HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 18, 15552-15557, May 3, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/18/15552    most recent M111476200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Golan, A. Articles by Hershko, A. Search for Related Content PubMed PubMed Citation Articles by Golan, A. Articles by Hershko, A. Social Bookmarking                      What's this?

The Cyclin-Ubiquitin Ligase Activity of Cyclosome/APC Is Jointly Activated by Protein Kinases Cdk1-Cyclin B and Plk^*

Amnon Golan, Yana Yudkovsky, and Avram Hershko

From the Unit of Biochemistry, the B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel

Received for publication, December 2, 2001, and in revised form, February 19, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A large, multisubunit complex, known as the cyclosome (1) or anaphase-promoting complex (APC¹; Ref. 2), 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 (1-3). 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. 4 and 5). 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 (6-9). 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. 10 and 11).

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 (6, 7) or cultured HeLa cells (12), 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 in Xenopus, which is required at several stages of the cell cycle, including in exit from mitosis (reviewed in Ref. 13). Indeed, depletion or inactivation of Plx1 in extracts of frog eggs blocks the degradation of cyclin B (14). Kotani et al. (15, 16) 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 (8) 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 and in 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 Cdc27 in vitro. However, the effects of cyclosome phosphorylation by Cdk1 or Cdc5 on its ubiquitin ligase activity have not been reported in this study (8).

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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Ubiquitin from bovine erythrocytes, carboxymethyl bovine serum albumin, soybean trypsin inhibitor, dephosphorylated -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 (17). p13^suc1 was expressed in bacteria and was purified as described (17). 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 (18). The affinity-purified -Cdc27 antibody was covalently coupled to Protein A beads (Affi-Prep Protein A Support; Bio-Rad), as described (19), at a concentration of ~4 mg/ml beads. Protein kinase Cdk1-glutathione S-transferase-88-cyclin B (referred to as "Cdk1-cyclin B") was prepared and purified as described (17). Units of Cdk1-cyclin B activity were as defined previously (17). 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 (20). This included incubation of insect cells with okadaic acid prior to harvesting, to phosphorylate and activate the kinase (20). 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 -casein, as described (21). One unit of Plk activity is defined as the casein-phosphorylating activity of 25 pg of purified recombinant Plk.

Purification of Cyclosome from Mitotically Arrested HeLa Cells-- Extracts from nocodazole-arrested HeLa S3 cells were prepared as described previously (12). 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 (17). 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 mM p-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%.

Assay of Cyclin-Ubiquitin Ligase Activity-- Reaction mixtures contained the following in a volume of 10 µl: 40 mM Tris-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 µM ubiquitin aldehyde, 1 pmol of E1, 5 pmol of E2-C, 1 µM okadaic 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 (7). 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 of ¹²⁵I-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.

