Proc. Natl. Acad. Sci. USAVol. 76, No. 10, pp. 5028-5032, October 1979Biochemistry

Characterization of the protein kinase activity of avian sarcomavirus src gene product

(src gene product p6Os/a-casein phosphorylation/immunoglobulin G/phosphatase/avian sarcoma virus-transformedmammalian cells)

PATRICIA F. MANESS, HANSJORG ENGESER, MICHAEL E. GREENBERG, MINNIE O'FARRELL,W. EINAR GALL, AND GERALD M. EDELMANThe Rockefeller University, 1230 York Avenue, New York, New York 10021

Contributed by Gerald M. Edelman, July 18, 1979

ABSTRACT The avian sarcoma virus src gene product,p6Osrc, has been purified 850-fold from cytoplasmic extracts ofthe rat tumor cell line RR1022 by using ammonium sulfatefractionation, hydrophobic chromatography on w-aminohexylagarose, and ion exchange chromatography on phosphocellu-lose. Partially purified p6Osrc is a monomer, with a native mo-lecular weight of about 60,000 and an apparent pI of 6.0.

In immunoprecipitates, p6Osrc catalyzed phosphorylation ofanti-p60sre IgG heavy chains within the variable (VH) domain,which contains the heavy chain portion of the antigen com-bining site. Crude preparations of p60sm contained phosphataseactivity able to cleave phosphate from IgG heavy chains; thisactivity was removed by the purification procedure, and par-tially purified p6Osrc could phosphorylate the heavy chain ofspecific antibody in solution. Furthermore, purified p6Osrccatalyzed phosphorylation in solution of the general proteinkinase substrate, a-casein, strengthening the hypothesis thatit may in fact function as a protein kinase in vivo.

Avian sarcoma viruses (ASV) are potent oncogenic agents thattransform fibroblasts in vitro and produce tumors in susceptiblehost animals. A single gene, src, of ASV is necessary both forinitiation and maintenance of transformation (for a review, seeref. 1). Genetic studies have suggested that the sarc gene productis a single protein (2). In accord with this interpretation, antiserafrom rabbits bearing ASV-induced tumors were found byBrugge and Erikson (3) to immunoprecipitate a single, nonvi-rion, transformation-specific protein from ASV-infectedchicken embryo fibroblasts and hamster cells. The protein,termed p6Osrc, migrated on sodium dodecyl sulfate (Na-DodSO4)/polyacrylamide gels with a molecular weight of60,000. An apparently identical protein has been translated invitro from the src portion of the ASV genome, providing ad-ditional evidence that p6Osrc is a product of the sarc gene (4).

p6Osrc in immunoprecipitates catalyzes the phosphorylationof specific IgG heavy chains (5, 6), but not of the classical pro-tein kinase substrates, a-casein and histones, when added toimmunoprecipitates (5). Furthermore, this heavy chain kinaseactivity is not observed in crude cell extracts, raising questionsof whether p60src-catalyzed phosphorylation is obscured bycompeting kinases, phosphatases, or inhibitors, or whether theheavy chain kinase activity is unrelated to the function of thesrc gene product in the cell and its role in transformation.To clarify this issue, we have partially purified p6Osrc from

extracts of the rat tumor cell line RR1022 and examined itskinase activities. The fractionation removed a phosphataseactivity, and both anti-p60src antibody and a-casein werephosphorylated in solution by partially purified p6Osrc. Theseresults suggest that p6Osrc may function as a protein kinase invivo.

MATERIALS AND METHODS

Cells and Antisera. The RR1022 cell line, derived from atumor induced in the inbred Amsterdam rat after injection ofthe Schmidt-Ruppin strain of ASV (7), was obtained from theAmerican Type Culture Collection and grown in Dulbecco'smodified Eagle's medium containing 10% calf serum in plasticroller bottles. Cells were harvested when the cultures wereapproximately 80% confluent, and stored at -70'C. Antiserawere obtained from rabbits bearing tumors (TBR sera) inducedby inoculation with ASV Schmidt-Ruppin strain D (3).

