JOURNAL OF VIROLOGY, Jan. 1989, p. 291-3020022-538X/89/010291-12$02.00/0Copyright X) 1989, American Society for Microbiology

Deletions and Insertions within an Amino-Terminal Domain ofpp6Ov-src Inactivate Transformation and Modulate

Membrane StabilityHWA-CHAIN R. WANG AND J. THOMAS PARSONS*

Department of Microbiology and University of Virginia Cancer Center, University of Virginia School of Medicine,Charlottesville, Virginia 22908

Received 5 July 1988/Accepted 21 September 1988

We previously showed (V. W. Raymond and J. T. Parsons, Virology 160:400-410, 1987) that variants of thePrague A strain of Rous sarcoma virus containing large deletions impinging on a region of the src gene encodingamino acid residues 143 to 169 were defective for transformation of chicken cells in culture. Here we report thatintroduction of small (tri-and tetrapeptide) deletions into a region of pp6O-src containing amino acid residues155 to 175 was found to inactivate transformation. In addition, insertion of four, but not one, amino acidresidues at position 161 also inhibited transformation. Biochemical analysis of the src proteins encoded byindividual transformation-defective variants revealed that the structural alterations introduced into thisdomain had only marginal effects upon src tyrosine-specific protein kinase activity. However, the src proteinsencoded by defective variants exhibited a significantly shorter half-life within the cell, although these proteinsefficiently and rapidly associated with cellular membranes. Our results suggest that the structural domainencompassing residues 155 to 177 may influence the stability of pp60srC in the cellular membrane, possibly viathe interaction of src with a cellular membrane component(s) or substrate(s).

The v-src gene of Rous sarcoma virus (RSV) encodes a60-kilodalton phosphoprotein, pp6O-src, which has tyrosine-specific protein kinase activity (5, 7, 12, 29, 39). Expressionof pp60v-src rapidly leads to morphological transformation ofchicken embryo (CE) cells as well as cells from a variety ofother species (26, 49). pp60vsrc contains two major sites ofphosphorylation, serine 17 and tyrosine 416, and severalminor sites (11, 44, 62). Phosphorylation of serine 17 appearsto result from the activity of cyclic AMP-dependent proteinkinase (11), whereas phosphorylation of tyrosine 416 resultsfrom autophosphorylation (48). Increased phosphorylationof either serine 17 or tyrosine 416 in vivo by treatment withcyclic AMP or vanadyl ion increases the protein kinaseactivity of pp60v-src (2, 10, 45, 54). The tyrosine-specificprotein kinase activity of pp60v-src is essential for initiatingthe events leading to cellular transformation (7). Expressionof pp60v-src leads to an increase in the total cellular phos-photyrosine levels in transformed cells (28, 58). Elevenpresumptive cellular substrates of pp60v-src which showincreased phosphotyrosine levels have been identified: p36(20), p42 (13), p50 (24), p81 (25), enolase, phosphoglyceratemutase, lactate dehydrogenase (15), Ca2+-binding proteincalmodulin (22), vinculin (57), talin (18), and fibronectinreceptor (27). Although the presence of these phosphopro-teins may not be a prerequisite for morphological transfor-mation, individually they could be involved in the appear-ance of specific features of transformation (14, 32, 41, 52,69). In addition, other phosphotyrosine-containing proteinscan be identified by using two-dimensional gels or Westernblotting (immunoblotting) with antiserum directed to phos-photyrosine (34, 53, 59, 67).

pp60v-src contains at least three distinct functional do-mains (38). The catalytic domain resides within the carboxy-terminal half of the molecule (38). The amino acid sequenceof this region shares homology with other known tyrosine

* Corresponding author.

kinases (1, 28, 56, 60, 61, 66) and a sequence motif, contain-ing lysine 295, which defines a canonical ATP-binding site(35, 63). Mutations within the catalytic domain inactivateprotein kinase activity and concomitantly inactivate virustransformation (7, 33, 42, 43, 62, 70). A proteolytic fragmentof pp6Ov-src consisting of the carboxy-terminal half of theprotein exhibits tyrosine kinase activity in vitro, whichindicates that these sequences constitute a domain capableof functional enzymatic activity (3).The second domain, comprising the first seven residues, is

involved in plasma membrane association (17, 30, 36). Thisregion is required for efficient pp60v-src myristylation, whichoccurs on the amino-terminal residue, glycine 2 (31).Nonmyristylated v-src proteins appear to be fully functionalas tyrosine kinases and phosphorylate protein substrates invitro (8, 17, 32). Myristylation of pp60src is required formembrane association, and plasma membrane association ofpp60V-src is correlated with transformation (30-32), whichsuggests that a critical substrate(s) for pp60vsrc resides at theplasma membrane.A third domain of pp6Ovsrc influences morphological

