Similarities Between a Phosphoprotein (Pp60src

Home , Phosphoprotein

[CANCER RESEARCH 40, 1733-1741, May 1980] 0008-5472/80/0040-OOOOS02.00 Similarities between a Phosphoprotein (pp60src)-associated Protein Kinase of Rous Sarcoma Virus and a Cyclic Adenosine 3':5'-Monophosphate- independent Protein Kinase That Phosphorylates Pyruvate Kinase Type M21

Peter Presek, Hartmut Glossmann,2 Erich Eigenbrodt, Wilhelm Schoner, Helga RÃ¼bsamen,Robert R. Friis, and Heinz Bauer

Rudolf Buchheim-Institut fÃ¼rPharmakologie [P. P., H. G.] and Institut fÃ¼rVirologie [H. R., R. R. F., H. B.Â¡.Fachbereich Humanmedizin, and Institut fÃ¼rBiochemie und Endokrinologie, Fachbereich VeterinÃ¤rmedizin [E. E., W. S.], Justus Liebig-UniversitÃ¤t G/essen, Frankfurter Strasse, D-6300 Giessen, Federal Republic of Germany

ABSTRACT INTRODUCTION

Chicken embryo cells (CEC) contain the pyruvate kinase Transformation of CEC3 by the Rous sarcoma virus is de isoenzyme type M2 (M2-PK). This enzyme was partially purified pendent on the expression of the src gene (21 ). The src gene from these cells by affinity chromatography on Sepharose- product is a phosphoprotein, called pp60src, which has cAMP- coupled inhibitor protein (M.W. 33,500) of M2-PK. Character independent phosphotransferase activity in immune complexes istic changes in the kinetic properties of M2-PK were observed formed with antisera from Rous sarcoma virus tumor-bearing when CEC were transformed by the Rous sarcoma virus. Trans rabbits (9). pp60src is closely related to a normal cellular phos formation leads to a lower affinity for phosphoenolpyruvate, a phoprotein, termed pp60sarc, which also has protein kinase high stimulation by fructose 1,6-diphosphate, and a rapid in- activity (10). Although other enzymic functions for pp60src have activation by magnesium adenosine triphosphate which is abol not been ruled out, it is likely that cellular transformation by ished by fructose 1,6-diphosphate. Rous sarcoma virus can be attributed in part to about 100-fold These characteristic changes of M2-PK in Rous sarcoma higher levels of a cAMP-independent protein kinase. virus-transformed CEC could be mimicked in vitro by adding to One of the most consistent biochemical characteristics of homogeneous M2-PK from chicken liver a partially purified proliferating cells and of tumor cells, including Rous sarcoma cyclic adenosine 3':5'-monophosphate-independent protein virus-transformed cells, is an elevated rate of aerobic glycolysis kinase. This enzyme is known to phosphorylate and inactivate (8). Tumor cells contain M2-PK (11, 16, 26, 42, 44) which is M2-PK. A protein with a molecular weight of 64,000 can be also found in nondifferentiated embryonic cells (22, 24). M2- phosphorylated in immune complexes, formed with monospe- PK is phosphorylated by a cAMP-independent protein kinase cific M2-PK antisera and extracts from Rous sarcoma virus- (M2-PK kinase)4 (6, 12), and its kinetic properties are influ transformed CEC, but not from nontransformed CEC. This enced by regulatory proteins (15). Since pyruvate kinase is a phosphoprotein has the same electrophoretic mobility as does control point of glycolysis in tumor cells (18), it is possible that M2-PK. It is assumed that transformation of CEC by Rous the altered kinetic properties of M2-PK in tumor cells (40) sarcoma virus may affect the regulatory system of M2-PK which reside in alterations of the above-mentioned cAMP-independ- includes inhibitory proteins and a cyclic adenosine 3':5'-mono- ent protein kinase-regulatory protein system which controls phosphate-independent protein kinase. The transforming gene M2-PK and is, besides other changes, responsible for the high src of Rous sarcoma virus is coding for a phosphoprotein, aerobic glycolysis in tumor cells. We investigated therefore pp60src, which is associated with protein kinase activity. This whether M2-PK kinetics is changed after expression of the src pp60src kinase activity was copurifying on affinity columns with gene as in other tumor cells. We will report similarities between M2-PK, hexokinase, and 3-phosphoglycerate kinase. Evidence the cAMP-independent kinase which phosphorylates M?-PK is presented which suggests functional similarities between and the pp60src-associated protein kinase, and we provide pp60src kinase and the protein kinase which participates in the evidence suggesting that M2-PK may be among the targets of M2-PK regulation. Both protein kinases cannot be stimulated pp60sfc. by cyclic adenosine 3':5'-monophosphate; they use guanosine MATERIALS AND METHODS triphosphate and adenosine triphosphate as phosphate donors. Both are inhibited by P',P5-di(adenosine-5')pentaphosphate Biochemicals and chemicals of analytical grade were ob and M2-PK inhibitor protein. The inhibition by fructose 1,6- tained from Boehringer Mannheim GmbH, Mannheim, W. Ger- diphosphate of both protein kinases is noncompetitive with respect to adenosine triphosphate. 3 The abbreviations used are: CEC. chicken embryo cells; cAMP, cyclic adenosine 3':5'-monophosphate; SR-D. Schmidt-Ruppin strain of Rous sarcoma virus, subgroup D; M2-PK. pyruvate kinase isoenzyme type M2; M2-PK kinase. cAMP-independent protein kinase which phosphorylates M2-PK; RAV. Rous- associated virus (RAV 50); pp60"c kinase, protein kinase activity in immune complexes formed with tumor-bearing rabbit sera; FDP. fructose 1,6-diphos 1 Supported by a grant from Stiftung Volkswagenwerk (37/731), Deutsche phate; P-ribose-PP, 5-phospho-a-D-ribose 1-pyrophosphate; Ap5A, P'.P5- Forschungsgemeinschaft (Scho 139/13). and Deutsche Forschungsgemein di(adenosine-5')pentaphosphate. schaft SFB 47 (Virologie). " The term will not imply that M2-PK is the only substrate for this cAMP- 2 To whom requests for reprints should be addressed. independent protein kinase. At the highest known purification state, low-molec Received November 25. 1979; accepted January 29. 1980. ular-weight endogenous proteins. M2-PK and o-casein, are substrates (6. 12).

