced

transforming activity is

not

due

to

reduced

specific

kinase

activity

(Table 1)

but

might

reflect altered

interaction(s)

with cellular

proteins resulting

from a

muta-tion-inducedconformational

change.

The

V5 cleavage

siteis located near the

boundary

of the src

homology

3

(SH3)

region, indicating

that the conformation ofat least part of this

region

is altered.

Furthermore,

oneof thetwomutations

flankingthe novelsite, Leu-124(SRA-SF)-* Val(SRD),lies

withintheSH3

region.

It has

previously

been

suggested

that the SH3

region

governs interactions of

pp6Osrc

and cellular

proteins (8, 10, 23, 24).

The functional difference between SRD and SRA-SF v-src may

provide

an

example

of a

correlated effect on both

pp6Osrc

structure and function mediated

by

this

region.

Parsimony

and

polymorphism

analysis.

Figure

4

shows

an

evolutionary

map ofseven v-src

coding

sequences

derived

fromcladistic

parsimony analysis

(which

generates themap

containing

the minimal numberof

nucleotide

changes [20]).

The

consistency

index is

96%

(i.e., the analysis

is

compli-cated

by

<4% of back or

parallel mutations).

The map is consistent with the limited

information

that is available

describing

the

evolutionary

historiesof the srcvariants

(12,

27,

34)

and

provides

a

guide

to compare

experiments

with different v-srcstrains.

Thelocations of the

polymorphic

sitesareshown in

Fig.

5.

Except

forthe

Asn-301---

>ThrandThr-338->Ile substitutions

(the

latter alone can activate the transforming

potential

of

pp6Oc-src [11])

and the

carboxyl-terminal substitutions,

none

of the

coding

mutations relative to c-src are found in all of the v-src variants. This is consistent with the

assumption

thatmostof the

point

mutationsarose

independently

atlater stages of viral evolution.

Although

the small

sample

size

precludes

calculation of

statistically

significant

measures, the survival of

large

re-gions

without mutation suggests that

they

are necessary

for

activity.

The three

largest

nonpolymorphic

amino acid

sequences

(33

to 37 residues in

length)

include amino acids 125 to 158

(region I),

382 to 419

(region

II), and 427 to

460

(region III) (Fig. 5). Region

I

overlaps

the SH3 and

SH2 domains. While

deleting

amino acids 112 to 142 does not

destroy

Prague-A

v-src

transforming activity (25),

= v-src (SRD) coding

M

v-src(SRA-SF) coding

mc-src coding

=

RSV LTR

=

MoMLV LTR

Fe Splice donor

*I Splice acceptor

-3

rlp

q'i

-3

r- -1 m

0-1

=

... m

VOL.64, 1990

I.- -.6

r-al ^

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TABLE 1. src

activity

Specifickinaseactivityc'

Plasmid Expressed gene" _formingRelative

focus-activity Monoclonal EC10 Avg

antibody327 El v

RSV LTR src plasmids Nonchimeric

psrcll v(SRD)-src 0.008 x 1.3

pRvsrcA v(SRA-SF)-src 0.03 x_ 1.1

pRcsrc c-src <10-6

v-srclc-srcchimeras

pRS21 v(SRD)/c-src (515) <10-6

pRPB5 v(SRA-SF)/c-src(515) 0.002 x 1.6

MoMLV LTR srcplasmids Nonchimeric

pMvsrcD v(SRD)-src 1.1 x 1.4 0.2 0.4 0.3 x 1.4

pMvsrc v(SRA-SF)-src 1.0x 1.3 1 1 1 x 1.2

pMcsrc c-src 0.02 x. 2.1 0.1 0.1 0.1 x 1.4

v-srclc-srcchimeras

pMvcsrcD v(SRD)/c-src(431) 0.2 x 1.6 0.9 1.3 1.0x 1.2

pMBB4 v(SRA)/c-src(431) 1.1 x 1.4 1.4 1.3 1.3 x 1.2

v-srclv-srcchimeras

pMvsrcDA v(SRD)-src(V461) 0.9 x 1.4 1.1 1.2 1.1 x 1.1

pMvsrcAD v(SRA-SF)-src(M461) 0.9_x 1.5 0.4 0.2 0.3 x. 1.4

a V461,

Met-461-aVal

mutation. M461,

Val-461-'Met

mutation.Numbers inparentheses indicatethe codonpositionsatwhich thechimeric recombinations

weremade.

