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• W2014149852 abstract "The cyclosome/anaphase-promoting complex is a multisubunit ubiquitin ligase that targets for degradation mitotic cyclins and some other cell cycle regulators in exit from mitosis. It becomes enzymatically active at the end of mitosis. The activation of the cyclosome is initiated by its phosphorylation, a process necessary for its conversion to an active form by the ancillary protein Cdc20/Fizzy. Previous reports have implicated either cyclin-dependent kinase 1-cyclin B or polo-like kinase as the major protein kinase that directly phosphorylates and activates the cyclosome. These conflicting results could be due to the use of partially purified cyclosome preparations or of immunoprecipitated cyclosome, whose interactions with protein kinases or ancillary factors may be hampered by binding to immobilized antibody. To examine this problem, we have purified cyclosome from HeLa cells by a combination of affinity chromatography and ion exchange procedures. With the use of purified preparations, we found that both cyclin-dependent kinase 1-cyclin B and polo-like kinase directly phosphorylated the cyclosome, but the pattern of the phosphorylation of the different cyclosome subunits by the two protein kinases was not similar. Each protein kinase could restore only partially the cyclin-ubiquitin ligase activity of dephosphorylated cyclosome. However, following phosphorylation by both protein kinases, an additive and nearly complete restoration of cyclin-ubiquitin ligase activity was observed. It is suggested that this joint activation may be due to the complementary phosphorylation of different cyclosome subunits by the two protein kinases. The cyclosome/anaphase-promoting complex is a multisubunit ubiquitin ligase that targets for degradation mitotic cyclins and some other cell cycle regulators in exit from mitosis. It becomes enzymatically active at the end of mitosis. The activation of the cyclosome is initiated by its phosphorylation, a process necessary for its conversion to an active form by the ancillary protein Cdc20/Fizzy. Previous reports have implicated either cyclin-dependent kinase 1-cyclin B or polo-like kinase as the major protein kinase that directly phosphorylates and activates the cyclosome. These conflicting results could be due to the use of partially purified cyclosome preparations or of immunoprecipitated cyclosome, whose interactions with protein kinases or ancillary factors may be hampered by binding to immobilized antibody. To examine this problem, we have purified cyclosome from HeLa cells by a combination of affinity chromatography and ion exchange procedures. With the use of purified preparations, we found that both cyclin-dependent kinase 1-cyclin B and polo-like kinase directly phosphorylated the cyclosome, but the pattern of the phosphorylation of the different cyclosome subunits by the two protein kinases was not similar. Each protein kinase could restore only partially the cyclin-ubiquitin ligase activity of dephosphorylated cyclosome. However, following phosphorylation by both protein kinases, an additive and nearly complete restoration of cyclin-ubiquitin ligase activity was observed. It is suggested that this joint activation may be due to the complementary phosphorylation of different cyclosome subunits by the two protein kinases. anaphase-promoting complex cyclin-dependent kinase dithiothreitol polo-like kinase A large, multisubunit complex, known as the cyclosome (1Sudakin V. Ganoth D. Dahan A. Heller H. Hershko J. Luca F.C. Ruderman J.V. Hershko A. Mol. Biol. Cell. 1995; 6: 185-198Crossref PubMed Scopus (641) Google Scholar) or anaphase-promoting complex (APC1; Ref. 2King R.W. Peters J.M. Tugendreich S. Rolfe M. Hieter P. Kirschner M.W. Cell. 1995; 81: 279-288Abstract Full Text PDF PubMed Scopus (825) Google Scholar), plays a key role in the control of the eukaryotic cell cycle. It was discovered as a ubiquitin-protein ligase that targets mitotic cyclins for destruction and whose action is essential for exit from mitosis (1Sudakin V. Ganoth D. Dahan A. Heller H. Hershko J. Luca F.C. Ruderman J.V. Hershko A. Mol. Biol. Cell. 1995; 6: 185-198Crossref PubMed Scopus (641) Google Scholar, 2King R.W. Peters J.M. Tugendreich S. Rolfe M. Hieter P. Kirschner M.W. Cell. 1995; 81: 279-288Abstract Full Text PDF PubMed Scopus (825) Google Scholar, 3Irniger S. Piatti S. Michaelis C. Nasmyth K. Cell. 1995; 81: 269-278Abstract Full Text PDF PubMed Scopus (472) Google Scholar). Subsequent studies have shown that it is also involved in the degradation of inhibitors of sister chromatid separation known as securins and of some other cell cycle regulatory proteins that are destroyed at the end of mitosis (reviewed in Refs. 4Zachariae W. Nasmyth K. Genes Dev. 1999; 13: 2039-2058Crossref PubMed Scopus (573) Google Scholar and 5Morgan D.O. Nat. Cell Biol. 1999; 1: E47-E53Crossref PubMed Scopus (304) Google Scholar). The ubiquitin ligase activity of the cyclosome/APC itself is intricately regulated. It is inactive in the S and G2 phases of the cell cycle and becomes active in mitosis. The activation of the cyclosome in mitosis is initiated by its phosphorylation, a process necessary for its subsequent conversion to the active form by the ancillary protein Cdc20/Fizzy (6Lahav-Baratz S. Sudakin V. Ruderman J.V. Hershko A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9303-9307Crossref PubMed Scopus (156) Google Scholar, 7Shteinberg M. Protopopov Y. Listovsky T. Brandeis M. Hershko A. Biochem. Biophys. Res. Commun. 