Filter Assay for p60src-Associated Protein Kinase. Aliquotsof cell extracts to be assayed for enzymatic activity were in-cubated with 10 ,ul of TBR serum on ice for 1 hr. Protein A-Sepharose was added to absorb the immune complexes formed.The antigen-antibody complexes were washed three times with200 Ail of 50 mM Tris-HCI, pH 7.2/0.15 M NaCl; once with 2.5M KCI/60 mM Tris-HCI, pH 7.2; and once with 40 mM Tris-HCI, pH 7.2/10 mM MgCl2. The reaction was started by theaddition of 2.5 uCi of [y-32P]ATP (Amersham, 1000-3000Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) in 20 mM Tris-HCI,pH 7.2/5 mM MgCl2 and, after 10 min at 370C, was stoppedby washing the immunoprecipitates three times with 200 Al of20 mM Tris-HCI, pH 7.2/5 mM MgCl2 and boiling for 2 minin 5% NaDodSO4/1% 2-mercaptoethanol. After centrifugationto remove protein A-Sepharose, the supernatant was broughtto 10% in trichloroacetic acid and heated to 90'C for 15 minto hydrolyze ATP. After the precipitates were cooled, they werecollected on Whatman glass fiber filters. The filters werewashed with 0.1 M Na pyrophosphate/5% trichloroacetic acidanadwith methanol before drying. 32P was quantitated by liquidscintillation counting. This filter assay is a modification of theNaDodSO4/polyacrylamide gel assay developed by Collett andErikson (5). The recovery of radioactivity was comparable inboth assays.

Partial Purification of p60src-Associated Protein Kinase.Frozen RR1022 cells (30 ml packed volume) were suspendedin 60 ml of 10mM K phosphate, pH 7.0/5 mM MgCl2/10mMdithiothreitol/0.1 mM EDTA/0.34M sucrose/0.5mM ATP/1%(vol/vol) Trasylol/2 mM phenylmethylsulfonyl fluoride, andcells were lysed by homogenization for 5 min in a Potter-El-vejhem homogenizer followed by 40 strokes in a Dounce ho-mogenizer. The homogenate was centrifuged for 2 hr at 100,000X g. Nucleic acids were removed from the supernatant by theadditon of 1.25% (wt/vol) streptomycin sulfate and centrifu-gation. Material precipitating from this supernatant between20 and 40% saturation with ammonium sulfate contained ap-

Abbreviations: TBR serum, serum from tumor-bearing rabbits; ASV,avian sarcoma virus; NaDodSO4, sodium dodecyl sulfate; VH, variableregion of immunoglobulin heavy chain; CHI, constant region of im-munoglobulin heavy chain.

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proximately 90% of the p6Osrc kinase activity. The ammoniumsulfate precipitate was dialyzed against buffer A (10 miM Kphosphate, pH 7.0/5 mM MgCl2/0.1 mM EDTA/0.34 M su-crose/10mM dithiothreitol) and chromatographed on a column(1.5 X 10 cm) of w-aminohexylagarose (Miles) previouslyequilibrated with buffer A. The column was washed with 100ml of buffer A, and a 500-ml linear gradient of NaCl (0-1.0 M)in buffer A was applied. The active fractions, eluting between0.6 M and 0.7 M NaCl, were pooled and dialyzed against bufferB (buffer A containing 1 mM dithiothreitol) and chromato-graphed on a column (1.2 X 16 cm) of phosphocelluloseequilibrated with buffer B. The column was washed with 60ml of buffer B, and a 200-ml linear gradient of K phosphate,pH 7.0, (0.01-0.4 M) in buffer B was applied. The active frac-tions, eluting at 0.15 M K phosphate, were pooled and dialyzedagainst buffer A. All procedures were carried out at 4VC.

RESULTSDetection of p6Osrc and Protein Kinase Activity in Im-

munoprecipitates. To determine whether p6Osrc was presentin RR1022 cells, two-dimensional gel electrophoresis (8) wasused to analyze imffiunoprecipitates of detergent lysates of[35S]methionine-labeled rat cells prepared with TBR serum

(Fig. 1). A 62,000-dalton protein was immunoprecipitated bythe TBR serum but not by normal rabbit serum. This proteindid not focus sharply under the denaturing conditions of theisoelectric focusing dimension but spread across a pH range of5.6 to 6.1. ThIe amount of the 62,000-dalton protein was notchanged when TBR serum was previously absorbed with dis-rupted ASV Schmidt-Ruppin D particles, suggesting that theprotein was not a viral structural protein. The relative amountof the protein in the rat cells was estimated to be 5 ng/mg of cellprotein.