bNIH3T3 cells weretransfected (5x 105cellsper 60-mmplate) with0.002 ,ug(forsrcexpression plasmidscontainingtheMoMLVLTRs)or0.1ILg(forsrc

plasmids containing RSVLTRs) ofthe indicated plasmidsand20

jig

ofsonicated calfthymuscarrierDNA aspreviously described (9).Pilot experiments showed

thattheseamountsinduced foci in thelinearresponse range of 0 to 100fociper plate. DNAs werelinearized bycleavageat auniqueNruIin theirprocaryotic regionspriortotransfection. Relativefocus-forming activities aregeometricaverages of a minimum of threeexperiments withduplicate plates. Valuesare normalized to theaveragefocus-formingactivityofpMvsrc (1.4x105fociper pmol). Thehigheroverallfocus-forming activities ofthe MoMLV LTRplasmids

resultfromtheirinductionofhigherlevels of srcexpression (26).

'Massculturesofsrcexpressercellswerecoselectedin 400,ug of G418(GIBCO Laboratories)per ml aftercotransfecting 5x 105 cells per 60-mmplate with

5 ,ugoftheindicated srcexpression plasmid andneoexpression plasmidpSV2neo(30) in a10:1molarratio. G418-resistantcolonies(50to 100)were pooledand

grownforeach massculture. Activitiesof theindicatedsrcproteinsweremeasuredin theimmune complexkinaseassayby usingeither monoclonal antibody

327 (2 to 3 experiments; average value shown) or EC10 (1 experiment) with acid-denatured rabbit muscle enolase as substrate. The proteins were

immunoprecipitated fromthe mass cultures after 18 hof metaboliclabelingwith[35Slmethionine and were thenincubatedwith[y-32PIATPand substrateas

previously described(13). Specific kinase activities werecalculated fromthe amounts of

[35S1pp60'r'

and

[32P]enolase

in theexcised bands (determined by double-channel scintillation counting)and thespecific activitiesof metaboliclabeling(13). Thegeometric averages ofallexperimentswithbothantibodiesare shown.

A

B

C

D

Mw

.36.5

deletions that include the 3' part of

region I (e.g., 142

to

169

[1],

143

to

173

[25],

or

112

to

196

[25])

destroy

or

di-minish

v-src

transforming activity

in

accord with the

hypo-thesis that

at

least this

portion of region I has been

selec-tively

conserved.

Regions

II and III lie

within

the central

core

of the

catalytic domain,

which has

the greatest

fre-quency

of

highly

conserved

residues

(these

regions

include

subdomains VII and VIII described in

reference

7).

Numer-ous

studies have shown

that

mutation of

amino

acids

within these

regions

can

eliminate v-src kinase and

trans-forming

activities

(reviewed

in reference

23).

It is

interesting

that the Val 461- Met

mutation is

at

the

border

of

region

III.

None of the other

v-src

polymorphisms

occur at

amino

acids

that

are

highly

conserved amongst the

protein kinases

(7).

*- * *_11

V4--.O.

[image:4.612.57.560.64.276.2]

Vs-- .&

FIG. 3. Cleveland

digests

of SRDandSRA-SF

pp60src

proteins.

pp60src

proteins

were

immunoprecipitated

withanexcessof

mono-clonal

antibody

EC10from cells labeledwith

[32P]orthophosphate

for 16h,

partially digested

with

Staphylococcus

aureusV8 protease,

and

analyzed by

12.5% sodium

dodecyl sulfate-polyacrylamide

gel

electrophoresis

and

autoradiography

as

previously

described (13).