1999; 260: 193-198Crossref PubMed Scopus (114) Google Scholar, 8Rudner A.D. Murray A.W. J. Cell Biol. 2000; 149: 1377-1390Crossref PubMed Scopus (241) Google Scholar, 9Kramer E.R. Scheuringer N. Podtelejnikov A.V. Mann M. Peters J.-M. Mol. Biol. Cell. 2000; 11: 1555-1569Crossref PubMed Scopus (358) Google Scholar). Cdc20 is the target for inhibition of the cyclosome/APC by the spindle checkpoint system, a surveillance mechanism that monitors the correct attachment of chromosomes to the mitotic spindle (reviewed in Refs. 10Amon A. Curr. Opin. Genet. Dev. 1999; 9: 69-75Crossref PubMed Scopus (326) Google Scholarand 11Shah J.V. Cleveland D.W. Cell. 2000; 103: 997-1000Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). While the role of cyclosome phosphorylation in its mitotic activation by Cdc20 is well established, there is some confusion concerning the identity of the protein kinases involved in this process. Using partially purified preparations of cyclosomes from clam oocytes (6Lahav-Baratz S. Sudakin V. Ruderman J.V. Hershko A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9303-9307Crossref PubMed Scopus (156) Google Scholar, 7Shteinberg M. Protopopov Y. Listovsky T. Brandeis M. Hershko A. Biochem. Biophys. Res. Commun. 1999; 260: 193-198Crossref PubMed Scopus (114) Google Scholar) or cultured HeLa cells (12Yudkovsky Y. Shteinberg M. Listovsky T. Brandeis M. Hershko A. Biochem. Biophys. Res. Commun. 2000; 271: 299-304Crossref PubMed Scopus (98) Google Scholar), we have shown that the major mitotic protein kinase, Cdk1-cyclin B, activates cyclosome derived from interphase cells or mitotic cyclosome that has been inactivated by phosphatase treatment. However, since these studies were done with partially purified cyclosome preparations, indirect effects such as a cascade of protein kinases initiated by the action of Cdk1-cyclin B could not be ruled out. Another candidate is polo-like kinase (Plk), known as Cdc5 in S. cerevisiae and Plx1 inXenopus, which is required at several stages of the cell cycle, including in exit from mitosis (reviewed in Ref. 13Nigg E.A. Curr. Opin. Cell Biol. 1998; 10: 776-783Crossref PubMed Scopus (308) Google Scholar). Indeed, depletion or inactivation of Plx1 in extracts of frog eggs blocks the degradation of cyclin B (14Descombes P. Nigg E.A. EMBO J. 1998; 17: 1328-1335Crossref PubMed Scopus (195) Google Scholar). Kotani et al. (15Kotani S. Tugendreich S. Fujii M. Jorgensen P.-M. Watanabe N. Hoog C. Hieter P. Todokoro K. Mol. Cell. 1998; 1: 371-380Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 16Kotani S. Tanaka H. Yasuda H. Todokoro K. J. Cell Biol. 1999; 146: 791-800Crossref PubMed Scopus (119) Google Scholar) have reported that Cdk1-cyclin B does not phosphorylate directly cyclosome/APC but phosphorylates and activates Plk1, which on turn phosphorylates cyclosome. In these experiments, immunoprecipitated cyclosome from mammalian cells was used, whose interactions with other proteins could be hampered by binding to immobilized antibody. More recently, Rudner and Murray (8Rudner A.D. Murray A.W. J. Cell Biol. 2000; 149: 1377-1390Crossref PubMed Scopus (241) Google Scholar) have studied the roles of Cdk1-dependent phosphorylation of cyclosome/APC in the budding yeast. Three subunits of the yeast cyclosome (Cdc16, Cdc23, and Cdc27), which were directly phosphorylated by Cdk1-cyclin B in vitro, were also phosphorylated in vivo. Mutation of all potential Cdk phosphorylation sites of these three cyclosome subunits abolished their phosphorylation in vitro andin vivo and caused a delay in the degradation of substrates of Cdc20-activated cyclosome in vivo. It was also observed that Cdc5 (the yeast homologue of Plk) phosphorylated Cdc16 and Cdc27in vitro. However, the effects of cyclosome phosphorylation by Cdk1 or Cdc5 on its ubiquitin ligase activity have not been reported in this study (8Rudner A.D. Murray A.W. J. Cell Biol. 2000; 149: 1377-1390Crossref PubMed Scopus (241) Google Scholar). In the present investigation, we have examined this problem by the use of highly purified cyclosome preparation from HeLa cells. We find that both Cdk1-cyclin B and Plk1 can directly phosphorylate the cyclosome, but the pattern of the phosphorylation of the different cyclosome subunits by the two protein kinases is not similar. Each protein kinase activates only partially ubiquitin ligase activity, but an additive activation is observed following the phosphorylation of the cyclosome by both protein kinases. Ubiquitin from bovine erythrocytes, carboxymethyl bovine serum albumin, soybean trypsin inhibitor, dephosphorylated α-casein, staurosporine, and p-nitrophenyl phosphate were obtained from Sigma, and okadaic acid was from Roche Molecular Biochemicals. E1, E2-C, ubiquitin aldehyde, and Suc1-Sepharose were prepared as described earlier (17Sudakin