JOURNAL OF VIROLOGY, Dec. 1986, p. 849-8570022-538X/86/120849-09$02.00/0Copyright X 1986, American Society for Microbiology

Biochemical Properties of p6oV-src Mutants That Induce DifferentCell Transformation Parameters

RICHARD JOVE,* ELLEN A. GARBER, HIDEO IBA,t AND HIDESABURO HANAFUSAThe Rockefeller University, New York, New York 10021

Received 25 June 1986/Accepted 26 August 1986

PA101 and PA104 are Rous sarcoma virus variants that are differentially temperature sensitive in celltransformation parameters, including stimulation of cell proliferation, morphological alteration, and anchor-age independence. To investigate the biochemical basis for the differential expression of these parameters, thetyrosine kinase activity and subcellular localization of the mutant p60VSrC proteins encoded in the variants wereexamined. Analysis of chimeric src proteins derived from the mutant proteins revealed that lesions in the kinasedomain inhibit in vitro kinase activity and confer temperature sensitivity on tyrosine phosphorylation ofcellular protein p34 in vivo. The amino-terminal portions of the mutant src proteins also influence tyrosinephosphorylation in vivo and in vitro, which is consistent with an interaction between an amino-terminal regionand the kinase domain. Large proportions of the mutant src proteins exist in soluble complexes with cellularproteins p50 and p90, even though the src proteins are myristylated. The formation of these soluble complexessegregates with lesions in the kinase domain and is independent of temperature. Our results demonstrate thatthe transformation parameters examined correlate to a limited extent with p34 phosphorylation but not withthe levels of in vitro kinase activity or soluble complex formation.

The transforming protein of Rous sarcoma virus (RSV),p60v-src, and its cellular homolog, p60c-src, are tyrosineprotein kinases associated with the plasma membrane (2, 7,8, 14, 24, 25, 29, 31, 32). The kinase activity of p60v-src,which is elevated in comparison with that of p60csrc (15, 25),is correlated with transforming activity (41, 42). Fatty acyla-tion of the amino terminus of p60v-src with myristic acid andplasma membrane association of p60vsrc are also correlatedwith transformation (17, 28, 38), suggesting that a criticalsubstrate for the kinase might reside at the plasma mem-brane. Another amino-terminal region has been proposed tobe involved in modulating kinase activity or substrate spec-ificity (16, 19, 37). Cellular proteins phosphorylated byp60v-src have been identified (10, 29), including a protein(p34) with a molecular weight of approximately 34,000 to38,000 (18, 40), although substrates causally related to trans-formation have not been demonstrated.

Infection of chicken cells with RSV results in stimulationof cell proliferation, morphological alteration, and anchorageindependence (23). PA101 and PA104 are RSV variants thatoriginally were selected for the capacity to stimulate cellproliferation without causing morphological alteration (5, 6).In addition, it was found that the mitogenic activity istemperature sensitive (ts) and does not require the inductionof anchorage independence (5). Further analysis revealedthat these variants are actually ts in all three transformationparameters, although temperature sensitivity is observed atdifferent temperatures for different parameters (27). In ac-companying papers (27, 34) we describe the molecularcloning and genetic analysis of the mutant PA101 and PA104v-src genes. These mutants provide a unique opportunity todiscern relationships among known biochemical propertiesand the transformation-related functions of p60v-src. We

* Corresponding author.t Present address: Department of Biophysics and Biochemistry,

University of Tokyo, Tokyo 113, Japan.

therefore examined the tyrosine kinase activities andsubcellular localization of the p60vsrc mutants and of chi-meric src proteins derived from them, and compared theseproperties with the corresponding cellular phenotypes.

MATERIALS AND METHODSCells and viruses. Chicken embryo fibroblasts (CEFs) and

chicken embryo neuroretina (NR) cells were prepared, main-tained, and infected as described previously (22, 39). Cellcultures were passaged once after infection to allow virusspread before the biochemical analyses.

All of the viruses used in this study were replication-competent RSV variants described in an accompanyingpaper (27). The parental viruses SRA, NYHB5, NYPA101,and NYPA104 contained the molecularly cloned wild-typeSchmidt-Ruppin A (SRA) strain RSV v-src, chicken c-src,PA101 v-src, and PA104 v-src sequences, respectively. Theviruses containing chimeric src genes derived by in vitrorecombination among the parental clones were NY101-SRA,NY104-SRA, NYSRA-101, NYSRA-104, NY101-HB5,NY104-HB5, NYHB5-101, NYHB5-104, NY104-101, andNY101-104 (27). The convention used here in designating thevariants encoding chimeric src proteins is as follows. Theorigin of the amino-terminal half of the src protein is indi-cated before the hyphen, and that of the carboxy-terminalhalf is indicated after the hyphen.

Isotopic labeling of cells. Cells were metabolically labeledwith [3H]leucine, [35S]methionine, 32Pi, or [3H]myristate asdescribed previously (17, 19, 25). Labeled cells were washedwith isotonic buffer and lysed in RIPA buffer containing 150mM NaCl, 10 mM Tris hydrochloride (pH 7.4), 1 mMEDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1%sodium dodecyl sulfate (SDS), 1 ,uM leupeptin, 1 ,uMantipain, and 0.1 p.M aprotinin. Similar results were obtainedwhen ionic detergents (SDS and sodium deoxycholate) wereexcluded from the RIPA buffer. The addition of 100 ,uMsodium orthovanadate (12) (Fisher Scientific Co., Pitts-

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850 JOVE ET AL.

burgh, Pa.) to RIPA buffer did not have a significant effect onany of the in vitro or in vivo results reported here and,therefore, was usually omitted.

