linker insertion-deletion mutagenesis of the v-src gene: isolation of

Vol. 63, No. 2JOURNAL OF VIROLOGY, Feb. 1989, p. 542-5540022-538X/89/020542-13$02.00/0Copyright C) 1989, American Society for Microbiology

Linker Insertion-Deletion Mutagenesis of the v-src Gene: Isolationof Host- and Temperature-Dependent Mutants

JEFFREY E. DECLUEt AND G. STEVEN MARTIN*

Department of Zoology, University of California, Berkeley, California 94720

Received 21 June 1988/Accepted 19 October 1988

The host cell regulators and substrates of the Rous sarcoma virus transforming protein pp6ov-src remainlargely unknown. Viral mutants which induce a host-dependent phenotype may result from mutations whichaffect the interaction of pp6Ovsrc with host cell components. To isolate such mutants and to examine the roleof different regions of src in regulating pp6ovsrc function, we generated 46 linker insertion and 5 deletionmutations within src. The mutant src genes were expressed in chicken embryo fibroblasts and in rat-2 cells byusing retrovirus expression vectors. Most linker insertions within the kinase domain (residues 260 to 512)inactivated kinase activity and transforming capacity, while most insertions in the N-terminal domain and atthe extreme C terminus were tolerated. A number of mutations generated a host-dependent phenotype.Insertions after residues 225 and 227, within the N-terminal regulatory domain (SH2), produced a fusiformtransformation in chicken embryo fibroblasts and abolished transformation in rat-2 cells; a similar phenotypealso resulted from two deletions affecting SH2 (residues 149 to 174 and residues 77 to 225). Insertionsimmediately C terminal to Lys-295, which is involved in ATP binding, also produced a conditional phenotype.Insertions after residues 299 and 300 produced a temperature-sensitive phenotype, while insertions afterresidues 304 and 306 produced a host cell-dependent phenotype. An insertion which removed the majortyrosine autophosphorylation site (Tyr-416) greatly reduced transformation of rat-2 cells, a property notpreviously observed with other mutations at this site. We conclude that mutations at certain sites within srcresult in conditional phenotypes. These sites may represent regions important in interactions with host cellcomponents.

The product of the Rous sarcoma virus (RSV) src gene,pp6Ov-src, is a protein-tyrosine kinase associated with theplasma membrane and cytoskeleton. The v-src protein se-quence contains five distinct regions (17, 37). Amino acids 1to 7 at the N terminus (region I) specify the addition of thefatty acid myristate to pp6Ov-src; myristylation of pp6Ov-src isrequired for association with cellular membranes and trans-formation (7, 18, 21). Region II, including amino acids 8 to87, is src specific. Region III encompasses the region encod-ing amino acids 88 to 259 and includes the SH2 domaindefined by Sadowski et al. (30); this region is believed tohave a regulatory function (9, 16, 17, 24, 26, 33, 37). Thecatalytic kinase domain (region IV) extending from aminoacids 260 to 514 is conserved among all the protein-tyrosinekinases (11, 36). Within the kinase domain are found aninvariant lysine residue (Lys-295) required for ATP bindingand transformation (20) and the major tyrosine phosphoac-ceptor site (Tyr-416). The C-terminal 12 amino acids (515 to526) of v-src (region V) represent the portion of the v-srccoding sequence which is distinct from the c-src sequence.Although the structure of pp6Ov-src has been extensively

characterized, little is known about the host cell proteinswith which pp6ov-src interacts. A number of cellular sub-strates for the protein-tyrosine kinase activity of pp6Ov-srchave been identified, but to date none of these substrateproteins has been shown to play a role in the transformationprocess (4, 5, 17). Most of these cellular substrates havebeen shown to be extensively phosphorylated at tyrosine innontransformed cells expressing myristylation-defectivepp6Ov-src (19), indicating that their phosphorylation is insuf-

* Corresponding author.t Present address: Dana-Farber Cancer Institute, Boston, MA

02115.

ficient for transformation. In addition, the cellular proteinsresponsible for targeting of pp6O-src to the inner surface ofthe plasma membrane and focal adhesion plaques (28) havenot been identified. Although the transmembrane protein-tyrosine kinases are known to be regulated by extracellularligands, the nature of the factors which regulate the activityof the non-transmembrane kinases remains unclear. Asan initial step in identifying the targets and regulators ofpp6Ov-src, we have sought to define regions of the proteinwhich are important in its interaction with host cell compo-nents. As previously discussed (9, 39), viral mutants whosetransforming capacity depends on the host cell type mayresult from mutations in the transforming protein whichaffect its interaction with host cell proteins. We thereforeisolated host-dependent transformation mutants of v-srcwhich transform avian cells, but which do not transformmammalian cells.We describe here the results of linker insertion-deletion

mutagenesis of the v-src gene. These studies were under-taken with two objectives: first, to define in greater detailimportant functional regions of the molecule; and second, togenerate host-dependent transformation mutants of the v-srcgene. Linkers were inserted into the v-src coding sequence,resulting in two- or four-amino-acid insertions, and deletionmutants were generated by eliminating the sequences be-tween linkers inserted at two different sites. The mutatedv-src genes were expressed in both avian and mammaliancells by the use of two different expression vectors. Wereport here the properties of the mutants generated by thisapproach, including both host range mutants and otherconditional transformation mutants. To begin to probe themolecular basis for the transformation phenotype of themutants, we examined the expression and in vitro kinaseactivity of the mutant pp6Ov-src proteins in infected chicken

542

LINKER INSERTION-DELETION MUTAGENESIS OF v-src 543

881 NC/48213.4kb

pIRLv-src4.1kb

* 19kb v-sr4ClaI fragment

a coding sequenceof v-si (1578bp)

-Clal

. ColEI originof replicotion

a pBR322

FIG. 1. Construction of the recombinant plasmid pIRLv-src. The 881NC/482 plasmid, which contains a permuted RSV genome with theClaI v-src insert, is shown on the upper right. The pIRL19 vector, into which the ClaI v-src fragment was cloned, is shown in the upper left.The resultant clone, pIRLv-src, is shown at the bottom. LTR, Long terminal repeat; kb, kilobases; bp, base pairs.

embryo fibroblasts (CEF). The results of these studiesindicate that in general the effects of the mutations on theability of v-src to transform CEF parallel their effects on thekinase activity of pp6Ov-src. In a subsequent paper (J. E. DeClue, E. Liebl, and G. S. Martin, manuscript in prepara-tion), we will describe in detail the characterization ofmammalian cells expressing the mutant proteins and theproperties of the host range mutants isolated in this study.