Extracts of RR1022 cells stimulated phosphorylation ofantibody heavy chains in immunoprecipitates prepared withTBR serum but not with normal rabbit serum. The reaction wasessentially complete within 1 min and was almost as efficientat 0°C as at 37°C. Little or no activity could be detected iI1similar extracts of normal rat kidney cells. These results suggestthat the 62,000-dalton protein from RR1022 cells is the ASV src

gene product, corresponding to that previously identified inother cells (3).

Site of Phbsphorylation of the IgG Heavy Chain. As shownpreviously (5, 6) only the heavy chain of specific antibody isphosphorylated by p6Osrc. To localize the site within the chain,32P-labeled immunoprecipitates on protein A-Sepharose beadswere treated with papain to cleave the IgG into Fab and Fc

fragments. Immunoelectrophoresis of the supernatant withanti-Fab and anti-Fc antibodies showed that equivalentamounts of the fragments were released but that only Fabfragments contained 32P (Fig. 2). These data restrict the site ofphosphorylation to the Fd region, which contains the constantdomain, CH1, and the variable domain, VH.To localize the site further, we took advantage of the dif-

ferential sensitivity of these domains to proteolysis in the ab-sence of light chains (9). Fd dimers were prepared from 32p-labeled Fab fragments and separated from immunoglobulinlight chains by gel filtration (Fig. 3A). Digestion with papainconverted the CH1 domain to small peptides, leaving the VHdomain intact (9). The VH fragment, separated from undigestedFd by gel filtration, contained 32p (Fig. 3B). No 32P was foundassociated with the lower molecular weight peptides derivedfrom CH1. These data indicate that phosphorylation by p6Osrctakes place within the VH domain, the region that, together withthe variable domain of the light chain, comprises the antigen-binding site.

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FIG. 1. Identification of p6Osrc by two-dimensional gel electro-phoresis. RR1022 cells were labeled for 4 hr with [35S]methionine(1400 Ci/mmol) in medium lackink methionine and lysed in 150mMNaCl/1% sodium deoxycholate/1% Triton X-100/0.1% NaDodSO/10mM Tris-HCl, pH 7.2/1% Trasylol. Cell lysates were centrifuged at100,000 X g for 1 hr. Supernatants were immunoprecipitated withTBR serum (Lower) or normal rabbit serum (Upper) and analyzedby isoelectric focusing in the first (horizontal) dimension and Na-DodSO4/polyacrylamide gel electrophoresis in the second (vertical)dimension (8). The positions of standard protein molecular weightmarkers (X 10-3) in the second dimension are indicated.

Partial Purification and Molecular Characteristics ofp6Osrc. Although p6Osrc from cell extracts phosphorylated spe-cific IgG in immunoprecipitates, it did not appear to phos-phorylate either specific IgG or any other substrates in solution.Its activity in extracts might be inhibited, however, by thepresence of cellular phosphatases, by competing kinases, or byother inhibitors. To eliminate such inhibitors, we carried outa limited fractionation of extracts from RR1022 cells using thefilter assay to screen fractions rapidly.

Cells were lysed in detergent-free buffer and centrifuged at100,000 X g. src kinase activity was distributed between thecytoplasmic supernatant fraction and the membrane-containingpellet. Although the pellet contained up to 70% of the src kinaseactivity, we chose to ffactionate the cytoplasmic fraction be-cause of the facility of using detergent-free solutions and be-cause the cytoplasmic fraction was active in a cellular micro-injection assay for p6Osrc that measures the dissolution of actincables (10). The supernatant was fractionated by the proceduresummarized in Table 1, and the results of chromatography on

c-aminohexylagarose and phosphocellulose are shown in Fig.

7pH

6 5

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anti-Fab

0anti-Fc

Table 1. Purification of p6O8rc from RR1022 rat tumor cellsTotal Total Specific

protein, activity, activity,Fraction mg units units/mg

Extract 1625 280 0.17Ammonium

sulfate (20-40%) 235 180 0.77w-Aminohexylagarose 15 80 5.3Phosphocellulose 0.19 21 110

One unit of activity incorporates 1 X 106 cpm Of 32p into IgG heavychains in 10 min at 370C under the conditions described for the filterassay in Materials and Methods.

FIG. 2. p60c phosphorylates the Fab fragment ofTBR antibody.IgG from TBR serum labeled with 32p by p6oarc was digested with0.15% papain in 50mM K phosphate, pH 7.0/10mM cysteine/2 mMEDTA for 60 min at 370C. After centrifugation to remove the proteinA-Sepharose, the supernatant was analyzed by immunoelectropho-resis at 1.7 V/cm for 2.5 hr in 0.05 M barbital, pH 8.5, followed byimmunodiffusion against sheep antisera against rabbit Fab fragment(upper trough) and Fc fragment (lower trough). (Upper) Amido blackstaining for proteins, showing precipitin lines for both Fab and Fc.(Lower) Autoradiography showing localization of 32p in the Fabfragment.