transformation and has been proposed to be involved inmodulating substrate recognition (6, 9, 16, 21, 37, 42, 43, 50).Deletion mutants within this domain of pp60v-src exhibitaltered transforming properties and encode src proteins withonly minor reductions in tyrosine-specific protein kinaseactivities (50). Deletion mapping indicates that the approxi-mate boundaries of this domain are delineated by amino acidresidues 143 to 169 (50). To further investigate the role of thisputative domain in transformation, we constructed a seriesof tri- or tetra-amino acid deletion mutations, and single- ortetra-amino acid insertion mutations in this region of the srcgene of the Prague A strain of RSV (PrA RSV). We reporthere that tri- or tetra-amino acid deletions or insertionswithin the region defined by residues 155 to 177 abrogatedpp60v-src transformation of CE cells. src proteins encoded bythese deletion variants exhibited a significantly reduced

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292 WANG AND PARSONS

half-life yet still efficiently associated with cellular mem-branes. Such structural alterations had little influence on srctyrosine-specific protein kinase activity. These results indi-cate that the domain encompassing residues 155 to 177 playsa role in mediating the interaction of pp6Ov-src with cellularmembrane components, possibly via the interaction with acellular membrane protein(s) or tyrosine kinase substrate(s).

MATERIALS AND METHODS

Cells, viruses, and plasmids. Cultures of primary CE cellswere prepared from gs-negative embryos (SPAFAS, Inc.,Norwich, Conn.) and transfected or infected as describedpreviously (7). Mutagenesis of viral DNA was carried out byusing a modified nonpermuted molecular clone of PrA RSVinserted into a pBR322 plasmid vector, pRL (51). Typically,1 ,ug of pRL DNA was used to transfect one 60-mm dish ofCE cells 24 h after trypsinization and plating. Changes in cellmorphology were routinely observed 6 to 8 days aftertransfection or 3 to 4 days after virus infection. Replicationof transformation-defective (td) virus was assessed by resis-tance to superinfection with PrA RSV as previously de-scribed (6). Virus stocks were harvested from overnightcultured medium and stored at -70°C.

Mutagenesis. The v-src gene from pRLv-src was clonedinto the single-stranded coliphage vector M13mp18 betweenthe HindIII and KpnI sites (51). Mutagenesis was carried outby using oligonucleotides (30-mers) to introduce specificdeletions or insertions (40). Mutant phage were identified byplaque hybridization or direct sequence analysis and wereplaque purified. The mutations were confirmed by dideoxyDNA sequencing, and the mutated src genes were reclonedinto the RSV vector pRL. Upon recloning, the mutated srcgenes from individual mutated pRL clones were reclonedback into M13 vector and resequenced 150 to 200 nucleo-tides both downstream and upstream of the mutagenizedregion to confirm the precise mutation.

Radiolabeling and pp6OSrc quantitation. For radiolabeling,cultures were incubated in labeling medium (Dulbecco mod-ified Eagle medium without methionine) containing 100 to200 ,uCi of [35S]methionine (Dupont, NEN Research Prod-ucts, Boston, Mass.) per ml. Cell extracts were prepared byusing a modified radioimmunoprecipitation assay (RIPA)lysis buffer (50 mM sodium phosphate buffer [pH 7.5], 150mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate,50 jig of leupeptin per ml, 1 mM sodium vanadate, 0.5%aprotinin, 1 mM phenylmethylsulfonyl fluoride, 4 mM p-nitrophenyl phosphate, 1 mM EDTA, a-2-macroglobulin).After determination of total cellular protein (BCA assay;Pierce Chemical Co., Rockford, Ill.), portions of the lysatescontaining equal amounts of cellular protein were immuno-precipitated with monoclonal antibody (MAb) EC10 topp60src. Immune complexes were adsorbed to Formalin-fixed Staphylococcus aureus (Pansorbin; Calbiochem-Behring, La Jolla, Calif.) and then washed and dissolved insample buffer as described previously (7, 23). The proteinswere resolved by sodium dodecyl sulfate (SDS)-poly-acrylamide gel electrophoresis (PAGE) and transferred fromthe polyacrylamide gel onto a nitrocellulose membrane, andpp6Osrc was detected by immunoblotting, using 125I-labeledMAb EC10 or 327 (0.1 ,uCi/ml). The labeled proteins weredetected by autoradiography and quantitated by densitom-etry.

