pp60src-Dependent protein phosphorylation in membranes from Rous sarcoma virus-transformed chicken embryo fibroblasts

VIROLOGY

143, 407-421 (19%)

pp60src-Dependent Protein Phosphorylation Rous Sarcoma Virus-Transformed Chicken PHILIP Department

of

DEHAZYA’ Zoology,

Received

AND

University October

in Membranes from Embryo Fibroblasts

G. STEVEN

of Califwnia,

22, 1984; accepted

Berkeley, January

MARTIN* Califmia

94720

7, lYX.5

The Rous sarcoma virus (RSV)-transforming protein, pp60 “‘, is a plasma membraneassociated tyrosine-specific protein kinase. A 36,000-Da cellular polypeptide (~36) which is phosphorylated at tyrosine in RSV-transformed chicken embryo fibroblasts (RSVCEF) is also plasma membrane associated. To determine if p36 is directly phosphorylated by ~~60”. and to examine the effects of mutations within STCon pp60”‘” phosphorylation and kinase activity in sit?* in the plasma membrane, src-dependent protein phosphorylation in membranes isolated from RSV-CEF has been characterized. These membrane preparations contained high ATPase and phosphoprotein phosphatase activities; but when sufficient concentrations of [y-32P]ATP were used, the phosphorylation of pp60”” and the phosphorylation of ~36 were linear for 1 min or more, and the initial rates of phosphorylation could therefore be determined. In membranes from RSV-CEF ~~60”” and ~36 became phosphorylated predominantly at tyrosine, while in membranes from uninfected cells p36 was phosphorylated at low levels at serine. When membranes from RSV-CEF were preincubated with tumor-bearing rabbit (TRR) serum, the IgG became phosphorylated while the phosphorylation of ~36 was inhibited, suggesting that p36 is directly phosphorylated by ~~60’~. Phosphorylation of pp60wr, ~36, and TBR-IgG was dependent on growth temperature in membranes from cells infected by a tcmperaturesensitive mutant, tsNY68, although some dcpcndence on growth temperature was observed even with membranes from wild-type RSV-infected cells. IIowever, at the nonpermissive temperature, tsNY68 ~~60”’ retained 20-4OYo of its kinase activity, providing supporting for the proposal (B. M. Sefton, T. Hunter, and K. Beemon (1980, J. Viral, 33, 220-229) that transformation may result from a small quantitative change were not in pp60”” activity. The phosphorylation of ~~60”” and its kinasp activity coordinately affected by growth temperature or mutations within STC, indicating that different factors affect the phosphoacceptor capacity and kinase activity of tho protein. $0 19X.5 Academic

Press. Inc

INTRODUCTION

Transformation by ROW sarcoma virus (RSV) is dependent on the expression of the src gene product, pp60”‘” (reviewed by Bishop and Varmus, 1982). pp60src and a number of other retrovirus-transforming proteins display tyrosine-specific protein kinase activity, and an increase in the cellular content of phosphotyrosine in

’ Present address: Fels Research Institute, Department of Pharmacology, 3420 North Broad St., Philadelphia, Pa. 19140. “To whom requests for reprints should be addrcsscd.

protein is observed in cells transformed by these viruses (reviewed by Cooper and Hunter, 1983). This activity may be required for transformation by this group of viruses, although it is possible that phosphorylation of nonprotein substrates such as phosphatidylinositols may also be involved (Macara et al., 1984; Sugimoto et al., 1984). A variety of proteins undergo phosphorylation at tyrosine as a result of pp60”” activity: these include vinculin, certain glycolytic enzymes, and a 36,000Da (36K) protein, p36 (Cooper and Hunter, 1983). As yet, however, no effect of phosphorylation on the function of these proteins has been detected.

DEHAZYA

408

Cell fractionation and immunocytochemical techniques have revealed pp60”” to be plasma membrane associated (reviewed by Krueger et al., 1983), and the properties of RSV mutants with mutations in the extreme N-terminal domain of pp60”‘” suggest that this membrane association may be necessary for transformation (Cross et ab, 1984). ~36, an abundant cellular protein which undergoes pp60”‘“-dependent phosphorylation, has also been found to fractionate with plasma membranes (reviewed in Cooper and Hunter, 1983). The interaction of ~~60”” with its substrates has been extensively studied using enzyme and substrate preparations that have been solubilized with detergent; however, detergent solubilization may affect functions of the protein which depend on its interaction with membrane components. The experiments reported here were performed to examine the in vitro activities of ~~60”‘” and its interaction with a presumed cellular substrate when both are associated with cell membranes. We have used a rapid cell fractionation procedure to prepare membrane fractions from transformed cells. In these membrane preparations pp60”” and p36 become phosphorylated at sites similar to those phosphorylated in viva. Under the appropriate conditions pp60”“‘-dependent phosphorylations occur with linear kinetics, and the initial rates of the reactions can therefore be determined. The use of these conditions has allowed us to quantitate the effects of anti-pp60”” antibodies, mutations within src, and cell growth temperature on the rates of phosphorylation of ~~60”” and the 36K protein. MATERIALS

AND

METHODS

Cells ati viruses. The wild-type SchmidtRuppin RSV subgroup A (SR-RSV-A) and the temperature-sensitive (ts) mutant tsNY68 have been described (Kawai and Hanafusa, 1971). tsNY68R3, a temperature-resistant partial revertant of tsNY68, was isolated as follows. Chicken embryo fibroblasts (CEF) were infected with tsNY68 and then mutagenized with 5-bromodeoxyuridine (100 pg/ml) as described

AND

MARTIN

by Bader and Brown (1971). Virus harvests were collected and used to infect fresh cultures of CEF, which were then plated in soft agar suspension. After incubation at 41.5” for 8-10 days, colonies were picked onto CEF monolayers and virus stocks grown at 41.5”. Temperature-resistant revertants were focus purified and retested for their ability to transform at both 36 and 41.5”. Unless otherwise indicated, secondary CEF were infected with the appropriate virus stock, grown at 36 or 41.5” and subcultured after 48 hr. Cells to be used for membrane preparations were re-fed with fresh medium 24 hr before use. Control experiments with uninfected cells were performed with cultures grown at least 48 hr at the appropriate temperature. Radiolabeling in vivo. Short-term (overnight) labeling was carried out as follows. Twenty-four hours after subculture, cultures growing in loo-mm dishes were rinsed twice with Tris-saline, pH 7.5. The cells were then incubated for 16-18 hr with 5 ml of methionine-free Dulbecco’s modified Eagle’s medium (DME), supplemented with 15% complete DME, 4% calf serum, 1% chicken serum, and 250 FCi [3”S]methionine (Amersham/Searle), or with 5 ml of phosphate-free DME supplemented with 4% calf serum, 1% chicken serum, and 0.5-3.0 mCi [32P]orthophosphate (ICN). For long-term (equilibrium) labeling with [35S]methionine this procedure was modified as follows (Sefton et (11., 1982). Eight hours after passage of infected cells, the growth medium was changed to a medium containing 20% of the normal concentration of methionine. Radiolabel was added and the cells were incubated for 24 hr at 36”. The cells were then given fresh radiolabeling medium and incubated for another 24 hr. Membrane preparation. Membranes were prepared by a modification of the procedure described by Radke et al. (1983). Cells were seeded at 2.5 X 10” per loo-mm plate for growth at 41.5”, or at 3 X 10” per plate for growth at 36”. Each membrane preparation used four loo-mm plates. The cells were washed with Trissaline at O”, scraped with a rubber police-

pp60’”