V. Shteinberg M. Ganoth D. Hershko J. Hershko A. J. Biol. Chem. 1997; 272: 18051-18059Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). p13suc1 was expressed in bacteria and was purified as described (17Sudakin V. Shteinberg M. Ganoth D. Hershko J. Hershko A. J. Biol. Chem. 1997; 272: 18051-18059Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Rabbit anti-phosphoserine and anti-phosphothreonine were purchased from Zymed Laboratories Inc. and were used at a concentration of 1 μg/ml for immunoblotting. Monoclonal anti-Cdc27 antibody was purchased from Transduction Laboratories, and polyclonal antibody against human Cdc27 was raised in rabbits and was affinity-purified as described (18Tugendreich S. Tomkiel J. Earnshaw W. Hieter P. Cell. 1995; 81: 261-268Abstract Full Text PDF PubMed Scopus (314) Google Scholar). The affinity-purified α-Cdc27 antibody was covalently coupled to Protein A beads (Affi-Prep Protein A Support; Bio-Rad), as described (19Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar), at a concentration of ∼4 mg/ml beads. Protein kinase Cdk1-glutathioneS-transferase-Δ88-cyclin B (referred to as “Cdk1-cyclin B”) was prepared and purified as described (17Sudakin V. Shteinberg M. Ganoth D. Hershko J. Hershko A. J. Biol. Chem. 1997; 272: 18051-18059Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Units of Cdk1-cyclin B activity were as defined previously (17Sudakin V. Shteinberg M. Ganoth D. Hershko J. Hershko A. J. Biol. Chem. 1997; 272: 18051-18059Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The baculovirus expression vectors of His6-Plx1 (referred to as “Plk”) and of its enzymatically inactive mutant N172A were generously provided by Dr. W. Dunphy. They were expressed in insect cells as described (20Kumagai A. Dunphy W.G. Science. 1996; 273: 1377-1380Crossref PubMed Scopus (468) Google Scholar). This included incubation of insect cells with okadaic acid prior to harvesting, to phosphorylate and activate the kinase (20Kumagai A. Dunphy W.G. Science. 1996; 273: 1377-1380Crossref PubMed Scopus (468) Google Scholar). Both wild-type and mutant His6-Plk were purified by chromatography on nickel-agarose. Examination of these purified Plk preparations by SDS-polyacrylamide gel electrophoresis and Coomassie staining showed the expected ∼70-kDa proteins, both of which were >95% homogenous. Activity of Plk was assayed by phosphorylation of α-casein, as described (21Qian Y.-W. Li E. Erikson C. Maller J.L. Mol. Cell. Biol. 1998; 18: 4262-4271Crossref PubMed Scopus (212) Google Scholar). One unit of Plk activity is defined as the casein-phosphorylating activity of 25 pg of purified recombinant Plk. Extracts from nocodazole-arrested HeLa S3 cells were prepared as described previously (12Yudkovsky Y. Shteinberg M. Listovsky T. Brandeis M. Hershko A. Biochem. Biophys. Res. Commun. 2000; 271: 299-304Crossref PubMed Scopus (98) Google Scholar). Affinity chromatography on Suc1-Sepharose was carried out by a modification of a previously described procedure for the isolation of cyclosomes from mitotic clam oocytes (17Sudakin V. Shteinberg M. Ganoth D. Hershko J. Hershko A. J. Biol. Chem. 1997; 272: 18051-18059Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Extracts form mitotic HeLa cells were first incubated with ATP to hyperphosphorylate cyclosomes by endogenous mitotic protein kinases. The reaction mixture contained the following in a volume of 4 ml: 250 mm HEPES-NaOH (pH 7.2), 5 mm MgCl2, 1 mm DTT, 2.5 mm ATP, 50 mm phosphocreatine, 500 μg/ml creatine phosphokinase, ∼20 mg of protein extract (100,000 × g supernatant) from mitotically arrested HeLa cells, and 1 μm okadaic acid. Following incubation at 30 °C for 60 min, the sample was mixed with 1 ml of Suc1-Sepharose beads (approximately 14 mg Suc1/ml of beads) that had been equilibrated with 20 mm Tris-HCl (pH 7.2) and 1 mm DTT (Buffer A). The sample was mixed with beads for 1 h at room temperature and then was transferred to a column (0.7-cm diameter). All subsequent operations were at 0–4 °C. The column was washed with 45 ml of Buffer A that contained 300 mm KCl and then with 15 ml of Buffer A. Cyclosome was eluted from Suc1-Sepharose beads with 20 ml of Buffer A that contained 50 mmp-nitrophenyl phosphate and 0.2 mg/ml soybean trypsin inhibitor. The eluate was concentrated to ∼1 ml by centrifuge ultrafiltration (Centriprep 10; Amicon), diluted 10-fold with Buffer A that contained 20% (v/v) glycerol, and concentrated again to a volume of ∼1 ml. Recovery of activity in this step was about 30%. The preparation was further purified by ion exchange chromatography on MonoQ as follows. A sample of 2 ml of the affinity eluate, containing about 200,000 units of cyclosome activity (see below), was applied to a MonoQ HR 5/5 column (Amersham Biosciences), which was equilibrated with 50 mm Tris-HCl (pH 7.3), 100 mm NaCl, and 1 mm DTT. The column was washed with 15 ml of the above buffer and then was subjected to a linear gradient of NaCl (100–700 mm) in the same buffer, for 35 min at a flow rate of 1 ml/min. Fractions of 1 ml were collected into tubes containing 0.2 mg of soybean trypsin inhibitor carrier protein. Fractions were concentrated by centrifuge ultrafiltration with