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. our laboratory (6% acrylamide separating gel, 3% acrylamide stacking gel; acrylamide N,N'-methylenebisacrylamide weight ratio of 37:1; 1.5 mm thick, 10 cm long). Immunoblotting of RESULTS pre-cast gels was described previously (19). Preparative gels Immunoprecipitation of El from Lysates of Metabolically were blotted according to Towbin et al (22). Immune com- Labeled HeLa Cells. HeLa cells were grown in medium plexes were detected with goat anti-rabbit IgG alkaline phos- containing [32P]phosphate and lysed. The lysate was preab- phatase conjugate and nitro blue tetrazolium/5-bromo-4- sorbed with nonimmune rabbit IgG and Pansorbin cells, and chloro-3-indolyl phosphate. El was immunoprecipitated with anti-El antiserum and Pan- Phospho Amino Acid Analysis. A confluent culture of HeLa sorbin cells. Immunoprecipitated proteins were separated by cells grown in a 25-cm2 dish was labeled for 3 hr in medium SDS/PAGE, and radioactive phosphoproteins were detected containing [32P]phosphoric acid at 1 mCi/ml. [32P]Phospho-E1 by autoradiography (Fig. 1A). Proteins immunoprecipitated was purified by immunoprecipitation and Western blotting with anti-El antiserum contained a radioactive phosphopro- onto a poly(vinylidene difluoride) membrane. The band of tein at 117 kDa. This band was absent in the control sample in [32P]E1 was excised and protein was hydrolyzed in 6 M HCl which preimmune serum was used for immunoprecipitation. (constant boiling; Pierce) at 100°C for 1 hr. Phospho amino The phosphoprotein at 117 kDa coincided exactly with the acid analysis was done according to the methods described by position of E11l7kDa detected by Coomassie staining (data not

A B C 1 2 3 1 2 1 2 3 4 1Op- ".9 top :. top ...... _ . F...

Z-111 -117 - 117 |-- ..., J-1107 \ 1 10 ;, :

.:. __ _-dIgG .f IgG - dye FIG. 1. Immunoprecipitation of El from metabolically labeled cell lysates. (A) Autoradiogram of 32P-labeled HeLa cell proteins resolved by SDS/PAGE. Lane 1, proteins precipitated by anti-El antiserum; lane 2, proteins precipitated by preimmune serum; lane 3, cell lysate. (B) Western blot from SDS/PAGE of proteins immunoprecipitated from 32P-labeled HeLa cell lysate by anti-El. Lane 1, autoradiogram of lane 2; lane 2, immunodetection of proteins in immunoprecipitate. Membrane was probed with rabbit anti-El and alkaline phosphate-conjugated goat anti-rabbit IgG; color was developed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. (C) Proteins immunoprecipitated from 32P-labeled cell lysates by anti-El and separated by SDS/PAGE. Lanes 1 and 2, autoradiogram of lanes 3 and 4; lane 3, Coomassie-stained gel of proteins precipitated from normal lung fibroblasts (CCL75); lane 4, Coomassie-stained gel of proteins precipitated from simian virus 40-transformed lung fibroblasts (CCL75.1). Tops of the running gels, which contained unresolved high molecular weight aggregates, are indicated. Apparent molecular masses are indicated in kilodaltons. Position of the heavy chain of IgG used for immunoprecipitation is shown. Dye fronts, which contained unresolved proteins, are indicated. Downloaded by guest on September 26, 2021 3456 Biochemistry: Cook and Chock Proc. Natl. Acad ScL USA 92 (1995) shown). Since the antiserum used in these studies is highly specific for El (14, 19), the presence of the phosphoprotein precipitated with anti-El and its absence in the control lane indicate that the phosphoprotein at 117 kDa is El. Omission of phosphatase inhibitors from the lysis buffer did not affect the amount of [32P]phosphoEl recovered by immunoprecipi- tation of the 117-kDa phosphoprotein (data not shown). Fig. 1B shows a Western blot of protein precipitated from metabolically labeled HeLa cell lysate with anti-El antiserum and protein A-agarose beads. The 117-kDa phosphoprotein I (detected by autoradiography) migrated at exactly the same