KINASE

ACTIVITY

IN MEMBRANES

409

man, and sedimented by low-speed cen- was shifted to the soluble fraction, and only 54% was recovered in the PlOO fractrifugation. The cells were then resustion. A somewhat more extensive shift of pended in homogenization buffer [20 mM tsNY68 pp60”” to the SlOO fraction in cells Tris-HCl, pH 7.1, 1 mM MgClz, 5 mM KCl, 1% (v/v) aprotinin (Sigma) and 0.1% grown at the nonpermissive temperature has been previously reported: the fraction (w/v) soybean trypsin inhibitor (Sigma)]; fraction in in certain experiments, as noted in the of pp60’” in the particulate cells grown at 41.5” was text, the homogenization buffer was also tsNY68-infected reported to be lo-20% (Courtneidge and supplemented with 200 pg/ml leupeptin Bishop, 1982) or 33% (Garber et al., 1983); (Sigma). The cells were allowed to swell is unfor 15 min at 0”, and then broken with 35 the reason for these variations known, but may reflect differences in the strokes of a tight fitting Dounce homoghomogenization and fractionation proceenizer. The homogenate was centrifuged dures or the low ionic strength of the at 350 g for 10 min to remove unbroken homogenization buffer used here. cells and nuclei. The pellet was resusIn vitro phosphorylation reactions. Unpended in homogenization buffer and recentrifuged. The supernatant fluids were less otherwise indicated, phosphorylation reactions were initiated by the addition combined as the post-nuclear supernatant of PlOO fraction (100 pg protein) to reac(PNS) fraction. The PNS was centrifuged tion mixtures (50 ~1 final volume) containat 100,000 g for 11 min in an Airfuge (Beckman Instruments) at 4”. The supering 320 FM [y-32P]ATP (sp act 50 Ci/ natant fluid (SlOO) was removed and the mmol) in kinase reaction buffer. After at 30” for 1 min for ~~60”“’ pellet fraction (PlOO) was resuspended in incubation kinase reaction buffer [50 mM Tris-HCl, phosphorylation, or 4 min for 36K phospH 7.5, 10 mM MgCIZ, 5 PM EGTA, 10 phorylation, 7 ~1 of 100 mM ATP and 57 ~1 of 2X RIPA buffer (Radke et al., 1983) mM NaF, and 0.1% (w/v) soybean tryspin inhibitor] for in vitro kinase assays. The were added sequentially at 0” to terminate of 32P and solubilize the amount of protein in the PlOO and SlOO the incorporation fractions was quantitated by the method membrane fraction. The reaction mixture was clarified by centrifugation and imof Bradford (1976). 5’-Nucleotidase activity of cell fractions was assayed as described munoprecipitated with the appropriate by Krzyzek et al. (1980). antiserum. For kinetic studies, reactions Immunoprecipitation of the PNS, the were carried out in loo-p1 volumes; samSlOO, and the PlOO fractions confirmed ples were removed at intervals and imthat the results of Airfuge centrifugation munoprecipitated. were similar to those of previously pubPhosphorylation of IgG heavy chain by lished centrifugation techniques. In con- pp60” in membrane fractions was assayed trol experiments with SR-RSV-A-transby the addition of tumor-bearing rabbit formed cells grown at 36”, at least 93% of (TBR) serum directly to PlOO fractions in the 5’-nucleotidase activity present in the the presence of 75 mM NaCl. After incuPNS was recovered in the PlOO fraction. bation at 0” for 30 min, an appropriate p36 was quantitatively recovered in the amount of concentrated kinase reaction PlOO fraction, in agreement with the findbuffer was added to the mixture to adjust ings of Courtneidge et al. (1983) and Radke it to reaction conditions. The reaction was et al. (1983). In SR-RSV-A-transformed initiated by the addition of [y-32P]ATP cells, 85% of the ~~60”” present in the and incubated at 30” for 1 min. After the PNS was recovered in the PlOO fractions, reaction had been terminated and the in agreement with previous data (see membranes solubilized, the IgG was colKrueger et al, 1983). pp60”‘” in tsNY68- lected with fixed Staphylococcus aureus infected cells grown at 36” had a distri(IgGsorb, The Enzyme Center). TBR used bution similar to that of pp60”” of SR- for kinase assays was heated to 50” for RSV-A grown at the same temperature, 30 min to reduce endogenous kinase acbut at 41.5“ some of the tsNY68 pp60”” tivity.

410

DEHAZYA

Phosphorylation of protein in the SlOO fraction was carried out by adjusting this fraction to the kinase buffer ion concentration, then adding this to the ATP mixture as described above. Immunoprecipitation and gel electrophoresis. Radiolabeled cells and membrane fractions phosphorylated in vitro were solubilized with RIPA buffer containing 2 mM EDTA (Radke et al., 1983). pp60”” and p36 were immunoprecipitated with TBR serum or rabbit anti-p36 serum (Radke et al., 1983), respectively. Procedures for immunoprecipitation, for electrophoretic separation of proteins on 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gels, and for autoradiography were as described (Radke et al., 1983). Preliminary titration experiments were carried out to confirm that excess antibody was present when antigen was to be quantitatively immunoprecipitated, and that a constant amount of antigen was immunoprecipitated when immunoprecipitation was to be carried out in antigen excess. Radioactivity in individual gel bands was quantitated by digestion of excised bands with NCS solubilizer (Amersham) at 50” for 2 hr, followed by liquid scintillation counting. For alkali treatment of proteins in polyacrylamide gels (Cooper and Hunter, 1981), the dried gels were incubated in 1.0 M KOH at 50” for 2 hr, rinsed with water and several changes of 10% isopropanol/lO% acetic acid, and dried again. Preparation of [T-‘~PMTP and analysis of ATP hydrolysis. [y-32P]ATP was synthesized by the method of Glynn and Chappell (1964) but was not phenol extracted. Hydrolysis of ATP in PlOO-containing reaction mixtures was quantitated as follows: At intervals, 5-~1 samples of reaction mixtures containing 320 PM ATP and 1 &i [y-32P]ATP were removed, added to 20 ~1 of 5% trichloroacetic acid at O”, and lyophilized. The lyophilized material was redissolved in water and l-p1 samples were spotted on cellulose thin plates (Boehringer-Mannheim). Ascending chromatography was performed in 0.1 M K,HP04, pH 6.8, containing 2% (v/v) lpropanol and 60% (w/v) ammonium sul-