MATERIALS AND METHODSRecombinant DNA methods. The dodecameric XhoI linker

(CTCGAGCTCGAG) was obtained from International Bio-technologies, Inc. (New Haven, Conn.). The dodecamericJS4 linker (CGACCTCGAGGT) was a gift of J. Stone.Manipulations were done by standard methods (23) withminor modifications. Ligations were done at 4°C overnightor at room temperature for 4 to 6 h. Restriction endonu-cleases were purchased from New England BioLabs, Inc.(Beverly, Mass.), and digestions were done as specified by

the manufacturer. Small- and large-scale plasmid prepara-tions were done by the alkaline-sodium dodecyl sulfate lysismethod (23). Linkers were phosphorylated with polynucle-otide kinase from New England BioLabs. DNA bands wereisolated from agarose gels either by elution onto NA-45paper (Schleicher & Schuell, Inc., Keene, N.H.) or byadsorption to activated glass (Glassmilk; Bio 101) and sub-sequent elution. Secondary structure predictions were doneby the method of Garnier et al. (12), using the PC/Genesoftware package from Intelligenetics.

Isolation of v-src linker-insertion mutants. The mutagenesisprotocol was based on the procedure described by Stone etal. (34; personal communication). Cesium chloride-bandedpIRLv-src (see Results and Fig. 1) was subjected to partialdigestion with one of nine different restriction enzymescontaining sites in the v-src coding sequence. Samples takenat various times during the digestion were analyzed byagarose gel electrophoresis. DNA from time points contain-ing abundant amounts of linear (singly cut) pIRLv-src was

VOL. 63, 1989

A%wm.cialciar

544 DECLUE AND MARTIN

pooled. DNA from these pooled samples was treated withcalf intestinal phosphatase (Boehringer Mannheim Biochem-icals, Indianapolis, Ind.) and ligated to phosphorylated XhoIlinkers. The ligation products were digested with an excessof XhoI and subjected to agarose gel electrophoresis. Thelinear DNA was eluted, redigested with an excess of XhoIenzyme, and precipitated. The mutated pIRLv-src mole-cules were recircularized by ligation with T4 DNA ligase(New England BioLabs).A portion of the ligation mix was used to transform

competent Escherichia coli DH5 (Bethesda Research Labo-ratories, Inc., Gaithersburg, Md.). Ampicillin-resistant col-onies were analyzed for linker insertions by digestion ofplasmid DNA with ClaI (which removes v-src from pIRLv-src) and XhoI. A large majority of the colonies screened(typically 85%) were found to be mutated. The location ofthe inserted linkers was determined by double digestions ofthe mutant DNA with XhoI and one of a number of enzymeswith a single site in pIRLv-src (e.g., EcoRI, HindIII, MluI)(Fig. 1), using known mutants generated with differentenzymes as standards.The mutants were designated with a letter and number

code based on that used by Stone et al. (34). The first letter,S, designates v-src; the second letter denotes the restrictionenzyme used to generate the mutant (A, AluI; D, DpnI; H,HpaII [for SHX1 to SHX11] and HaeIII [for SHX12 toSHX21]; N, NlaIV; P, PvuII; R, RsaI; S, SmaI; T, ThaI);the third letter, X, shows the introduced XhoI site; and thenumber denotes the site in the v-src coding sequence whichwas mutated. The precise location of the insertions is shownin Table 2; when necessary the site of the mutation (theresidue preceding the inserted linker) is included in bracketsafter the mutant designation.

Insertion of mutant v-src genes into retroviral expressionvectors. The effects of the mutations were analyzed byexpressing the mutant v-src genes in the Moloney murineleukemia virus-based vector fpGV-1 (27) and in the avianvector RCAN (15). The ClaI v-src fragment was subclonedfrom pIRLv-src into Clal-digested fpGV-1 and then sub-cloned again from the latter plasmid into ClaI-digestedRCAN. To eliminate the vector from which the v-src frag-ment was to be cloned, we digested the donor DNA with anenzyme with a site or sites in the donor vector, but not in thev-src insert. When subcloning from the pIRLv-src vector,PvuI, which has a single site in pIRL19, was used, whileHindIll, which has two sites in fpGV-1, was used to elimi-nate fpGV-1. The orientation of v-src fragments cloned intofpGV1 or RCAN was determined by digestion with anenzyme which cuts within the vector sequences and eitherXhoI (which cuts at the linker) or NcoI (which cuts at codon1 in src).

Construction of v-src deletion mutants. Deletion mutantswere generated by ligating DNAs derived from differentin-frame insertion mutants. fpGV-1 plasmids donating the 5'or 3' portion of v-src were digested with XhoI, which cuts atthe unique site of linker insertion, and BglII, which cutsfpGV-1 once, within the Tn5 transposon sequences. Di-gested DNAs were then ligated and used to transform E.coli. This strategy depends on the fact that to recircularizeand restore kanamycin resistance, a plasmid must containboth the XhoI-BglII and the BglII-XhoI fragments. Subse-quent digestion with ClaI revealed those plasmids whichcontained deletions in v-src. Deletion mutants were desig-nated XD1 to XD6 (Table 1).

Transfection of avian and mammalian fibroblasts. CEF andrat-2 cells (35) were cultured as described previously (9).

TABLE 1. Mutants created by deletion of v-src sequences

Donor of Donor of Amino AminoMutant 5' XhoI 3' XhoI acids acids

site site deleted inserteda

XD1 SHX1 SHX3 15-168 RPRGXD2 SHX3 SHX4 169-174 RPRGXD3 SHX4 SHX5 175-262 RPRGXD4 SHX2 SPX1 77-225 DLEXD6 SRX1 SHX4 149-174 SRG

a Amino acids encoded by the joined half-linkers.

One day prior to transfection, cells were plated at 36°C at adensity of 106/100-mm dish. The next day, the cells weretransfected with 10 jig of purified plasmid DNA by thePolybrene-dimethyl sulfoxide method (22). Three days afterthe CEF cultures were transfected, the cells weretrypsinized and replated. Typically, two passages posttrans-fection were required to achieve a high level of infection, andon day 9 the cells were plated for radiolabeling, virusharvests, and photomicroscopy. Two days after transfectionof the rat-2 line, cultures were trypsinized and replated intotwo plates. The cells were maintained in growth mediumcontaining 250 ,ug of G418 (geneticin; GIBCO Laboratories,Grand Island, N.Y.) per ml. Rat-2 cells were fed at 3- or4-day intervals, and 12 or 13 days after replating, the cellswere scored for transformation.