Protein kinase assay. The autophosphorylation activities ofsrc proteins encoded by individual RSV variants were mea-sured by using the immune complex kinase assay as de-

scribed previously (7). Both autophosphorylation and phos-phorylation of the exogenous substrate enolase weredetermined after the addition of 5 ,ug of acid-treated enolase(47) and 5 ,uCi of [-y-32P]ATP (3,000 Ci/mmol; Dupont, NEN)to washed immune complexes in kinase buffer [20 mMpiperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES; pH 7.0),10 mM MnCl2]. After incubation for 5 min at 22°C, thereactions were terminated by the addition of SDS-samplebuffer. Labeled samples were heated at 100°C and subjectedto electrophoresis on SDS-polyacrylamide gels, and thelabeled proteins were visualized by autoradiography. Quan-titation of src protein autophosphorylation and enolase phos-phorylation was carried out either by densitometry or byexcision of the enolase and pp60src bands and scintillationcounting of the labeled gel fragments.Membrane fractionation. Infected CE cells were labeled

for 1 h with 200 ,uCi of [35S]methionine per ml, and the cellswere scraped from the culture dishes, washed twice withphosphate-buffered saline buffer, suspended in 1 ml of hy-potonic lysis buffer (10 mM Tris hydrochloride [pH 7.0], 1mM MgCl2) (65) supplemented with protease inhibitors, andallowed to swell on ice for 5 min. The cells were lysed with30 strokes in a tight-fitting Dounce homogenizer, and NaClwas added to 150 mM. The lysates were centrifuged at90,000 x g for 30 min to obtain cytoplasmic and totalmembrane fractions. The membrane fraction was suspendedin RIPA lysis buffer and centrifuged at 600 x g for 10 min toremove nuclei. Both membrane and cytoplasmic fractionswere immunoprecipitated with MAb EC10 and subjected toSDS-PAGE, and the quantity of pp60v-src was measured byimmunoprecipitation as described above.

Sedimentation analysis of pp69Src. Infected CE cells werelysed in RIPA lysis buffer containing protease inhibitors.The lysates were centrifuged through 10 to 30% glycerolgradients containing RIPA lysis buffer (4) in a TsT 602 rotor(Ivan Sorvall, Inc., Norwalk, Conn.) for 20 h at 49,000 rpmat 4°C. Gradients were fractionated into 18 tubes. Eachfraction was immunoprecipitated by using MAb EC10, andpp6Ov-src in each fraction was quantitated by Western blot-ting as described above.

Glucose uptake assay. Cells infected with individual v-srcvariants were rinsed with phosphate-buffered saline andincubated for 5 or 7 min at room temperature with 1 ,uCi of[3H]2-deoxyglucose per ml (30 Ci/mmol; Dupont, NEN) (68,69). Cells were rinsed three times with ice-cold phosphate-buffered saline and lysed in 0.1 N NaOH-1% SDS. The celllysates were divided into equal portions for scintillationcounting and protein determination.

RESULTS

Isolation and characterization of deletion variants. Thesequences within the amino-terminal one-third of the v-srcprotein influence morphological transformation (37, 42).Previous studies have shown that a region of the v-srcprotein delineated by residues 143 to 169 appears to benecessary for cellular transformation (50). RSV variantscontaining deletions which impinge on this region are defec-tive for transformation. One such variant, td dll21 (Fig. 1),encodes a structurally altered src protein which exhibitstyrosine protein kinase activity in vitro and retains the abilityto phosphorylate the in vivo substrate p36, calpactin I heavychain (43). RSV variants containing deletions within se-quences adjacent to this critical region (ts d1119, a deletion ofamino acid residues 173 to 227, and ts d1120, a deletion ofresidues 169 to 225) (Fig. 1) are temperature sensitive with

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AMINO-TERMINAL MUTATIONS IN pp60v-src 293

v-src

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dl 145dl 155dl 161dl 165dl 171dl 175is 161Eis 161LELE

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LCQQA--LUYHGARIPRSEUNELLKYS.--GDFLURES-QG.. ..HASLTRAQAEHMLI1--F.L..... R......

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FIG. 1. Amino acid sequence of an amino-terminal domain of p60rc encoding residues 141 to 180. The deduced amino acid sequence ofPrA RSV src is compared with the sequence of cellular c-src, the SH2 region of Fujinama sarcoma virus-encoded v-fps (55), and the B domainof phospholipase C (64). Symbols: I, identical amino acids; *, residues that differ between v-src and c-src; ^, amino acids inserted in theindividual RSV variants. Residues within boxes are amino acids deleted in the individual RSV variants. The phenotype of each RSV variantis denoted at the right. T, Transforming; td, transformation defective; ts, temperative sensitive.