AND

MARTIN

fate. After autoradiography, spots corresponding to ATP and 32Pi were scraped into vials and the radioactivity was quantitated by liquid scintillation counting. Phosphoamino acid analysis. Proteins radiolabeled with 32P were eluted from SDS-polyacrylamide gels and precipitated with 20% trichloroacetic acid in the presence of carrier protein. The precipitate was washed with absolute ethanol and subjected to partial acid hydrolysis in 6 N HCl (llO”, 80 min). Phosphoamino acids were resolved by electrophoresis on cellulose thin-layer plates by electrophoresis at pH 1.9 in the first dimension and at pH 3.5 in the second (Hunter and Sefton, 1980). Peptide mapping. Partial protease cleavage of proteins with S. aureus V8 protease was performed as described by Cleveland et al. (1977). Tryptic peptide maps of 32P-labeled p36 were prepared as described (Beemon and Hunter, 1978; Radke et al., 1980). RESULTS

Kinetics of Phosphorylation from RSV-Transformed

in Membranes Cells

Membranes were prepared from RSVtransformed cells by a rapid procedure involving centrifugation in a Beckman Airfuge. These membranes contained the bulk of ~~60”” and the 36K cellular polypeptide p36 (see Materials and Methods). To identify conditions under which in vitro phosphorylation would proceed with linear kinetics, we first determined the stability of ATP in the phosphorylation mixture. ATP incubated with the PlOO fraction of SR-RSV-A-transformed cells grown at 36” was rapidly hydrolyzed. At a concentration of 20 PM or less, ATP was almost completely hydrolyzed after 1 min of incubation at 30” and fell below the reported K, (about 14 PM) of purified pp60”‘” (Collett et al., 1980; Levinson et al., 1980) within seconds after the addition of the PlOO fraction to the reaction mixture. However, at a concentration of 320 PM, the half-life of ATP was about 2 min and its concentration remained above the K,, of pp60”” for 8 min (Fig. 1). This concen-

~~60”’

KINASE

ACTIVITY

IN MEMBRANES

411

tards the proteolysis of ~~60”” in cell homogenates, and this inhibitor was included in later studies (see below). To determine what fraction of ~~60”” was phosphorylated under conditions of linear 32P incorporation, cultures of SRRSV-A-transformed cells were labeled to equilibrium with [35S]methionine. The PlOO fraction was prepared and the number of moles of ~~60”” in the standard reaction mixture determined. pp60”” was phosphorylated in a parallel reaction mixture and the amount of phosphate incorFIG. 1. Kinetics of phosphorylation of pp60” and In p36 and kinetics of ATP hydrolysis in the PlOO porated into pp60”” was determined. four experiments, between 0.18 and 0.4% fraction from SR-RSV-A-transformed cells. Kinase reactions were initiated by the addition of the PlOO of the molecules became labeled in vitro. Phosphorylation of p36 was examined fraction to reaction mixtures containing 320 FM [y“P]ATP (sp act 50 Ci/mmol). At intervals aliquots as described for pp60”‘“. Incorporation of were withdrawn, and the incorporation of 32P into 32P was linear for about 4 min, after pp60” and p36 (dashed lines) was determined by which it slowed and reached a plateau immunoprecipitation as described under Materials (Fig. 1). When reaction mixtures were and Methods. 0, pp60” phosphorylation; +, phosquenched after 4 min of incubation by the phorylation of ~36. The hydrolysis of ATP (solid addition of unlabeled ATP to 15 mM final line) was assayed in separate experiments. concentration and then incubated for up to 16 min, the amount of 32P incorporated tration of [y-“‘P]ATP (at a sp act of 50 into p36 remained constant in both Ci/mmol) was used for all further experquenched and control phosphorylation iments unless otherwise indicated. mixtures. Thus, the observed plateau in Incorporation of 32P into pp60”” was p36 labeling appears to result from a quantitated by terminating the reaction decline in the rate of phosphorylation as described under Materials and Methods rather than to turnover of the phosphate and immunoprecipitating the detergentafter incorporation. Inhibition of ~~60”“’ solubilized membranes with TBR serum. proteolysis by leupeptin did not extend Under the reaction conditions described the period of phosphorylation, indicating above, pp60”” phosphorylation was linear that the reaction ceases for reasons other for 1 min, but after 2 min, the amount of than proteolysis of ~~60”“’ (see below and 32P in pp60”” fell rapidly (Fig. 1). This Fig. 6). decline was due at least in part to proteThe IgG of TBR serum has been used olysis, which was observed when pp60”‘” extensively as a substrate to quantitate was immunoprecipitated from the PlOO immune complex kinase activity of deterfraction of [35S]methionine-labeled SR- gent-solubilized pp60”“. To determine if RSV-A-transformed cells incubated under this activity could be assayed when the the same reaction conditions. Some loss membrane association of pp60”” is preof radiolabel also may have been due to served, a PlOO preparation was incubated the action of membrane-associated phosfirst with TBR serum and then with [yphoprotein phosphatases (Foulkes, 1983), 32P]ATP. Phosphorylation of IgG heavy since after 4 min of incubation 70% of chain, like that of pp60”“, was linear for [35S]methionine-labeled molecules were 1 min, peaked, and then declined (not intact, whereas by this time point, the shown). The decrease in the level of phosamount of 32P incorporated into pp60”” phorylation was in this case probably due had decreased by about 50% (Fig. 1). While to phosphatase activity, in conjunction these studies were in progress, Wells and with the decline in ~~60”“‘ activity; it did Collett (1983) reported that leupeptin re- not appear to be due to proteolysis, since

412

DEHAZYA

stained bands corresponding to IgG heavy chain did not show obvious changes in staining intensity or the appearance of peptide fragments during incubation. As described under Materials and Methods, approximately 15% of the pp60” in the PNS was associated with the SlOO fraction. In vitro labeling of ~~60”” in this fraction was usually not detected (Fig. 2). Instead, several lower molecular weight polypeptides became phosphorylated. One of these, a polypeptide migrating slightly more rapidly than IgG heavy chain on SDS-polyacrylamide gels, appears to be the 52K proteolytic breakdown product of pp60”” described by Levinson et al. (1980). Incubation of [?S]methionine-labeled SlOO fractions under kinase reaction conditions for 1 min resulted in the proteolysis of about 50% of the pp60”‘” molecules present in this fraction. Thus, even in the presence of aprotinin and soybean trypsin inhibitor, nonsedimentable pp60”” seems to be more vulnerable to proteolytic breakdown than the membrane-associated form. This may account for the lack of detectable phosphorylation of intact ~~60”” in the SlOO fraction.

PP 60

FIG. 2. Phosphorylation of ~~60”’ in SlOO and PlOO fractions. Reaction mixtures contained [y=P]ATP plus 100 pg of protein from the SlOO fraction (lane 1) or the PlOO fraction (lane 2). Reactions were carried out for 1 min, and pp60”” was then immunoprecipitated with TBR serum. Arrowhead indicates the position of ~~60~‘.