position as El117kDa (detected by immunostaining). Fig. 1C shows that El phosphorylation is not unique to HeLa cells, since it was observed in normal and simian virus y 40-transformed lung fibroblasts as well. Demonstration That Phosphorylation ofEl Occurs in Cells. FIG. 3. Phospho amino acid analysis of acid-hydrolyzed [32P]El. To show that the phosphorylated El was formed in the cells Positions of the phospho amino acid standards are circumscribed by rather than in the lysate, we measured the incorporation of 32p dashed lines. S, phosphoserine; T, phosphothreonine; Y, phosphoty- into El as a function of the duration of the labeling period. A rosine. series of identical cultures were incubated with 32P-labeling dephosphorylated enzymatically to see whether E11l7kDa medium for various amounts of time. Lysates were prepared would be converted to ElllokDa. and quick-frozen in liquid nitrogen until all samples were Lysates prepared from 32P-labeled cells were dialyzed collected. The samples were thawed and then clarified by and treated with potato acid phosphatase. After dephospho- filtration, and the extent of El phosphorylation was deter- rylation, El was immunoprecipitated and the immunoprecipi- mined by immunoprecipitation, SDS/PAGE, and autoradiog- tated proteins were separated by SDS/PAGE, stained with raphy. Fig. 2 shows that the amount of radiolabel present in the Coomassie blue, and analyzed by autoradiography and densi- immunoprecipitated El was dependent on the amount of time tometry. The results in Fig. 4A show that El was 94% the cells were exposed to the labeling medium, thus demon- dephosphorylated by phosphatase treatment. If E11l7kDa were strating that El phosphorylation is a process which occurs in indeed phosphorylated ElllOkDa, then dephosphorylation the living cell. should have converted 94% of Coomassie-detectable El117kDa Phosphe Amino Acid Analysis. Phospho amino acid analysis into ElllokDa. Such a conversion would alter the ratio of the of the hydrolyzed [32P]phospho-El is shown in Fig. 3. Radio- activity was present in the phosphoserine spot detected with 1 1 7kDa ninhydrin, indicating that El is phosphorylated on a serine A residue. No radioactivity was detected in the phosphothreo- nine or phosphotyrosine spots. The absence of phosphoty- rosine in phosphorylated El was confirmed by Western blot- ting with anti-phosphotyrosine antibodies (data not shown). cJ Dephosphorylation of [32P]El. El exists as two isoforms -_ which migrate at 110 kDa and 117 kDa. Since phosphorylation of proteins often affects their migration in SDS/polyacryl- 0 amide gels, we were interested in determining whether phos- co phorylation may be the structural difference between the two isoforms-i.e., that El117kDa may be a phosphorylated form of ElillokDa. To test this hypothesis, phosphorylated El was hours 1.7 2.3 4.0 relative distance B .+ phosphatase -phosphatase tn C 700 117kDa 11 OkDa 4-0 0 600 500 3.0 co .CD Cu L- 400 oCu 300 2.0 0 0 L- 0 200 Cu

100 U) 0 c 1.0 =- 0) co r] C- L. .0 0 2 4 6 8 10 duration of labeling (hours) 0 relative distance relatived..distancea.n. FIG. 2. Time course of incorporation of 32p into phosphorylated El. HeLa cells were labeled for various times before lysis. El was FIG. 4. (A) Densitometric scan of the autoradiogram in the region immunoprecipitated and resolved by SDS/PAGE, and the amount of of E11l7kDa with and without phosphatase treatment. (B) Densito- 32p incorporation was determined by autoradiography. (Inset) Region metric scans of Coomassie-stained gel over the region containing the of the autoradiogram showing phosphorylated El. El isoforms, with and without phosphatase treatment. Downloaded by guest on September 26, 2021 Biochemistry: Cook and Chock Proc. Natl. Acad. Sci. USA 92 (1995) 3457 isoforms (E1jl7kM,/ElllOkDa) from 1:1.7 (control sample) to On a cellular level, phosphorylation may target El to a 1:49 (phosphatase-treated