Centricon-30 concentrators (Amicon), diluted 10-fold in Buffer A, and concentrated again to a volume of 100 μl. Glycerol was added to 20% (v/v). The central peak fractions of the purified cyclosome preparation were pooled and were stored at −70 °C in small samples. Recovery of activity in this step was around 25–30%. Reaction mixtures contained the following in a volume of 10 μl: 40 mmTris-HCl (pH 7.6), 1 mg/ml carboxymethyl bovine serum albumin, 1 mm DTT, 5 mm MgCl2, 10 mm phosphocreatine, 50 μg/ml creatine phosphokinase, 0.5 mm ATP, 50 μm ubiquitin, 1 μmubiquitin aldehyde, 1 pmol of E1, 5 pmol of E2-C, 1 μmokadaic acid, 1–2 pmol of 125I-labeled cyclin B-(13–91)/protein A (referred to as 125I-cyclin; 1–2 × 105 cpm), source of cyclosome as specified, and 0.4 μl of Cdc20/Fizzy produced by in vitro translation in reticulocyte lysate, as described (7Shteinberg M. Protopopov Y. Listovsky T. Brandeis M. Hershko A. Biochem. Biophys. Res. Commun. 1999; 260: 193-198Crossref PubMed Scopus (114) Google Scholar). Following incubation at 30 °C for 1 h, samples were subjected to electrophoresis on a 12.5% polyacrylamide-SDS gel. Results were quantified by PhosphorImager and were expressed as the percentage of125I-cyclin converted to ubiquitin conjugates. A unit of activity is defined as that converting 1% of 125I-cyclin to ubiquitinylated derivatives under the above described assay conditions, in the range of assay linear with cyclosome concentration. Reaction mixtures contained the following in a volume of 10 μl: 40 mm Tris-HCl (pH 7.6), 1 mm DTT, 10% (v/v) glycerol, 2 mg/ml soybean trypsin inhibitor, 0.1 mm MnCl2, 3–7 μl of purified cyclosome (containing ∼500–700 units of activity), and 10 units/μl λ phosphatase (New England Biolabs). Following incubation at 30 °C for 30 min, phosphatase action was stopped by the addition of EGTA to a final concentration of 10 mm. To rephosphorylate cyclosome, [γ-32P]ATP (1 mm, ∼200 cpm/pmol) was added, along with 5 mm MgCl2 10 μm p13suc1 and protein kinase as specified. Following a second incubation at 30 °C for 30 min, protein kinase action was stopped by the addition of staurosporine (10 μm). We have previously observed that incubation of partially purified preparations of cyclosome from clam oocytes or human cells with protein kinase Cdk1-cyclin B converted them to phosphorylated forms that could be activated by Cdc20/Fizzy (7Shteinberg M. Protopopov Y. Listovsky T. Brandeis M. Hershko A. Biochem. Biophys. Res. Commun. 1999; 260: 193-198Crossref PubMed Scopus (114) Google Scholar, 12Yudkovsky Y. Shteinberg M. Listovsky T. Brandeis M. Hershko A. Biochem. Biophys. Res. Commun. 2000; 271: 299-304Crossref PubMed Scopus (98) Google Scholar). However, since these experiments were carried out with partially purified preparations, it was possible that Cdk1-cyclin B phosphorylated and activated another protein kinase, which in turn phosphorylated the cyclosome. Indeed, it has been suggested that Cdk-dependent phosphorylation of Polo-like kinase 1 (Plk1) activates the latter to phosphorylate the cyclosome (15Kotani S. Tugendreich S. Fujii M. Jorgensen P.-M. Watanabe N. Hoog C. Hieter P. Todokoro K. Mol. Cell. 1998; 1: 371-380Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 16Kotani S. Tanaka H. Yasuda H. Todokoro K. J. Cell Biol. 1999; 146: 791-800Crossref PubMed Scopus (119) Google Scholar). To examine this problem, we have highly purified cyclosome/APC from HeLa cells by a combination of affinity chromatography and fast protein liquid chromatography procedures. We preferred this type of purification to immunoprecipitation, as done by other investigators (8Rudner A.D. Murray A.W. J. Cell Biol. 2000; 149: 1377-1390Crossref PubMed Scopus (241) Google Scholar, 9Kramer E.R. Scheuringer N. Podtelejnikov A.V. Mann M. Peters J.-M. Mol. Biol. Cell. 2000; 11: 1555-1569Crossref PubMed Scopus (358) Google Scholar, 15Kotani S. Tugendreich S. Fujii M. Jorgensen P.-M. Watanabe N. Hoog C. Hieter P. Todokoro K. Mol. Cell. 1998; 1: 371-380Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 22Yu G. Fang H. Kirschner M.W. Mol. Cell. 1998; 2: 163-171Abstract Full Text Full Text PDF PubMed Google Scholar), because immunoprecipitated cyclosome/APC has very low enzymatic activity, and its interactions with protein kinases or other proteins may be sterically hindered by binding to immobilized antibody. Affinity purification of HeLa cyclosomes on p13suc1-Sepharose was adapted from a procedure developed previously for mitotic clam oocyte cyclosomes (17Sudakin V. Shteinberg M. Ganoth D. Hershko J. Hershko A. J. Biol. Chem. 1997; 272: 18051-18059Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The cell cycle regulatory protein p13suc1 contains, in addition to its Cdk binding site, also a phosphate-binding site (23Pines J. Curr. Biol. 1996; 6: 1399-1402Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) that preferentially binds proteins phosphorylated on Ser/Thr-Pro motifs (24Landrieu I. Odaert B. Wieruszeski J.M. Drobecq H. Rousselot-Pailley P. Inze D. Lippens G. J. Biol. Chem. 2001; 276: 1434-1438Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Mitotically phosphorylated cyclosomes bind strongly to Suc1-Sepharose and can be eluted