Immunoprecipitation of proteins. The preparation of anti-serum raised against p60Src produced in Escherichia coli(anti-p60 serum) and tumor-bearing rabbit (TBR) serum havebeen described previously (2, 25). Monoclonal antibody 327against p6Osrc was the generous gift of J. Brugge (33).Antiserum raised against purified cellular protein p34 (anti-p34 serum) was generously provided by M. E. Greenbergand G. M. Edelman (21). Proteins solubilized in RIPA bufferwere reacted with an excess of antibodies, and the immunecomplexes were bound to protein A-Sepharose beads andwashed with RIPA buffer as described previously (25). Forimmunoprecipitation with the monoclonal antibody, rabbitanti-mouse immunoglobulins were used together with theprotein A-Sepharose as described previously (25).

Cell fractionation. Cells were lysed and fractionated bydifferential centrifugation into particulate and soluble frac-tions as described previously (19). The results were notsignificantly affected by altering the NaCl concentrationfrom 10 to 300 mM during the fractionation procedure. Forthe sedimentation analyses of src proteins, cell lysates werecentrifuged through 10 to 30% glycerol gradients as de-scribed previously (3, 19).

In vitro protein kinase assay. Immunoprecipitates preparedas described above were further washed with 40 mM Trishydrochloride (pH 7.2), and autophosphorylation by p60Orcproteins in immune complexes was assayed in 30 ,ul ofreaction buffer containing 20 mM Tris hydrochloride (pH7.2), 5 mM MgCl2, and 0.1 ,um [-y-32P]ATP (3,000 Ci/mmol) at4°C for 30 min (25). In some experiments heat- and acid-denatured enolase (5 ,ug; Sigma Chemical Co., St. Louis,Mo.) was added to the reaction mixture as exogenoussubstrate (9). Similar results were obtained when the ATPconcentration was increased to 20 ,uM or when the reactiontemperature was increased to 30°C.

Protein analysis. Immunoprecipitates were analyzed byelectrophoresis through 10% SDS-polyacrylamide gels (30),and labeled proteins were detected by fluorography orautoradiography. Some gels were treated with 1 M KOH at55°C for 2 h as described previously (11) to enrich for32P-labeled phosphotyrosine. Gels were exposed at roomtemperature without intensifying screens for quantitation orat -70°C with screens. The amount of radioactivity associ-ated with a gel band was determined by using a scintillationcounter or estimated by densitometric scanning of the ex-posed films.

RESULTS

In vitro kinase activity. Results of previous studies showedthat the tyrosine kinase activities of the mutant src proteinsencoded in PA101 and PA104 are low, as determined by invivo and in vitro kinase assays (26, 39). Figure 1 shows thatthe p60 proteins encoded in NYPA101 and NYPA104, whichcontain the molecularly cloned src genes of PA101 andPA104, possess extremely low in vitro autophosphorylatingactivities in an immune complex kinase assay. The levels ofautophosphorylation observed with these two mutant pro-teins are less than 2% that of the wild-type p60v-src of SRA,as reported previously (26). To investigate the structuralbasis for the reduced kinase activities, the levels of in vitroautophosphorylation by chimeric proteins derived from themutant and wild-type viral src proteins were examined (Fig.1A and Table 1). The p6Os of NYSRA-101 and NYSRA-104,

which contain the amino-terminal half of the wild-typeprotein and the carboxy-terminal halves of the mutant pro-teins, display low levels of kinase activity comparable tothose of the parental mutant proteins. These results demon-strate that the carboxy-terminal halves of the NYPA101 andNYPA104 proteins, which comprise the tyrosine kinasedomains, contain lesions that inhibit kinase activity.The NY101-SRA chimeric p60 protein, which has the

amino-terminal half of the NYPA101 protein and thecarboxy-terminal half of the wild-type protein, also displaysa reduced level of kinase activity in comparison with that ofSRA p60vsrc (Fig. 1A). This result demonstrates that lesionswithin the amino terminus of the NYPA101 p60 protein,located outside of the kinase domain (27, 34), can inhibit thekinase activity. Chimeric proteins involving exchanges ofamino- and carboxy-terminal halves between the NYPA101and NYPA104 p60 proteins (the NY104-101 and NY101-104proteins) possess kinase activities comparable to those of theparental mutants (Fig. 1B). As expected, the NY104-SRAp60 protein had the same level of activity as the wild-typep60v-src (Fig. 1A) because this chimeric protein does notcontain any amino acid substitutions relative to the SRAwild-type protein (27, 34).The autophosphorylating activity of the cellular src pro-

tein encoded in NYHB5 is also very low in vitro, as reportedpreviously (25, 26). Chimeric proteins involving p60csrc andthe mutant p60 proteins, which induce a variety of transfor-mation phenotypes (27), exhibit a wide range of kinaseactivities. Figure 1B and Table 1 show that the amino-terminal halves of the mutant proteins (contained in theNY101-HB5 and NY104-HB5 p6Os) activate the kinase ac-tivity relative to that of p60csrc. These results demonstratethat amino-terminal lesions can activate the kinase domainof p60csrc.One of the most striking features of these results is the

lack of correlation between the level of in vitro kinaseactivity of the proteins and the cellular phenotypes inducedby them (Table 2) (27). Results similar to those obtainedfrom the autophosphorylation data also were obtained whenenolase was present in the in vitro kinase assay as anexogenous substrate (data not shown). This suggests that thelevels of in vitro autophosphorylation do not simply reflectdifferences in the occupancy of the tyrosine residue at aminoacid 416 (Tyr-416), the phosphoacceptor site for the in vitroreaction. To exclude potential artifact in the in vitro assay, avariety of conditions for the extraction and assay of the srcproteins was tested. It has been reported previously that thepresence of ionic detergents (16, 39) or sodium orthovanad-ate (12) in RIPA buffers can affect the in vitro kinaseactivities of src proteins. However, the results of the kinaseassays were not significantly different when ionic detergentswere omitted or when sodium orthovanadate (100 ,uM) wasadded to the buffers (data not shown). The results also werenot significantly affected by altering the concentration ofATP in the assay (0.1 to 20 ,uM) or the temperature of the invitro reaction (4 to 30°C). Moreover, the incubation temper-ature (34 to 41°C) of the cells from which the proteins wereextracted did not influence the in vitro kinase activity (datanot shown). Thus, altering the conditions of this in vitroassay did not yield results that correlated with the transfor-mation parameters induced by the src proteins.