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1980 American Association for Cancer Research. P. Presek et al. many, E. Merck AG, Darmstadt, W. Germany, and Serva Fein- versible and that no associated phosphatase was removing the biochemica, Heidelberg, W. Germany. y-[32P]ATP and [35S]- label (tested for up to 20 min of an ATP chase). This phospho- methionine were obtained from Amersham Buchler, Braun transferase activity in the immunocomplex will be referred to schweig, W. Germany. as pp60src kinase activity. The activity of this kinase is given in Cells and Viruses. CEC were prepared from 11-day-incu fmol 32PÂ¡incorporated per mg of extract protein or per ml of bated White Leghorn specific-pathogen-free eggs generously column fraction. provided by E. Vielitz, Lohmann-Tierzucht GmbH (Cuxhaven, Preparation of Affinity Gel. The M.W. 33,500 protein inac W. Germany). Culture techniques and media have been de tivating M2-PK of chicken liver and the inhibitor fraction were scribed (1). The Rous sarcoma virus used in this study was prepared according to the method of Eigenbrodt and Schoner SR-D. The nontransforming avian retrovirus used was RAV-50, (15). Phosvitin was used as a spacer between Sepharose CL- subgroup D. 4B beads (Pharmacia) and the inhibitor proteins, because Radioactive Labeling of Cells. Nearly confluent cell cultures fixation of the inhibitor proteins directly to CNBr-activated were labeled for 2 hr with 0.2 to 0.4 mCi of [35S]methionine per Sepharose gave gels with low pyruvate kinase-binding capac ml (300 to 800 Ci/mmol). One Ci = 3.7 x 10'Â°becquerels). ity. The highest binding capacity as well as a purification factor Cell Lysis and Preparation of Extracts. Infected (RAV-50) of about 15-fold (from crude extracts) for M2-PK was found if or transformed (SR-D) CEC were washed twice with phosphate- the affinity gel was prepared as follows. Dephosphorylated buffered saline and extracted with ice-cold Buffer A [100 HIM phosvitin (200 mg) and 64 mg of inhibitor protein in 80 ml of 6 sodium phosphate buffer, pH 7.0-10 mM EDTA-40 mM NaF- M guanidine hydrochloride-1 M 2-mercaptoethanol were stirred trypsin-kallikrein inhibitor (Trasylol; Bayer AG; 0.4 mg/ml)- gently at room temperature for 12 hr. The proteins were de 0.05% (v/v) Triton X-100-10 mM 2-mercaptoethanol], 1 ml/ salted on a Sephadex G-25 column (Pharmacia), and 180 mg 106 cells by homogenization (Dounce homogenizer, 6 strokes). of the lyophilized protein were coupled to CNBr-activated Triton X-100 was included in the extraction medium to remove Sepharose CL-4B (37). The gel was extensively washed sub the enzymes from particulate fractions. The homogenate was sequently with 0.1 M KHCO3 and 0.5 N acetic acid until no centrifuged for 60 min at 50,000 x g. The supernatant was inhibitor protein was eluted. This procedure resulted in the kept at 4Â°and was used immediately after extraction, because fixation of 4 mg of the M.W. 33,500 inhibitor protein to 1 g of of the instability of pyruvate kinase and hexokinase. Sepharose CL-4B, which had a binding capacity of 120 units Antisera. Antisera against pp60src were obtained from rab of M2-PK. To evaluate the effect of phosvitin in the character bits by injecting purified SR-D into newborn animals and bleed istics of chromatography, 16 mg of dephosphorylated phosvitin ing the tumor-bearing animals starting at 6 weeks of age (36). were bound to Sepharose CL-4B under the same conditions. Antisera against M?-PK from chicken liver were obtained as One g of this (phosvitin-Sepharose) gel can bind 8 units M2- described (33). PK, and a purification factor of 3 was achieved. Phosphotransferase Activity in Immunoprecipitates. Rab Affinity Chromatography. Eight ml of the affinity gel were bit antiserum against pp60SICwas adsorbed for 2 hr at 0Â°onto packed in a 10- x 1-cm column and equilibrated with 50 ml 5 formaldehyde and heat-inactivated Staphylococcus aureus mM Tris-HCI, pH 7.0, before use. Extract (6 to 16 mg of protein) Cowan I (a gift from Dr. Schaeg, Institut fÃ¼rBakteriologie, in 2.5 to 9.0 ml of Buffer A was applied with a flow rate of 0.2 Giessen, W. Germany) or onto protein A-Sepharose (Deutsche ml/min. Elution was started with 10 ml 5 mM Tris-HCI, pH 7.0, Pharmacia, Freiburg, W. Germany). Five fi\ antiserum were followed by 30 ml 5 mw Tris-HCI, pH 7.0, containing 0.15 M used for 25 jul of a 10% bacteria suspension or for 4 mg (dry NaCI. A linear salt gradient from 0.15 to 1.0 M NaCI in 5 mM weight) of the substituted Sepharose. Protein A-IgG complexes Tris-HCI (pH 7.0) was applied thereafter. Fractions of 1.2 ml were washed 3 times with 1 ml ice-cold Buffer B (Buffer A were collected. The column was regenerated with 100 ml without 2-mercaptoethanol). Antigenic material was incubated unbuffered 2 M (NH4)2SO4, followed by 100 ml of 5 mM Tris- with the antiserum in solid phase for 1 hr at 0Â°.The immune HCI, pH 7.0, containing 0.1% NaN3. The entire procedure was complexes were extensively washed and blotted dry. The phos- carried out at 4Â°. photransferase reaction was started at 0Â°by adding 5 or 10 Preparation of M2-PK and M2-PK Kinase. M2-PK from fi\ of 100 mw Tris-HCI (pH 7.4)-100 mM MgCI2 buffer containing chicken liver was prepared as reported previously (13), but 0.1 about 1 to 0.5 ftCi y-pPJATP (2000 to 3000 Ci/mmol). When mM phenylmethyl-sulfonylfluoride and 2 mM e-aminocaproic additions were present, the reaction volume was kept at 30 p\. acid were included in all buffers. Under these conditions, The reaction was terminated after 3 min by addition of 30 jul proteolysis was minimized and the subunit molecular weight of sample buffer (10% SDS-1.4 M 2-mercaptoethanol-20% glyc- M2-PK was found to be about 10,000 higher than the M.W. erol-6 M urea-0.125 M Tris-HCI, pH 6.8) followed by heating at 51,000 reported before (6, 13). 96Â°for 5 min. The supernatant after centrifugation was applied The procedure for the isolation of M2-PK kinase has been to 11 % polyacrylamide slab gels, and 32Pradioactivity from the described (15). The partially purified preparations of this pro heavy-chain region of IgG was determined as described (36). tein kinase still contained the inhibitory proteins of M2-PK and Incorporation of 32Pi into the heavy-chain region under these when the influence of M2-PK kinase on phosphoenolpyruvate conditions was complete after 3 min and linear with respect to affinity of purified M2-PK from chicken liver was studied (Table a more than 20-fold range of different amounts of extract from 4), also some phosphoprotein phosphatase. SR-D-transformed CEC at a given amount of antiserum. The Enzyme Assays. Hexokinase (EC 2.7.1.1), phosphofructo- phosphorylated reaction product could be also released with kinase (EC 2.7.1.11), glucose-6-phosphate dehydrogenase acetic acid (1 M) and was identified after neutralization as IgG (EC 1.1.1.49), and lactate dehydrogenase (EC 1.1.27) were by double immunodiffusion with anti-rabbit IgG. ADP and ATP tested as described by Singh er al. (38), and 3-phosphoglyc- chase experiments revealed that the kinase reaction was re erate kinase (EC 2.7.2.3) was tested according to the method