with a phosphate-containing compound such as p-nitrophenyl phosphate (17Sudakin V. Shteinberg M. Ganoth D. Hershko J. Hershko A. J. Biol. Chem. 1997; 272: 18051-18059Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). HeLa cells were arrested in mitosis with nocodazole, and extracts of these cells were further incubated with MgATP and okadaic acid (see “Experimental Procedures”), to hyperphosphorylate cyclosome subunits. Affinity chromatography on Suc1-Sepharose resulted in a 50–70-fold enrichment of mitotic cyclosomes from HeLa cells with a recovery of ∼30% of activity. The eluate of the affinity column, which contains many other phosphorylated proteins, was subjected to fast protein liquid chromatography ion exchange chromatography on a MonoQ column. As shown in Fig. 1A, phosphorylated cyclosome bound strongly to the anion exchange column and was eluted at a rather high salt concentration (∼500–520 mm NaCl). Chromatography on MonoQ separated the cyclosome from most other proteins from the previous purification step, which were eluted at lower salt concentrations. Thus, MonoQ chromatography separated the cyclosome essentially completely from residual Cdk1-cyclin B and Plk that were eluted at around 320 and 400 mm NaCl, respectively (data not shown). Several proteins in the molecular mass region of 60–220 kDa eluted in coincidence with the peak of cyclosome activity in fractions 39–40 (Fig. 1B). As shown and discussed in detail below, these bands are subunits of the cyclosome/APC. To examine whether all proteins present in the purified preparation are indeed subunits of the cyclosome, it was necessary to subject it to phosphatase treatment, since several phosphorylated subunits of the mitotic cyclosome have retarded and somewhat heterogeneous electrophoretic migration. Treatment of purified mitotic cyclosome preparation with λ phosphatase effectively dephosphorylated all subunits, as shown by the loss of all immunoreactivity with a mixture of anti-phosphoserine and anti-phosphothreonine antibodies (Fig.1C, lane 2). In the untreated sample (Fig. 1C, lane 1), two prominently phosphorylated proteins with molecular mass of ∼125 and ∼70 kDa, were tentatively identified as phosphorylated Cdc27 and Cdc16, respectively. Silver staining showed that phosphatase treatment caused the conversion of four proteins to sharper bands of markedly more rapid electrophoretic migration (Fig. 1D). These were identified as APC1, Cdc27, Cdc16, and Cdc23 (lane 2), by the exact correspondence of their molecular mass to those reported for these subunits of unphosphorylated human cyclosome/APC (4Zachariae W. Nasmyth K. Genes Dev. 1999; 13: 2039-2058Crossref PubMed Scopus (573) Google Scholar, 25Peters J.-M. Exp. Cell Res. 1999; 248: 339-349Crossref PubMed Scopus (106) Google Scholar). Other protein bands present in the purified, phosphatase-treated cyclosome preparation were identified as APC2, APC5, and APC7 (Fig.1D, lane 2), again by the exact correspondence of their molecular mass to those of subunits of the human cyclosome/APC (4Zachariae W. Nasmyth K. Genes Dev. 1999; 13: 2039-2058Crossref PubMed Scopus (573) Google Scholar, 25Peters J.-M. Exp. Cell Res. 1999; 248: 339-349Crossref PubMed Scopus (106) Google Scholar). Thus, phosphatase treatment of the purified cyclosome preparation allowed us to conclude that the preparation is close to homogeneity, since all protein bands encompass those expected to be present in human cyclosome/APC. We next asked whether purified, dephosphorylated cyclosome preparation can be rephosphorylated by protein kinases Cdk1-cyclin B and/or Plk1. For this purpose, purified cyclosome preparation was subjected to treatment with λ phosphatase, as described above, and then phosphatase action was terminated by the addition of EGTA. This chelator effectively removes the Mn2+ ion, which is required for the activity of λ phosphatase (26Zhuo S. Clemens J.C. Hakes D.J. Barford D. Dixon J.E. J. Biol. Chem. 1993; 268: 17754-17761Abstract Full Text PDF PubMed Google Scholar). Subsequently, dephosphorylated cyclosome was incubated with [γ-32P]ATP, in the presence of protein kinase Cdk1-cyclin B, Plk, or both. All incubations contained p13suc1, since this protein was shown to be required for the action of Cdk1-cyclin B to phosphorylate and activate the cyclosome (27Patra D. Dunphy W.G. Genes Dev. 1998; 12: 2549-2559Crossref PubMed Scopus (121) Google Scholar, 28Shteinberg M. Hershko A. Biochem. Biophys. Res. Commun. 