In vivo phosphorylation of p34. To obtain an independentmeasure of the kinase activities of the mutant proteins, wesought to determine the levels of tyrosine phosphorylation ininfected cells. Because the increase in phosphotyrosine intotal cellular protein induced by the parental NYPA101 and

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BIOCHEMICAL PROPERTIES OF p60v-src MUTANTS 851

< - __ <

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cocM co cO; O a3 " 'o' aV) m - - - } CC: _ _F

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a

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pO6D & p60-

A

3H-LEU 32pFIG. 1. In vitro kinase activities of src proteins assayed by autophosphorylation. Infected CEFs maintained at 37°C were metabolically

labeled with [3H]leucine (3H-LEU) for 4 h, and the p6AYrc proteins were immunoprecipitated from equivalent amounts of cell lysates withanti-p60 serum. One-half of each immunoprecipitate was directly loaded onto a gel, and 3H-labeled proteins were detected by fluorography.The other half of each immunoprecipitate was incubated in the autophosphorylation reaction mix with [y-32P]ATP prior to loading on a parallelgel, and 32P-labeled proteins were detected by autoradiography. Cell lysates were prepared from cells infected with the viruses indicatedabove the corresponding lanes or were mock infected. The molecular weight (MW) standards are (from top to bottom): 200,000, 97,000,68,000, 43,000, and 26,000.

NYPA104 mutants (39) was extremely low (less than 0.1% ofthe total phosphoamino acids), and thus difficult to reliablymeasure, we assayed the levels of phosphotyrosine in thecellular protein p34 (18, 40). It has been shown that theincrease in phosphorylation of p34 induced by p60v-src is theresult of tyrosine phosphorylation, although p34 also con-tains phosphoserine (10, 18, 25). The low levels of p34tyrosine phosphorylation in NYPA101- and NYPA104-infected cells (less than 25% that of SRA-infected cells)maintained at 37°C was abolished when the cells were shiftedto 41°C (Fig. 2), as previously reported for PA101 and PA104(39). The results shown in Fig. 2 and Table 2 demonstratethat temperature sensitivity in p34 phosphorylation at 41°Csegregates with the kinase domains of the mutant proteins.Cells infected with NYHB5-101 display partial temperaturesensitivity in p34 phosphorylation, which is consistent withthe observation that this mutant appears to be only partiallyts in transformation at 41°C (27). These results suggest that

lesions within the kinase domains of the mutant proteinsconfer temperature sensitivity on the kinase activity.Comparison of the levels of p34 phosphorylation in in-

fected cells maintained at 34 and 37°C revealed that thephosphorylation pattern was essentially identical at bothtemperatures (Fig. 2). This result contrasts with the obser-vation that the cellular phenotypes of cells infected withseveral of the mutants are very different at 34 and 37°C (forexample, NYPA101, NYPA104, NY101-HB5, and NYHB5-101) (27). From a comparison of the transformation param-eters with the levels of p34 phosphorylation induced by themutants as a function of temperature (Table 2), it appearsthat p34 phosphorylation does not correlate strongly withmorphological transformation or anchorage independence.Temperature sensitivity in p34 phosphorylation correlatedwith temperature sensitivity in mitogenic activity. However,the mitogenic activity was not incompatible with extremelylow levels of p34 phosphorylation, as observed with the

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852 JOVE ET AL.

TABLE 1. In vitro autophosphorylating activities of src proteinsin immune complexesa

Virus Relativeactivity (%)b

SRA ......................................... 100NYHBS ......................................... 4NYPA101 ......................................... 2NYPA104 ......................................... <1NY101-SRA ......................................... 12NY104-SRA ......................................... 100NYSRA-101 ......................................... 4NYSRA-104 ......................................... <1NY101-HB5 ......................................... 22NY104-HB5 ......................................... 40NYHB5-101 ......................................... 11NYHB5-104 ......................................... <1NY104-101 ......................................... 2NY101-104 ......................................... <1

a In vitro kinase assays were carried out as described in the legend toFig. 1.

b The autophosphorylating activity of the SRA wild-type p60v-src proteinwas taken as 1009o.

mitogenic viruses NY101-HB5 and NY101-104 (Table 2).These two viruses induced similar levels of p34 phosphory-lation in CEFs and rapidly proliferating NR cells (data notshown).Overexpression of p60csrc in NYHB5-infected cells did

not induce a detectable increase in phosphorylation of p34 atany temperature, which is consistent with results of previousstudies (25). The same results were obtained when sodiumorthovanadate (100 ,uM) was added to the extraction buffers,suggesting that cellular phosphatases do not appreciablyaffect p34 phosphorylation during extraction from culturedchicken cells (data not shown). By contrast, p60csrc exhib-ited low but detectable in vitro kinase activity. The resultsshown in Fig. 1 and 2 indicate that the levels of p34phosphorylation in vivo generally do not correspond to thelevels of in vitro kinase activity in this study. These resultsdemonstrate that in vivo and in vitro measurements of the

kinase activities of src proteins are not necessarily compa-rable. Despite the differences, however, the data show thatthe level of tyrosine phosphorylation as measured by eitherassay is determined not only by the kinase domain but alsoby the amino-terminal portion of the chimeric src protein(Tables 1 and 2).