respect to morphological transformation (43, 50). To furtherinvestigate the function of this region of the src protein intransformation, six deletion mutations and two insertionmutations were individually introduced into this region ofthe v-src gene by oligonucleotide-directed mutagenesis. Fig-ure 1 summarizes the amino acid residues deleted (boxed) inthe deletion variants d1145, d1155, d1161, d1165, d1171, andd1175. Insertion variants isl61E and isl6l(LE)2 contained a

single Glu residue or the tetrapeptide Leu-Glu-Leu-Gluinserted after amino acid 160. Transfection of cells withwild-type (wt) RSV DNA or the variant d1145 or isl61Eresulted in typical morphological alteration of cells 6 to 8days posttransfection (Fig. 1 and Fig. 2B, D, and F).However, cells transfected with d1155, d1161, d1165 (Fig.2C), d1171, d1175, and isl6l(LE)2 (Fig. 2E) exhibited nodistinguishable alteration in morphological features after 14days (Fig. 1) and were resistant to superinfection with wtPrA RSV, which indicated complete infection of the cellpopulation. Cells infected with the previously characterizedts d1119 exhibited a transformed phenotype when grown at35°C but reverted to a normal morphology when shifted to41°C (Fig. 2H and G). These results demonstrate that se-

quence alterations created by specific tri- and tetra-aminoacid deletions or a tetra-amino acid insertion within the

region defined by residues 155 to 177 alter v-src transforma-tion potential.As an independent measure of the extent of cellular

transformation, the rate of glucose uptake was measured incells infected with the individual v-src variants (Table 1).Cells infected with wt RSV, transformation-competent var-iants d1145 and isl61E, and ts d1119 (35°C, permissivetemperature) exhibited elevated rates of glucose uptake. Incontrast, cells infected with the td variants d1165 andisl6l(LE)2 exhibited rates of glucose uptake similar to thoseof normal CE cells. Cells infected with ts d1119 and main-tained at the nonpermissive temperature (41°C) showed asubstantial reduction in glucose transport compared withcells grown at the permissive temperature (35°C), in agree-ment with the previous measurements (14). These data areconsistent with the morphological observation that the tdRSV variants are defective for cellular transformation.

Synthesis and stability of src proteins. To measure the levelof v-src protein expression in cells transfected with individ-ual variants, virus-infected cells were lysed in RIPA bufferand cell extracts were immunoprecipitated with the src-specific MAb EC10. The relative amount of src protein inimmune complex was measured by SDS-PAGE and immu-noblotting with the 1251I-labeled MAb 327. The amount of src

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J. VIROL.294 WANG AND PARSONS

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AMINO-TERMINAL MUTATIONS IN pp60v-src 295

TABLE 1. Measurement of [3H]2-deoxyglucose uptake ininfected cells

Uptake (cpm recovered/mg ofCE cells Mutant cell protein per min)phenotypea

Expt 1 Expt 2

Uninfected 1,004 (1.0)b 723 (1.0)Infected with:

v-src T 3,372 (3.4) 4,446 (6.0)d1145 T 3,280 (3.3) 2,946 (4.0)isl61E T 3,004 (3.0) 3,410 (4.6)d1165 td 1,014 (1.0) 1,314 (1.8)isl6l(LE)2 td 1,226 (1.2) 1,294 (1.7)ts d1119 T350C 3,358 (3.3) 3,919 (5.2)410C td 2,314 (2.3) 2,729 (3.7)

a T, Transformed; td, transformation defective.b Values in parentheses are expressed relative to the value observed for

uninfected cells.

protein expressed in cells infected with the individual RSVvariants differed substantially (Fig. 3). In general, the levelof src protein expressed in cells infected with td variants(Fig. 3, lanes 4 through 8 and 10) was less than the amount ofsrc protein in wt-, ts d1119-, or ts d1120-infected cells (lanes2, 11, and 12), with isl6l(LE)2-infected cells exhibiting thelowest amount of src protein and d1165-infected cells show-ing the highest amount. In fact, to detect appreciable levelsof src protein by immunoblotting, it was necessary toincrease fourfold the amount of cell extract from cellsinfected with the td variants (Fig. 3). Control experimentsusing other src-specific monoclonal antibodies or polyclonalrabbit serum confirmed that the deletions did not alter theability of individual antisera to immunoprecipitate the vari-ant src proteins (data not shown).To determine whether the reduced level of src protein