Lanes A

through

D contain

digests

of

pp60v(sRD)-src

(A),

pp60v(SRA-SF)-src

(B),

pp6Ov(SRD)-src(V461)

(C),

and

pp60v(SRA-SF)-src(M461)

(D)

derivedfromcoselectedmassculturesof NIH 3T3cells thatweretransfected withsrc

expression plasmids

(Fig.

2)

and

pSV2neo (30).

The

positions

of the 34-kDa amino

fragment (V1),

26-kDa

carboxyl

fragment (V2),

18-kDa amino-terminal

fragment

(V3),

16-kDaamino-terminal

fragment (V4),

and novelamino-terminal9.5-kDa

V5

fragment

areindicated. The posi-tionsofmolecular

weight

standards areindicatedin kilodaltons in the

Mw

column.

3548 -8

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NOTES 3549

C-SRC

FIG.

4. Cladistic

parsimony

analysis

of seven v-src strains.

Sequences of thec-src(15,32), SRD,SRA-NY (18, 31),SRA-SF (2,

15), Prague strain of RSV, subgroup C (PrC)(28), Prague strain of

RSV, subgroup A (PrA) (4), Bryan high titer (BH-RSV) (18), and

B77 (33)v-src sequences were analyzed with thePAUP (Phyloge-netic Analysis Using Parsimony) 2.4.2 program from the Illinois

Natural HistorySurvey. Themostparsimoniousevolutionarytreeis shown. Branch lengths(marked above thebranches)correspondto

thenumberofnucleotide changes between the different srcgenes. 31

8

81

18 2 1.2

SRA(s SRA(ny)

4

SRD

B77 BH-RSV

PRA PRC

10 20 30 40 50 60 70 80 90 100

II

...X...XX... ....X...S.X..X...X...S...X...X X...X

S S X S

SH3 ->||<- SH2

110 120 130 140 150 160 170 180 190 200

...s...X...X... X Xs...X...

-- I

---SH2

-I

||c- CATALYTIC

210 220 230 240 250 260 270 280 290 300

.X. s. ...s...s.. X...X... . ....Xs...

X

310 320 330 340 350 360 370 380 390 400

X...X.s...s..X.X ..sX..X s.X.

410 420 430 440 450 460 470 480 490 500

. sX..X..X.... X.X.s. ....s..Xs

- I--- s--- III--- X

CATALYTIC

>1

510 520

l

l

XX s.X X.XXXXODX)O(X

FIG. 5. Polymorphism ofsrcvariants.Thesequences of SRD, SRA-SF, SRA-NY, PraguestrainofRSV, subgroupA(PrA), Praguestrain

of RSV, subgroup C (PrC), Bryan high titer(BH)-RSV, and B77 v-src andchicken c-src are compared. The numbers identify amino acid

positions. Dots represent amino acids thatareconserved in all variants. Silent mutations are indicatedbys;amino acid substitutions are

indicated by X. Multiple symbols represent multiple mutations within the same codon in different variants. With the exception ofthe

mutationsat301,338,and the carboxylterminus,noneofthesubstitutionsrelativetoc-srcareconservedinallv-srcstrains.Thewigglylines

and Roman numerals identify the threelargestnonpolymorphicregions.The boundariesof theconservedcatalytic, SH2(14),andSH3 regions

aremarked. The positionof Met-461 is marked withanasterisk.

VOL. 64, 1990

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3550 NOTES

We areindebtedto T. Whittamfor cladisticparsimony analysis andhelpfuldiscussions; K.Lynch, S. Haggerty,and R. F.Frisque for assistance in sequencing; H. Hanafusa, G.M. Cooper, and A. D.Zelenetzfor plasmids;and J. S.Bruggeand S. J. Parsonsfor antibodies.

This workwas supported by Public HealthService grants RO1-CA32317 andRO1-CA47333 fromthe National InstitutesofHealth andanRCDA (K04-CA01139)toD.S.

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