Stability of mutant proteins. The temperature sensitivity inp34 phosphorylation could be explained by increased degra-dation of the mutant src proteins at 41°C. We thereforeexamined the stabilities of the NYPA101 and NYPA104 p60proteins at 37 and 41°C in a pulse-chase experiment. Bothmutant p60 proteins were equally stable at either tempera-ture throughout the course of a 24-h chase period followinga 1-h pulse with label (Fig. 3). The apparent half-lives of theNYPA101 and NYPA104 p60s were approximately 4 to 6 h,as determined by this pulse-chase experiment. For compar-ison, wild-type p60vsrc and p60csrc previously were found tohave apparent half-lives of approximately 8 and 24 h, respec-tively (25). We did not determine the half-lives of thechimeric proteins. However, all of the src proteins in thisstudy incorporated similar amounts of radioactivity during a4-h metabolic labeling period (Fig. 1), suggesting that none ofthese proteins is significantly more unstable than the wild-type p6Ov-sc protein.Complex formation with p50 and p90. The mutant src

proteins of certain ts RSV variants have been shown to beassociated with cellular proteins p50 and p90 to a greaterextent than wild-type p60vsrc (3, 4, 13). In contrast, stableassociation of p60csrc with these cellular proteins has notbeen detected (25). To investigate the significance of thisassociation, we examined complex formation with p50 andp90 by the mutant src proteins. Figure 4A shows that a

greater amount of p50 and p90 is coimmunoprecipitated withsrc protein from NYPA104-infected cell lysates than fromSRA-infected cell lysates, which is consistent with results ofstudies of other ts mutants (3, 4, 13). The increased coim-munoprecipitation with p50 and p90 was also observed withNYPA101-infected cell lysates (data not shown). Analysis ofimmunoprecipitates from cell lysates containing chimeric src

proteins revealed that increased complex formation segre-

TABLE 2. Comparison of p34 phosphorylation and transformation parameters induced by RSV variants as a function of temperaturea

340C 370C 410CVirus

P M A p34b p M A p34b p M A p34b

SRA NAC + + 1.0 + + + 1.0 + + + 1.0NYHB5 - - UD - - - UD - - + d UDNYPA101 + + 0.2 + + e + 0.2 - - - UDNYPA104 + + 0.2 + - - 0.2 - - - UDNY101-SRA +f + 0.5 + +f 0.4 + +f + 0.2NY104-SRA + + 1.3 + + + 1.2 + + + 1.0NYSRA-101 + + 1.3 + + + 0.9 - - - UDNYSRA-104 + + 0.2 + - - 0.2 - - - UDNY101-HB5 - - UD + - + UD + - + UDNY104-HB5 + + 0.2 + + + 0.4 + + + 0.2NYHB5-101 + + 0.9 + + + 0.7 + - +g 0.2NYHB5-104 + + 0.3 + + - 0.3 - - - <0.1NY104-101 + + 1.4 + + + 0.9 - - - <0.1NY101-104 - - 0.2 + - - <0.1 - - - UD

a Abbreviations: P, stimulation of cell proliferation; M, morphological alteration; A, anchorage independence; p34, level of phosphotyrosine in p34.b The phosphotyrosine levels in p34 were determined as described in the legend to Fig. 2, and all values are relative to the level induced by SRA at each

temperature (UD, undetectable levels).c NA, Not assayed.d See an accompanying paper (27) for an explanation of this result.e Morphological alteration was subtle.f Cells exhibited a fusiform morphology.8 The ts property was observed at 42°C.

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BIOCHEMICAL PROPERTIES OF p60v-src MUTANTS 853

340

GOOC o co 0~I,,sS.Ssstw-sstwsss w~~~~~~E

370

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410Ln mLO v <rOc <5

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FIG. 2. Phosphorylation of cellular protein p34 in infected cells as a function of temperature. CEFs were metabolically labeled with 32P,for 4 h, and p34 was immunoprecipitated from equivalent amounts of cell lysates with anti-p34 serum. Following electrophoresis the gels weretreated with alkali at a high temperature to enrich for phosphotyrosine in proteins, and the 32P-labeled proteins were visualized byautoradiography. Cell cultures were maintained at 34, 37, or 41°C prior to and during the labeling. Cells were infected or mock infected, as

indicated above the corresponding lanes. The molecular weight (MW) standards are shown in the right panel, and the arrowheads point top34.

gates with the kinase domains of the mutant proteins (Fig.4A). Chimeric proteins containing the wild-type p60v-srckinase domain exhibited low levels of complex formation,whereas proteins containing the kinase domain of p60c-src didnot form detectable complexes (Fig. 4A; data not shown).To verify that the src proteins are associated in stable

complexes with the coimmunoprecipitated cellular proteins,the sedimentation behavior of these proteins was examinedin glycerol gradients (3, 19). Figure 4B shows that themajority of wild-type p60v-src (approximately 90%) sedimentsin the gradient with a velocity similar to that of the 76,000-molecular-weight viral precursor structural protein Pr76(which serves as an internal marker for the monomeric formof p60) as reported previously (3, 13, 19). By contrast, the

101

0 2 6 12 24

3741 3741 3741 37 47 37 41 M

_--_

p60_- 4-M _

F~~~~

majority of the NYPA104 p60 sediments in the gradient witha velocity slightly greater than that of the 180,000-molecular-weight viral precursor structural protein Pr180. We estimatefrom this analysis that greater than 90% of the NYPA104 p60is associated in tight complexes with p50 and p90. Thechimeric src proteins containing the carboxy-terminal halvesof the NYPA101 and NYPA104 p60 proteins displayedsedimentation profiles indistinguishable from that of theNYPA104 p60 protein (data not shown), which is in agree-ment with the coimmunoprecipitation results using unfrac-tionated cell lysates (Fig. 4A). These data suggest that p50and p90 interact primarily with the kinase domain and thatlesions within this domain greatly affect the stability of thecomplexes.

104

0 2 6 12 24

37 41 37 41 37 41 37 41 37 41 M

. .

p60- If___d - o- - - -

FIG. 3. Relative stabilities of the NYPA101 and NYPA104 src proteins. Infected CEFs were pulse-labeled with [35S]methionine for 1 h andthen chased with an excess of unlabeled methionine for increasing lengths of time. The time of chase (0, 2, 6, 12, and 24 h) and the incubationtemperature of the cultures (37 or 41°C) are indicated above the corresponding lanes. The p6&rrc proteins were immunoprecipitated fromequivalent amounts of cell lysates with monoclonal antibody 327. The molecular weight standards (M) are the same as described in the legendto Fig. 1.