Phosphatase Treatment of Purified Cyclosome and Rephosphorylation with [-³²P]ATP-- 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  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, [-³²P]ATP (1 mM, ~200 cpm/pmol) was added, along with 5 mM MgCl₂ 10 µM p13^suc1 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).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purification of Cyclosome/APC from Mitotic HeLa Cells-- 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 (7, 12). 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 (15, 16). 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 (8, 9, 15, 22), 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 p13^suc1-Sepharose was adapted from a procedure developed previously for mitotic clam oocyte cyclosomes (17). The cell cycle regulatory protein p13^suc1 contains, in addition to its Cdk binding site, also a phosphate-binding site (23) that preferentially binds proteins phosphorylated on Ser/Thr-Pro motifs (24). Mitotically phosphorylated cyclosomes bind strongly to Suc1-Sepharose and can be eluted with a phosphate-containing compound such as p-nitrophenyl phosphate (17). 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.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Purification of cyclosome/APC from mitotic HeLa cells and its dephosphorylation with  phosphatase. A, chromatography on MonoQ. Cyclosomes from mitotic HeLa cells were purified by affinity chromatography on Suc1-Sepharose followed by ion exchange chromatography on MonoQ, as described under "Experimental Procedures." Cyclin-ubiquitin ligation activity was assayed in 1-µl samples of MonoQ column fractions. B, 5-µl samples of the peak fractions from the same MonoQ column were separated on an 8% polyacrylamide-SDS gel and were stained with silver. Numbers at the top indicate column fractions, and numbers on the right show the position of molecular mass marker proteins (kDa). Protein bands that co-migrate with cyclosome activity are indicated by dots between fractions 39 and 40. The central peak fractions of cyclosome/APC, indicated at the top, were pooled and used for subsequent experiments. C, phosphatase treatment of purified cyclosome. Samples of 7 µl of purified cyclosome were treated (or not) with  phosphatase, as described under "Experimental Procedures," and then were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting with a mixture of anti-phosphoserine and anti-phosphothreonine antibodies. D, purified cyclosome preparation was treated (or not) with  phosphatase as in C, separated on 8% polyacrylamide-SDS gel, and stained with silver.  phosphatase (25 kDa) has run off the gel. The arrows on the left and right indicate tentative identification (according to molecular mass) of phosphorylated and dephosphorylated subunits of the cyclosome, respectively. Numbers on the right indicate the positions of molecular mass marker proteins (kDa).

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  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 (4, 25). 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 (4, 25). 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.

Phosphorylation of Different Subunits of the Cyclosome by Protein Kinases Cdk1-Cyclin B and Plk1-- 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  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  phosphatase (26). Subsequently, dephosphorylated cyclosome was incubated with [-³²P]ATP, in the presence of protein kinase Cdk1-cyclin B, Plk, or both. All incubations contained p13^suc1, since this protein was shown to be required for the action of Cdk1-cyclin B to phosphorylate and activate the cyclosome (27, 28). 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 with ³²P-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 and 3, 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).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Phosphorylation of different subunits of the cyclosome/APC by protein kinases Cdk1-cyclin B and Plk. A, incorporation of [32P]phosphate. Phosphatase treatment of purified cyclosome and rephosphorylation with [-32P]ATP were carried out as described under "Experimental Procedures." Where indicated, 1000 units of Cdk1-cyclin B or 2000 units of Plk were supplemented in the second incubation. Subsequently, samples were mixed with 10 µl of anti-Cdc20-protein A beads, at 4 °C for 2 h. The beads were washed four times with 1-ml portions of radioimmune precipitation buffer (19) and were eluted with electrophoresis sample buffer. The samples were subjected to electrophoresis on 8% polyacrylamide-SDS gel and radioautography. Numbers on the left show the positions of molecular mass marker proteins (kDa), and numbers on the right indicate tentative identification of phosphorylated cyclosome subunits. B, effects of phosphorylation by protein kinases Cdk1-cyclin B and Plk on electrophoretic migration of Cdc27 subunit of the cyclosome/APC. Phosphatase treatment and subsequent incubation with protein kinases are indicated at the top. Experimental conditions were as in A, except that unlabeled ATP was used in the rephosphorylation reaction. Samples were separated on an 8% polyacrylamide-SDS gel, transferred to nitrocellulose, and blotted with an anti-Cdc27 antibody. Numbers on the right indicate the migration position of molecular mass marker proteins (kDa).

Protein Kinases Cdk1-Cyclin B and Plk Jointly Activate the Cyclin-Ubiquitin Ligase Activity of the Cyclosome-- 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 (6, 7, 12). 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.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Joint activation of dephosphorylated cyclosome by protein kinases Cdk1-cyclin B and Plk. A, activation by each protein kinase. Purified cyclosome preparation from mitotic HeLa cells was subjected to phosphatase treatment as described under "Experimental Procedures." Following termination of phosphatase action with EGTA, samples were incubated with increasing concentrations of protein kinases Cdk1-cyclin B or Plk, as indicated, in the presence of unlabeled ATP. Protein kinase action was terminated with staurosporine, and then cyclin-ubiquitin ligase activity was assayed in the presence of Cdc20/Fizzy as described under "Experimental Procedures." Results are expressed as the percentage of the activity of a control treatment, which was subjected to similar incubations, but phosphatase action was prevented by the prior addition of EGTA, and protein kinases were not supplemented. B, additive activation by the two protein kinases. Incubation conditions were similar to those described in A, except that the indicated amounts of Cdk1-cyclin B were added in the absence (open squares) or presence (filled circles) of Plk (2000 units).