Radiolabeling and virus harvests. CEF were plated in35-mm wells for radiolabeling as described previously (9).The next day, the cells were labeled for 18 h with[35S]methionine in a medium containing 2.6 mg of methio-nine per liter (20% of the level in growth medium); after 18 hthe cells were lysed. For virus harvests, infected CEF wereplated at 2.5 x 106/100-mm dish. Three days later, virus washarvested, clarified by centrifugation, and frozen.

Immunoprecipitation and kinase assays. Immunoprecipita-tion procedures were as described previously (9). For immu-noprecipitation of 35S-labeled proteins, portions of lysatescontaining equal numbers of acid-precipitable radioactivitywere compared. Tumor-bearing rabbit serum and a goatanti-p279'9 serum (Research Resources, National CancerInstitute, Bethesda, Md.) were used in excess to immuno-precipitate pp60v-src and Pr769'9, respectively.For immune complex kinase assays, one-half of the mock-

labeled lysates was immunoprecipitated with 10 ,ul of tumor-bearing rabbit serum. The immune complexes were collectedon 20 volumes of 10% fixed Staphylococcus aureus andwashed as described previously (9). The kinase reactionbuffer used for analyses of pp60vsrc contained 5 mM MgCl2and 5 mM MnCI2. The protein concentration of duplicateportions of the mock-labeled lysates was determined by thebicinchoninic acid assay (31) with reagents from PierceChemical Co. (Rockford, Ill.). To calculate the specificactivities of the mutant pp60v-src molecules, the valuesobtained for total kinase activity (kinase activity per micro-gram of cellular protein) were divided by the amount ofradiolabel in pp6Ovsrc immunoprecipitated from parallel[35S]methionine-labeled cultures. The values obtained fortotal kinase activity and specific activity were both normal-ized to the values obtained for cells expressing wild-typeSchmidt-Ruppin RSV subgroup A (SR-A) pp6Ovsrc.

RESULTS

Generation of linker insertion-deletion mutants of v-src.The procedure used to generate v-src mutants was similar to

J. VIROL.


that used by Stone et al. (34) to mutate the v-fps oncogene.In this procedure, 6- or 12-base-pair XhoI linkers are in-serted at a variety of restriction enzyme sites, resulting intwo- or four-amino-acid insertions, as well as the generationof a novel XhoI site, which serves as a marker for themutation.To facilitate the isolation of v-src mutants, we transferred

the v-src coding sequences to a small (2.2-kilobase) pBR322-derived vector, pIRL19 (29). A 1.9-kilobase ClaI fragmentisolated from the plasmid 881NC/482 (13, 14), a derivative ofthe SR-A clone described by DeLorbe et al. (10), was clonedinto the unique ClaI site in pIRL19 (Fig. 1). The resultingclone, termed pIRLv-src, comprises 4,138 base pairs, ofwhich 1,578 make up the coding sequences of v-src.A total of 46 insertion mutants were derived by partial

digestion of pIRLv-src with nine different restriction en-zymes (Table 2). Eight of the enzymes yield blunt double-stranded molecules, which were mutated with a blunt,self-complementary dodecameric linker (CTCGAGCTCGAG). Following digestion with XhoI, this linker producedtwo-amino-acid insertions (or a one-amino-acid deletion witha three-amino-acid insertion). Eleven mutants were pro-duced from pIRLv-src cut with the enzyme HpaII. Thisenzyme, which cleaves DNA to yield a 3' G-C overhang,was used in conjunction with a partially self-complementarydecanucleotide (CGACCCTGAGGGT) to generate four-ami-no-acid insertions (Table 2). In addition, five different dele-tion mutants were generated by deleting segments betweenXhoI linker insertions in the same reading frame (Table 1).To analyze the transformation potential of the v-src mu-

tants in mammalian and avian cells, we cloned the wild-typeand mutant v-src ClaI fragments into retroviral vectors. Forexpression in mammalian cells, the Moloney murine leuke-mia virus-derived vector fpGV-1 (27) was used (Fig. 2). Thisvector contains a selectable marker, the TnS neomycinphosphotransferase gene, which provides resistance in bac-terial cells to kanamycin and in eucaryotic cells to neomycin(G418). The v-src ClaI fragments were cloned into theunique ClaI site located at the 3' end of fpGV-1, so that thesplice acceptor site within the v-src fragment lay 5' to thecoding sequences, resulting in the expression of v-src from asubgenomic mRNA. For expression in CEF, v-src ClaIfragments were cloned into the avian expression vectorRCAN (15) (Fig. 2). This vector contains an intact, nonper-muted copy of an avian leukosis virus genome with a uniqueClaI site. A spliced subgenomic mRNA is produced whichencodes pp60vsrc.

Transformation of rat-2 cells by mutant v-src genes. Rat-2cells were transfected with fpGV-1 plasmids, and stabletransfectants were selected with G418. Neor rat-2 cell clonesderived from cultures transfected with fpGV-1 were uni-formly flat and nonrefractile, like the parental rat-2 line.Upon transfection of rat-2 with the wild-type v-src-con-taining fpGV-1 construct, approximately 55% of the coloniesdisplayed alterations in morphology and a loss of densityinhibition of growth, characteristic of v-src-transformedfibroblasts. Of 51 fpGV-1 derivatives encoding linker inser-tion-deletion mutant v-src genes, 21 retained the ability totransform rat-2 cells (Table 2; Fig. 3). The majority of these(15 of 21) bear mutations in the amino-terminal 200 aminoacids of ppv`src. However, several mutants with altera-tions in region III displayed a reduced frequency of trans-formation (insertions after amino acids 138, 148, 152, and203), while others were entirely defective (SPX1[225],SHX13[228], XD4 [dl77-225], and XD6 [dl49-174]). In thegreat majority of cases (24 of 27), mutations in the kinase

domain (region IV; amino acids 260 to 513) abolished trans-formation (Table 2; Fig. 3). Two of the three transformation-competent mutants with mutations in the kinase domain(SHX6[229] and SNX3[300]) contained insertions just Cterminal to the conserved lysine residue (Lys-295) (Table 2;Fig. 3). The third transformation-competent mutant with amutation in the kinase domain, SRX4, encoded an insertionafter amino acid 356. Mutations in the last 12 amino acids ofpp60-src did not abolish transformation.