observed in cells infected with individual td variants re-

1 2 3 4 5 6

p60 _

flected differences in expression of src mRNA or differencesin the stability of variant src proteins, we measured thesynthesis and turnover of src protein in cells infected with wtRSV, d1145, d1155, d1165, d1175, isl6lE, isl6l(LE)2 and tsd1119. Infected cultures were labeled with [35S]methioninefor 2 h and then incubated for 4 or 8 h in complete culturemedium. At each time point, the src proteins were immuno-precipitated from the same amount of total cellular protein,immune complexes were resolved by SDS-PAGE, and srcprotein was detected by autoradiography (Fig. 4). After 2 hof labeling with [35S]methionine, similar amounts of labeledsrc protein were detected in cells infected with wt RSV, theindividual td variants, or ts d1119 maintained at the permis-sive or nonpermissive temperature. However, after a 4- or8-h chase period, considerably less src protein was detectedin cells infected with the td variants than in cells infectedwith wt RSV, d1145, isl6lE, or ts d1119 (Fig. 4). Theseresults indicate that the turnover of src protein in the cellsinfected with td variants was significantly greater than theturnover rate of src protein expressed in cells infected withwt RSV, transformation-competent variants, or ts d1119. Therelative amount of src proteins immunoprecipitated fromextracts during the labeling and chase periods was quanti-tated by densitometry, and the approximate half-life of eachv-src variant protein was estimated (Table 2). Comparedwith wt src protein, src proteins encoded by d1155, d1165,d1175, and isl6l(LE)2 appfared to turn over more rapidly,having an apparent half-life ranging from 2 to 3 h. It shouldbe noted that this estimate of half-life may represent anoverestimate since we cannot readily distinguish the lowlevel of v-src protein present after the 4- and 8-h chaseperiod from the small amount of resident pp6Ocsrc. srcproteins encoded by the transforming variants d1145 andisl6lE exhibited a half-life (4 to 4.5 h) closer to that of wtRSV-encoded src protein. src protein expressed in ts d1119-infected cells appeared to turn over slightly faster at thenonpermissive temperature (4 h) than at the permissive

7 8 9 10 11 12

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_- -.6--P53

FIG. 3. Immunoprecipitation of pp6Orc from infected cells. Extracts were prepared from infected cells, and either 100 or 400 ,ug of extractprotein was immunoprecipitated with the src MAb EC10 and analyzed by SDS-PAGE. pp6Ov-src protein was quantitated by immunoblottingwith "25I-labeled MAb 327. Lanes: 1, CE cells; 2, wt RSV; 3, d1145; 4, d1155; 5, d1161; 6, d1165; 7, d1171; 8, d1175; 9, isl61E; 10, isl6l(LE)2;11, ts d1119 (35°C); 12, ts d1120 (350C). Lanes 1, 4 through 8, and 10 contain immune complexes immunoprecipitated from 400 ,ug of cellprotein.

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296 WANG AND PARSONS

AwtRSV40 wtRSV36 dl 145 dl155 d11651 2 3 4 5 6 7 8 9 10 11 12 13 14 15

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Bdl 175 iS161E isl6l(LE)2 ts 11936 ts 1194

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

p60_-' j . -*

_--p53

FIG. 4. Analysis of pp60v-s' synthesis in wt- and mutant-infected cells. Infected cells were labeled with [35S]methionine for 2 h and thenincubated with growth medium containing unlabeled methionine for 4 or 8 h. Lanes: 1, 4, 7, 10, 13, 16, 19, 22, 25 and 28, 2-h label; 2, 5, 8,11, 14, 17, 20, 23, 26 and 29, 4 h with unlabeled methionine; 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30, 8 h with unlabeled methionine. Labeledpp60v-src was immunoprecipitated with the src MAb EC10 and analyzed by SDS-PAGE as described in Materials and Methods.

temperature (6 h). These data indicate that sequence alter-ations within the region defined by residues 155 to 177appear to influence the stability of pp6Ov-src and lead to anincreased rate of degradation of the mutant src protein.

Kinase activity of src variants. The in vitro kinase activityof individual src variant proteins was measured by usingimmune complexes prepared from cells infected with wtRSV, d1145, isl61E, td variants d1165, and ts d1119 (Fig. 5and Table 3). The src proteins encoded by d1145 exhibitedthe highest level of both autophosphorylation and exogenousphosphorylation activities, 60% greater than was found for

TABLE 2. Estimate of the half-life of variant src proteins

Half-life (h)bMutant Phenotype"

36°C 400C

v-src T 6.0 6.0dl145 T 4.5 NDisl61E T 4.0 NDd1155 td 2.5 NDdl165 td 3.0 NDd1175 td 2.5 NDisl6l(LE)2 td 2.0 NDts d1119 ts 6.0 4.0

" T, Transforming; td, transformation defective; ts, temperature sensitive.b pp6f,Src was immunoprecipitated as described in the legend to Fig. 4, and

the relative amount of src protein in each lane was quantitated by densitom-etry. The half-life of each variant was estimated by plotting the relativeamount of src protein (in arbitrary units) on a semilogarithmic scale. ND, Notdone.