AND

MARTIN

Sites of Phosphorylation of pp6F and ~36 in Vitro The sites of in vitro phosphorylation of ~~60”” were examined by partial proteolysis and phosphoamino acid analysis. Partial V8 protease cleavage of SR-RSVA pp60”‘” produces two major peptides, a 34K N-terminal fragment and a 26K Cterminal fragment (Collett et al., 1979). The 34K fragment of metabolically labeled pp60”” contains the majority of the phosphoserine (Collett et al., 1979); phosphotyrosine is recovered both from the 26K fragment (Collett et ah, 19’79), and, if phosphoprotein phosphatase activity is inhibited, from the 34K fragment (Collett et al, 1984). Both fragments were phosphorylated in vitro, but the majority of the label was recovered in the 34K Nterminal fragment (Fig. 3). Further digestion of the 34K phosphopeptide by increasing amounts of V8 protease resulted in the appearance of lower molecular weight fragments, which were also apparent in metabolically labeled ~~60”‘” (Fig. 3). Phosphoamino acid analysis of ~~60”” labeled in vitro (Fig. 4) revealed the presence of phosphotyrosine (ea. 85%), phosphoserine (ea. 10%) and traces of phosphothreonine (ca. 5%). Phosphoamino acid analysis of the V8 peptides revealed that the 34K fragment contained phosphotyrosine and phosphoserine plus traces of phosphothreonine, while the 26K fragment contained only phosphotyosine (data not shown). These results indicate that tyrosine is the major phosphoacceptor residue in this system, and suggest that the sites of phosphorylation of pp60”‘” in I&U) are similar to those observed in vivo; however, further analysis would be required to determine if the N-terminal tyrosine phosphorylation site detected here is identical to that described by Collett et al. (1983, 1984). To examine the specificity of p36 phosphorylation, tryptic digests were prepared from p36 from SR-RSV-A-transformed cells, labeled either by metabolic labeling with [32P]orthophosphate in vivo or by phosphorylation in vitro in membrane preparations as described above. The di-

pp60”” 1

2

KINASE 3

ACTIVITY

4

34 >

26 >

FIG. 3. Partial cleavage with S. ~UWUS protease V8 of pp60”” phosphorylated in vitro and in vim SR-RSV-A pp60”” labeled in viva (lanes 1, 2) or in vitro (lanes 3, 4 was cleaved with 5 ng (lanes 1, 3) or 50 ng (lanes 2, 4) of V8 protease and the cleavage products were separated by electrophoresis on a 15% SDS-polyacrylamide gel. Arrowheads (labeled with molecular weights in kilodaltons) indicate the positions of uncleaved pp60”” and the 34K and 26K cleavage products. Several smaller fragments derived from more complete cleavage of the 34K peptides are also evident.

gests were analyzed by electrophoresis and chromatography on cellulose thinlayer plates. Both digests were found to contain a single major phosphorylated peptide; the major phosphopeptides in the two digests comigrated when the digests were mixed and analyzed under these conditions (Fig. 5). However, as noted previously (Erikson and Erikson, 1980), digests of metabolically labeled p36 frequently contained a minor phosphopeptide, with a lower Rf value after chromatography in the second dimension (Fig. 5C), which was not observed in the protein labeled in vitro. Phosphoamino acid analysis of p36 phosphorylated in vitro in membranes from SR-RSV-A-transformed cells revealed the presence of phosphotyrosine (at least 80% from inspection of

413

IN MEMBRANES

autoradiograms) and phosphoserine only (Fig. 4). In membranes from uninfected cells, p36 became phosphorylated at low levels (Fig. 6A, lane 1) at serine (data not shown). These findings agree with previously published phosphoamino acid and phosphopeptide analyses of metabolically phosphorylated p36 (Radke et al., 1980; Erikson and Erikson, 1980) and suggest that the kinase(s) which phosphorylates p36 in vitro is the same as that which phosphorylates it in vivo. Inhibition of p36 Phosphorylation by Antibodies to ~~60’” The phosphorylation of p36 at tyrosine could be due either to direct phosphorylation by ~~60”” or to the activation of cellular, tyrosine-specific protein kinases. To distinguish between these possibilities, PlOO fractions from SR-RSV-A-transformed cells were preincubated with (Fig. 6A, lane 2) or without (Fig. 6A, lane 3) an excess of TBR serum prior to the addition of [y-32P]ATP. For comparison, phosphorylation of p36 in uninfected cell membranes is shown in Fig. 6A, lane 1. p36 was immunoprecipitated and the incorporation of 32P quantitated. TBR serum inhibited phosphorylation by 91% as com-

PP60SrC

36K’

FIG. 4. Phosphoamino acid analysis of ~~60”’ and p36 phosphorylated in vitro. Phosphorylated proteins eluted from SDS-polyacrylamide gels were subjected to partial acid hydrolysis and analyzed by twodimensional electrophoresis on cellulose thin-layer plates. First dimension, left to right, electrophoresis at pH 1.9; second dimension, bottom to top, electrophoresis at pH 3.5. S, phosphoserine; T, phosphothreonine; Y, phosphotyrosine. The two hydrolysates were run on the same plate and the phosphoamino acids derived from the hydrolysate of p36 are indicated by asterisks

414

DEHAZYA

r.

AND

MARTIN

_

FIG. 5. Tryptic phosphopeptides of ~36 phosphorylated in viva or irk vitro. p36 was radiolabeled in viva by metabolic labeling with [a’P]orthophosphate or in vitro by phosphorylation in PlOO fractions. The immunoprecipitated ~36 was eluted from SDS-polyacrylamide gels and digested with L-1-ptosylamino-Z-phenyl ethylchloromethyl ketone trypsin. Phosphopeptides were separated by electrophoresis at pH 8.9 (right to left) followed by chromatography (bottom to top) as described by Radke et ccl. (1980). (A) p36 phosphorylated in vitro; (B) mixture of metabolically labeled and in vitro labeled p36 with one major phosphopeptide; (C) metabolically labeled ~36, showing second more slowly migrating phosphopeptide evident in some preparations.

pared to the level observed in the absence of antibody; no inhibition was observed with nonimmune serum (not shown). The A

1

2

3

'123

36Kw

FIG. 6. Inhibition of phosphorylation of ~36 by TBR serum. (A) The PlOO fractions of uninfected (lane 1) or SR-RSV-A-transformed cells (lanes 2, 3) were preineubated for 30 min at 0” with an excess of (lane 2) or without (lanes 1, 3) TBR serum. After reaction for 4 min with 320 wcM [-)J-~‘P]ATP, ~36 was immunoprecipitated and analyzed by SDS-polyacrylamide gel electrophoresis. (B) Autoradiogram of the same gel as in A after alkali digestion. Arrowheads indicate the positions of ~36 and of pp608”, which coprecipitates with ~36 following the kinase reaction.