1999; 257: 12-18Crossref PubMed Scopus (36) Google Scholar). Samples were precipitated with a polyclonal antibody directed against Cdc27 and were resolved by SDS-polyacrylamide gel electrophoresis. Immunoprecipitation of cyclosomes was necessary to remove labeled, “autophosphorylated” cyclin B and Plk1 proteins. Without added protein kinase, there was no significant labeling of any protein band (data not shown), indicating the absence of protein kinases in the purified cyclosome preparation. Following incubation with Cdk1-cyclin B, several protein bands became labeled with32P-phosphate, most prominently a broad band that corresponds to the migration position of phosphorylated Cdc27 (Fig.2A, lane 1). The identification of this band was verified by immunoblotting with a monoclonal antibody directed against Cdc27. As shown in Fig. 2B, lanes 2 and3, the incubation of purified, phosphatase-treated cyclosome preparation with Cdk1-cyclin B caused a marked retardation in electrophoretic mobility of the Cdc27 subunit. Cdk1-cyclin B also caused the phosphorylation of some other cyclosome subunits. As opposed to the robust phosphorylation of Cdc27 by Cdk1-cyclin B, this protein kinase phosphorylated to a much lesser degree some lower molecular mass subunits, such as a ∼70-kDa protein that corresponds to phosphorylated Cdc16 (Fig. 2A,lane 1). This is in contrast to the marked phosphorylation of the 70-kDa band (relative to that of Cdc27) in “naturally” phosphorylated, purified cyclosome preparation (Fig.1C, lane 1). These observations suggested that some other protein kinase(s) may be involved in the phosphorylation of Cdc16 and possibly of some other subunits of the cyclosome. In fact, incubation of purified, dephosphorylated cyclosome with Plk1 caused a much more prominent phosphorylation of Cdc16 and of a lower molecular mass subunit that corresponds to the expected position of Cdc23 (Fig. 2A, lane 2). By contrast, Cdc27 was phosphorylated by Plk1 to a much lesser degree, as shown either by the low incorporation of [32P]phosphate (Fig. 2A, lane 2) or by the less prominent electrophoretic mobility shift of the Cdc27 protein (Fig. 2B, lane 4). It thus seems that both protein kinases phosphorylate the same subunits of the cyclosome, but the relative degree of the phosphorylation of the different subunits is quite dissimilar. In addition, protein kinase Cdk1-cyclin B phosphorylates several proteins at the high molecular mass region of 200–250 kDa, which may represent different phosphorylated forms of APC1 (Fig. 2A,lane 1). These high molecular mass proteins are not phosphorylated significantly by Plk (Fig. 2A,lane 2). Incubation of dephosphorylated cyclosome with both protein kinases showed a phosphorylation pattern of the different subunits of the cyclosome expected by the additive action of the two kinases (Fig. 2A, lane 3). We have next examined the effects of cyclosome phosphorylation by the two protein kinases on its cyclin-ubiquitin ligase activity in the presence of Cdc20. Purified, dephosphorylated cyclosomes were incubated with various protein kinases or their combination, and then protein kinase action was stopped by the addition of staurosporine. Subsequently, Cdc20 was added, and the cyclin-ubiquitin ligation activity of cyclosomes was determined in a further incubation. Using partially purified cyclosome preparations, we have previously observed nearly complete restoration of cyclin-ubiquitin ligase activity by protein kinase Cdk1-cyclin B (6Lahav-Baratz S. Sudakin V. Ruderman J.V. Hershko A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9303-9307Crossref PubMed Scopus (156) Google Scholar, 7Shteinberg M. Protopopov Y. Listovsky T. Brandeis M. Hershko A. Biochem. Biophys. Res. Commun. 1999; 260: 193-198Crossref PubMed Scopus (114) Google Scholar, 12Yudkovsky Y. Shteinberg M. Listovsky T. Brandeis M. Hershko A. Biochem. Biophys. Res. Commun. 2000; 271: 299-304Crossref PubMed Scopus (98) Google Scholar). With the purified preparation, we now find that Cdk1-cyclin B significantly increases the activity of dephosphorylated cyclosome. However, restoration of activity was only partial and did not exceed ∼40% of the control value even at high concentrations of Cdk1-cyclin B (Fig.3A). Incubation with Plk also significantly stimulated cyclin-ubiquitin ligase activity, but the extent of maximal stimulation was even less than that observed with Cdk1-cyclin B and did not exceed ∼20% of the control value even at high concentrations of Plk (Fig. 3A). When dephosphorylated cyclosome was incubated with increasing concentrations of Cdk1-cyclin B in the presence of a constant amount of Plk, an additive activation could be seen, which greatly exceeded the limits observed with each separate protein kinase. Thus, up to 75% of cyclin-ubiquitin ligation activity was restored following incubation of dephosphorylated cyclosome with both Cdk1-cyclin B and Plk1 (Fig. 3B). This joint action of Cdk1-cyclin B and Plk required protein kinase activity, since restoration of cyclosome activity was completely prevented by the addition of the protein kinase inhibitor staurosporine (10 μm), prior to the supplementation of the two protein kinases (data not shown). Other control experiments showed that a similar preparation of catalytically inactive (N172A) mutant of Plk, when added in concentrations similar to those of wild-type Plk, had no significant action on either the phosphorylation or activation of dephosphorylated cyclosome (data not shown). This suggests that the observed effects of Plk are not due to some contaminating kinases, because such kinases would also contaminate the N172A mutant of Plk. A possible explanation for the joint action of the two protein kinases is that Cdk1-cyclin B phosphorylates and activates Plk, which in turn phosphorylates the cyclosome, as suggested by Kotani et al. (15Kotani S. Tugendreich S. Fujii M. Jorgensen P.