1734 CANCER RESEARCH VOL. 40

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1980 American Association for Cancer Research. pp60src Kinase and a Kinase Phosphorylating M2-PK of BÃ¼cher(7). The activity of pyruvate kinase (EC 2.7.1.40) to the column, and only 5 to 20% eluted in the void volume. was determined as described previously (13). All enzymatic Very little activity appeared with the 0.15 M NaCI wash. pp60src activities and kinetic parameters were measured at 37Â°. One kinase could be eluted with a salt gradient and appeared in 3 unit of enzyme is defined as the amount of protein catalyzing peaks. Peaks I and II (0.49 M NaCI; 0.56 M NaCI) could not the consumption of 1 /Â¿molof substrate per min under the always be as sharply resolved as shown in the experiment in above-mentioned conditions. Chart 1. We have been unable to separate pyruvate kinase and Inactivation of M2-PK by M2-PK Kinase. The inactivation of the other glycolytic enzymes coeluting with it from the pp60src M2-PK by M2-PK kinase preparations was measured in a 2- kinase on the affinity column, e.g., by changing the salt gra step assay. In the first step, M2-PK, M2-PK kinase, and MgATP dient. The minor Peak III (0.75 M NaCI) of pp60src kinase activity were preincubated in 50 mw Tris-HCI (pH 7.4), 4 rtiM 1,2- to be seen in each experiment was free from pyruvate kinase diaminocyclohexatetraacetic acid, 10 mw 2-mercaptoethanol, activity. By immunoprecipitation, however, it was found that and bovine serum albumin (1 mg/ml) at 25Â°. M2-PK activity Peak III contained traces of enzymically inactive pyruvate ki was 5 units/ml. MgCI2 and ATP were added to start the nase (results not shown). inactivation. The final concentrations were 10 rriM MgCI2 and It was observed that the pooled peak fractions (I and II) from 0.2 rriM ATP. In a second step, 10-/Â¿laliquots of the preincu SR-D CEC precipitated spontaneously when kept at 4Â°for 3-5 bated mixture were transferred every second min into the hr. Protein kinase activities as well as endogenous substrates pyruvate kinase assay mixture. The preincubation time, at other than pp60src kinase were present in the peak fractions as which half-maximal inhibition of M2-PK was observed, was seen by endogenous phosphorylation and sodium dodecyl recorded. This value correlates under optimal conditions with sulfate-polyacrylamide electrophoresis (not shown). Protein ki the phosphorylating capacity of M2-PK kinase (12, 14). nase activity phosphorylating M2-PK from chicken liver was Protein Determination. Protein was measured by the method eluted in Peak III. of Lowry ef al. (29) after precipitation by 15% trichloroacetic Kinetic Properties of M2-PK in Uninfected CEC, CEC Trans acid or by precipitation of [35S]methionine-labeled extracts with formed with SR-D, or CEC Infected with RAV. In Tables 2 and trichloroacetic acid on filter discs. 3, the kinetic data for M2-PK in extracts from uninfected CEC, RAV-infected CEC, or SR-D-transformed CEC are compared. The mean specific activities of M2-PK were on the average RESULTS more than 3 times higher in extracts from SR-D-transformed Affinity Chromatography of Pyruvate Kinase from CEC. CEC than in uninfected CEC. Infection of CEC with RAV also CEC contain M2-PK. The isoenzyme has been determined by increased the specific activity of M2-PK but much less than double gel immunodiffusion with monospecific antibodies di seen with the transforming virus. Whereas the K05 of M2-PK rected against types ML L, and M2 from chicken muscle, lung, for phosphoenolpyruvate (with ADP as substrate) was 0.25 rriM and liver, respectively, as well as by starch gel electrophoresis for uninfected CEC, it was increased to 0.38 mw in RAV- (33, 41). It was desirable to purify M2-PK from CEC extracts infected cells and to 0.53 mw in SR-D-transformed CEC. since (see below) the kinetic properties of M2-PK might be An increase of the PEP affinity was observed by addition of influenced by metabolites as allosteric effectors in crude cell FDP. The increase was 4.4-fold in extracts from RAV-infected extracts. The purification method had to be rapid because M2- CEC and 13-fold in extracts from SR-D-transformed CEC. PK is unstable, especially in extracts from SR-D-transformed Serine, which is a known activator of tumor and placenta M2- CEC. For this purpose, we have prepared an affinity column PK (40), increased the phosphoenolpyruvate affinity of M2-PK which contained the inhibitor protein of pyruvate kinase. at low concentrations (50% activation around 50 Â¡Â¿M)inextracts When CEC extracts were chromatographed over the affinity from all 3 types of CEC. column, a highly reproducible elution pattern was found for Alanine and phenylalanine strongly inhibited M2-PK, the en protein, pyruvate kinase, other glycolytic enzymes, and pp60src zyme from CEC transformed by SR-D being most sensitive with kinase (Chart 1), and 85% of the applied protein was recovered respect to phenylalanine inhibition (Table 3). GTP had no effect in the void volume. The remaining adsorbed protein could be on M2-PK activity in extracts from uninfected CEC but partially eluted between 0.27 and 1.0 M NaCI; the main fraction ap inhibited M2-PK in extracts from CEC infected with RAV or peared between 0.35 and 0.4 M NaCI. Pyruvate kinase eluted transformed by SR-D. ATP partially inhibited M2-PK in all at 0.49 M NaCI together with the glycolytic enzymes hexoki- extracts. nase and 3-phosphoglycerate kinase. Identical activity elution Thus, it appears that infection by RAV caused certain patterns were found with extracts from RAV-infected or SR-D- changes in the properties of pyruvate kinase compared to transformed CEC. The purification factors in the peak fractions uninfected cells. Although the changes are interesting and (0.49 M NaCI) for pyruvate kinase from both extracts were unexplained, we have not further studied this phenomenon and similar (Table 1). focused instead on a comparison between M2-PK from CEC Copurification of pp60'rc with Pyruvate Kinase upon Affin infected by RAV and transformed by SR-D. ity Chromatography. The biological differences between RAV- It is known that FDP in micromolar concentrations increases infected and SR-D-transformed CEC are caused by the expres the phosphoenolpyruvate affinity of M2-PK (25). Since FDP sion of the src gene of the sarcoma virus. pp60src kinase, which levels in CEC transformed with RSV compared to nontrans- is coeluted with pyruvate kinase activity (Chart 1) when extracts formed cells are 10-fold higher (3), it was possible that M2-PK of SR-D-transformed CEC were run over the affinity column, in the cell extracts (especially from SR-D CEC) was prestimu- could not be measured in the extracts from CEC infected with lated by FDP. We therefore isolated M2-PK from extracts of RAV or in the column fractions from these extracts with our RAV-infected as well as from SR-D-transformed CEC with the tumor-bearing rabbit antisera. The major fraction was adsorbed affinity column procedure as outlined above, and we investi-