inhibition of phosphorylation was also apparent when the same gel was treated with alkali to reduce the background due to incorporation at residues other than tyrosine (Fig. 6B). These data suggest that the presence of phosphotyrosine in p36 is due to direct phosphorylation by pp60” rather than the activation of cellular tyrosine kinases. We also noted that a 60K phosphoprotein was coprecipitated with p36 immunoprecipitated from kinase reaction mixtures (Fig. 6, arrowheads). This coprecipitation was more readily observed in PlOO fractions from cells grown at 36” than in membrane fractions from cells grown at 41.5”. The 60K protein had the same phosphoamino acid content and V8 protease digestion profile as pp60’” phosphorylated in vitro and immunoprecipitated with TBR serum (not shown). The coprecipitation of pp60”‘” by anti-p36 antibody suggests that pp60”” and p36 can become associated under these reaction conditions. Effects of Growth Temperature and Mutations in src on the in Vitro Phosphorylation of ~36, ppW”, and TBR I!JG To examine the effects of mutations within src on the rates of protein phos-

pp60”

KINASE

ACTIVITY

415

IN MEMBRANES

performed on the same infected cell population, but varied from embryo preparation to embryo preparation. So that the results of different infections can be compared, the data in Table 1 are presented as ratios of counts incorporated in PlOO fractions from cells grown at 41.5” to counts incorporated in PlOO fractions from the same cells grown at 36” (“relative incorporation” values). It should be noted, however, that even in cells grown at 36” src-dependent phosphorylations were generally lower in membrane fractions from tsNY68-infected cells than in membrane fractions from SR-RSV-A-infected cells. The rate of phosphorylation of pp60”“, ~36, and TBR IgG was lower in membranes from SR-RSV-A-infected cells grown at 41.5” than in membranes from the same cells grown at 36”. Thus, the phosphoacceptor activity of SR-RSV-A pp60”” was about 2.8-fold lower in membranes from cells grown at 41.5” (relative incorporation, 0.36); phosphorylation of

phorylation in vitro, cells were infected with SR-RSV-A, tsNY68, or a temperature-resistant partial revertant derived from tsNY68, tsNY68-R3 (R3). The infected cells were grown at 36 or 41.5” and PlOO fractions prepared. Phosphorylation reactions were carried out using the PlOO fractions or PlOO fractions preincubated with TBR serum, and the incorporation of 32P into pp60”“, ~36, or TBR IgG was quantitated. The amount of pp60”‘” in transformed cells is known to be variable (Sefton et ab, 1982), and the fraction of pp60”‘” which is membrane associated is affected by mutations within src (Courtneidge and Bishop, 1982; Garber et al., 1983). For these reasons, ~~60”” was immunoprecipitated both in antibody excess (total phosphoacceptor activity) and in antigen excess (specific phosphoacceptor activity); this latter measurement allowed the phosphorylation of equivalent amounts of pp60”” to be assayed. The absolute levels of 32P incorporation were very reproducible when separate assays were

TABLE

1

EFFECTS OFGROWTHTEMPERATURE ON~~~O~~~-DEPENDENTPHOSPHORYLATION INMEMBRANES OF WILD-TYPEANDMUTANT-INFECTED CELLS' Relative

incorporation

(41.5/36”)b

pp60”

Virus

Specific activity”

SR-RSV-A tsNY 68 tsNY68-R3

0.36 (1.0) 0.13 (0.36) 0.31 (0.86)

Total activityd 0.36 (1.0) 0.10 (0.27) 0.33 (0.91)

~36”

TBR IgGf

0.71 (1.0) 0.22 (0.31) 0.38 (0.54)

0.77 (1.0) 0.31 (0.40) 0.45 (0.58)

a Phosphorylation reactions were carried out under conditions of linear incorporation and the radioactivity in immunoprecipitated proteins was determined as described under Materials and Methods. ‘Ratio of incorporation in PlOO fractions from cells grown at 41.5 to incorporation in PI00 fractions from cells grown at 36. Figures in parentheses represent the adjusted relative incorporation values; that is, the relative incorporation ratio for each virus divided by the corresponding ratio for wild-type SR-RSV-A. ‘Specific ~~60”” phosphoacceptor activity was obtained by immunoprecipitation of ~~60”” in antigen excess, and represents the phosphorylation of the same amount of pp60” protein in each membrane preparation. d Total pp60” phosphoacceptor activity was obtained by immunoprecipitation in antibody excess. ’ p36 was quantitatively immunoprecipitated in antibody excess. fThe amount of TBR serum used in IgG kinase assays was sufficient to quantitatively immunoprecipitate ~~60”;” in the reaction mixture.

416

DEHAZYA

p36 and IgG in membranes from SR-RSVA-infected cells also showed some growth temperature dependence (relative incorporation, 0.70.8), although the effect was not as large. The basis of the growth temperature dependence observed with wild-type SR-RSV-A is at present unclear. In PlOO fractions from tsNY68-infected cells pp60”” phosphoacceptor activity and the phosphorylation of p36 and TBR IgG were markedly inhibited when the infected cells were grown at 41.5” (Table 1): the relative incorporation value for pp60”” phosphorylation was approximately 0.1, and for p36 and TBR IgG phosphorylation 0.2-0.3. Since pp60”“-dependent phosphorylations were to some extent growth temperature dependent even in membranes from wild-type RSV-infected cells (see above), we also present in Table 1 the relative incorporation values for the mutant virus divided by the values obtained for the wild-type virus. The adjusted relative incorporation values for phosphorylation of pp60”“, ~36, and TBR IgG were all approximately 0.3-0.4 (Table 1). These data confirm that the phosphorylation of p36 and TBR IgG is arc dependent and indicate that the mutant protein is partially but not completely inactivated at the nonpermissive temperature. It should be noted, however, that we have not distinguished here between phosphorylation at tyrosine and phosphorylation at serine; if phosphorylation at serine is not src dependent then temperature dependence of tyrosine phosphorylation would be slightly greater than indicated by the relative incorporation values shown in Table 1. Our findings are consistent with previous data on the temperature-dependent phosphorylation and immune-complex kinase activity of tsNY68 ~~60”” (Collett et ab, 1979; Sefton et oh, 1980). The in vitro phosphoacceptor and kinase activities of the ~~60”” encoded by the revertant tsNY68-R3 were also examined (Table 1). The phosphoacceptor activity was restored to levels close to those of wild-type virus, with an (adjusted) relative incorporation of approximately 0.9. In contrast, p36 and TBR IgG phosphorylation was only partially restored, with (ad-