sample). Fig. 4B shows that dephos- particular intracellular organelle. Grenfell et at (24) reported phorylation caused little or no conversion of El117kDa into a cell cycle-dependent redistribution of the enzyme within the ElillOkDa. Thus, phosphorylation is not the structural feature cell. Could phosphorylation of El be a signaling event which responsible for the different migration of the El isoforms leads to the redistribution of this enzyme? If so, the level of El observed in SDS/PAGE. Some loss of both El isoforms was phosphorylation might be expected to change in a cell cycle- seen in the phosphatase-treated sample relative to the control, dependent manner. Numerous cell cycle events are controlled which may have been due to lower recovery in the immuno- by phosphorylation or dephosphorylation (25). El has been precipitation step. shown to play roles in S, G2, and M phases of the cell cycle (3). Perhaps phosphorylation of El by a cell cycle-specific kinase is the means by which the ubiquitin-conjugation pathway is DISCUSSION functionally linked to the cell cycle. Indeed, serine-835 is We have demonstrated that ubiquitin-activating enzyme is located within a consensus sequence specific for CDC2 kinase, phosphorylated in living cells. The time course of radiolabel (S/T)-P-X-R/K, where X is a polar residue (26). In addition, incorporation (Fig. 2) shows a lag phase, and after 9 hr of based on the consensus sequence analysis, human El also labeling time, the extent of 32p incorporation had not yet contains 1 specific substrate site for cAMP-dependent protein reached its maximum. Therefore, the intensity of the bands kinase, 3 for cGMP-dependent protein kinase, 5 for Ca2+/ shown in Fig. 1, which was obtained after 3 hr or less of calmodulin-dependent protein kinase II, 17 for casein kinase incubation with [32P]phosphoric acid, represents only a small II, and 16 medium-specific sites for protein kinase C. However, fraction of the phosphorylatable El present in the cell. the accessibility of these sites for kinases is determined by the Previously, we demonstrated the existence of two isoforms of quaternary structure of El. El in human cells, which we designated E11l7kDa and ElllokDa to Elucidation of the mechanisms controlling ubiquitin conju- reflect their apparent molecular sizes determined by SDS/PAGE gation continues to be a major challenge and an important (14). These two isoforms were immunologically crossreactive, goal. The discovery of El phosphorylation in living cells showed essentially identical peptide maps, and possessed El provides the basis for new hypotheses concerning potential activity. Pulse-labeling studies revealed that turnover rates were regulatory mechanisms of ubiquitin conjugation that may lead inconsistent with E11l7kDa being the precursor of EllokDa. Fur- to better understanding of this important pathway. ther, E11l7kDa was not immunoreactive with anti-ubiquitin anti- serum, indicating that E11l7kDa is not ubiquitinated ElllokDa. We thank Ms. Natalie A. Davis for technical assistance, Drs. Do Since the data in Fig. 1 showed that only the band Joon Park and Chang Won Lee for instructions in the use of the El17iDa Hunter thin-layer peptide mapping system, and Dr. Francesca Santini contained radioactive phosphate after metabolic labeling, we for helpful discussions and for a critical of the investigated the possibility that Elii17iDa was the phosphorylated reading manuscript. form of Ellol,kDa. Enzymatic dephosphorylation with potato acid 1. Rechsteiner, M., ed. (1988) Ubiquitin (Plenum, New York). phosphatase did not significantly alter the ratio of the two 2. Hershko, A. (1988) J. Biol. Chem. 263, 15237-15240. isoforms detected by Coomassie staining (Fig. 4), which indicates 3. Finley, D. & Chau, V. (1991) Annu. Rev. Cell Bio. 7, 25-69. that removal ofthe phosphate group did not convert E11i7kDa into 4. Wiebel, F. F. & Kunau, W.