Transformation of CEF by v-src mutants. CEF were trans-fected with the RCAN-v-src plasmids. After two or threepassages, the cells became fully infected and expressedabundant levels of viral structural proteins (e.g., Pr769ag; seeFig. 6 and Table 3) and pp6Ov-src. CEF infected with theRCAN vector with no insert (Fig. 4) resembled uninfectedCEF; the cells remained in a monolayer and were uniformlynonrefractile. Cells infected with the wild-type v-src-con-taining RCAN virus (Fig. 4) displayed a transformed mor-phology, characterized by rounding, piling of cells on oneanother, and a generally disorganized pattern of growth.The properties of the v-src mutants expressed in CEF

were in general similar to those observed in rat-2 cells, butsome significant differences were noted (Table 2). As previ-ously observed in the transfected rat-2 cells, mutations in theamino-terminal src-specific domain (amino acids 8 to 87) didnot affect the transforming capacity of v-src, while mostinsertions in the kinase domain abolished transformation.Mutations affecting SH2 resulted in a variety of alterations inthe transformation potential of v-src. Four mutants withmutations in this domain (two insertions, SPX1[225] andSHX13[228], and two deletions, XD4 [dM77-225] and XD6[d1149-174]) previously identified as transformation defec-tive in rat-2 cells were nevertheless able to transform CEF.All these mutants, as well as one (SDX1[152]) which trans-formed rat-2 cells at a reduced frequency, produced afusiform morphology in CEF that was distinct from thatinduced by the wild type. Another mutant with a mutation inthe SH2 domain (SDX2[203]) produced a typical roundedshape in CEF cultured at 36°C (Fig. 5), yet when these cellswere cultured at 41.5°C, they were fusiform. These resultsindicate that the SH2 domain exerts a strong influence on thetransforming activity of v-src. Mutations in this region arecapable of conferring a host-dependent phenotype on v-srcand of altering the morphology of transformation in CEF.

Several mutations close to the ATP-binding site within thekinase domain resulted in a conditional transformation phe-notype. The mutant SHX7, which contains an insertion afteramino acid 304, was found to transform CEF despite beingtransformation defective in rat-2 cells. CEF transformed bySHX7-v-src resembled cells transformed by the wild-typegene product (Fig. 4); this was in contrast to CEF cellstransformed by the other host-dependent mutants. A secondinsertion in this region, SHX15, with an insertion after aminoacid 306, resulted in a disorganized growth pattern in in-fected CEF but had little effect on the morphology ofindividual cells. Additional conditional mutants were iso-lated in the same region of v-src; both SHX6[299] andSNX3[300] elicited a temperature-sensitive transformationof CEF (Fig. 5; Table 2). At 36°C, CEF expressing thesemutants exhibited a fully rounded shape, while at 41.5°C, thecells reverted to a nontransformed morphology (Fig. 5).These mutants also were temperature sensitive for transfor-mation of rat-2 cells (data not shown).Another mutation, SRX5[415], which contains an inser-

tion that deletes the major tyrosine phosphoacceptor site(tyrosine 416) within pp60V-src also resulted in a conditional

VOL. 63, 1989


TABLE 2. Transformation of rat-2 cells and CEF by v-src insertion-deletion mutants

Residue Amino Rat-2 cell CEF Virus titerMutant preceding acids transformationa transformation' (FFU/ml)'

mutation inserted

101452555976138148152168168174203

206225228259263263272293299

SNX1SHX1SNX2SHX12SAX1SHX2STX1SRX1SDX1SHX3SSx1SHX4SDX2

(41.50C)SAX2SPx1SHX13STX2SHX5SSX2SAX3SHX14SHX6

(41.50C)SNX3

(41.5°C)SHX7SHX15SAX4SHX8SAX5SRX2SRX3SRX4SAX6SHX16SHX17SRX5SHX18SHX9SAX7SHX19SHX10SNX4SHX11SHX20SRX6SHX21

(41.50C)SPX2

300

304306316317321325339356362375390415437437454459459461473506509514

515

dllS-168dl169-174dl175-262d177-225d/149-174

XD1XD2XD3XD4XD6

Vector alone(41.50C)

Wild-type v-src(41.5°C)

SsRPRGLERALEDLEVRAdIY::SRDdlI: :TRVRPRGdlR: :PRGRPRGdlI: :TRV

LELERARARPRGdlR: :PRGLERADLEV

LE

TSRSRALERPRGLEdlY::SRDdlY::SRDdlY: :SRDLERARAdlY::SRDSsRPRGLESSRPRGSRRPRGLEdY: :SRDRA

LE

0.990.970.990.970.900.920.650.760.371.060.981.120.77

0.940.010.011.01

<2.9 x 10-3<2.9 x 10-3<2.9 x 10-3<2.9 x 10-30.96

0.97

<2.9 x 10-'<2.9 x 10-3<2.9 x 10-3<2.9 x 10-3<2.9 x 10-3<2.9 x 10-3<2.9 x 10-31.04

<2.9 x 10-3<2.9 x10-3<2.9 x 10-30.13

<2.9 x 10-3<2.9 x 10-3<2.9 x 10-3<2.9 x 10-3<2.9 x 10-3<2.9 x 10-3<2.9 x 10-3<2.9 x 10-3<2.9 x 1o-30.90

0.97

++F++F

++F

++F

++++++F

<2.9 x 10-30.93

<2.9 x 10-3<2.9 x 10-3<2.9 x 10-3

<2.5 x 10-4

2.9 x1052.5 x1052.7 x1053.0x 1044.0 x 1043.0 x1051.5 x 1047.0x 1045.0 x 1042.3 x 1051.0 X 1058.0 x1041.1 X1055.0 x 1043.0 x1054.5 x 1041.0 X 1053.0x 104<5 x 100<5 x 1002.0x 102<5 x 1003.0 x 1057.0 x 1023.5 x 1055.0 x 1021.2 x 1051.4 x 104<5 x 100<5 x 100<5 x 100<5 x 100<5 x 1004 x 105

<5 x 100<5 x 1002 x 101

3.0x 105<5 x 101<5 x 100<5 x 100<5 x 100<5 x 100<5 x 100<5 x 100<5 x 1002.5 x 1017.0x 1045.0 x 102

5 x 105

3 x 1011.4 x 105<5 x 1002 x1055 x 105

<5 x 100<5 x 100

9X 1051.1 X 106

1.00

a Frequency of transformation by fpGV-1-v-src plasmids as a fraction of the value obtained with wild-type v-src (0.55).b Morphological transformation of infected CEF. F, Fusiform. +++, Morphology indistiguishable from CEF infected with wild-type v-src-RCAN; ++,

morphology as wild type but less than 100% transformation; +, morphological transformation less distinct than wild type; +, morphology much less distinct thanwild type and/or minority of cells transformed.

' Titer of RCAN-v-src constructs in CEF. Assays were done at 36°C except when indicated. FFU, Focus-forming units.