wild-type pp6O-src. In contrast, the src proteins encoded bythe transformation-competent variant isl61E and the tdvariant d1165 exhibited similar specific activities, the levelsof exogenous phosphorylation activities being approxi-mately 40 to 50% of the level of wt src protein and the levelsof autophosphorylation activities being approximately 80 to90% of the level of wt src protein. Comparison of these dataindicates that there is no correlation between the effects ofsequence alterations which inhibit the transforming functionof src within the domain defined by residues 155 to 177 andthe specific activity of the src protein kinases. The ts d1119v-src protein showed a reduced level of autophosphorylationand exogenous phosphorylation activities (compared with wtsrc protein) when measured at either the permissive or thenonpermissive temperature. Together, these findings suggestthat there is no direct correlation between in vitro tyrosineprotein kinase activity of variant src proteins and the lack oftransforming activity of these proteins.

Subcellular localization of newly synthesized src protein.The association of src protein with the cytoplasmic mem-brane is required for cell transformation (17, 32). To deter-mine the effects of the structural alterations on the associa-tion of individual variant src proteins with cellularmembranes, wt RSV-, d1145-, td variant d1165; and ts d1119-infected cells were labeled with [35S]methionine for 1 h andthe labeled membrane and cytoplasmic fractions were pre-pared. Each fraction was immunoprecipitated with MAbEC10, and labeled proteins were resolved by SDS-PAGE(Fig. 6). A majority of v-src protein was detected in mem-brane fractions from wt RSV-, d1145-, and td variant d1165-

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AMINO-TERMINAL MUTATIONS IN pp6fV-src 297

dl 1653 4

S-_A o I

EI_P

sogo--p53- Enola se

FIG. 5. Immune complex kinase activity of pp6O-src variants. Cell extracts were prepared from uninfected and infected cells, and immunecomplexes were prepared with MAb EC10. One half of each immune complex preparation was used to determine the tyrosine kinase activityin a standard immune complex kinase activity assay; the remaining half was used to determine the amount of src protein by the immunoblotassay (see legend to Fig. 3). Exogenous phosphorylation activity was measured by addition of denatured rabbit muscle enolase(even-numbered lanes). Autophosphorylation activity was measured by incubation of immune complexes in the absence of enolase(odd-numbered lanes).

infected cells as well as from ts d1119-infected cells withinthe 1-h labeling period, which suggested that newly synthe-sized src proteins localized to cellular membranes irrespec-tive 6f their transformation activity.

Association of src protein with the p9O-p5O proteins. Soonafter synthesis, the src protein enters a cytoplasmic complexwith cellular proteins p90 and p50 (4). To determine whetherthe defect in transformation correlated with the increasedassociation of src proteins with p90-p50 proteins, whole-celllysates from wt RSV-, isl6lE-, td variant d1165-, and tsd1119-infected cells were analyzed on 10 to 30% glycerolgradients. Individual fractions were immunoprecipitatedwith MAb EC10 and analyzed by SDS-PAGE, and the srcprotein was quantitated by immunoblotting with 125I-labeledMAb 327. An example of such an analysis for wt RSV- andd1165-infected cells is shown in Fig. 7. To quantitate therelative amount of src protein complexed with p50 and p90,the level of src protein present in each gradient fraction was

TABLE 3. In vitro kinase activity of mutant src proteins

Relative sp act of phosphorylation of:aMutant

pp60src Enolase

v-src 1.0 1.0d1145 1.6 ± 0.1 1.4 ± 0.2d1165 0.8 ± 0.1 0.4 ± 0.1isl61E 0.9 ± 0.1 0.5 ± 0.1ts d1119

370C 0.6 ± 0.1 0.5 ± 0.1410C 0.5 ± 0.1 0.4 ± 0.1

a Kinase activity was measured by the immune complex kinase assay. Theextent of phosphorylation of pp6OC or enolase was determined by autoradi-ography and densitometry. The amount of p6src present in each immunecomplex preparation was determined by immunoblotting with 251I-labeledMAb 327 (see legend to Fig. 5). The specific activity for each preparation ofsrc protein was then calculated by dividing the amount (counts per minute) of32P incorporated into the kinase substrate by the amount of 125I-labeled MAb327 bound to src protein in the immunoblot. The value for wt p6Osr, has beenset to 1. Values shown are averages of three independent determinations.

measured by densitometry, and the relative amount of srcprotein was plotted as a proportion of the total src protein inthe gradient (Fig. 8). Compared with cells infected with wtsrc (Fig. 8A) or the transformation-competent variant isl61E(Fig. 8C), cells infected with td variant d165 contained aslightly higher but still minor proportion of src protein (30%)that sedimented rapidly in the gradient (Fig. 8B). When thedistribution of src was measured in cells infected with tsd1119 and grown at the permissive and the nonpermissivetemperatures (Fig. 8E and D), similar amounts of rapidlysedimenting src protein were observed at the two tempera-tures. Therefore, it appears unlikely that the direct associa-tion of src protein with p90-pSO plays a major role in theinhibition of cell transformation by td variants such as d1165or the alteration in phenotype observed when ts d1119 cellsare shifted to 41°C.

p6 0,,-.. . 40 Q_ d_4

W,q4i lo i'w_ _ ; .....-9X.