-M. Watanabe N. Hoog C. Hieter P. Todokoro K. Mol. Cell. 1998; 1: 371-380Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 16Kotani S. Tanaka H. Yasuda H. Todokoro K. J. Cell Biol. 1999; 146: 791-800Crossref PubMed Scopus (119) Google Scholar). However, several observations argue against this possibility. (a) While these authors have used bacterially expressed, unphosphorylated Plk1, we have used enzymatically active, phosphorylated Plk1 produced in baculovirus-infected insect cells subjected to okadaic acid treatment (see “Experimental Procedures”). This preparation phosphorylates well at least some subunits of the cyclosome/APC (Fig. 2A). (b) casein is phosphorylated by polo-like kinases (21Qian Y.-W. Li E. Erikson C. Maller J.L. Mol. Cell. Biol. 1998; 18: 4262-4271Crossref PubMed Scopus (212) Google Scholar) but not by Cdk1-cyclin B. We find that phosphatase treatment significantly reduced casein kinase activity of Plk1. However, subsequent incubation of this preparation with Cdk1-cyclin B did not restore casein kinase activity (Table I). (c) Qian et al. (29Qian Y.-W. Erikson E. Maller J.L. Mol. Cell. Biol. 1999; 19: 8625-8632Crossref PubMed Google Scholar) have identified two amino acid residues inXenopus Plk1, Ser-128 and Thr-201, as the phosphorylation sites responsible for the activation of this enzyme. These highly conserved phosphorylation sites are also present in human Plk1. Both residues are followed by a hydrophobic amino acid residue (and not by Pro) and thus cannot be targets for phosphorylation by a cyclin-dependent kinase. The cumulative evidence thus strongly indicates that the joint action of protein kinases Cdk1-cyclin B and Plk1 on cyclosome activity is not due to the activation of Plk1 by Cdk1. It seems more reasonable to assume that it is due to the complementary pattern by which the different subunits of the cyclosome are phosphorylated by each protein kinase (see “Discussion”).Table IProtein kinase Cdk1-cyclin B does not stimulate the activity of polo-like kinaseIncubation 1Incubation 2Casein phosphorylated, percentage of control%Plk100Cdk1-cyclin B2Plk + phosphatase24Plk + phosphataseCdk1-cyclin B15In the first incubation, 200 units/μl Plk were incubated (or not) with 10 units/μl λ phosphatase, under conditions similar to those described for phosphatase treatment of cyclosome (see “Experimental Procedures”). Phosphatase treatment was terminated by the addition of EGTA (10 mm) to all samples. In the second incubation, 100 units/μl of Cdk1-cyclin B were added along with [γ-32P]ATP (1 mm), MgCl2 (5 mm) and dephosphorylated α-casein (0.3 mg/ml), and incubation was continued for another 60 min at 30 °C. Samples were separated on a 12.5% polyacrylamide-SDS gel, and the incorporation of [32P]phosphate into α-casein was quantified by PhosphorImager analysis. Open table in a new tab In the first incubation, 200 units/μl Plk were incubated (or not) with 10 units/μl λ phosphatase, under conditions similar to those described for phosphatase treatment of cyclosome (see “Experimental Procedures”). Phosphatase treatment was terminated by the addition of EGTA (10 mm) to all samples. In the second incubation, 100 units/μl of Cdk1-cyclin B were added along with [γ-32P]ATP (1 mm), MgCl2 (5 mm) and dephosphorylated α-casein (0.3 mg/ml), and incubation was continued for another 60 min at 30 °C. Samples were separated on a 12.5% polyacrylamide-SDS gel, and the incorporation of [32P]phosphate into α-casein was quantified by PhosphorImager analysis. In this study, we have used highly purified preparations of cyclosome/APC from HeLa cells to define the roles of protein kinases Cdk1-cyclin B and Plk in the activation of its ubiquitin ligase activity. Following the last step of chromatography on MonoQ, the purified cyclosome preparation is well separated from most other proteins (Fig. 1) and has no detectable protein kinase or protein phosphatase activities. The purity of the preparation was indicated by the observation that following phosphatase treatment, the molecular mass of all proteins exactly corresponded to those of subunits of human cyclosome/APC (Fig. 1D). The development of this purification procedure was necessary because previous studies have used either partially purified preparations, in which indirect effects are possible, or immunoprecipitated cyclosome/APC preparations. We found that the cyclin-ubiquitin ligase activity of cyclosome immunoprecipitated by a procedure similar to that used by other authors (8Rudner A.D. Murray A.W. J. Cell Biol. 2000; 149: 1377-1390Crossref PubMed Scopus (241) Google Scholar, 15Kotani S. Tugendreich S. Fujii M. Jorgensen P.-M. Watanabe N. Hoog C. Hieter P. Todokoro K. Mol. Cell. 1998; 1: 371-380Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 22Yu G. Fang H. Kirschner M.W. Mol. Cell. 1998; 2: 163-171Abstract Full Text Full Text PDF PubMed Google Scholar) is 50–100-fold lower than that of our purified, soluble cyclosome preparations, when compared at similar levels of the Cdc27 subunit (data not shown). This estimate agrees with the amounts of HeLa cell extracts (1.5–3 mg of protein) used by other workers for immunoprecipitation (8Rudner A.D. Murray A.W. J. Cell Biol. 2000; 149: 1377-1390Crossref PubMed Scopus (241) Google Scholar, 22Yu G. Fang H. Kirschner M.W. Mol. Cell. 1998; 2: 163-171Abstract Full Text Full Text PDF PubMed Google Scholar), which yielded cyclin-ubiquitin ligation activity in immunoprecipitates comparable with that observed in ∼100-fold smaller amounts of crude extracts of HeLa cells (12Yudkovsky Y. Shteinberg M. Listovsky T. Brandeis M. Hershko A. Biochem. Biophys. Res. Commun. 