AND

MARTIN

justed) relative incorporation values of 0.5-0.6. Thus although R3 was isolated by selection for transformation at 41.5”, the kinase activity of R3 pp60”” remained to some extent temperature sensitive. Temperature-shift experiments were also performed to examine the kinetics of reactivation of tsNY68 pp60”‘“. tsNY68infected cells were cultured at 41.5”, then shifted to 36” in the presence of cycloheximide. After 60 and 120 min at 36”, PlOO fractions were prepared, and the phosphorylation of pp60”” and p36 was examined. The phosphorylation of these proteins in PlOO fractions from cells maintained at either 36 or 41.5” was also assayed. As shown in Fig. 7, by 60 min after the shift to 36” the in vitro phosphorylation of p36 had returned to the level seen in membranes from cells maintained continuously at 36”; phosphorylation of p36 in vivo is also restored by this time (Radke and Martin, 1979). In conA

B

C

0

36K t

FIG. ‘7. Renaturation of pp60”” phosphoacceptor and p36-directed kinase activities in tsNY68-infected cells. tsNY68-infected cells grown at 41.5” were shifted to 36” for 60 min (lane B) or 120 min (lane C) in the presence of cycloheximide. As controls, cells infected with the same virus were maintained at 41.5” (lane A) or 36” (lane D). PlOO fractions were prepared and phosphorylation reactions carried out for 1 min at 30” in duplicate aliquots. ~~60”” was immunoprecipitated in antigen excess to quantitate specific phosphoacceptor activity; p36 was quantitatively immunoprecipitated in antibody excess. Arrowheads indicate the positions of the two proteins.

pp60”

KINASE

ACTIVITY

trast, although an increase in pp60”” phosphorylation was apparent 60 min after the shift to 36”, the phosphoacceptor activity reached the control level only 120 min after the shift. Thus the phosphoacceptor activity of pp60” was apparently restored more slowly than its p36-directed kinase activity. This may reflect the growth temperature dependence of phosphoacceptor activity which was observed even with wild-type RSV-infected cells. Activation of ~36 Phosphorylation Concentrations of A TP

at High

Purchio et al. (1983) have reported that preincubation of purified pp60”‘” with 100 ph4 ATP increased the kinase activity of the enzyme significantly. To determine if the kinase activity of membrane-assoin a ciated pp60”” could be stimulated similar manner, p36 was phosphorylated for 16 min at 0.32 or 3.2 mM ATP, using the same specific activity of [y-32P]ATP at both concentrations. The phosphorylation mixture also contained 200 pg/ml leupeptin, which has been shown to retard proteolysis of pp60”‘” in cell homogenates (Wells and Collett, 1983). The results are shown in Fig. 8: the presence of 3.2 m&f

2

4

8 Ttme-min.

16

FIG. 8. Stimulation of p36 phosphorylation at high concentrations of ATP. PlOO fractions from SRRSV-A-transformed cells were incubated with 0.32 mM (0) or 3.2 mM (m) [y-32P]ATP at 30” in reaction mixtures containing leupeptin (200 pg/ml). The kinetics of incorporation of “P into p36 was quantitated by immunoprecipitation as described under Materials and Methods.

IN MEMBRANES

417

ATP stimulated the phosphorylation of p36 about eightfold over the level observed at 0.32 mM ATP. Although the initial rate and extent of the reaction are increased, incorporation ceased after approximately 8-16 min under both conditions. DISCUSSION

The results reported here indicate that in membranes from RSV-CEF, p36 becomes phosphorylated at the same site as that phosphorylated in vivo, and suggest that it is directly phosphorylated by pp60”“. Moreover, measurements of the kinase activity of ~~60”‘” encoded by tsNY68 and the revertant tsNY68-R3 indicate that transformation can result from small changes in total pp60”” kinase activity. Finally, the effects of growth temperature and mutations in src on ~~60”” phosphorylation and on its kinase activity indicate that different factors affect phosphoacceptor capacity and kinase activity. These and other conclusions are discussed in more detail below. Our initial objective in these studies was to establish conditions under which arc-dependent phosphorylations in membrane preparations could be quantitated, so that the effects of anti-pp60”” antibodies and mutations within src could be examined. By using high concentrations of ATP we were able to observe linear incorporation of phosphate into protein for at least short periods. Thus although these membrane preparations contain high concentrations of ATPases, proteases, and phosphoprotein phosphatases, it is possible to quantitate pp60”” phosphorylation and kinase activity. Our results are in general agreement with previous studies of pp60”‘” and p36 phosphorylation in membranes (Garber et al., 1983; Amini and Kaji, 1983), in which concentrations of ATP much lower than the K,, for pp60”” were used. However, it should be noted that with the cell fractionation technique used here about 50% of tsNY68 pp60”” is retained in the membrane fraction at the nonpermissive temperature, whereas others (Courtneidge and Bishop,

418

DEHAZYA

1982; Garber et ab, 1982) have reported that most of pp60”‘” is soluble under these conditions; the retention of tspp60”” in membranes may be due to the low ionic strength of the homogenization buffer used in these experiments. We did not detect any phosphorylation of ~~60”‘” in the soluble (SlOO) fraction, as also noted previously by Garber et al. (1983). Soluble pp60”‘” is known to be largely complexed to two cellular polypeptides, and this may in part account for its low phosphoacceptor activity (Brugge et al., 1983); in addition, as described here, soluble pp60”‘” appears more susceptible to proteolysis than the membrane-associated form. The major tryptic phosphopeptide of p36 phosphorylated in PlOO fractions from transformed cells was identical to the major phosphopeptide of p36 phosphorylated in vivo. This peptide is also phosphorylated in vitro by purified pp60”” (Erikson and Erikson, 1980). p36 phosphorylation occurred predominantly at tyrosine in membranes from transformed cells and was growth temperature dependent in membranes from tsNY68-infected cells. Furthermore, p36 phosphorylation was inhibited by precincubation with TBR serum containing antibodies against pp60”“; a similar observation was made by Kobayashi et al. (1981) for p36 phosphorylation in cell homogenates. These observations suggest that p36 is phosphorylated directly by pp60”“, rather than by a cellular kinase activated in transformed cells by ~~60”“. If such a cascade mechanism did exist, one would have to postulate that the continued activity of the cellular kinase in PlOO fractions required the continuous function of pp60”‘” in vitro, since preincubation with anti-pp60”” antibody must inactivate the hypothetical cellular kinase; this postulate seems unlikely but cannot be rigorously excluded. Recently Clinton and Roskowski (1984), using angiotensin as a substrate, have reported that antisrc serum reduces the total tyrosyl kinase activity in extracts of RSV-transformed BHK cells to the levels of control extracts and have, similarly, concluded that cellular tyrosyl kinases are not activated by

