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Phosphorylation of Ubiquitin-Activating Enzyme in Cultured Cells

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Phosphorylation of Ubiquitin-Activating Enzyme in Cultured Cells Proc. Natl. Acad. Sci. USA Vol. 92, pp. 3454-3457, April 1995 Biochemistry Phosphorylation of ubiquitin-activating enzyme in cultured cells JAMES C. COOK* AND P. BOON CHOCKt Laboratory of Biochemistry, Section on Metabolic Regulation, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 3, Room 202, Bethesda, MD 20892 Communicated by Earl R. Stadtman, National Institutes of Health, Bethesda, MD, January 3, 1995 ABSTRACT Ubiquitin-activating enzyme, El, is the first protein ligase. Isoforms of all three enzymes have been enzyme in the pathway leading to formation of ubiquitin- described (11-14, 19). protein conjugates. El exists as two isoforms in human cells Obviously, ubiquitin conjugation must be a regulated pro- which are separable by electrophoresis. These isoforms mi- cess. Only certain proteins are recognized by the conjugating grate with apparent molecular sizes of 110 kDa and 117 kDa enzymes, and some are targets for conjugation only under in SDS/polyacrylamide gels. Immunoprecipitation ofEl from certain environmental conditions (15, 16) or during specific lysates of HeLa cells metabolically labeled with [32P]phos- phases of the cell cycle (17). Yet little is known about how this phate indicated the presence of a phosphorylated form of El regulation is accomplished. which migrates at 117 kDa. Phospho amino acid analysis Previous reports demonstrated that El can undergo in vitro identified serine as the phosphorylated residue in El. Phos- phosphorylation (18) and that it exists as two isoforms in phorylated El was also detected in normal and transformed human cells (19). These were designated "ElllOkDa" and cells from another human cell line. Phosphatase-catalyzed "Eli7IkDa" to reflect their apparent molecular masses on SDS/ dephosphorylation ofEl in vitro did not eliminate the 117-kDa polyacrylamide gels. Discovery of a second isoform suggested the El isoform detected by Coomassie staining after SDS/poly- possibility that El may be a target for differential regulation of acrylamide gel electrophoresis, thereby demonstrating that ubiquitin conjugation and that specificity could, in principle, be phosphorylation is not the sole structural feature differenti- controlled through El. ating the isoforms of El. These observations suggest new In this paper we demonstrate that El is phosphorylated in hypotheses concerning mechanisms ofmetabolic regulation of normal and transformed human cells, that El is phosphory- the ubiquitin conjugation pathway. lated on a serine residue, and that phosphorylation is not the structural feature which differentiates the two isoforms of El. Ubiquitin i's a small (76 amino acids), highly conserved glob- ular protein found in all eukaryotic cells (1). Covalent binding EXPERIMENTAL of ubiquitin to other proteins is the means by which ubiquitin PROCEDURES accomplishes its many varied functions. Levels of ubiquitin- Materials. Alkaline phosphatase-conjugated goat anti- conjugated proteins are controlled by competing conjugating rabbit IgG, Dulbecco's modified Eagle's medium (DMEM), and deconjugating enzyme systems; thus, ubiquitin can be methionine-free DMEM, phosphate-free DMEM, heat- thought of as a functional group in a reversible posttransla- inactivated calf serum, penicillin, and streptomycin were all tional modification of proteins. from GIBCO-BRL. Nonidet P-40 (protein-grade), Tween 20 Some ubiquitin-protein conjugates are rapidly broken down (protein-grade), 4-(2-aminoethyl)benzenesulfonyl fluoride, by an ATP-dependent protease, with the ubiquitin moiety Pansorbin (formalin-fixed Staphylococcus aureus cells), and acting as the signal for proteolysis (for review, see ref. 2). protein A-agarose were from Calbiochem. Anti-phosphoty- However, other conjugates appear to be quite stable, and the rosine antibodies (PY 20) and [32P]phosphoric acid were from function of ubiquitin in these conjugates is not known. Cellular ICN. Nitrocellulose blotting membrane (BA-S83, 0.2-,um pore processes which involve ubiquitin include cell cycle regulation, size) was from Schleicher & Schuell. Poly(vinylidene difluo- DNA replication, DNA repair, protein turnover, chromatin ride) blotting membrane (Immobilon-P) was from Millipore. remodeling in mitosis, ribosome biogenesis, heat shock re- Potato acid phosphatase (20 units/mg), aprotinin, bestatin, sponse, transmembrane protein transport, red blood cell mat- leupeptin, and pepstatin A were from Boehringer Mannheim. uration, and choline uptake by neurons (for review, see ref. 3), All other chemicals were reagent-grade or better. as well as peroxisome biogenesis (4). In addition, ubiquitin has Tissue Culture. HeLa S3 cells (CCL2.2), WI-38 cells been identified as a component of abnormal protein deposits (CCL75), and WI-38VA13 subline 2RA cells (CCL75.1) were in several chronic neurodegenerative diseases including Alz- obtained from the American Type Culture Collection. Cells heimer disease, Parkinson disease, and amyotrophic lateral were grown in monolayer