-H. (1992) Nature (London) 359, 73-76. EllokDa. Thus, the structural feature responsible for the different 5. Mayer, R. J., Lowe, J. & Landon, M. (1991) J. Pathol. 163, migration of the isoforms observed in SDS/PAGE experiments 279-281. remains to be identified. 6. Ciechanover, A., Heller, H., Katz-Etzion, R. & Hershko, A. it that (1981) Proc. Natl. Acad. Sci. USA 78, 761-765. Although appears E11l7kDa is the isoform which is 7. Hershko, A., Heller, H., Elias, S. & Ciechanover, A. (1983) J. phosphorylated, we cannot rule out the possibility that Biol. Chem. 258, 8206-8214. ElllOkDa is the phosphorylated species. However, for this to be 8. Pickart, C. M. & Rose, I. A. (1985)J. Bio. Chem. 260,1573-1581. true, only a small proportion of ElllokDa would be phosphor- 9. Haas, A. L., Warms, J. V. B., Hershko, A. & Rose, I. A. (1982) ylated (Fig. 4), and phosphorylated EllolkDa would comigrate J. Biol. Chem. 257, 2543-2548. perfectly with E11i7kDa in our SDS/PAGE system (Fig. 1). 10. Haas, A. L. & Rose, I. A. (1982) J. Biol. Chem. 257,10329-10337. The existence of two isoforms of El suggests the possibility 11. Heller, H. & Hershko, A. (1990) J. Bio. Chem. 265, 6532-6535. that individual El isoforms may be targets of metabolic 12. Jentsch, S., Seufert, W., Sommer, T. & Reins, H.-A. (1990) Trends and could a role in the differential Biol. Sci. 15, 195-198. regulation play regulation 13. Hatfield, P. M. & Vierstra, R. D. (1992) J. Bio. Chem. 267, of ubiquitin conjugation to specific target proteins. That El is 14799-14803. phosphorylated in the cell suggests a possible mechanism for 14. Cook, J. C. & Chock, P. B. (1992) J. Biol. Chem. 267, 24315-24321. regulating ubiquitin conjugation through El. 15. Carlson, N., Rogers, S. & Rechsteiner, M. (1987) J. CellBio. 104, On a molecular level, phosphorylation of El could alter the 547-555. efficiency of ubiquitin activation, thus affecting the pathway 16. Jabben, M., Shanklin, J. & Vierstra, R. D. (1989) J. Biol. Chem. downstream from this isoform. Kong and Chock (18) reported 264, 4998-5005. that in vitro phosphorylation of El (from rabbit reticulocytes) by 17. Glotzer, M., Murray, A. W. & Kirschner, M. W. (1991) Nature protein kinase C enhanced exchange 2-fold. (London) 349, 132-138. ATP/PPi activity 18. Kong, S.-K & Chock, P. B. (1992)J. Biol. Chem. 267, 14189-14192. However, phorbol ester has little effect on the level of phosphor- 19. Cook, J. C. & Chock, P. B. (1991) Biochem. Biophys. Res. Com- ylation (unpublished results), suggesting that protein kinase C mun. 174, 564-571. may not be responsible for the observed phosphorylation of El 20. Harlow, E. & Lane, D. (1988) Antibodies: A Laboratory Manual in living cells. Phosphorylation could also inhibit or enhance (Cold Spring Harbor Lab. Press, Plainview, NY). physical interactions between El and other macromolecules, such 21. Laemmli, U. K (1970) Nature (London) 227, 680-685. as the E2 isoenzymes. It is also possible that phosphorylation of 22. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl. Acad. El may affect its interaction with different isozymes of E2 Sci. USA 76, 4350-4354. differently. Since each isozyme of E2 appears to have a preferred 23. Cooper, J. A., Sefton, B. M. & Hunter, T. (1983) Methods substrate Enzymol. 99, 387-402. protein for ubiquitin conjugation (12), El phosphory- 24. Grenfell, S. J., Trausch, J. S., Handley-Gearhart, P., Ciechanover, A. lation could be a mechanism for differential regulation of ubiq- & Schwartz, A. L. (1992) Mol. Biol. Cell Suppi. 3, 178 (abstr.). uitin conjugation to specific target proteins. Intermolecular in- 25. Nigg, E. A., Krek, W. & Peter, M. (1991) Cold Spring Harbor teractions between El and as yet unidentified regulators could Symp. Quant. Biol. 56, 539-547. also be affected by phosphorylation. 26. Kennelly, P. J. & Krebs, E. G. (1991)J Biol. Chem. 266,15555-15558. Downloaded by guest on September 26, 2021