4 A and B. Overall, fractionation resulted in a 650-fold puri-fication of p6Orc kinase activity with an 8% yield, but the finalphosphocellulose fraction was not homogeneous as judged byNaDodSO4/polyacrylamide gel electrophoresis. Because of thelow yields after phosphocellulose chromatography, the bulk ofthe molecular characterization was carried out on the fractionof p6Osrc obtained from the w-aminohexylagarose chromato-graphic step.

Attempts to determine the native molecular weight of p6Offcin crude extracts by molecular sieve chromatography andglycerol gradient centrifugation indicated extensive aggregationof p6Osrc. However, p6Osrc kinase no longer aggregated after

A

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purification by hydrophobic chromatography and exhibiteda sedimentation behavior on glycerol gradients similar to thatof bovine serum albumin (68,000 daltons) (Fig. 4C). When thesame fraction was applied to a calibrated column of SephacrylS-200, the src kinase migrated as a protein with an apparentmolecular weight of about 60,000 daltons (Fig. 4D). Eventhough these methods do not have sufficient resolution to de-termine the molecular weight precisely, they do indicate thatthe native molecular weight of purified p6osrc (about 60,000daltons) is similar to that observed by NaDodSO4/polyacryl-amide gel electrophoresis. Purified p6Osrc is therefore amonomeric protein.

Isoelectric focusing under nondenaturing conditions (12)indicated an apparent pl of 6.0 for p6Osrc kinase activity puri-fied by w-aminohexylagarose chromatography. In some ex-periments, a second peak of kinase activity with apparent pIof 5.0 was seen, possibly representing either p60src bound toprotein components that migrate at this position or p6osrc

40- A

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0

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I- -So

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50 100 50 100Fraction Fraction

FIG. 3. Preparation ofVH fragment of 32P-labeled IgG. The su-

pernatant (2 mg) from a papain digest of an immunoprecipitate la-beled with 32P by p6O1rc (as described in the legend of Fig. 2) was

mixed with carrier Fab fragment (15 mg) of IgG from an unimmunizedrabbit. Fd dimer was prepared (9) by reduction and alkylation of theinterchain disulfide bonds, dialysis against 1 M acetic acid, and gelfiltration on a column (1 X 161 cm) of Sephadex G-100 (A). Materialin the fractions indicated by the bar was digested with 0.5% papainat 371C for 20 min at pH 5.5, dialyzed against 1 M acetic acid, andfractionated on the same Sephadex column (B). The VH fragment hasan apparent molecular weight of 14,000 and contains 32p. The identityof the fragment was confirmed by NaDodSO4/polyacrylamide gelelectrophoresis. The column void volume (Vo), total volume (VT), andelution volume of ribonuclease (14K) are indicated.

~0.8.50.6 M NaCI 06-680

0.40.-"040

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30 60 90 j

109K 68K 15K14 i 4

2

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0.15 M P1

0.20

0.16-

0.12-

0.02

0.04in

40 80 120

DV0 68K43K 13K4 44 4

10 20 30 80 120Fraction

160

FIG. 4. (A) Chromatography of the 20-40% ammonium sulfatefraction on w-aminohexylagarose. (B) Phosphocellulose chromatog-raphy of the active p6Osrc fractions obtained from w-aminohexyl-agarose chromatography. (C) Glycerol gradient centrifugation. Theactive p60src fractions obtained from w-aminohexylagarose chroma-tography were layered on a linear gradient of 10-30% (wt/vol) glycerolin 10 mM K phosphate, pH 7.0/0.1 mM EDTA/1 mM MgCl9 andcentrifuged for 40 hr at 40,000 rpm in an SW 40 Beckman rotor (11).The gradient was calibrated with Escherichia coil DNA polymeraseI (109K), bovine serum albumin (68K), and lysozyme (15K). (D)Sephacryl S-200 chromatography. The p6&src fraction obtained fromw-aminohexylagarose chromatography was applied to a column ofSephacryl S-200 (101 X 1.4 cm), previously equilibrated in 10mM Kphosphate, pH 7.0/5 mM MgCl2/0.1 mM EDTA/0.34 M sucrose/0.5M NaCl/1 mM dithiothreitol. The column was calibrated with bluedextran (Vo), bovine serum albumin (68K), ovalbumin (43K), andcytochrome C (13K).