J. VIROL.


fpGV- 1 vector6.9 kb (with v-srcfrag ment)

RCAN vector13.4kb (with v-src)

FIG. 2. Expression vectors used in analysis of v-src mutants.The mammalian fpGV-1 vector is shown at the top, while the avianexpression vector RCAN is depicted at the bottom. The 1.9-kilobase(kb) ClaI v-src fragment is shown at the positions of the unique ClaIsites into which the fragments were cloned. The sizes of the vectorsplus src inserts are given. For clarity, the polyadenylation site in

fpGV-1, which is actually within the long terminal repeat (LTR), isshown at the right; the vector does not contain any bacterialsequences outside the retrovirus genome. S.A., Splice acceptor site;S.D., splice donor site; P.L., polylinker. Symbols: J, ColElorigin of replication; O, simian virus 40 origin of replication; ,

Tn5 transposon sequences.

phenotype. This mutant, which displayed a greatly reducedtransformation potential in rat-2 cells, nevertheless was fullytransforming in CEF at 36°C (Fig. 5). Following a shift to41.5°C, SRX5-infected CEF were found to be somewhat lesstransformed, although this effect was variable and the cellsnever reverted to a completely normal phenotype (Fig. 5).Thus, transformation by this mutant is host dependent andpartially temperature dependent. One other temperature-dependent mutant, SHX21, was isolated (Fig. 5). The inser-tion in SHX21 lies after amino acid 514, in the extremecarboxy terminus of pp60v-srcTo confirm the properties of v-src mutants in CEF,

v-src-containing RCAN viruses were harvested from cul-tures of infected CEF and tested for focus induction on

indicator CEF monolayers. Without exception, RCAN de-rivatives encoding transformation-competent v-src mutantsyielded virus stocks which contained 104 to 106 focus-forming units per ml (Table 2). In contrast, RCAN virusesencoding transformation-defective mutant v-src genes

formed no foci, or very few foci. The titer of wild-typev-src-RCAN virus was not affected when the assay was

conducted at 41.5°C (Table 2). In contrast, the titers of thetemperature-sensitive mutants (SDX2[203]. SHX6[299],SNX3[300], and SHX21[514]) were reduced 100- to 1,000-fold (relative to the titer obtained at 36°C) when assayed at

41.5°C. In all cases, foci were induced with single-hit kinet-ics with respect to the inoculated dose.

Expression of viral proteins in mutant-infected CEF. Toexamine the expression of viral proteins in infected CEF, weimmunoprecipitated Pr76zaz and its cleavage products withanti-p279'9 antiserum from lysates of [35S]methionine-la-beled cells (Fig. 6B, odd-numbered lanes; and Table 3). Inalmost every case, the expression of Pr76zaz in CEF infectedwith mutant v-src-RCAN viruses was within 25% of the levelof parallel cultures infected with the unmodified RCANvector, indicating that the cultures were fully infected. Toexamine the expression of pp60v-src in the infected cultures,we immunoprecipitated the protein from lysates of [35S]me-thionine-labeled cells with a tumor-bearing rabbit serumwhich immunoprecipitates both viral structural proteins andpp6Ov-src (Fig. 6B, even-numbered lanes; Table 3). The levelof expression of pp6Ov-src encoded by many of the mutantswas equal to that observed with the wild-type virus (Table3); this was the case with both transformation-competentand transformation-defective mutants. In some cases, re-duced levels of pp60v-src were detected; pulse-chase exper-iments indicated that this was due to decreased stability ofthe mutant proteins (data not shown).

Kinase activity of mutant pp6O-src molecules. The kinaseactivity of the mutant pp60v-src molecules expressed in CEFwas determined by the immune complex kinase assay (Fig.6A; Table 3). Lanes 1 to 10 of Fig. 6A represent thephosphorylation of immunoglobulin heavy chain in immuno-precipitates prepared in parallel with the immunoprecipitatesfrom [35S]methionine-labeled cells shown in Fig. 6B. Lane 1represents cells infected with RCAN vector alone, lane 10shows cells infected with the wild-type v-src-RCAN con-struct, and lanes 2 to 9 show cells infected with a series ofRCAN viruses with mutant v-src genes. The in vitro immunecomplex kinase assay may not always accurately reflect theability of pp6Ov-src to phosphorylate exogenous substrates.Nevertheless, the kinase activity per microgram of cellularprotein (Table 3) correlated very well with the ability totransform CEF. Thus, mutants which induced kinase activ-ities in excess of 3 to 4% of the wild-type level were ingeneral transforming for CEF, while mutants which inducedkinase activities below this level were nontransforming.(There were a few minor exceptions to this rule; for exam-ple, CEF expressing SHX21[514]-v-src at 41.5°C containedover 10% of the kinase activity of cells expressing wild-typev-src.) These data confirm that transformation by v-srcdepends on pp6Ov-src kinase activity and that the level ofkinase activity in wild-type SR-A v-src-transformed CEF farexceeds the threshold required for transformation.To investigate the effects of the individual mutations on

the specific activity of the pp60v-src kinase, we calculated therelative specific activity of pp60vsrc molecules using valuesobtained for the level of expression of pp60v-src in parallel[35S]methionine-labeled cultures. Mutants with insertionswithin the first 76 amino acids and at the extreme carboxyterminus retained 50 to 110% of the activity of the wild type.Most insertions and deletions in the SH2 domain reduced therelative specific activity of pp60v-src two- to sixfold, whilemutants bearing insertions in the kinase domain that abol-ished transformation in most cases retained only 2% or lessof the wild-type activity. These data indicate that the effectsof the insertion-deletion mutations on the transforming ca-pacity of v-src bear a strong relation to their effects on the invitro kinase activity of pp6Ov-src.