2000; 271: 299-304Crossref PubMed Scopus (98) Google Scholar). It is thus possible that binding to immobilized antibody sterically hinders interactions of immunoprecipitated cyclosome/APC with ancillary proteins, substrates, or protein kinases. With the use of purified, dephosphorylated cyclosome preparations, we found that both protein kinases Cdk1-cyclin B and Plk directly phosphorylate different subunits of the cyclosome, but the pattern of phosphorylation of different subunits by the two protein kinases was not similar. Whereas Cdk1-cyclin B phosphorylated cyclosome subunits Cdc27 and APC1 to a much greater extent than did Plk, the opposite is the case with subunits Cdc16 and Cdc23 (Fig. 2). Each protein kinase can restore only partially cyclin-ubiquitin ligase activity of dephosphorylated cyclosome, to limits that cannot be exceeded even at high concentrations of the protein kinases (Fig. 3A). However, following phosphorylation by both Cdk1-cyclin B and Plk, an additive and nearly complete restoration of cyclin-ubiquitin ligase activity was observed (Fig. 3B). It appears reasonable to assume that this joint action is due to complementary phosphorylation of the different subunits of the cyclosome by the two protein kinases, probably at different phosphorylation sites. Alternative explanations, such as the activation of Plk by Cdk1-dependent phosphorylation (15Kotani S. Tugendreich S. Fujii M. Jorgensen P.-M. Watanabe N. Hoog C. Hieter P. Todokoro K. Mol. Cell. 1998; 1: 371-380Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 16Kotani S. Tanaka H. Yasuda H. Todokoro K. J. Cell Biol. 1999; 146: 791-800Crossref PubMed Scopus (119) Google Scholar), have not been confirmed (Table I). The present report clears up some of the confusion in the literature concerning the identity of the protein kinases involved in the activation of the cyclosome/APC at the end of mitosis. In agreement with our previous results with partially purified preparations (6Lahav-Baratz S. Sudakin V. Ruderman J.V. Hershko A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9303-9307Crossref PubMed Scopus (156) Google Scholar, 7Shteinberg M. Protopopov Y. Listovsky T. Brandeis M. Hershko A. Biochem. Biophys. Res. Commun. 1999; 260: 193-198Crossref PubMed Scopus (114) Google Scholar,12Yudkovsky Y. Shteinberg M. Listovsky T. Brandeis M. Hershko A. Biochem. Biophys. Res. Commun. 2000; 271: 299-304Crossref PubMed Scopus (98) Google Scholar) as well as with recent genetic and biochemical data of Rudner and Murray in yeast (8Rudner A.D. Murray A.W. J. Cell Biol. 2000; 149: 1377-1390Crossref PubMed Scopus (241) Google Scholar), it seems that Cdk1-cyclin B has a direct and major (Fig. 3A) role in the phosphorylation and activation of the cyclosome. This provides a negative feedback loop, by which Cdk1-cyclin B triggers its own inactivation at the end of mitosis. An additional regulatory mechanism is provided by synergistic action of Plk. It is notable that levels of Plk also oscillate in the cell cycle, with a peak in mitosis (13Nigg E.A. Curr. Opin. Cell Biol. 1998; 10: 776-783Crossref PubMed Scopus (308) Google Scholar). Thus, the timely activation of cyclosome/APC in exit from mitosis may be ensured by its complementary phosphorylation by both mitotic protein kinases. We thank Clara Segal for devoted technical assistance and W. Dunphy for the baculovirus expression vectors of wild-type and mutant His6-Plx1." @default.
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• W2014149852 title "The Cyclin-Ubiquitin Ligase Activity of Cyclosome/APC Is Jointly Activated by Protein Kinases Cdk1-Cyclin B and Plk" @default.
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• W2014149852 cites W1871272574 @default.
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• W2014149852 cites W1974310284 @default.
• W2014149852 cites W1974559056 @default.
• W2014149852 cites W1979983744 @default.
• W2014149852 cites W1985009222 @default.
• W2014149852 cites W1995011897 @default.
• W2014149852 cites W2003906429 @default.
• W2014149852 cites W2005717642 @default.
• W2014149852 cites W2012939857 @default.
• W2014149852 cites W2016274030 @default.
• W2014149852 cites W2033141310 @default.
• W2014149852 cites W2035759917 @default.
• W2014149852 cites W2036390681 @default.
• W2014149852 cites W2077278757 @default.
• W2014149852 cites W2078164898 @default.
• W2014149852 cites W2078611167 @default.
• W2014149852 cites W2084343743 @default.
• W2014149852 cites W2090017838 @default.
• W2014149852 cites W2106869142 @default.
• W2014149852 cites W2112854488 @default.
• W2014149852 cites W2114480956 @default.
• W2014149852 cites W2132300009 @default.
• W2014149852 cites W2138817040 @default.
• W2014149852 cites W2143105402 @default.
• W2014149852 cites W2146626810 @default.
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