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5030 Biochemistry: Maness et al.

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Table 2. Dephosphorylation of IgG heavy chain by fractionatedcell extracts

32p incorporation, cpmFraction added Fraction added

Fraction added before reaction after reaction

Buffer 73,500 84,800Ammonium sulfate 1,000 500w-Aminohexylagarose 76,200 50,000

Equal amounts of p6Osrc (0.08 units) were immunoprecipitated froman w-aminohexylagarose fraction with TBR serum. Either buffer (20mM Tris-HCl, pH 7.2/5 mM MgClJ/10 mM dithiothreitol), an aliquotof the ammonium sulfate fraction of p6Wsrc (0.0006 units), or an aliquotof' the w-aminohexylagarose fraction of p6Osrc (0.0006 units) wasadded. The kinase reaction was initiated by adding ['y-32P]ATP. Aftei10 min at 370C, samples were washed to remove [,y-32P]A'T'P. An al-iquot of either the ammonium sulfate fraction (0.0006 units) or thew-aminohexylagarose fraction (0.0006 units) was then added tosamples that had received buffer initially. All samples were incubatedfor a further 10 min at 370C before assay by the filter method. Resultswere confirmed by NaDodSO4/polyacrylamide gel electrophoresis.

modified by, for example, limited proteolysis or phosphoryl-ation.

Kinase Activity of p6Osrc in Solution. As indicated above,p60src from crude extracts catalyzed phosphorylation of specificantibody in immunoprecipitates, but phosphorylation of thesame IgG added to the crude extracts was not observed in so-lution. To test for inhibitors in the extracts, ammonium sulfatefractions and p6Osrc fractions purified by the w-aminohexyl-agarose chromatographic step were added to immunoprecip-itates containing p6osrc, and the kinase reaction was initiatedwith [y-32P]ATP (Table 2). Addition of the crude ammoniumsulfate fraction but not of the purified p6Osrc resulted in a sig-nificant decrease in the amount of 32p incorporated into im-munoglobulin heavy chains. Furthermore, incubation of theammonium sulfate fraction with immunoprecipitates previ-ously labeled with 32P resulted in marked dephosphorylationof the immunoglobulin heavy chains. In contrast, the purifiedp6Osrc fraction contained little dephosphorylating activity.These results suggested that a phosphatase activity acting onphosphorylated IgG was present in crude extracts but was al-most completely removed by hydrophobic chromatography.Remtval of the inhibitory activity made it possible to detect

src kinase activity in solution. The p6Osrc purified by thephosphocellulose chromatographic step phosphorylated IgGheavy chains from TBR serum in solution (Fig. 5, lane 2) butnot IgG from normal rabbit serum (Fig. 5, lane 1). In contrast,addition of [y-32P]ATP to the ammonium sulfate fraction didnot lead to incorporation of 32P into added IgG, although it didresult in phosphorylation of various proteins in the fraction,suggesting the presence of other protein kinases.The phosphocellulose fraction of p6osrc was found to phos-

phorylate ae-casein in solution (Fig. 5, lane 3). Removal of thep6Osrc by prior immunoprecipitation with TBR serum de-creased the ability of this fraction to phosphorylate a-casein(Fig. 5, lane 4). These results suggest that it is the p6osrc itselfthat is primarily responsible for phosphorylation of a-caseinin solution. Under similar conditions, calf thymus histones werenot phosphorylated by the same preparations of p6Osrc, evenin the presence of cyclic AMP.

DISCUSSIONTo date, the protein kinase activity of p6osrc has been observedonly in specific immunoprecipitates (5, 6). As we and others (5,6) have noted, the phosphorylation of IgG heavy chain is ex-tremely rapid and proceeds almost as well at 00C as at 370C,

Ug.

o- H chain

< Casein

1 2 3 4 5FIG. 5. Phosphorylation of IgG heavy chains and a-casein in

solution. A phosphocellulose fraction containing p6Osrc was incubatedat 370C for 10 min in kinase buffer (20 mM Tris-HCl, pH 7.2/5 mMMgCl2/10 mM dithiothreitol/50 pCi of Ly-32PIATP per ml) withnormal rabbit serum IgG (lane 1), TBR serum IgG (lane 2), and bovinea-casein (lane 3), each at 1 mg/ml. p6osrc was depleted from thephosphocellulose fraction by addition of TBR serum IgG (1 mg/ml)and protein A-Sepharose beads. The supernatant was incubated witha-casein at 1 mg/ml (lane 4) in kinase buffer and kinase buffer alone(lane 5) to detect phosphorylation of IgG due to residual immunecomplexes in the supernatant. Samples were electrophoresed in Na-DodSO4/polyacrylamide gels and processed for autoradiography.