Association of mutant pp6Ov-src molecules with cellularproteins pp5O and pp9O. pp6Ov-src transiently associates with

VOL. 63, 1989


XD2169 H174

SHX3 SSXIRPRG ARPRG

RPRG RA (D-.DLEV AY-SRD SDXI SHX4 AI:TRV RA

SSRPR| SLEA|TRV RPRG

SNXI ISTXI LllSEX

SS LE RA

3N

15 XDI 168

175 262

,5 SAX7 \SNX SHX20iD LE \SR LE

SHXIS SHX9 S SRX6SS RPRG RPRG AY:SRD

SHX1O SHX19RPRG SS

77F- XD4 225

149 174

FIG. 3. Positions of linker insertion-deletion mutations and their effects on transformation of rat-2 cells. The sites of the mutations areshown on a schematic map of the v-src coding sequence. For the linker insertion mutations, the positions of the residues preceding theinsertions are given within the circles; below these are listed the names of the mutants and the amino acids inserted. The deletions are drawnin as bars, with the positions of the first and last amino acids deleted at the ends. Those mutations which do not abolish transforming functionin rat-2 cells (as judged by the transfection assay; see text and Table 2) are drawn above the bar. Mutations listed below the bar result in atransformation-defective phenotype in rat-2 cells. Mutations denoted by triangles transform rat-2 cells but at a reduced frequency.

two cellular phosphoproteins, ppSO and pp90, prior to itsassociation with cellular membranes (1). This associationcan be detected in immunoprecipitates of pp6Ov-src from[35S]methionine-labeled cells (Fig. 6B). In several cases,pp60vsrc molecules encoded by transformation-defectivev-src genes have been shown to be more extensively asso-ciated with the pp50-pp9O complex (37). We noted that mostinsertions in the region from residues 263 to 362 resulted ina strong association of pp6Ov-src with the complex (Table 3).Two temperature-sensitive mutants with mutations in thisregion (SHX6[299] and SNX3[300]) showed a stronger asso-ciation at 41.5°C than at 36°C. These mutations may preventthe release of pp6Ov-src from the complex by disruptingdeterminants which specify interactions of pp60vsrc withthese proteins.

DISCUSSION

Effects of mutations within different regions of v-src. Theconstruction and analysis of 46 insertion mutants and 5deletion mutants spanning the v-src coding sequence permit-ted a systematic comparison of the effects of mutations indifferent regions of pp6Ov-src. The effects of an insertion on

transforming activity were clearly dependent on the site ofinsertion and the domain of the protein which was altered.Mutations within the region between amino acids 8 and 77(region II), as well as a mutation in the extreme carboxyterminus (region V), had little effect on the transforming or

kinase activities of pp60vsrc. Mutations within region IIIresulted in different phenotypes depending on the site of themutation. Insertions in the region 138 to 152 and after aminoacid 203 reduced the frequency of transformation in rat-2cells, while other insertions (after 168, 174, 206, and 259) had

little effect on transformation. Insertions after amino acids225 and 228 and the deletions XD4 (d177-225) and XD6(d1149-174) produced a host-dependent phenotype.With few exceptions, mutations throughout the kinase

domain, encompassing amino acids 260 to 514, abolishedtransforming ability and kinase activity, indicating thattransformation and enzymatic activity require the entiirekinase domain to be intact. Mutations in the kinase domainalso affected the formation or stability or both of the com-plex of pp6Ov-src with the cellular phosphoproteins pp5O andpp90. Most (but not all) of the insertions between residues263 and 362 led to accumulation of the pp60-pp50-pp90complex. In contrast, most of the mutations in the region 375to 509 led to decreased levels of the complex. These findingsconfirm and extend previous reports that deletions or pointmutations in src can affect the formation or dissociation ofthe complex (1, 17, 37).

Conditional mutations in region III. The sequences ofregion III have previously been proposed to represent amodulatory domain (6, 16, 17, 24, 26, 30, 33, 37). A numberof v-src mutants have been described with deletions oralterations in this region which exhibit altered transformingcapacity or kinase activity. The SR-A v-src deletion mutantNY320, which lacks amino acids 149 to 169, was found toinduce a fusiform morphology in infected CEF (6). The RSVPrague A strain v-src mutants CH119 and CH120 lack aminoacids 173 to 227 and 169 to 225, respectively, and bothinduce a temperature-sensitive transformation of CEF (2).Further mutagenesis of the Prague A strain v-src (26) hasdefined amino acids 149 to 172 as essential for transforma-tion by that gene. In addition, mutations in the correspond-ing region of c-src at amino acids 95 and 96 can activate

J. VIROL.


RCAN

WT

XD4

SHX7

SRX5

I-Il~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~IFIG. 4. Phase-contrast photomicrographs of CEF infected with host-dependent transformation mutants. Fully infected CEF were plated

at a density of 2 x 106/100-mm dish 3 days prior to photomicroscopy. All cells were cultured at 36°C. WT, wild-type.

transforming capacity (17). We have previously shown that amutation in the SH2 region of v-fps induces a host-depen-dent phenotype, arguing that this region of the v-fps proteinmay direct its interaction with cellular components (9).The sequences of region III, in particular the SH2 segment

or B-C regions (residues 140 to 250) and the SH3 segment orA region (residues 88 to 140), show marked homology withthe sequences of other members of the protein-tyrosinekinase family that are membrane associated but not trans-membranous (24, 30, 33). A phosphatidylinositol-specificphospholipase C, C-148, has recently been shown to containa large region of homology with region III of the src gene(33). The sequences within phospholipase C that are relatedto region III may serve a regulatory function in this enzyme.The recently described v-crk gene, which is expressed as afusion to viral gag sequences, also contains blocks ofhomology to region III (24). Expression of this gene elevatesthe phosphorylation of cellular proteins on tyrosine, despite

the fact that the crk gene does not itself encode a kinasedomain; this suggests that these sequences can activatecellular protein-tyrosine kinases, perhaps by titration of acellular inhibitor (24). These findings support the idea thatregion III has a regulatory function.Two linker insertions within SH2 resulted in host-depen-

dent transformation and a fusiform morphology in CEF. Theinsertions in the mutants SPX1 (after residue 225) andSHX13 (after residue 228) lie in a region which is highlyconserved among the cytoplasmic protein-tyrosine kinases.Secondary structure prediction (12) suggests that the seg-ment from 223 to 231 forms an extended region, which mightbe a site at which pp6ov-src interacts with host cell protein(s).Two deletion mutations affecting SH2 also resulted in a

host-dependent phenotype. The mutant XD4, lacking resi-dues 77 to 225, and the XD6 mutant, lacking residues 149 to174, exhibited similar transformation properties; both in-duced a fusiform transformation in CEF but did not trans-

VOL. 63, 1989


form rat-2 cells. In XD6, some of the most highly conservedsequences in SH2 are deleted, while in XD4, the entireregion homologous to phospholipase C-148 (including bothSH2 and SH3) is deleted. Despite the similarity in theirphenotypic effects on CEF and rat-2 cells, these two dele-

360

RCAN :N- -,X

; w~;

WT 4

SDX22

SHX6 X

SNX3

SRX2S

SHX21 ,*,'' .,.