MAY 1980 1735

Chart 1. Affinity chromatography of pyruvate kinase. In A, 5.5 ml of SR-D CEC extract (1.57 mg of protein per ml) were loaded on the column and eluted as described (see "Materialsand Methods ), and the activity of pp60"c kinase was recorded from the indicated fractions using a 200-/J aliquot. Part of the pp60"' kinase activity appeared in the void volume of the column. Three peaks (I. II, III) of pp60s'c kinase activity were eluted with 0.49 M NaCI (I), 0.56 M NacÃ(II), and 0.75 M NaCI (III). Protein recovery was 107%; protein elution profile is shown, ÃŸ.elution pattern of phosphofructokinase (PFK), glucose-6-phosphate dehydrogenase (G/-6-PDH), 3-phosphoglycerate kinase (3-PGK). pyruvate ki nase (PK). and hexokinase (HK) from the experiment shown in A. 3-Phosphoglycerate kinase, pyruvate kinase, and hexokinase were eluted at 0.49 M NaCI; phosphofructokinase and glucose-6-phosphate de hydrogenase were eluted in the void volume to gether with about 3% of pyruvate kinase activity. In C, 7.0 ml of RAV CEC extract (1.13 mg of protein per ml) were loaded on the column and eluted the same as was the SR-D CEC extract. The protein profile was almost identical with that shown for the SR-D CEC extract in A and has been omitted for reasons of clarity only. Hexokinase, pyruvate ki nase, and 3-phosphoglycerate kinase eluted at 0.49 M NaCI. Protein recovery was 110%.

JO 20 30 40 50 60 TO

FRACTION NUMBER

Table 1 Purification of pyruvate kinase and pp60"c kinase from cellular extracts by affinity chromatography Specific activities and purification factors of the peak fractions from the Table 2 experiment shown in Chart 1. Recovery of pp60"c kinase was estimated to be Kinetic properties of M2-PK in extracts from CEC 121 % (S.D.) of the activity applied, and that of pyruvate kinase was 85% for both Ko s (mM) for phosphoenolpy- RAV and SR-D runs. ruvate in the presence of protein)CEC Activity (units/mg ac tivity (units/ mM mM infectedwithSR-DRAV-50Enzymepp60"c crudeextracts12.611.50.011.7InpeakfractionI. CECUninfectedInfected mg)3.551.515.722.5111.46.9None0.250.300.38NO0.56ND0.2FDPND6ND0.087ND0.038ND1L-serineNDND0.050ND0.038ND tionfactor111714.717.40.014.8 kinasePyruvate 136.0sII. 216.0III. 185.0I. withRAV-50Transformedwith kinasepp60"c 200.40.0163.0Purifica kinasePyruvate kinaseIn SR-DSubstrateADP"GDPADPGDPADPGDPapÃ©eme " The activity of pp60"c kinase is given in fmol 32Pincorporated into the heavy- Concentration of the nucleotide diphosphates was 0.75 mM. chain region of IgG. ' ND, not done.

1736 CANCER RESEARCH VOL. 40

Table 3 Effects of amino acids and nucleotides on M2-PK in extracts from CEC with ADP as substrate

byCECUninfected 50% inhibition (mM) by5of inhibition activa mM mM tion (mM) by alanineL-Alanine0.54 ATP44 GTP0202050% L-serine0.040.0270.052 1.00 Infected with 0.250.580.13 0.410.41GTP>200.790.39% 46465 RAV-50 Transformed 0.58ATP1.12 with SR-DL-Phenyl- gated the effects of FDP on the phosphoenolpyruvate affinity of the partially purified enzymes. The Michaelis-Menten graph (Chart 2) compares the phosphoenolpyruvate affinity of these partially purified M2-PK's. The K05's for phosphoenolpyruvate with ADP and GDP as nucleotide substrates (determined from Hill plots, not shown) are given in Table 4. If these data are compared with the values obtained in cell extracts (Table 2), a decrease of the phosphoenolpyruvate affinity after purification is obvious. The decrease is more marked (3-fold) for the enzyme partially purified from extracts of SR-D-transformed CEC compared with the enzyme isolated from extracts of RAV- infected CEC (1.5-fold). This may be explained by the loss of an activator, which is most probably FDP. In accordance with this assumption, FDP increased the phosphoenolpyruvate af finity 22-fold in the partially purified preparation from SR-D- transformed CEC, whereas only a 13-fold stimulation was seen in the crude extract. We furthermore measured the inactivation of the partially ao 25 3.0 200 purified M2-PK preparations by MgATP (Table 4). MgATP in activated M2-PK from SR-D-transformed CEC 3 times faster PEP [mM] than the enzyme from RAV-infected CEC. FDP (0.2 mM) Chart 2. Michaelis-Menten kinetics of M2-PK partially purified by affinity chro- blocked the inactivation. Thus, in comparison with RAV-in matography. The peak fractions (from the experiments shown in Tables 4 and Chart 1) were used for the kinetic studies with ADP (1.5 mM) as substrate. The fected CEC, pyruvate kinase activity from SR-D-transformed specific activities of M2-PK in extracts from RAV-infected CEC O and SR-D- CEC displays the highest specific activity, the lowest phospho transformed CEC (O) were 163 Â± 15 and 200 Â± 10 units/mg of protein, respectively. Results obtained in the presence of 0.2 mM FDP are: â€¢¿RAV;â€¢¿. enolpyruvate affinity, the highest stimulation by FDP, and a SR-D. PEP. phosphoenolpyruvate. faster rate of inactivation by MgATP. Similarities between pp60s" Kinase and M2-PK Kinase. It phorylate (with ATP) at 0Â°within 3 min their protein substrates. has been shown above that low phosphoenolpyruvate affinity Neither cAMP (10 /IM) nor the Walsh inhibitor (100 jug/ml) is one of the characteristic properties of partially purified M2- affected phosphate incorporation. Both kinases are inhibited PK from SR-D CEC extracts (Table 4). Since these partially by low concentrations of ADP whether GTP or ATP were the purified fractions were also enriched in pp60src kinase activity, phosphate donors. Sugar phosphates like P-ribose-PP and FDP inhibited both kinases. The K0s's for FDP were 1.2 mw it is possible that the presence of this kinase influenced the kinetic properties of M2-PK. The most obvious experiment (pp60sfc kinase) and 0.2 mM (M?-PK kinase), respectively. Both would be to completely free M2-PK from pp60SICkinase and to kinase activities are inhibited by Ap6A in /ÃŒMconcentrations. reevaluate its kinetic properties. Unfortunately, although nu The inhibitory protein in M2-PK, which was used as ligand in merous procedures have been tried, this has not yet been the affinity chromatography, inhibited both kinase activities. successful. Half-maximal inhibition of both kinase activities was observed We have therefore performed in vitro experiments with highly in the same concentration range. purified homogeneous M2-PK from chicken liver and the par M2-PK kinase has an apparent molecular weight around tially purified M2-PK kinase from chicken liver. We wanted to 60,000 upon sodium dodecyl sulfate electrophoresis (15), obtain insight into the mechanism by which the characteristic slightly less than that of M?-PK (M.W. 64,000). When the change, especially in the phosphoenolpyruvate affinity, caused apparent molecular weights of the pp60SICkinase were deter by the expression of the src gene after transformation of CEC mined by molecular sieve chromatography on Sepharose CL- by SR-D could occur. Table 4 shows that the partially purified 4B, 60% of the affinity eluted at a ve/v0 volume corresponding M?-PK kinase which phosphorylates M2-PK confers low phos to a molecular weight of 160,000. Ninety-five % of the total phoenolpyruvate affinity to purified M2-PK. It is therefore pos pyruvate kinase activity applied eluted in the same fractions. sible that the pp60src kinase exerts a similar effect. Therefore, Three additional peaks of pp60src kinase activity were observed the molecular and kinetic characteristics of both kinases were at apparent molecular weights of -300,000 (together with 5% compared. The data are summarized in Table 5. Both kinases of pyruvate kinase activity), >500,000 (no pyruvate kinase use GTP and ATP as phosphate donors and maximally phos- activity found), and <100,000 (no pyruvate kinase activity