- 97 kd

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854 JOVE ET AL.

BSRA

1 3 5 7 9 11104

1 3 5 7 9 11

_ ,o

a:O £o o C

Prl80

p90*Pr76 -

p6Op5O

p27

FIG. 4. The association of src proteins with cellular proteins p50 and p90. Infected CEFs maintained at 37°C were metabolically labeledwith [3H]leucine as described in the legend to Fig. 1. (A) Coimmunoprecipation of p60rc proteins with p50 and p90 from equivalent amountsof cell lysates with TBR serum. Cells were infected with the viruses indicated above the corresponding lanes. The viral precursor proteinsPr18O and Pr76 and the viral structural protein p27 are also recognized by this TBR serum and serve as internal size standards. (B) Cell lysateswere fractionated by sedimentation through glycerol gradients, and proteins were immunoprecipitated from the gradient fractions indicatedabove the lanes. The direction of sedimentation is from left to right in each panel. The results shown here with cells infected with either SRAor NYPA104 are representative, respectively, of src proteins that are predominantly present either in the monomeric form or in the complexedform with p50 and p90.

To investigate the relationship between the transformationphenotypes and complex formation with p50 and p90, wecompared the sedimentation behavior of the various srcproteins extracted from infected cells incubated at 34, 37, or41°C. This analysis revealed that the association with p50and p90 is independent of temperature in all cases (data notshown). These data suggest that even though the mutantkinase domains of the src proteins are responsible for theincreased stable association with the cellular proteins,changes in the levels of complex formation apparently do notaccount for the temperature sensitivity in kinase activity orcell transformation parameters.

Subcellular localization of p60 mutants. The majority ofwild-type p60v-src and p6fC-src is localized at the inner surfaceof the plasma membrane (25, 29). This membrane localiza-tion, which involves myristic acid attachment to the aminoterminus, is highly correlated with transformation by p60v-src(17, 28, 38). We examined the subcellular localization of themutant src proteins by biochemical fractionation experi-ments to determine whether a correlation exists betweenmembrane association and the cellular phenotypes inducedby these mutants. Figure 5 shows that at least 50% of theNYPA104 p60 in cells fractionates as soluble protein. Thissuggests that, in contrast to the behavior of the wild-typep60v-src (4, 13), both the soluble and membrane-associatedfractions of the mutant proteins are complexed with p50 andp90. These results were not significantly affected by alteringthe NaCl concentration (10 to 300 mM) during the extraction(data not shown). The majority of wild-type p60v-src (80 to90%) fractionates with the insoluble membrane pellet underthe same conditions, as reported previously (17, 19). Anal-ysis of the chimeric src proteins revealed that the fraction-ation patterns of the proteins containing the carboxy-terminal halves of the NYPA101 and NYPA104 p60s areindistinguishable from that ofNYPA104 (data not shown). Inaddition, this anomalous fractionation behavior is indepen-dent of the temperature (from 34 to 41°C) at which infectedcells are incubated prior to the protein analyses (data notshown).The fractionation behavior of these proteins is paralleled

by the increased p50 and p90 complex formation in that bothproperties segregate in a temperature-independent manner

with the mutant kinase domains. These observations suggestthat complex formation affects the subcellular localization ofthe src proteins such that a greater proportion becomessoluble. This suggestion is further supported by analysis of amutant of NYPA104, which was isolated from a tumor in achicken that had been injected with NYPA104 (27) andwhich has wild-type transforming properties (NYPA104Ta).The majority of this mutant transforming proteitn is presentin the insoluble membrane pellet and sediments as a mono-mer in a glycerol gradient, similar to authentic wild-typep60v-src (Fig. 5; data not shown). Thus, restoration of wild-type subcellular fractionation behavior was accompanied byloss of the elevated levels of p50 and p90 association.

Myristylation of mutant proteins. The unusual subcellularfractionation properties of the mutant proteins could be

SRA 104 104TaP S P S P S

Pr1 80-

Pr 76- h

p60--

p27-_ -m

FIG. 5. Subcellular fractionation of src proteins. Infected CEFsmaintained at 37°C were metabolically labeled with [3H]leucine asdescribed in the legend to Fig. 1 and then fractionated into insolubleparticulate (P) and soluble (S) fractions. The src proteins wereimmunoprecipitated from the fractions in RIPA buffer with TBRserum. This TBR serum recognizes the viral precursor and struc-tural proteins described in the legend to Fig. 4. The virusNYPA104Ta (104Ta) is a transforming mutant isolated from a tumorin a chicken that originally had been injected with NYPA104.

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BIOCHEMICAL PROPERTIES OF p60v-src MUTANTS 855

explained by lack of amino-terminal myristylation. Wetherefore examined the incorporation of [3H]myristate intothe NYPA101 and NYPA104 proteins in vivo. The resultsshown in Fig. 6 demonstrate that the mutant proteins were

myristylated. This is consistent with the finding that theseproteins do not contain mutations within the amino-terminalsequences known to be required for myristylation (17, 38).Because this labeling experiment was carried out in NRcells, these results also show that myristylation of src

proteins is not unique to CEFs (17). The data presentedabove suggest that even though the mutant src proteins aremyristylated, unusually large proportions of these proteinsare in soluble complexes with p50 and p90.