_ p53

1 2 3 4 5 6 7 8 9 10FIG. 6. Subcellular localization of newly synthesized pp60'vsrc.

Cells infected with individual RSV variants were labeled with[35S]methionine for 1 h, and membranes and cytoplasmic fractionswere prepared as described in Materials and Methods. src proteinpresent in each fraction was quantitated by immunoprecipitationwith MAb EC10 and SDS-PAGE. Lanes: 1 and 2, RSV; 3 and 4,d1145; 5 and 6, td variant d1165; 7 and 8, ts d1119 grown at 35°C; 9 and10, ts d1119 grown at 41°C. Odd-numbered lanes represent mem-brane fractions, and even-numbered lanes denote cytoplasmic frac-tions.

isI6lLE)256

wtRSV7 8

CE1 2

p60-m.

d11459 10

is 161E11 12

ts1193713 14

tS11 94115 16

I,

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298 WANG AND PARSONS

A

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

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FIG. 7. Sedimentation analysis of pp6O-src proteins. Whole-cell lysates were applied to a 10 to 30% glycerol gradient; after centrifugation,individual fractions were immunoprecipitated with MAb EC10 and pp6v-src was quantitated as described in Materials and Methods.Sedimentation was from top to bottom. Arrows indicate positions of the bovine serum albumin marker (67 kilodaltons). (A) wt RSV, (B) d1165.

DISCUSSION

We reported previously that RSV variants that containdeletions within the src gene which remove amino acidresidues 38 to 142 readily induce cellular transformation,whereas variants containing deletions which impinge on theregion of the src gene encoding residues 143 to 169 lack theability to mediate cellular transformation (50). In addition,we have observed that variants containing a deletion ofamino acid residues 169 to 225 or 173 to 227 are temperaturesensitive for transformation (6, 50), providing additionalevidence for an important functional domain within thisregion of pp6Ov-src. Site-directed mutagenesis techniqueshave been used to introduce tri- and tetrapeptide deletionsinto a region from residues 145 to 177 (Fig. 1) and single-residue and tetrapeptide insertions at residue 161 (Fig. 1).CE cells infected with d1155, d1161, d1165, d1171, d1175, orisl6l(LE)2 exhibited no distinguishable alteration in mor-phological features (Fig. 2) and virtually no change in therate of glucose uptake (Table 1). However, cells infectedwith d1145 or isl61E became fully transformed and were

indistinguishable from wt RSV-infected cells (Fig. 2). Theseresults, considered together with our previous data, indi-cated that the region from approximately residues 155 to 177is critical for v-src transforming activity. The results re-

ported here confirm and extend the observations of Cross etal. (16), who showed that deletion of sequences encoding

residues 149 to 169 within the src gene of the Schmidt-Ruppin strain of RSV significantly altered the morphologicalfeatures of infected cells.We have investigated a number of parameters that may

influence the activity of proteins encoded by td variants,including the level of src protein, in vitro tyrosine proteinkinase specific activity, and synthesis and transport tocytoplasmic membranes. Our results clearly show that thelevel of src protein expressed in cells infected with tdvariants was less than the amount of src protein in transfor-mation-competent RSV variants or ts d1119-infected cells.Among the td variant-infected cells, d1165-infected cellsconsistently exhibited the highest level of src protein, al-though this level was approximately 25 to 50% of the levelobserved in wt RSV-infected cells. The lower level of srcprotein in td variant-infected cells appeared to be the resultof an increased turnover of src protein, as determined on thebasis of measurement of the half-life of src protein in cellsinfected with wt RSV, transformation-competent variantsd1145 and is161E, td variants d1155, d1165, d1175, andisl61(LE)2, and ts d1119. The relationship between the tdphenotype and the steady-state level of src variant proteinsis not simple. In cells infected with RSV variants encodingsrc proteins that are rapidly degraded, the level of src protein(possibly in a specific membrane complex) may be below thethreshold necessary to initiate cellular transformation. How-

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AMINO-TERMINAL MUTATIONS IN pp6v-src 29