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Table 4 Comparison of kinetic parameters of partially purified M2-PK of SR-D-transformed and RA V-infected CEC with homogeneous chicken liver M2-PK (in the presence and absence of a partially purified M2-PK kinase isolated from chicken liver) The effects of the additional presence of M2-PK kinase on the kinetics of purified chicken liver M2-PK were studied in the optical assay, whereas the inactivation of M2-PK kinase was measured in a 2-step assay (see "Materials and Methods").

5 (mM) of phospho- inac Specific activity (HIM) of phos- enolpyruvate (0.2 mM tivation after min (units/mg)Pyruvate phoenolpyruvateADP0.631.50.311.6aGDP0.581.50.28Ko FDPpresent)ADP0.1950.0660.0660.46aGDP0.0631.000.083Half-maximalwithMgATP4514002bMgATP+(f,, 2) at 25Â°

kinase prepa 0.2mw rationRAV-50-infected FDP21000008" CECSR-D-transformed CECChicken

liverMinus kinasePlusM2-PK M2-PK kinaseADP160200600GDP60160408KOS a The preparation of M2-PK kinase used was contaminated with a phosphoprotein phosphatase. The preparation of M2-PK kinase was almost free of contaminations by a phosphoprotein phosphatase.

Table 5 Comparison of properties of pp60"c kinase and chicken liver M,-PK kinase

5 (flM)Inhibi- by byPhosphate Reverse tion inhibi M2-PK inhibi mMcorporationin- reaction3 1 tionbyFDP(mM)1.20.2Type torproteinfraction GDPwith K0 5 (JIM) FDPinhibitionNoncompetitiveof (fig/ml)20 (%)y-(32P]ATP ATP GTP ApsA with ADP chain)M2-PK,(heavy Â±10Â°15 pp60Â°"kinaseM2-PK 90y-{32P]ATP 3-5 1-3 1 1-2 withrespect ATPNoncompetitiveto 3-6 <10 <10 <10 50Half-maximal kinasePhosphateacceptorsIgG6casein.low-molecular-weight withrespect Â±10 to ATPHalf-maximalinhibition endoge nous substratesKo " The velocity of the dephosphorylation of the phosphoprotein (formed from ATP) was measured at 0Â°at different ADP concentrations. b Phosphorylated in the immune complex formed with tumor-bearing rabbit sera. c Mean Â±S.D. found). Recoveries for the pp60src kinase and pyruvate kinase rum is used for the entire procedure. Since purified M2-PK has activity were around 100%. The M2-PK kinase has apparent a molecular weight of 64,000, it is highly probable that the molecular weights of about 240,000 and 160,000 when chro- phosphoprotein with a molecular weight of 64,000 represents matographed on the same column. phosphorylated pyruvate kinase. Endogenous phosphorylation of the peak fractions isolated DISCUSSION from the affinity column indicated the presence of protein kinases as well as endogenous substrates of these kinases. Transformation of CEC by SR-D leads to characteristic Although we suspected that M2-PK was among the proteins changes in the kinetic properties of M2-PK. These changes which were phosphorylated, our attempts to precipitate phos- include a lowering of phosphoenolpyruvate affinity (Tables 2 phoproteins from phosphorylated fractions with M2-PK antibod and 4), an increase in the activation ratio by FDP (with ADP as ies failed since the fractions precipitated as a complex spon substrate) (Tables 2 and 4) and an increase in the rate of taneously after 3-5 hr. We have therefore used another ap inactivation of partially purified M2-PK in the presence of ATP proach to demonstrate a possible phosphorylation of M2-PK in (Table 4). Such changes in the kinetics of M2-PK after viral an immune complex formed with antisera against chicken liver transformation can be mimicked in vitro by the addition of high M?-PK. The procedure was done as described for pp60src concentrations of partially purified M2-PK kinase to purified M2- kinase (see "Materials and Methods"), except that monospe- PK from chicken liver (Table 4). The latter finding suggests that cific Mj-PK rabbit antiserum was used instead of tumor-bearing phosphorylation of M2-PK might be responsible for the ob rabbit serum. The stained gel showed a protein band at M.W. served alterations in the kinetics. Consistent with this assump 64,000 specifically precipitated by M2-PK rabbit antiserum. tion, a protein with a molecular weight of 64,000 was phospho This band was of equal stain intensity using extracts from CEC rylated in the immune complex formed with M2-PK antisera and infected with RAV or transformed with SR-D. This M.W. 64,000 extracts from SR-D-transformed CEC. It is highly probable that band is phosphorylated in immune complexes formed with M2- this phosphoprotein represents phosphorylated M2-PK. No PK antiserum and SR-D CEC extracts (Fig. 1). such phosphorylation was seen with extracts from RAV-in- Neither preimmune serum (either with SR-D or RAV CEC fected CEC (Fig. 1). This finding suggests that a protein kinase extracts) nor RAV CEC extracts with M2-PK antiserum showed which is present in SR-D-transformed CEC preferably is in the phosphorylated M.W. 64,000 protein. Two other phospho- volved in the alteration of the regulatory system of M2-PK. proteins (M.W. 48,000 and 56,000) represent cellular proteins A lowering of phosphoenolpyruvate affinity as it occurs in but not phosphorylated heavy chains from IgG. They are also SR-D-transformed CEC (Tables 2 and 4) and an enhanced seen when protein A-Sepharose without preadsorbed antise- inactivation of M2PK by MgATP (Table 4) should lead to a