DISCUSSIONThe tyrosine kinase activity and subcellular localization of

p60v-src appear to be the most important biochemical prop-erties of this protein with respect to transformation. Here wehave reported an examination of the relationships amongthese properties and the stimulation of cell proliferation,morphological alteration, and anchorage independence in-duced by p60v-src. For this purpose we used RSV mutantsdescribed in accompanying papers (27, 34) that differentiallyexpress these transformation parameters as a function oftemperature. The biochemical properties of these mutantproteins are summarized in Table 3.The chimeric src proteins derived from the NYPA101 and

NYPA104 p60 proteins exhibit a wide range of kinaseactivities, as measured in the immune complex kinase assay.The results of these experiments indicate that lesions withinthe kinase domains of the mutant proteins severely reducethe in vitro kinase activities. Lesions within the mutantkinase domains also affect all of the transformation param-eters in infected cells (27). In addition, mutations within theamino-terminal region of the NYPA101 p60 affect both the invitro kinase activity and cell morphology. Nevertheless,there is no consistent correlation between the levels of invitro kinase activity and the expression of any of the

3r LEU 3 Yi

_

-60

FIG. 6. Myristylation of the NYPA101 and NYPA104 src pro-

teins. Parallel cultures of infected chicken embryo NR cells were

metabolically labeled with [3H]myristate (3H-MYR) or [3H]leucine

(3H-LEU) for 4 h. The p6Osrc proteins were immunoprecipitatedfrom equivalent amounts of cell lysates with anti-p60 serum.

TABLE 3. Effects of amino- and carboxy-terminal mutations inthe PA101 and PA104 src proteins on biochemical properties

of p60V-SrC

Chimeric src proteinsa:Biochemical property

104/V V/104 101/V V/101

In vitro kinase wt Low Low Lowp34 phosphorylation wt ts Low tsComplex formation wtb >90% wt >90%Membrane association wtc <50% wt <50%

a Origin of the amino-terminal half of the chimeric src protein/origin of thecarboxy-terminal half. wt, Wild-type levels; ts, temperature-sensitive prop-erty. Values refer to percentage of total src protein.bLess than 10%o of the wild-type protein was present in the complexed form

with p5O and p90.c Greater than 80%o of the wild-type protein fractionated with the insoluble

membrane pellet.

transformation parameters. It should be emphasized thatalthough the levels of in vitro kinase activity were found tobe relatively invariant even when different assay conditionswere tested, we cannot exclude the possibility that this assaydoes not accurately reflect the levels of in vivo kinaseactivity. For example, the in vitro kinase activity might beaffected by the binding of antibody or by instability of themutant src proteins during extraction. Therefore, it is dif-ficult to evaluate the significance of the differences observedamong the mutant proteins by the conventional in vitrokinase assay.A different measure of the kinase activities was obtained

by determining the levels of tyrosine phosphorylation of p34in infected cells. This analysis revealed that temperaturesensitivity in p34 phosphorylation segregates with lesions inthe kinase domains of the chimeric src proteins. Theseresults are consistent with the finding that temperaturesensitivity in the transformation parameters segregates withthe mutant kinase domains (27, 34). There is also a generalcorrelation between decreased p34 phosphorylation andincreased transformation defectiveness, as reported previ-ously in studies of other mutants (11, 35, 36, 43). In addition,temperature sensitivity in p34 phosphorylation specificallycorrelates with temperature sensitivity in mitogenic activity.We note, however, the lack of an absolute correlationbetween the level of p34 phosphorylation and any of thetransformation parameters examined here. For example,NYPA101 and NYPA104 induced similar levels of p34phosphorylation and yet they were different in their capaci-ties to induce morphological alteration and anchorage inde-pendence at 37°C. Moreover, cells infected with NY101-HB5 were positive for mitogenic activity and anchorageindependence but did not contain detectable levels ofphosphotyrosine in p34. Therefore, although p34 phosphor-ylation might reflect to some extent the in vivo kinaseactivities of the mutant proteins, it is possible that p34 isinvolved in the expression of a cellular phenotype that wasnot studied here. However, we cannot exclude the possibil-ity that a minimum level of p34 phosphorylation is necessarybut not sufficient for the expression of any transformationparameter examined.The results of this study demonstrate that the levels of in

vitro and in vivo phosphorylation, as measured by theseassays, are not directly comparable. In addition, althoughthe kinase activities of the NYPA101 and NYPA104 p60appear to be equally thermolabile in vivo (Fig. 2), only thekinase activity of the NYPA104 p60 protein is morethermolabile than that of wild-type p60v-src in vitro (39). The

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856 JOVE ET AL.

apparent discrepancies between results of the two assaysmight be explained by intrinsic differences in the assaysystems, including the fact that the phosphorylation ofdifferent substrates is being examined. There is, however, animportant feature of the results that is common to bothassays. The levels of phosphorylation are determined notonly by the kinase domain but also by a region outside of thisdomain. For example, an amino-terminal region of theNYPA101 p60 protein appears to inhibit the activity of thewild-type p60vsrc kinase domain both in vivo and in vitro. Bycontrast, the amino-terminal portion of the SRA p60 con-tained in the NY104-HB5 protein appears to potentiate theactivity of the p60csrc kinase domain in vitro or in vivo.These results are consistent with the proposed modulatoryfunction of a putative amino-terminal domain (16, 19, 37) andsuggests an interaction between the amino-terminal andkinase domains.The NYPA101 and NYPA104 p6Os exhibit an unusual

behavior in biochemical fractionation that previously has notbeen described. The increased solubility of these proteinscannot be attributed to the lack of myristic acid at theiramino termini. This unusual fractionation behavior segre-gates with lesions in the mutant kinase domains, as doescomplex formation with p50 and p90. The data suggest thatlesions in the kinase domain result in a more stable interac-tion with these soluble cellular proteins, which in turnincreases the proportion of soluble versus membrane-associated p60. This suggested interaction of p50 and p90with the kinase domain is consistent with the possibility thatthese cellular proteins might regulate the kinase activity (3,4, 13). However, the level of p34 phosphorylation appears tobe independent of complex formation, suggesting that p50and p90 might not be involved in regulating substrate spec-ificity or kinase activity. For example, both the NYSRA-101and NYPA104 p6Os were present predominantly in thecomplex and yet the two proteins induced very differentlevels of p34 phosphorylation. Previous studies of ts mutantshave demonstrated changes in the levels of complex forma-tion and subcellular distribution as a function of temperature(4, 13, 20). In contrast, these properties of the mutant srcproteins investigated here do not vary significantly withtemperature. This suggests that the differences in cellularphenotypes observed at the different temperatures are notmediated simply by changes in the proportions of the srcproteins in soluble complexes. It should be noted, however,that relatively small changes in the fractionation behavior orthe levels of complex formation might not be detected by themethods used in these experiments.The lack of a strong correlation among the cellular phe-