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. (15, 16). 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 (21) 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. (29) have identified two amino acid residues in Xenopus 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").

View this table: [in this window] [in a new window]   Table I Protein kinase Cdk1-cyclin B does not stimulate the activity of polo-like kinase In the first incubation, 200 units/µl Plk were incubated (or not) with 10 units/µl  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 [-32P]ATP (1 mM), MgCl2 (5 mM) and dephosphorylated -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 -casein was quantified by PhosphorImager analysis.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (8, 15, 22) 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 (8, 22), which yielded cyclin-ubiquitin ligation activity in immunoprecipitates comparable with that observed in ~100-fold smaller amounts of crude extracts of HeLa cells (12). 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 (15, 16), 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 (6, 7, 12) as well as with recent genetic and biochemical data of Rudner and Murray in yeast (8), 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 (13). Thus, the timely activation of cyclosome/APC in exit from mitosis may be ensured by its complementary phosphorylation by both mitotic protein kinases.

	ACKNOWLEDGEMENTS

We thank Clara Segal for devoted technical assistance and W. Dunphy for the baculovirus expression vectors of wild-type and mutant His₆-Plx1.

	FOOTNOTES

^* This work was supported by a grant from the United States-Israel Binational Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 9724-829-5344; Fax: 9724-853-5773; E-mail: hershko@tx.technion.ac.il.

Published, JBC Papers in Press, February 21, 2002, DOI 10.1074/jbc.M111476200

	ABBREVIATIONS

The abbreviations used are: APC, anaphase-promoting complex; Cdk, cyclin-dependent kinase; DTT, dithiothreitol; Plk, polo-like kinase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				E. Eytan, I. Braunstein, D. Ganoth, A. Teichner, J. C. Hittle, T. J. Yen, and A. Hershko Two different mitotic checkpoint inhibitors of the anaphase-promoting complex/cyclosome antagonize the action of the activator Cdc20 PNAS, July 8, 2008; 105(27): 9181 - 9185. [Abstract] [Full Text] [PDF]

				R. Rahal and A. Amon Mitotic CDKs control the metaphase-anaphase transition and trigger spindle elongation Genes & Dev., June 1, 2008; 22(11): 1534 - 1548. [Abstract] [Full Text] [PDF]

				A. M. Bentley, G. Normand, J. Hoyt, and R. W. King Distinct Sequence Elements of Cyclin B1 Promote Localization to Chromatin, Centrosomes, and Kinetochores during Mitosis Mol. Biol. Cell, December 1, 2007; 18(12): 4847 - 4858. [Abstract] [Full Text] [PDF]

				J.-Y. Huang, G. Morley, D. Li, and M. Whitaker Cdk1 phosphorylation sites on Cdc27 are required for correct chromosomal localisation and APC/C function in syncytial Drosophila embryos J. Cell Sci., June 15, 2007; 120(12): 1990 - 1997. [Abstract] [Full Text] [PDF]

				I. Braunstein, S. Miniowitz, Y. Moshe, and A. Hershko Inhibitory factors associated with anaphase-promoting complex/cylosome in mitotic checkpoint PNAS, March 20, 2007; 104(12): 4870 - 4875. [Abstract] [Full Text] [PDF]

				P. Marangos, E. W. Verschuren, R. Chen, P. K. Jackson, and J. Carroll Prophase I arrest and progression to metaphase I in mouse oocytes are controlled by Emi1-dependent regulation of APCCdh1 J. Cell Biol., January 1, 2007; 176(1): 65 - 75. [Abstract] [Full Text] [PDF]