1738 CANCER RESEARCH VOL. 40

Fig. 1. Phosphorylation of a protein in immune complexes using monospecific M2-PK rabbit antisera instead of tumor- bearing rabbit sera. The immunoadsorption and the phosphotransferase reaction was as described for tumor-bearing rabbit sera. Extracts from CEC. transformed by SR-D (Track 1 + 2) or infected with RAV (Track 3 + 4) (100 fig of protein each), were adsorbed to protein A-Sepharose loaded with M2-PK antiserum. As a control, rabbit preimmune serum was used (Track 5 + 6 with SR-D and Track 7 + 8 with RAV). A band at M.W. 64.000 (64K) is phosphorylated in immune complexes formed from SR- D CEC but not from RAV CEC extracts. The bands at M.W. 48,000 and 56,000 (48K, 56K) do not represent phosphoryl ated heavy chains from IgG since bands with identical mobility are phosphorylated if protein A-Sepharose without adsorbed antiserum is used for the entire procedure (not shown).

decrease of glycolysis under cellular conditions rather than to (2). A mainly cytosolic localization of pp60src is suggested by the observed 2- to 5-fold increase in the flux from glucose to experiments with immunofluorescence (35, 45) and cell frac- lÃ¡clate after viral transformation. When RSV-transformed CEC tionation studies (5). It is of interest in this context that the 2 are incubated in glucose, the levels of FDP are found to be 3- key regulatory enzymes of glycolysis, namely, phosphofructo- to 10-fold higher than in nontransformed cells (3). This finding kinase and pyruvate kinase, are also cytosolic and controlled points to a limited capacity under cellular conditions of a by cAMP-independent protein kinases (4, 12, 13). However, glycolytic enzyme involved in the conversion of FDP to lÃ¡clate. membrane-bound forms of pyruvate kinase (1 7) and of pp60src M2-PK might be Ihis conlrol point, since the enzyme is inhibited kinase have also been reported (45). An attractive working by ATP (26, 27) and via phosphorylation by a cAMP-independ- hypothesis linking the observed effects on the catalytic prop ent protein kinase. FDP activates M2-PK by increasing its erties of M2-PK with the events leading to transformation is that affinity for phosphoenolpyruvate (Chart 2; Table 4). Moreover, either M?-PK itself or one of the regulatory proteins of M?-PK FDP has been found to decrease the velocity of phosphoryla are among the targets of pp60SIC. Support for this hypothesis tion and inactivation of a purified M?-PK by a cAMP-independ- comes from the following findings. pp60s'c kinase was purified ent protein kinase (14) and to decrease the rate of MgATP- by affinity chromatography on matrix-bound M.W. 33,500 in dependent inactivation of M?-PK isolated from SR-D CEC ex hibitor protein together with M?-PK (Chart 1), and pp60src tracts more effectively than of the enzyme isolated from ex kinase and M2-PK could not be separated from each other in tracts of RAV-infected CEC (Table 4). The rise of cellular FDP enzymic active form by many other methods. This property of concentrations in RSV-transformed CEC (3) could thus over pp60s" kinase is reminiscent of the behavior of M^-PK kinase, come the bottleneck of pyruvate kinase in the sense of a feed which has a high affinity for M?-PK (1 5). forward activation (39) and lead to an increase in lactate Other similarities between M2-PK kinase and pp60src kinase formation from glucose. were found. Both kinases display a broad nucleotide specificity It is yet an unsolved question as to which events may lead to and a high affinity for ADP in the reverse reaction (;.e., de- the alterations of the regulatory system of M?-PK in SR-D- phosphorylation of phosphoprotein) (Table 5). This implies that transformed CEC, preferably. Transformation of the CEC by under cellular conditions, if there is no special blockade mech the Rous sarcoma virus is dependent on the expression of the anism for the reverse reaction with nucleotide diphosphates, src gene (21, 43). The src gene product is a phosphoprotein, both kinases have as a side reaction a nucleotide diphosphate pp60src, with protein kinase activity (9, 36). It is closely related kinase activity (34). The phosphate acceptor site in M2-PK is to a phosphoprotein from normal vertebrate cells (pp60sarc or probably an ester phosphate, indicated by its acid stability (6), pp60) (30). The about 100-fold higher levels of this cAMP- and not phosphohistidine as reported for the phosphoenzyme independent protein kinase caused by the expression of the intermediates from nucleotide diphosphate kinases (31). The src gene in SR-D-infected CEC could trigger the cellular trans broad nucleotide specificity and the high rate of reverse reac formation (30). The natural substrates of pp60safc and the tion could enable both kinases to regulate the degree of phos targets of pp60src are yet not known. One or more of the targets phorylation of their targets (and the physiological response in of pp60src appear to reside in the cytoplasm since enucleated the case of M2-PK kinase) to the nucleotide tri- and diphosphate cells can express the specific morphological transformation ratio, without wasting energy.

MAY 1980 1739

NON-TRANSFORMED CELL TRANSFORMED CELL

GLUCOSE Protein ^biosynthesis Chart 3. Control of carbohydrate metabo lism at Mj-PK by interconversion catalyzed by 'T a cAMP-independent protein kinase and pos Nucleic acid sibly by pp60src. Elevated tissue levels com biosynthesis ÃŽ pared to nontransformed cells are indicated P-RIBOSE-PPN by capitals. Dashed arrows, effectors altering the activities of pyruvate kinase and protein kinase. respectively; + or -, direction of the alteration. We have omitted for reasons of clarity the regulation of glycolysis by oxygen. Oxygen governs glycolysis most probably via ATP inhibition of phosphofructokinase (Pas teur effect). In Rous sarcoma virus-trans formed cells, the oxygen control (among other reasons) is impaired by the high FDP levels which overcome the ATP inhibition of phos phofructokinase.