notypes and the biochemical properties of the src proteinsstudied here indicates that these analyses do not address thephosphorylation of critical cellular targets involved in trans-formation. This is perhaps most strikingly evidenced by theobservation that the various src proteins induced similarlevels of p34 phosphorylation at 34 and 37°C, even thoughthe cellular phenotypes induced by several of the mutantswere very different at these two temperatures. These resultsstrongly suggest that the substrate specificities of the kinasedomains, rather than the absolute levels of kinase activity,are changing between these temperatures. This is consistentwith the hypothesis that the dissociation of transformationparameters reflects differential phosphorylation of multiplecellular substrates involved in transformation (1, 10, 11, 35,44). Further support for this hypothesis is derived from thefindings that the different parameters have different require-ments for the amino-terminal domains (16, 17; G. Calothy,

D. Laugier, F. R. Cross, R. Jove, T. Hanafusa, and H.Hanafusa, submitted for publication), which might primarilybe involved in determining substrate specificity. It is attrac-tive to postulate that the substrate specificities of the tsmutants described here become more restricted with in-creasing temperature, resulting in the expression of fewertransformation parameters. Therefore, mutants such asthese might conditionally phosphorylate fewer cellular sub-strates than wild-type p60vsrc and thereby could facilitate thesearch for substrates causally related to transformation.

ACKNOWLEDGMENTS

We thank J. Brugge for monoclonal antibody 327, M. E.Greenberg and G. M. Edelman for anti-p34 serum, and the membersof our laboratory for helpful discussions and suggestions. We aregrateful to C. Grandori, B. Mayer, M. Sudol, L.-H. Wang, and H.Yu for critical comments on the manuscript.

R.J. is the recipient of Damon Runyon-Walter Winchell CancerFund Fellowship DRG-786, and E.A.G. is the recipient of a MerckFellowship. This work was supported by Public Health Servicegrant CA14935 from the National Cancer Institute and by grantMV128C from the American Cancer Society to H.H.

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2. Brugge, J. S., and R. L. Erikson. 1977. Identification of atransformation-specific antigen induced by an avian sarcomavirus. Nature (London) 269:346-348.

3. Brugge, J. S., E. Erikson, and R. L. Erikson. 1981. The specificinteraction of the Rous sarcoma virus transforming protein,pp60src, with two cellular proteins. Cell 25:363-372.

4. Brugge, J. S., W. Yonemoto, and D. Darrow. 1983. Interactionbetween the Rous sarcoma virus transforming protein and twocellular phosphoproteins: analysis of the turnover and distribu-tion of this complex. Mol. Cell. Biol. 3:9-19.

5. Calothy, G., F. Poirier, G. Dambrine, P. Mignatti, P. Combes,and B. Pessac. 1980. Expression of viral oncogenes in differen-tiating chick embryo neuroretinal cells infected with aviantumor viruses. Cold Spring Harbor Symp. Quant. Biol.44:983-990.

6. Calothy, G., F. Poirier, G. Dambrine, and B. Pessac. 1978. Atransformation defective mutant of Rous sarcoma virus inducingchick embryo neuroretilial cell proliferation. Virology 89:75-84.

7. Collett, M. S., and R. L. Erikson. 1978. Protein kinase activityassociated with the avian sarcoma virus src gene product. Proc.Natl. Acad. Sci. USA 75:2021-2024.

8. Coliet, M. S., A. F. Purchio, and R. L. Erikson. 1980. Aviansarcoma virus protein p60Src shows protein kinase activityspecific for tyrosine. Nature (London) 285:167-169.

9. Cooper, J. A., F. S. Esch, S. S. Taylor, and T. Hunter. 1984.Phosphorylation sites in enolase and lactate dehydrogenaseutilized by tyrosine protein kinases in vivo and in vitro. J. Biol.Chem. 259:7835-7841.

10. Cooper, J. A., and T. Hunter. 1984. Regulation of cell growthand transformation by tyrosine-specific protein kinases: thesearch for important cellular substrate proteins. Curr. Top.Microbiol. Immunol. 107:125-162.

11. Cooper, J. A., K. D. Nakamura, T. Hunter, and M. J. Weber.1983. Phosphotyrosine-containing proteins and expression oftransformation parameters in cells infected with partial trans-formation mutants of Rous sarcoma virus. J. Virol. 46:15-28.

12. Courtneidge, S. A. 1985. Activation of the pp6csrc kinase bymiddle T antigen binding or by dephosphorylation. EMBO J.4:1471-1477.

13. Courtneidge, S. A., and J. M. Bishop. 1982. The transit ofpp60v-src to the plasma membrane. Proc. Natl. Acad. Sci. USA79:7117-7121.

14. Courtneidge, S. A., A. D. Levinson, and J. M. Bishop. 1980. The

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protein encoded by the transforming gene of avian sarcomavirus (pp6(Yrc) and a homologous protein in normal cells (pp-60Prol0-src) are associated with the plasma membrane. Proc. Natl.Acad. Sci. USA 77:3783-3787.

15. Coussens, P. M., J. A. Cooper, T. Hunter, and D. Shalloway.1985. Restriction of the in vitro and in vivo tyrosine proteinkinase activities of pp604-src relative to pp6O-src. Mol. Cell. Biol.5:2753-2763.

16. Cross, F. R., E. A. Garber, and H. Hanafusa. 1985. N-terminaldeletions in Rous sarcoma virus p60sr: effects on tyrosinekinase and biological activities and on recombination in tissueculture with the cellular src gene. Mol. Cell. Biol. 5:2789-2795.