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ever, in the case of cells infected with td variant d1165, thelevel of src protein is reduced approximately 40%. In addi-tion, preliminary quantitation of phosphotyrosine-containingproteins in cells infected with dl165 (using phosphotyrosineantibodies) indicates that many of the proteins phosphory-lated by wt pp60v-src are phosphorylated on tyrosine ind1165-infected cells (R. Wang, unpublished observations).We suggest, therefore, that at least in dl165-infected cells,the src protein must be defective in the interaction with somecellular component required for transformation.The analysis of in vitro kinase activity summarized in

Table 3 is consistent with earlier observations that deletionswithin the amino-terminal domain containing residues 155 to177 do not greatly reduce the specific activity of the src

protein kinase. Levels of both autophosphorylation andexogenous phosphorylation are similar in immune com-

plexes containing either td variant d1165 src protein, trans-

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FIG. 8. Relative distribution of pp6Ov-src after glycerol gradientfractionation. Whole-cell lysates were analyzed by centrifugation ona 10 to 30% glycerol gradient, and src protein was quantitated byimmunoblotting as described in the legend to Fig. 7. The relativeamount of src protein in each fraction was estimated by densito-

15 metry and expressed as a percentage of the total src protein presentin the gradient fractions. (A) RSV; (B) td variant d1165; (C) isl61E;(D) ts d1119 grown at 41°C; (E) ts d1119 grown at 35°C.

formation-competent variant isl61E src protein, or ts d1119src protein prepared from cells maintained at permissive andnonpermissive temperatures. Therefore, there appears to beno direct correlation between in vitro tyrosine protein kinaseactivity of variant src proteins and their lack of transformingactivity. Sadowski et al. (55) have reported that insertions ina region of the v-fps oncogene designated SH2 (src homol-ogy 2 [Fig. 1], residues 137 to 241 of v-src) impaired theability of Fujinami avian sarcoma virus to transform Rat-2cells. Analysis of the gag-fps protein revealed that SH2mutants were deficient in tyrosine protein kinase activity inrat cells but, when expressed in bacteria, retained tyrosineprotein kinase activity. These authors suggested that theSH2 region was not required for catalytic activity but couldprofoundly influence the adjacent kinase region, possibly byinteraction with cellular factors (55, 19). Our results providesupport for the idea that the analogous region of the src

204

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300 WANG AND PARSONS

protein modulates the interaction of src with cellular factorsrequired for transformation, whereas the RSV variants re-ported here retain tyrosine kinase activity.Both the subcellular localization of src in cytoplasmic

membranes and activation of tyrosine kinase are crucial forcellular transformation (30-32). Mutation of Gly-2 to Ala orGlu or deletion of the first 14 amino-terminal residues yieldsnonmyristylated src proteins which are defective in theirability to associate with cellular membranes and retain highlevels of in vivo and in vitro kinase activity but are transfor-mation defective (16, 31, 32, 46). Our data indicate thatsignificant amounts of newly synthesized src proteins en-coded by td variants translocate to the membrane. However,their inability to transform cells and the increased turnoverof the variant src proteins provide evidence for an alteredinteraction of variant src proteins with other cellular mem-brane components. Recently, Stahl et al. reported a partialhomology within the amino-terminal region of pp60src (resi-dues 147 to 188) with phospholipase C (the B region) (64).Although the structural implications of this homology areunclear, an intriguing possibility is that the src protein andphospholipase C interact with a common family of mem-brane components.

In summary, we have shown that deletions within residues155 to 177 appear to alter the interaction of pp60src withmembrane components necessary to stabilize src protein inthe cytoplasmic membrane and perturb the ability of srcprotein to interact with cellular factors required to trigger thecell transformation. These specific membrane componentsmay or may not be substrates for pp60src phosphorylation.Additional experiments to investigate the patterns of phos-photyrosine-containing proteins in cells infected with dele-tion variants of RSV are in progress.

ACKNOWLEDGMENTS

We thank Betty Creasy for preparation of CE cells. We acknowl-edge the many helpful comments and advice of W. Potts, A.Reynolds, S. Kanner, A. Bouton, S. Parsons, and M. Weber, as wellas T. Lansing and B. Cobb. We are indebted to S. Parsons and J.Brugge for providing antibodies to pp6Osrc. We thank B. Nordin forhelp in preparation of the manuscript.

This investigation was supported by Public Health Service grantsCA29243 and CA40042, awarded by the National Cancer Institute,and by grant NP462 from the American Cancer Society.

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3. Brugge, J. S., and D. Darrow. 1984. Analysis of the catalyticdomain of phosphotransferase activity of two avian sarcomavirus transforming proteins. J. Biol. Chem. 259:4550-4557.

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