Â»LACTATE

We investigated the FDP sensitivity of pp60src kinase, since FDP (39) and P-ribose-PP (23) must then overcome the block FDP restored the high phosphoenolpyruvate affinity and ade of M2-PK, presumably by inhibition of pp60src kinase. As a blocked the ATP-caused inactivation in the partially purified consequence glycolysis can run only at the expense of high M2-PK preparation isolated from SR-D-transformed CEC. It was FDP and P-ribose-PP levels. found that FDP inhibited the phosphotransfer reaction in the Elevated levels of FDP favor protein biosynthesis (28). An immune complex as was observed for M2-PK kinase when M2- increased synthesis of Ap4A is observed at high ATP/ADP PK served as substrate (14). Since both kinase activities as ratios during protein biosynthesis (32). Ap4A binds to DMA well as M2-PK are adsorbed by the affinity column with the polymerase A and leads to DNA replication (19, 20). It is M.W. 33,500 inhibitor protein of M2-PK as ligand, we tested interesting in this context that Ap5A (which is structurally re the inhibitor protein fraction and found that it inhibited pp60src lated to Ap4A) inhibits M2-PK kinase and pp60src kinase in vitro. kinase. A schematic representation of some metabolic differences We do not know at the present time whether M2-PK kinase between normal (nonproliferating) cells and Rous sarcoma is a phosphoprotein as has been shown for pp60src or pp60sarc virus-transformed cells, the suggested interaction of M2-PK because separation of M2-PK kinase from phosphorylated M2- and pp60src and their possible role in transformation are shown PK has not been possible thus far. Our current antisera pre in Chart 3. pared against M2-PK kinase coprecipitate M2-PK. Several properties of pp60src kinase are remarkably similar ACKNOWLEDGMENTS to those of M2-PK kinase. This does not imply that M2-PK kinase is structurally identical to pp60 (or pp60sarc) and that The authors gratefully acknowledge the excellent technical assistance of pp60stc is a modified M2-PK kinase. The results suggest, how Cornelia Konrad, Rita Schlusche, Eleonore HÃ¶lle, Michael Weil, Hans GÃ¶bler, and Karin Volz for help in typing the manuscript. We are grateful to Dr. M. ever, that the 2 kinases have some common functional prop Reinacher, Institut fÃ¼rVeterinÃ¤r-Pathologie. Giessen, for the M2-PK antisera and erties. to Bayer AG, especially Dr. Haberland, for Trasylol. We have presented mainly indirect evidence for the hypoth esis that M2-PK and/or its regulatory proteins are among the REFERENCES targets of pp60src. This hypothesis is nevertheless especially 1. Becker, D., Kurth, R., Critchley, D., Friis, R. R., and Bauer, H. Distinguishable attractive if the consequences for metabolism are taken into transformation-defective phenotypes among temperature-sensitive mutants account. RSV-transformed CEC show an expansion of all in of Rous sarcoma virus. J. Viral., 21: 1042-1055, 1977. termediates of the pentose phosphate pathway, the glycolytic 2. Beug, H., Petus, J. H., and Graf, T. Expression of virus specific morpholog ical cell transformation induced in enucleated cells. Z. Naturforsch. Teil C intermediates above triÃ³se phosphates, and increased levels Biochem. Biophys. Biol. Viral., 31: 766-768, 1976. of P-ribose-PP. This is accompanied by high rates of lactic acid 3. Bissel, M. J., White, R. C.. Hatie, C., and Bassham, J. A. Dynamics of production when glucose is the substrate under aerobic con metabolism of normal and virus-transformed chick cells in culture. Proc. Nati. Acad. Sei. U. S. A., 70: 2951-2955, 1973. ditions. Aerobic glycolysis may be a prerequisite for P-ribose- 4. Brand, J. A., and Soling, H. D. Activation and inactivation of rat liver PP and nucleic acid synthesis. In order to meet the increased phosphofructokinase by phosphorylation-dephosphorylation. FEBS Lett., demands for nucleic acid synthesis precursors, RSV-trans 57. 163-168. 1975. 5. Brugge, J. S., Steinbaugh, P. J., and Erikson, R. L. Characterisation of the formed cells must selectively channel carbohydrates to P-ri avian sarcoma virus protein p60"c. Virology, 91: 130-140, 1978. bose-PP. The preferential channeling may be achieved by high 6. Brunn, H., Eigenbrodt, E., and Schoner, W. Isolation of an acidic peptide from pyruvate kinase type M2 of chicken liver containing the phosphate glycolytic activities and, as a first step, via a blockade of M2- acceptor site for a cyclic AMP independent protein kinase. Hoppe-Seyler's PK by pp60src kinase. The rise of cellular concentrations of Z. Physiol. Chem., 360. 1257-1261, 1979.