17. Cross, F. R., E. A. Garber, D. Pellman, and H. Hanafusa. 1984.A short sequence in the p6OsrC N terminus is required for p6((rcmyristylation and membrane association and for cell transfor-mation. Mol. Cell. Biol. 4:1834-1842.

18. Erikson, E., and R. L. Erikson. 1980. Identification of a cellularprotein substrate phosphorylated by the avian sarcoma virustransforming gene product. Cell 21:829-836.

19. Garber, E. A., F. R. Cross, and H. Hanafusa. 1985. Processingof p6OV-src to its myristylated membrane-bound form. Mol. Cell.Biol. 5:2781-2788.

20. Garber, E. A., J. G. Krueger, H. Hanafusa, and A. R. Goldberg.1983. Temperature-sensitive membrane association of pp6Osrc inNY68-infected cells correlates with increased tyrosine phos-phorylation of membrane-associated proteins. Virology126:73-86.

21. Greenberg, M. E., and G. M. Edelman. 1983. The 34 kd pp6(Yrcsubstrate is located at the inner surface of the plasma mem-brane. Cell 33:767-779.

22. Hanafusa, H. 1969. Rapid transformation of cells by Roussarcoma virus. Proc. Natl. Acad. Sci. USA 63:318-325.

23. Hanafusa, H. 1977. Cell transformation by RNA tumor viruses.Comp. Virol. 10:401-483.

24. Hunter, T., and B. W. Sefton. 1980. Transforming gene productof Rous sarcoma virus phosphorylates tyrosine. Proc. Natl.Acad. Sci. USA 77:1311-1315.

25. Iba, H., F. R. Cross, E. A. Garber, and H. Hanafusa. 1985. Lowlevel of cellular protein phosphorylation by nontransformingoverproduced p60csrc. Mol. Cell. Biol. 5:1058-1066.

26. Iba, H., R. Jove, and H. Hanafusa. 1985. Lack of induction ofneuroretinal cell proliferation by Rous sarcoma virus variantsthat carry the c-src gene. Mol. Cell. Biol. 5:2856-2859.

27. Jove, R., B. J. Mayer, H. Iba, D. Laugier, F. Poirier, G. Calothy,T. Hanafusa, and H. Hanafusa. 1986. Genetic analysis of p60v-srcdomains involved in the induction of different cell transforma-tion parameters. J. Virol. 60:840-848.

28. Kamps, M. P., J. E. Buss, and B. M. Sefton. 1985. Mutation ofNH2-terminal glycine of p605c prevents both myristoylation andmorphological transformation. Proc. Natl. Acad. Sci. USA82:4625-4628.

29. Krueger, J. G., E. A. Garber, and A. R. Goldberg. 1983.Subcellular localization of pp60gYc in RSV-transformed cells.Curr. Top. Microbiol. Immunol. 107:51-124.

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31. Levinson, A. D., H. Oppermann, L. Levintow, H. E. Varmus,and J. M. Bishop. 1978. Evidence that the transforming gene ofavian sarcoma virus encodes a protein kinase associated with aphosphoprotein. Cell 15:561-572.

32. Levinson, A. D., H. Oppermann, H. E. Varmus, and J. M.Bishop. 1980. The purified product of the transforming gene ofavian sarcoma virus phosphorylates tyrosine. J. Biol. Chem.255:11973-11980.

33. Lipsich, L. A., A. J. Lewis, and J. S. Brugge. 1983. Isolation ofmonoclonal antibodies that recognize the transforming proteinsof avian sarcoma viruses. J. Virol. 48:352-360.

34. Mayer, B. J., R. Jove, J. F. Krane, F. Poirier, G. Calothy, andH. Hanafusa. 1986. Genetic lesions involved in temperaturesensitivity of the src gene products of four Rous sarcoma virusmutants. J. Virol. 60:858-867.

35. Nakamura, K. D., and M. J. Weber. 1982. Phosphorylation of a36,000 Mr cellular protein in cells infected with partial transfor-mation mutants of Rous sarcoma virus. Mol. Cell. Biol.2:147-153.

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37. Parsons, J. T., D. Bryant, V. Wilkerson, G. Gilmartin, and S. J.Parsons. 1984. Site-directed mutagenesis of Rous sarcoma viruspp60kr: identification of functional domains required for trans-formation, p. 37-42. In G. F. Vande Woude, A. J. Levin, W. C.Topp, and J. D. Watson (ed.), Cancer cells, vol. 2. Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.

38. Pellman, D., E. A. Garber, F. R. Cross, and H. Hanafusa. 1985.Fine structural mapping of a critical NH2-terminal region ofp6(Src. Proc. Natl. Acad. Sci. USA 82:1623-1627.

39. Poirier, F., G. Calothy, R. E. Karess, E. Erikson, and H.Hanafusa. 1982. Role of p605Y kinase activity in the inductionof cell proliferation by Rous sarcoma virus. J. Virol. 42:780-789.

40. Radke, K., and G. S. Martin. 1979. Transformation by the Roussarcoma virus: effects of src gene expression on the synthesisand phosphorylation of cellular phosphopeptides. Proc. Natl.Acad. Sci. USA 78:5212-5216.

41. Sefton, B. M., T. Hunter, and K. Beemon. 1980. Temperature-sensitive transformation by Rous sarcoma virus and tempera-ture-sensitive protein kinase activity. J. Virol. 33:220-229.

42. Sefton, B. M., T. Hunter, K. Beemon, and W. Eckhardt. 1980.Evidence that the phosphorylation of tyrosine is essential fortransformation by RSV. Cell 20:807-816.

43. Stoker, A. W., P. J. Enrietto, and J. A. Wyke. Functionaldomains of pp60-src proteins as revealed by analysis of temper-ature-sensitive Rous sarcoma virus mutants. Mol. Cell. Biol.4:1508-1514.

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