AND

MARTIN

RSV transformation. In addition, the coprecipitation of pp60”‘” with p36 immunoprecipitated from membrane reaction mixtures (Fig. 6) suggests that the two polypeptides become associated under these in vitro reaction conditions. This complex between pp60”” and p36 might represent a stable association of enzyme with substrate. Indeed, such stable associations of protein kinases and their substrates have been observed in a number of other systems, for example, between cyclic-AMP-dependent protein kinase and glycogen phosphorylase kinase (Walsh et al., 1968), and with polyoma middle T antigen and pp60”-“” (Courtneidge and Smith, 1983). However, it is possible that the formation of a p~6O”~“-p36 complex is artifactual and does not occur in vivo. Sefton et al. (1980) reported that in cells infected by tsNY68 at the nonpermissive temperature, pp60”‘” retained 30-40% of the immune-complex kinase activity present in wild-type virus-transformed cells, provided that ionic detergents were avoided during the immunoprecipitation procedure. They suggested, as one possible interpretation of these findings, that the transformed state of the cell might be determined by small changes in pp60”” kinase activity above or below some critical threshold. The findings reported here are consistent with this proposal. Thus in membranes from tsNY68-infected CEF grown at 41.5”, the nonpermissive temperature, the rate of phosphorylation of p36 and of TBR IgG was 20-40% of that observed in membranes from the same cells grown at the permissive temperature. Furthermore in membranes from cells transformed at 41.5” by the revertant tsNY68-R3 the rate of phosphorylation of p36 and TBR IgG was only partially restored to the wild-type level. The same conclusion, namely, that transformation requires expression of pp60”” above a critical threshold level, has also been reached on the basis of studies on the expression of a recombinant src gene transcriptionally linked to an inducible promoter (Jakobovits et al, 1984). It is not clear at present whether the kinase activity of

~~60”’

KINASE

ACTIVITY

pp60”‘” is regulated by phosphorylation. Tyrosine-416 of WC can be substituted without substantially altering kinase activity (Cross and Hanafusa, 1983; Snyder et al., 1983). However, it has been reported that phosphorylation of a tyrosine residue at the N-terminus of pp60”‘” occurs in vitro when purified pp60”” is incubated with high concentrations of ATP. This Nterminal tyrosine phosphorylation, which can also be detected under certain conditions in metabolically labeled pp60”“, appears to be associated with a significant increase in the activity of the enzyme (Purchio et al., 1983; Collett et al., 1983, 1984). In the studies reported here, the phosphorylation of pp60”” and its kinase activity were differentially affected by growth temperature and by mutations within src. Thus, even with wild-type RSV, the phosphoacceptor activity of ~~60”‘” was strongly inhibited by growth at 41.5”, whereas its kinase activity with p36 or IgG as substrate was only slightly inhibited. In temperature-shift experiments with tsNY68-infected cells, ~~60”‘” phosphorylation was restored more slowly than p36 kinase activity. Furthermore, in the revertant R3, phosphorylation of ~~60”” showed a growth temperature dependence similar to that of wild-type RSV, while its kinase activity showed a temperature sensitivity intermediate between that of the wild type and that of the ts parent: tsNY68 is known to be multiply mutant (Kawai et ah, 197’7) and it is possible that in R3 reversion has occurred at only one of the mutant sites. These observations indicate that the rate of phosphorylation of pp60’” can be affected by factors which do not affect its kinase activity, but do not directly address the issue of whether the level of phosphorylation can regulate activity. However, we have also observed that phosphotyrosine is recovered in the N-terminal V8 protease fragment of pp60”” phosphorylated in vitro in PlOO fractions, and that p36 phosphorylation is stimulated at high (mM) ATP concentrations; these observations are consistent with the proposal (Collett et al., 1984) that N-terminal phosphorylation at tyro-

IN MEMBRANES

419

sine can regulate the kinase activity of pp60”“. The membrane phosphorylation system described here should prove useful in analyzing the kinase activity of other members of the WC family. For example, although the avian erythroblastosis virus e&B gene product has been reported to lack kinase activity in immunoprecipitates, we have recently observed that both the erbB gene product and the cellular substrate p36 can be phosphorylated at tyrosine in PlOO fractions from cells transformed by avian erythroblastosis virus (Gilmore et al, 1985). This membrane phosphorylation system may also prove useful in characterizing the interaction of pp60”‘” with other protein substrates or with nonprotein substrates such as phospholipids (Macara et al, 1984; Sugimoto et ah, 1984). ACKOWLEDGMENTS We thank Masae Namba for excellent technical assistance,and Kathryn Radke, Tom Gilmore, Judy Young, and Jeff Prior for helpful suggestions and critical comments on the manuscript. This work was supported by NIH Grant CA17542. P.D. was supported by NIH Training Grants CA09141 and CA07170. REFERENCES AMINI, S., and KAJI, A. (1983). Association of pp36, a phosphorylated form of the presumed target protein for the src protein of Rous sarcoma virus, with the membrane of chicken cells transformed by Rous sarcoma virus. Proc. Natl. Acad. Sci. USA 80, 960-964. BADER, J. P., and BROWN, N. R. (1971). Induction of mutations in an RNA tumour virus by an analogue of a DNA precursor. Nature New Bid. 234, 11-12. BEEMON, K., and HUNTER, T. (1978). Characterization of Rous sarcoma virus STCgene products synthesized in vitro. J. Vid. 28, 551-566. BISHOP, J. M., and VARMUS, H. E. (1982). Functions and origins of retroviral transforming genes. In “RNA Tumor Viruses” (R. Weiss, N. Teich, H. E. Varmus, and J. Coffin, eds.), pp. 999-1108. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. BRADFORD, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal B&hem. 72, 248-254.

420

DEHAZYA

BRLJGGE, J. S., YONEMOTO, W., and DARROW, D. (1983). Interaction between the Rous sarcoma virus transforming protein and two cellular phosphoproteins: Analysis of the turnover and distribution of this complex. Mol. Cell. Biol. 3, 9-19. CLEVELAND, D. W., FISCHER, S. G., KIRSCHNER, M. W., and LAEMMLI, U. K. (1977). Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Ch,em. 252, 1102-1106. CLINTON, G., and ROSKOWSKI, R. (1984). Tyrosyl and cyclic AMP-dependent protein kinase activities in BHK cells that express viral pp60m”. Mol. Cell. Biol. 4, 9’73-977. COLLETT, M. S., BELZER, S. K., and PURCHIO, M. F. (1984). Structurally and functionally modified forms of pp60’-s” in Rous sarcoma virus-transformed cell lysates. Mol. Cell. Biol. 4, 1213-1220. COLLETT, M. S., ERIKSON, E., and ERIKSON, R. L. (1979). Structural analysis of the avian sarcoma virus transforming protein: Sites of phosphorylation. J. Viral. 29, 770-781. COLLETT, M. S., PURCHIO, A. F., and ERIKSON, R. L. (1980). Avian sarcoma virus transforming protein, pp60”” shows protein kinase activity specific for tyrosine. Nature (London) 285, 167-169. COLLETT, M. S., WELLS, S. K., and PURCIIIO, A. F. (1983). Physical modification of purified Rous sarcoma virus pp60’-” protein after incubation with ATP/Mg=. Virology 128, 285-297. COOPER, J. A., and HUNTER, T. (1981). Changes in protein phosphorylation in Rous sarcoma virus transformed chicken embryo cells. Mol. Cell. Biol. 1, 165-178. COOPER, J. A., and HUNTER, T. (1983). Regulation of cell growth and transformation by tyrosine-specific protein kinases: The search for important cellular substrates. Curr. Top. Microbial. Immunol. 107, 125-161. COURTNEIDGE, S. A., and BISHOP, J. M. (1982). The transit of ~~60’.” to the plasma membrane. Proc. Natl. Acad. Sci. USA 79, 7117-7121. COURTNEIDGE, S. A., RALSTON, R. K., ALITALO, K., and BISHOP, J. M. (1983). Subcellular location of an abundant substrate (~36) for tyrosine specific protein kinases. Mol. Cell. Biol. 3, 340-350. COURTNEIDGE, S. A., and SMITH, A. (1983). Polyoma virus transforming protein associates with the product of the c-src cellular gene. Nature (London) 303, 435-439. CROSS, F. R., GARBER, E. A., PELLMAN, D., and HANAFUSA, H. (1984). A short sequence in the ~~60’” N terminus is required for pp60”” myristylation and membrane association and for cell transformation. Mol. Cell. Biol. 4, 1834-1842. CROSS, F. R., and HANAFUSA, H. (1983). Local mutagenesis of Rous sarcoma virus: the major sites