41.50

-AiNt

.00W.W

161,c

tions differed greatly in their effect on the kinase activity ofthe v-src gene product. In XD6-infected CEF, the kinaseactivity is approximately 20% of the wild-type level. Incontrast, XD4 expresses levels of kinase activity in excess ofthe wild-type levels. This indicates that the fusiform pheno-type is not simply a consequence of reduced kinase activity.The finding that XD4 pp60v-src, which contains an extensivedeletion (dn77-225), exhibits higher kinase activity than XD6pp60v-src, which contains a smaller deletion (d1149-174),suggests that the additional sequences deleted in XD4 nor-mally function to inhibit pp60v-src kinase activity. This isconsistent with the finding that mutations in SH3 (the Aregion) can activate the transforming capacity and kinaseactivity of pp60c-src (17; J. Brugge, personal communica-tion).The mutant SRX1, which has an insertion after amino acid

148, is the analog of the v-fps mutant RX15m (30), which wasfound to be devoid of transforming function and to have agreatly decreased kinase activity. By contrast, SRX1-v-srcwas found to transform both CEF and rat-2 cells (the latter ata reduced frequency), and the pp60v-src encoded by SRX1exhibited a relative specific activity of 30.6%. Thus, theeffects of mutations in SH2 of the SR-A v-src may differ fromthose of the analogous mutations in other protein-tyrosinekinases. This may reflect at least in part the greater levels ofprotein-tyrosine phosphorylation induced by SR-A pp60v-src(5); with SR-A a large decrease in protein kinase activity isnecessary to reduce the level of activity below the thresholdrequired for transformation.

Mutations in the kinase domain. A cluster of conditionalmutations mapped to a small area four to nine amino acids Cterminal to lysine 295. This region precedes a sequence of 21amino acids (304 to 324) which secondary structure predic-tions suggest may form a stretch of alpha-helix (data notshown). Two of these mutants (SHX6[229]) and SNX3[300])were found to have a temperature-dependent phenotype inboth rat-2 cells and CEF, while SHX7[304] and SHX15[306]exhibited a host-dependent phenotype. The lesion responsi-ble for the temperature-sensitive phenotype of the RSVmutant tsLA32 was found to be at amino acid 300 (37). Thekinase activity of pp60v-src encoded by tsLA32 is low relativeto the wild-type activity and is not significantly temperaturesensitive; the same is true for the SNX3 mutant. Thus, thissmall area appears to be a hot spot for the generation ofconditional mutants. It is possible that this region containsdeterminants involved in the binding of substrates, as theywould be expected to lie in close apposition to the ATPmolecule.One insertion mutant in the kinase domain, SRX4, which

contains an insertion after amino acid 356, retained fulltransforming function. Secondary structure predictions (12)suggest that this site lies within a six-amino-acid stretch ofextended conformation between two alpha-helical regions.Thus, this insertion may lie in a less rigid, hingelike regionand may exert only minimal effects on protein conformation.

FIG. 5. Phase-contrast photomicrographs of CEF infected withtemperature-sensitive mutants and grown at 36 or 41.5°C. CEF weretransfected with RCAN and derivatives encoding wild-type (WT) ormutant v-src genes and passaged at 36°C. Three days beforephotomicroscopy, the cells were replated and one set was shifted to41.50C. The left panel in each row represents cells maintainedcontinuously at 36°C; the right panel represents the same cell typeafter 3 days at 41.5°C. Row 1, CEF infected with RCAN with noinsert; row 2, CEF expressing wild-type v-src; rows 3 to 7, CEFexpressing mutant v-src genes.

J. VIROL.


A97-

68-

-IgG

43-

36-

2 3 4 5

_? _

2345w4w *9 *

6 7 8 9 10

:w w'q

_ _ _- _

_- _1

-pp50

36-...... w

\/ \/ \/\ /

1 2 3 4 5 6 7\/ \/ \/

8 9 10

FIG. 6. pp6Ov-src immune complex kinase activity (A) and expression of Pr76gag and pp60v-src (B) in mutant-infected CEF. (A)Phosphorylation of the immunoglobulin heavy chain in immune complexes from mutant-infected CEF. pp6oV-rc was immunoprecipitated fromlysates of mock-labeled CEF and incubated with [y-32P]ATP. Radiolabeled proteins were analyzed by sodium dodecyl sulfate-polyacrylamidegel electrophoresis. Autoradiography was for 30 min at room temperature. CEF were infected with RCAN vectors expressing the followingv-src genes: lane 1, none; lane 2, SAX7[454]; lane 3, SHX8[317]; lane 4, SHX13[228]; lane 5, STX1[138]; lane 6, STX2[259]; lane 7,SDX1[152]; lane 8, SAX1[59]; lane 9, SHX12[55]; lane 10, wild-type SRA. IgG, Immunoglobulin G. (B) Expression of RCAN-encoded Pr769'9and pp6Ov-src in mutant-infected CEF. Lysates of CEF labeled for 18 h with [35S]methionine were immunoprecipitated with goat anti-p279'9or tumor-bearing rabbit serum. Immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis andautoradiography (3 days at -70°C). The order of the samples is the same as in panel A; the left lane of each pair is the anti-p27immunoprecipitate, the right lane the tumor-bearing rabbit serum immunoprecipitate. Numbers on left show molecular weight (x 103).

The mutant SRX5[415], which encodes a three-amino-acidinsertion and deletes tyrosine 416, was found to transformrat-2 cells at a greatly reduced frequency. However, tyrosine416 itself is not required for transformation of mammaliancells by v-src. A mutant in which tyrosine 416 of v-src was

changed to phenylalanine was shown to transform NIH-3T3cells and to encode a fully active kinase (32). (Cross andHanafusa [8] reported a mutation in which amino acids 412to 416 were replaced with linker sequences. This mutant wasfully transforming in CEF, but its transforming properties inmammalian cells were not reported.) Thus, it is clear that thehost-dependent properties of SRX5 do not result simplyfrom a loss of tyrosine 416, but must be related to the aminoacid insertion at this site. Strikingly, the in vitro kinaseactivity of SRX5-pp60vsrc equaled or exceeded that of thewild type; we have found that this is also true whenSRX5-pp60v-src is expressed in rat-2 cells (J. E. DeClue, E.Liebl, and G. S. Martin, unpublished data).The temperature-sensitive mutant SHX21[514] bears an

insertion located at the junction of regions IV and V. Thephenotype of this mutant was somewhat leaky; a full rever-

sion was not achieved following upshift of CEF to 41.5°C.The SHX21 insertion is predicted to fall near the end of analpha-helix which contains the last eight amino acids foundin both v-src and c-src. Bryant and Parsons (3) have useddeletion and frameshift analysis to show that alterations ofthe amino acids predicted to make up this alpha-helix resultin a transformation-defective phenotype. A series of dele-tions in the C terminus of the v-src gene of the SR-D strainwere constructed by Yaciuk and Shalloway (38), and theeffects were measured by transfection of the mutant src

genes into 3T3 cells. These studies showed that leucine 516,which is present in both v-src and c-src and which isconserved among other protein-tyrosine kinases, is requiredfor transformation. This leucine would be moved from itsnormal position by the insertion in SHX21.Ng and Privalsky (25) have previously suggested that

in-frame insertional mutagenesis is of general use in the

I

B a

97- _ft-PP90-Pr76gag

VOL. 63, 1989


TABLE 3. Expression of viral proteins in mutant-infected CEF

Site of Level of Level Total src Kinase sp pp5O-pp90Mutant mutation Pr76gaga of b kinase actd complex'ppfjfjSrC activityc