perhaps because of a particular steric arrangement of the pro-teins bound together in the immunoprecipitate. We have shownthat the region of phosphorylation is in the VH domain, whichcontains the heavy chain portion of the antigen binding site.These data are consistent with the interpretation that p6Osrcphosphorylates the antibody molecule at or near where it isbound in the antigen-combining site.We were not able to detect phosphorylation of specific

antibody by crude extracts without prior immunoprecipitation.On the assumption that specific immunoprecipitation separatedp60src from inhibitors in the crude extract, we carried out apartial purification of p6Osrc from the rat tumor line RR1022.A 650-fold purification of src kinase activity was achieved withan 8% yield by using ammonium sulfate fractionation, hydro-phobic chromatography on c-aminohexylagarose, and ionexchange chromatography on phosphocellulose. The resultingpartially purified p6Osrc fraction was not homogeneous, but wasfree of phosphoprotein phosphatase activity as demonstratedby its inability to cleave phosphate from phosphorylated im-munoglobulins.The purification allowed us to demonstrate that p6Osrc

phosphorylated a-casein and specific immunoglobulin heavychains without prior immunoprecipitation. Histones, on theother hand, did not serve as substrates even in the presence ofcyclic AMP. Nonspecific IgG was not phosphorylated by p6Osrc,suggesting that the phosphorylation of IgG from TBR serumrequires a specific interaction between the two proteins,probably resulting from their proximity in the antigen-com-bining site and a fortuitous, appropriate acceptor site in the VHdomain. The phosphorylation of a-casein, however, suggeststhat p60src is indeed a protein kinase and that this activity couldcontribute to its role in cellular transformation. Any in vivotargets of this activity remain to be elucidated.We have previously shown that microinjection of cytoplasmic

extracts of ASV-transformed chicken embryo fibroblasts into

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normal cells causes a disruption of actin-containing stress fibers(10). Components of the cytoskeleton, some of which are knownto be phosphorylated, are possible in vivo targets of the src ki-nase. The combined use of in vitro phosphorylation assays andthe cellular microinjection assay should help to determinewhether the protein kinase activity of p6sc is directly requiredfor the cytoskeletal alteration.

We thank Dr. H. Hanafusa and Mr. R. Karess for supplying the virusand for their generous help. We acknowledge with gratitude thetechnical assistance of Ms. Catherine Volin, Ms. Robin Evans, Ms. LisaPratt, and Ms. Mary Coogan. This work was supported by GrantsCA19040, AI11378, and AM04256 from the National Institutes ofHealth.

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Proc. Nati. Acad. Sci. USA 76 (1979)

2. Wyke, J., Bell, J. & Beamand, J. (1974) Cold Spring Harbor Symp.Quant. Biol. 39,897-905.

3. frugge, J. S. & Erikson, R. L. (1977) Nature (London) 269,346-348.

4. Purchio, A. F., Erikson, E., Brugge, J. S. & Erikson, R. L. (1978)Proc. Nati. Acad. Sci. USA 75, 1567-1571.

5. Collett, M. S. & Erikson, R. L. (1978) Proc. Nati. Acad. Sci. USA75,2021-2024.

6. Levinson, A. D., Oppermann, H., Levintow, L., Varmus, H. E.& Bishop, J. M. (1978) Cell 15,561-572.

7. Ahlstrom, C. G. & Jonsson, N. (1961) Acta Pathol. Microbiol.Scand. 54, 145-172.

8. O'Farrell, P. H. (1975) J. Biol. Chem. 250,4007-4021.9. Mole, L. E., Geier, M. D. & Koshland, M. E. (1975) J. Immunol.

114, 1442-1448.10. McClain, D. A., Maness, P. F. & Edelman, G. M. (1978) Proc.

Natl. Acad. Sci. USA 75,2750-2754.11. Freifelder, D. (1973) Methods Enzynol. 27, 140-150.12. Holtlund, J. & Kristensen, T. (1978) Anal. Biochem. 87, 425-

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