[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 Rübsamen,Robert R. Friis, and Heinz Bauer Rudolf Buchheim-Institut fürPharmakologie [P. P., H. G.] and Institut fürVirologie [H. R., R. R. F., H. B.¡.Fachbereich Humanmedizin, and Institut fürBiochemie und Endokrinologie, Fachbereich Veterinärmedizin [E. E., W. S.], Justus Liebig-Universität 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). MAY 1980 1733 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 32P¡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'°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 4°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 0°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.