101452555976138148152168168174203206225228259263263272293299

300

SNX1SHX1SNX2SHX12SAX1SHX2STX1SRX1SDX1SHX3SSX1SHX4SDX2SAX2SPx1SHX13STX2SHX5SSX2SAX3SHX14SHX6

(41.50C)SNX3

(41.5°C)SHX7SHX15SAX4SHX8SAX5SRX2SRX3SRX4SAX6SHX16SHX17SRX5SHX18SHX9SAX7SHX19SHX10SNX4SHX11SHX20SRX6SHX21

(41.50C)SPX2

304306316317321325339356362375390415437437454459459461473506509514

515

XD1XD2XD3XD4XD6

RCAN

Wild-type v-src(41.50C)

d115-168d1169-174dl175-262dn7-225d1149-174

0.861.271.070.651.280.790.660.811.300.781.260.340.900.870.951.130.861.080.951.151.221.220.991.371.490.830.680.701.070.801.661.171.411.471.780.980.980.661.311.101.071.251.091.321.071.440.821.521.42

1.391.390.830.661.28

0.930.530.771.212.311.420.911.240.800.510.830.150.451.520.330.531.911.091.130.880.220.350.380.580.380.320.630.281.140.390.180.220.760.240.120.511.450.500.260.640.710.230.330.160.550.120.340.470.53

0.030.470.990.720.41

105.040.987.076.0187.063.485.638.014.260.190.13.68.5

113.35.4

17.284.21.51.51.60.055.92.34.64.3

31.23.20.40.90.81.20.5

44.20.40.10.4

224.51.11.90.52.40.30.20.30.40.5

34.911.440.3

1.810.03.3

130.68.3

112.577.9113.463.181.044.494.230.617.8

118.6108.223.819.071.516.332.544.11.41.31.90.2

16.56.17.9

11.396.75.11.30.82.06.52.1

58.21.60.70.8

154.72.17.50.73.41.20.71.80.64.0

102.124.275.7

54.621.53.4

181.420.0

++

+

+

++

++++++++++++++++

++++

++++++++++

++++++

++

++

+++++

1.00

1.051.22

1.001.00

100.0100.0

100.0100.0

+

a [35S]methionine radiolabel in Pr769af following an 18-h labeling of RCAN-v-src-infected CEF, expressed as a fraction of the radiolabel obtained with CEFinfected with the RCAN vector without an insert.

b [35S]methionine radiolabel in pp60`-rc following an 18-h labeling of RCAN-v-src-infected CEF, expressed as a fraction of the radiolabel obtained with CEFinfected with the RCAN vector with the wild-type SR-A v-src insert.

c Total immune complex (immunoglobulin heavy chain) kinase activity of pp60C-S"C per microgram of cellular protein, expressed as a percentage of the valueobtained with wild-type SR-A v-src.

d Specific activity of the src kinase, obtained by dividing the total immune complex kinase activity (footnote c) by the abundance of pp6iv-seC (footnote b).I The level of the pp6O-ppSO-pp9O complex was assessed by inspection of autoradiograms of immunoprecipitates prepared with tumor-bearing rabbit serum from

[35S]methionine-labeled CEF. The values for the pp5O-pp9O complex reflect the ratio of pp5O and pp9O to pp60i-Src in the immunoprecipitates.

J. VIROL.


creation and analysis of conditional mutants. The resultsdescribed here bear out this suggestion and indicate thatboth host- and temperature-conditional mutations can beisolated by this type of mutagenesis. Previous studies (17,37) on mutants of v-src and c-src have utilized either CEF or

mammalian cells as hosts, but generally not both; however,it is clear from the findings reported here that the two celltypes may respond differently to mutations in src. Host-dependent phenotypes may result from alterations in theability of the mutant transforming proteins to interact withhost cellular components. Characterization of such mutantsmay provide information about the host components re-

quired for transformation.

ACKNOWLEDGMENTS

We thank Masae Namba and Michael Argyres for excellenttechnical assistance; Jim Stone for advice and linkers; Steve Hughesand Terry Robins for vectors; and Barbara Weiss and Jim Ferrell forreview of the manuscript.

This work was supported by Public Health Service grant CA17542from the National Institutes of Health. J.D.C. was the recipient ofpredoctoral fellowships from the National Science Foundation andthe University of California at Berkeley.

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2. Bryant, D., and J. T. Parsons. 1982. Site-directed mutagenesisof the src gene of Rous sarcoma virus: construction andcharacterization of a deletion mutant temperature sensitive fortransformation. J. Virol. 44:683-691.

3. Bryant, D., and J. T. Parsons. 1984. Amino acid alterationswithin a highly conserved region of the Rous sarcoma virus src

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4. Cooper, J. A., and T. Hunter. 1983. Regulation of cell growthand transformation by tyrosine-specific protein kinases: thesearch for important cellular substrate proteins. Curr. Top.Microbiol. Immunol. 107:125-161.

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6. Cross, F. R., E. A. Garber, and H. Hanafusa. 1985. N-terminaldeletions in Rous sarcoma virus p6Oirc: effects on tyrosinekinase and biological activities and on recombination in tissueculture with the cellular src gene. Mol. Cell. Biol. 5:2789-2795.

7. Cross, F. R., E. A. Garber, D. Pellman, and H. Hanafusa. 1984.A short sequence in the pp6Osrc N terminus is required forpp6fSrc myristylation and membrane association. Mol. Cell.Biol. 4:1834-1842.

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18. Kamps, M. P., J. E. Buss, and B. M. Sefton. 1985. Mutation ofN-terminal glycine of pp6Ov-src prevents both myristoylation andmorphological transformation. Proc. Natl. Acad. Sci. USA82:4625-4628.

19. Kamps, M. P., J. E. Buss, and B. M. Sefton. 1986. Rous sarcomavirus transforming protein lacking myristic acid phosphorylatesknown polypeptide substrates without inducing transformation.Cell 45:105-112.

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