				H.-J. Yoon, A. Feoktistova, J.-S. Chen, J. L. Jennings, A. J. Link, and K. L. Gould Role of Hcn1 and Its Phosphorylation in Fission Yeast Anaphase-promoting Complex/Cyclosome Function J. Biol. Chem., October 27, 2006; 281(43): 32284 - 32293. [Abstract] [Full Text] [PDF]

				F. Eckerdt and K. Strebhardt Polo-like kinase 1: target and regulator of anaphase-promoting complex/cyclosome-dependent proteolysis. Cancer Res., July 15, 2006; 66(14): 6895 - 6898. [Abstract] [Full Text] [PDF]

				M. Schmidt, H.-P. Hofmann, K. Sanders, G. Sczakiel, T. L. Beckers, and V. Gekeler Molecular alterations after Polo-like kinase 1 mRNA suppression versus pharmacologic inhibition in cancer cells. Mol. Cancer Ther., April 1, 2006; 5(4): 809 - 817. [Abstract] [Full Text] [PDF]

				E. Eytan, Y. Moshe, I. Braunstein, and A. Hershko Roles of the anaphase-promoting complex/cyclosome and of its activator Cdc20 in functional substrate binding PNAS, February 14, 2006; 103(7): 2081 - 2086. [Abstract] [Full Text] [PDF]

				W. Zhang, L. Fletcher, and R. J. Muschel The Role of Polo-like Kinase 1 in the Inhibition of Centrosome Separation after Ionizing Radiation J. Biol. Chem., December 30, 2005; 280(52): 42994 - 42999. [Abstract] [Full Text] [PDF]

				J. B. Casaletto, L. K. Nutt, Q. Wu, J. D. Moore, L. D. Etkin, P. K. Jackson, T. Hunt, and S. Kornbluth Inhibition of the anaphase-promoting complex by the Xnf7 ubiquitin ligase J. Cell Biol., April 11, 2005; 169(1): 61 - 71. [Abstract] [Full Text] [PDF]

				B. C. M. van de Weerdt, M. A. T. M. van Vugt, C. Lindon, J. J. W. Kauw, M. J. Rozendaal, R. Klompmaker, R. M. F. Wolthuis, and R. H. Medema Uncoupling Anaphase-Promoting Complex/Cyclosome Activity from Spindle Assembly Checkpoint Control by Deregulating Polo-Like Kinase 1 Mol. Cell. Biol., March 1, 2005; 25(5): 2031 - 2044. [Abstract] [Full Text] [PDF]

				A. Schmidt, P. I. Duncan, N. R. Rauh, G. Sauer, A. M. Fry, E. A. Nigg, and T. U. Mayer Xenopus polo-like kinase Plx1 regulates XErp1, a novel inhibitor of APC/C activity Genes & Dev., February 15, 2005; 19(4): 502 - 513. [Abstract] [Full Text] [PDF]

				D. V. Hansen, A. V. Loktev, K. H. Ban, and P. K. Jackson Plk1 Regulates Activation of the Anaphase Promoting Complex by Phosphorylating and Triggering SCF{beta}TrCP-dependent Destruction of the APC Inhibitor Emi1 Mol. Biol. Cell, December 1, 2004; 15(12): 5623 - 5634. [Abstract] [Full Text] [PDF]

				M. A. T. M. van Vugt, B. C. M. van de Weerdt, G. Vader, H. Janssen, J. Calafat, R. Klompmaker, R. M. F. Wolthuis, and R. H. Medema Polo-like Kinase-1 Is Required for Bipolar Spindle Formation but Is Dispensable for Anaphase Promoting Complex/Cdc20 Activation and Initiation of Cytokinesis J. Biol. Chem., August 27, 2004; 279(35): 36841 - 36854. [Abstract] [Full Text] [PDF]