1740 CANCER RESEARCH VOL. 40

7. BÃ¼cher,T. Phosphoglycerate kinase from brewer's yeast. Methods En- kinase. II. Purification of M2-type pyruvate kinase from Yoshida ascites zymol., 7. 415-422, 1955. hepatoma 130 cells and comparative studies on the enzymological and 8. Burk, D. M., Woods, M., and Hunter, J. On the significance of glycolysis for immunological properties of the three types of pyruvate kinases, L., Mi and cancer growth with special reference to Morris rat hepatoma. J. Nati. M2. J. Biochem. (Tokyo), 72: 1001-1015, 1972. Cancer. Inst., 38: 839-863, 1967. 28. Lenz, J. R., Chatterjee. G. E., Maroney, A., and Baglioni. C. Phosphorylated 9. Collett, M. S., and Erikson, R. L. Protein kinase activity associated with the sugars stimulate protein synthesis and Met-tRNAf binding activity in extracts avian sarcoma virus src gene product. Proc. Nati. Acad. Sei. U. S. A., 75. of mammalian cells. Biochemistry, 77: 80-87, 1978. 2021-2024, 1978. 29. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein 10. Collett, M. S., Erikson, E., Purchio, A. F., Brugge, J. S., and Erikson, R. L. measurement with the Polin phenol reagent. J. Biol. Chem., 793: 265-275, A normal cell protein similar in structure and function to the avian sarcoma 1951. virus transforming gene product. Proc. Nati. Acad. Sei. U. S. A., 76: 3159- 30. Oppermann, H., Levinson. A. D., Varmus, H. E., Levintow, L.. and Bishop, J. 3164, 1979. M. Uninfected vertebrate cells contain a protein that is closely related to the 11. Criss, W. E. A review of isoenzymes in cancer. Cancer Res., 37. 1523- product of the avian sarcoma virus transforming gene (src). Proc. Nati. 1542, 1971. Acad. Sei. U. S. A., 76: 1804-1808, 1979. 12. Eigenbrodt, E., Abdel-Fattah Mostafa, M., and Schoner, W. Inactivation of 31. Parks, R. E., and Agarwal, R. P. Nucleosidediphosphokinases. In: P. D. pyruvate kinase type M2 from chicken liver by phosphorylation, catalyzed Boyer (ed.). The Enzymes, Vol. 8, pp. 307-333. New York: Academic Press, by a cAMP-independent protein kinase. Hoppe-Seyler's Z. Physiol. Chem., Inc., 1973. 358: 1047-1055, 1977. 32. Rapoport, E., and Zamecnik, P. C. Presence of diadenosine 5',5'"-P'.P4- 13. Eigenbrodt, E., and Schoner, W. Purification and properties of the pyruvate tetraphosphate (Apâ€žA)in mammalian cells in levels varying widely with kinase isoenzymes type L and M2 from chicken liver. Hoppe-Seyler's Z. proliferative activity of the tissue: a possible positive "pleiotypic activator." Physiol. Chem., 358. 1033-1046, 1977. Proc. Nati. Acad. Sei. U. S. A., 73: 3984-3988, 1976. 14. Eigenbrodt, E., and Schoner, W. Modification of the pyruvate kinase inter- 33. Reinacher, M.. Eigenbrodt, E.. Schering, B., and Schoner, W. Immunohis- conversion of pyruvate kinase type M2 from chicken liver by fructose-1.6- tochemical localization of pyruvate kinase isoenzymes in chicken tissues. diphosphate and L-alanine. Hoppe-Seyler's Z. Physiol. Chem., 358: 1055- Histochemistry, 64: 145-161, 1979. 1067. 1977. 34. Richer!, N. D., Davies. P. J. A., Jay, G., and Pastan, l. H. Characterization 15. Eigenbrodt, E., and Schoner, W. Modification of pyruvate kinase activity by of an immune complex kinase in immunoprecipitates of avian sarcoma virus- proteins in chicken liver. Hoppe-Seyler's Z. Physiol. Chem., 360. 1243- transformed fibroblasts. J. Virol., 37: 695-706, 1979. 1252, 1979. 35. Rohrschneider, L. R. Immunofluorescence on avian sarcoma virus-trans 16. Farina, A. F., Shatton, J. B., Morris, H. P., and Weinhouse, S. Isoenzymes formed cells: localization of the src gene product. Cell, 76: 11-24, 1979. of pyruvate kinase in liver and hepatoma of the rat. Cancer Res., 34: 1435- 36. RÃ¼bsamen,H., Friis, R. R., and Bauer, H. src gene product from different 1446, 1974. strains of avian sarcoma virus: kinetics and possible mechanism of heat 17. Fossel, E. T., and Solomon. A. K. Ouabain-sensitive interaction between inactivation of protein kinase activity from cells infected by transformation- human red cell membrane and glycolytic enzyme complex in cytosol. defective, temperature-sensitive mutant and wild-type virus. Proc. Nati. Biochim. Biophys. Acta, 570. 99-111, 1978. Acad. Sei. U. S. A.. 76: 967-971, 1979. 18. Gonsalvez, M., Garcia, Suarez S.. and LÃ³pez-Alceron, L. Metabolic control 37. Sharma, R. K., Desai, R., Thompson, T. R., and Wang, J. H. Purification of of glycolysis in normal and tumor permeabilized cells. Cancer Res., 38: the heat-stable inhibitor protein of the Ca2*-activated cyclic nucleotide 142-148, 1978. phosphodiesterase by affinity chromatography. Can. J. Biochem., 56: 598- 19. Grummt, F. Diadenosine 5',5'", P'.PMetraphosphate triggers initiation of in 604, 1978. vitro DNA replication in baby hamster kidney cells. Proc. Nati. Acad. Sei. U. 38. Singh, M.. Singh, V. N., August, J. T., and Horecker, B. L. Alterations in S. A., 75: 371-375, 1978. glucose metabolism in chick embryo cells transformed by Rous sarcoma 20. Grummt, F., Paul, D., and Grummt, J. Regulation of ATP pools, rRNA and virus. Arch. Biochem. Biophys., 765: 240-246, 1974. DNA synthesis in 3T3 cells in response to serum or hypoxanthine. Eur. J. 39. Singh, V. W., Singh, J., August, J. T., and Horecker, B. L. Alterations in Biochem., 76: 7-12, 1977. glucose metabolism in chick embryo cells transformed by Rous sarcoma 21. Hanafusa, H. Cell transformation by RNA tumor viruses. In: H. Fraenkel- virus: intracellular levels of glycolytic intermediates. Proc. Nati. Acad. Sci. Conrat and R. P. Wagner (eds.). Comprehensive Virology. Vol. 10, pp. 401 - U. S. A., 77: 4129-4132, 1974. 483. New York: Plenum Press, 1977. 40. Spellman, C. M., and Fottrell, P. F. Similarities between pyruvate kinase 22. Harris, W., Days, R., Johnson, C., Finkelstein, I., Stallwarth, J., and Hubert, from human placenta and tumours. FEBS Lett., 37: 281-284. 1973. C. Studies on avian heart pyruvate kinase during development. Biochem. 41. Strandholm, J. J., Cardenas, J. M., and Dyson, R. D. Pyruvate kinase Biophys. Res. Commun., 75. 1117-1121, 1977. isozymes in adult and fetal tissues of chicken. Biochemistry. 74: 2242- 23. Hovi, T., Vaheri, A., and Allison, A. C. Transformation-associated increase 2246, 1975. of phosphoribosyl pyrophosphate concentration in chick embryo fibroblasts. 42. Van Veelen, C. W. M., Verbiest, H., Vlug, A. M. C., Rijksen. G., and Staal. G. FEBS Lett., 703. 43-46, 1979. E. J. Isozymes of pyruvate kinase from human brain, meningiomas and 24. Ibsen, K. H. Interrelationships and functions of the pyruvate kinase isoen- malignant gliomas. Cancer Res., 38: 4681 -4687, 1978. zymes and their variant forms: a review. Cancer Res., 37: 341-353, 1977. 43. Vogt, P. K. The genetics of RNA tumor viruses. In: H. Fraenkel-Conrat and 25. Ibsen, K. H., Murray, L., and Maries, S. W. Electrofocusing and kinetic R. P. Wagner (eds.), Comprehensive Virology, Vol. 9, pp. 341-455. New studies of adult and embryonic chicken pyruvate kinases. Biochemistry, 75. York: Plenum Press, 1979. 1064-1073, 1976. 44. Weinhouse, S. Metabolism and isozyme alterations in experimental hepato- 26. Imamura, K., and Tanaka, T. Multimolecular forms of pyruvate kinase from mas. Fed. Proc., 32: 2162-2167, 1973. rat and other mammalian tissues. I. Electrophoretic studies. J. Biochem. 45. Willingham, M. C., Jay, G.. and Pastan, J. Localization of the ASV src gene Tokyo, 77: 1043-1051, 1972. product to the plasma membrane of transformed cells by electron micro 27. Imamura, K., Taniuchi, K., and Tanaka, T. Multimolecular forms of pyruvate scopic immunocytochemistry. Cell, 78: 125-134, 1979.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1980 American Association for Cancer Research. Similarities between a Phosphoprotein (pp60src)-associated Protein Kinase of Rous Sarcoma Virus and a Cyclic Adenosine 3 ′:5′-Monophosphate-independent Protein Kinase That Phosphorylates Pyruvate Kinase Type M 2

Peter Presek, Hartmut Glossmann, Erich Eigenbrodt, et al.

Cancer Res 1980;40:1733-1741.

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