cultures at 37°C in a humidified sclerosis (for review, see ref. 5). Any advances in our under- atmosphere of 5% C02/95% air. Growth medium was DMEM standing of ubiquitin metabolism will undoubtedly contribute supplemented with 10% calf serum (heat-inactivated), peni- to understanding the pathogenesis of these diseases. cillin (50 units/ml), and streptomycin (50 ,ug/ml). Formation of ubiquitin-protein conjugates requires the ac- Antiserum. Preparation and characterization of antiserum tion of at least two, and sometimes three, enzymes: El, E2, and raised against El were described previously (14, 19). E3 (6-8). El, ubiquitin-activating enzyme, activates ubiquitin Metabolic Cell Labeling. Monolayer cultures were metabol- by forming a high-energy thioester bond between the C ically labeled with. [32P]phosphate by incubation with medium terminus of ubiquitin and a sulfhydryl moiety on El (9, 10). containing [32P]phosphoric acid in phosphale-free growth me- Activated ubiquitin is transferred to one of a family of ubiq- dium supplemented with 10% phosphate-free calf serum. 32p uitin-conjugating isoenzymes collectively known as E2. Most labeling medium was 1 mCi/ml for phospho amino acid analysis isoenzymes of E2 conjugate ubiquitin to target proteins di- and 0.03-0.2 mCi/ml for all other experiments (1 mCi = 37 rectly, whereas some require the action of E3, ubiquitin- MBq). The labeling period was 1-3 hr unless specified otherwise. The publication costs of this article were defrayed in part by page charge *Present address: Department of Virus and Cell Biology, Merck payment. This article must therefore be hereby marked "advertisement" in Research Laboratories, West Point, PA 19486. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 3454 Downloaded by guest on September 26, 2021 Biochemistry: Cook and Chock Proc. Nati. Acad. Sci. USA 92 (1995) 3455 Immunoprecipitation of El. A monolayer culture of cells Cooper et al (23) with a Hunter thin-layer peptide mapping grown in a 175-cm2 culture dish was rinsed with ice-cold system (model HTLE-7000; CBS Scientific). Tris-buffered saline (TBS: 10 mM Tris-HCl, pH 7.6/150 mM Dephosphorylation with Potato Acid Phosphatase. One NaCl) and lysed with 7 ml of Nonidet P-40 lysis buffer [1% 175-cm2 culture dish of HeLa cells was labeled with [32P]phos- (vol/vol) Nonidet P-40/0.15 M NaCl/0.05 M Tris-HCl, pH 7.4] phate, lysed, and clarified as above. The clarified lysate was (20) with protease inhibitors: 4-(2-aminoethyl)benzenesulfo- dialyzed into 0.1 M Tris-HCl, pH 7.0/0.15 M NaCl/1 mM nyl fluoride (180 ,tg/ml), aprotinin (80 ,tg/ml), leupeptin (14 dithiothreitol, and concentrated to 5 ml by ultrafiltration in a ,ug/ml), and bestatin (56 ,tg/ml). Phosphatase inhibitors were Centricon-100 filtration unit (Amicon). The retentate was included in the lysis buffer in the early experiments of this treated with potato acid phosphatase by adding 0.1 ml of 0.2% study but were omitted in later experiments (see Results): (wt/vol) potato acid phosphatase/0.01 M MgCl2/0.1 M so- p-nitrophenyl phosphate (2.5 mM), sodium orthovanadate (1 dium citrate, pH 5.8, to 2.5 ml of lysate. A mock reaction mM), and sodium fluoride (50 mM). The lysate was clarified without phosphatase served as a control. Dephosphorylation by centrifugation at 14,000 x g for 10 min at 4°C or by filtration proceeded for 12 hr at 4°C, an additional 20 units of the with a Millex-GV 0.22-,um filter unit (Millipore). For the phosphatase was added, and the reaction was allowed to experiment shown in Fig. 1A, the lysate was preabsorbed with continue for another 2 hr at room temperature. The solutions nonimmune rabbit IgG and solid-phase protein A (Pansorbin were centrifuged to remove particulate material. El was cells) as described (20). El was immunoprecipitated by adding immunoprecipitated, solubilized in Laemmli sample buffer, anti-El antiserum (or preimmune serum for control) and and separated by SDS/PAGE. The gel was stained with Pansorbin cells (20). All other immunoprecipitations were Coomassie blue and dried. Radioactivity was detected and performed without the preabsorption step and by adding the quantified by autoradiography, and Coomassie-stained protein lysate to protein A-agarose beads which had been preloaded was quantified by densitometry. with IgG from anti-El antiserum (0.2-5 mg of IgG per ml of Autoradiography and Densitometry. Radiolabeled proteins packed beads). Precipitated protein was solubilized prior to were detected by x-ray film autoradiography without enhance- electrophoresis by adding Laemmli sample buffer and heating ment and by storage phosphor screen autoradiography. Quan- at 100°C for 2 min (21). titation was accomplished with a Molecular Dynamics model Electrophoresis and Immunoblotting. SDS/polyacrylamide 400 Phosphorlmager using IMAGEQUANT software, version 3.0. gel electrophoresis (PAGE) of proteins was done according to Quantitation of protein on Coomassie-stained gels was done the method of Laemmli (21) with either pre-cast slab gels with a Molecular Dynamics personal densitometer, also with (Novex; 1 mm thick, 8 cm long) or preparative slab gels cast in IMAGEQUANT software.
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