AND

MARTIN

of tyrosine and serine phosphorylation of ~~60”” are dispensible for transformation. Cell 34, 597607. ERIKSON, E., and ERIKSON, R. L. (1980). Identification of a cellular protein substrate phosphorylated by the avian sarcoma virus transforming gene product. Cell 21, 829-836. FOUI,KES, J. G. (1983). Phosphotyrosyl-protein phosphatases. Curr. Ton. Microbial. Immunol. 107, 163180. GARBER, E. A., KRUEGER, J. G., HANAFIJSA, H., and GOI,DBERG, A. R. (1983). Temperature-sensitive membrane association of ~~60~ in tsNY68-infected cells correlates with increased tyrosine phosphorylation of membrane associated proteins. Virology 126, 73-86. GILMORE, T., DECLUE, J. E., and MARTIN, G. S. (1985). Protein phosphorylation at tyrosine is induced by the v-erbB gene product in vivo and in vitro. CelZ, in press. GLYNN, I. M., and CHAPPELL, F. B. (1964). A simple method for the preparation of 3”P-labelled adenosine triphosphate of high specific activity. B&hem. J. 90, 147-149. HUNTER, T., and SEFTON, B. M. (1980). The transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc. Natl. Acad. Sci. USA 77, 1311-1315. JAKOBOVITS, E. B., MAJORS, J. E., and VARMUS, H. E. (1984). Hormonal regulation of the Rous sarcoma virus src gene via a heterologous promoter defines a threshold dose for cellular transformation. Cell 38, 757-765. KAWAI, S., DUESBERG, P. H., and HANAFUSA, H. (1977). Transformation-defective mutants of Rous sarcoma virus with STCgene deletions of varying length. J. Viral. 24, 910-914. KAWAI, S., and HANAFUSA, H. (1971). The effect of temperature on the transformed state of cells infected with a Rous sarcoma virus mutant, Virolog~ 46, 470-479. KOBAYASHI, N., TANAKA, A., and KAJI, A. (1981). In vitro phosphorylation of the 36K protein in extract from Rous sarcoma virus transformed chicken fibroblasts. J. Biol. Gem. 256, 3053-3058. KRUEGER, J. G., GARBER, E. A., and GOLDBERG, A. R. (1983). Subcellular localization of pp60” in RSV-transformed cells. Curr. Top, Microbial. Immunol. 107, 51-124. KRZYZEK, R. A., MITCHELL, R., LAIJ, A., and FARAS, A. (1980). Association of pp60”” and src protein kinase activity with the plasma membrane of nonpermissive and permissive avian sarcoma virus infected cells. J. Viral. 36, 805-815. LEVINSON, A. D., OPPERMANN, H., LEVINTOW, L., VARMUS, H. E., and BISHOP, J. M. (1980). The purified product of the transforming gene of avian

pp60””

KINASE

ACTIVITY

sarcoma virus phosphorylates tyrosine. J. BioL Chem. 255, 11973-11980. MACARA, I., MARINETTI, G., and BALDUZZI, P. (1984). Transforming protein of avian sarcoma virus UR2 is associated with phosphatidylinositol kinase activity: Possible role in tumorigenesis. Proc. Natl. Acad. Sci. USA 81, 2728-2732. PURCHIO, A. F., WELLS, S. K., and COLLETT, M. S. (1983). Increase in the phosphotransferase specific activity of purified Rous sarcoma virus pp60’~“‘” protein after incubation with ATP plus Me. Mol. Cell. BioL 3, 1589-159’7. RADKE, K., CARTER, V. C., Moss, P., DEHAZYA, P., SCHLIWA, M., and MARTIN, G. S. (1983). Membrane association of a 36,000-dalton substrate for tyrosine phosphorylation in chicken embryo fibroblasts transformed by avian sarcoma viruses. J. Cell BioL 97, 1601-1611. RADKE, K., GILMORE, T., and MARTIN, G. S. (1980). Transformation by Rous sarcoma virus: A cellular substrate for transformation-specific protein phosphorylation contains phosphotyrosine. Cell 21, 821-828. RADKE, K., and MARTIN, G. S. (1979). Transformation by Rous sarcoma virus: Effects of the STC gene product on the synthesis and phosphorylation of

IN MEMBRANES

421

cellular polypeptides. Proc. NatL Acad. Sci. USA 76, 5212-5216. SEFTON, B. M., HUNTER, T., and BEEMON, K. (1980). Temperature sensitive transformation by Rous sarcoma virus and temperature sensitive protein kinase activity. J. Viral. 33, 220-229. SEFTON, B. M., PATSCHINSKY, T., BERDOT, C., HUNTER, T., and ELLIOT, T. (1982). Phosphorylation and metabolism of the transforming protein of Rous sarcoma virus. J. ViroL 41, 813-820. SNYDER, M. A., BISHOP, J. M., COLBY, W. W., and LEVINSON, A. D. (1983). Phosphorylation of tyrosine-416 is not required for transforming property and kinase activity of ~~60’“. Cell 32, 891-909. SUGIMOTO, Y., WHITMAN, M., CANTLEY, L., and ERIKSON, R. (1984). Evidence that the Rous sarcoma virus transforming gene product phosphorylates phosphatidylinositol and diacylglycerol. Proc. NatL Acad. Sci. USA 81, 2117-2121. WALSH, D. A., PERKINS, J. P., and KREBS, E. G. (1968). An adenosine 3’5’-monophosphate dependent protein kinase from rabbit skeletal muscle. J. Biol. Chem. 243, 3763-3774. WELLS, S. K., and COLLETT, M. S. (1983). Specific proteolytic fragmentation of pp60’~s” in transformed cell lysates. J. Viral. 47, 253-258.