Ubiquitin-dependent proteolysis of Cut2/Pds1 and Cyclin B is required for sister chromatid separation and exit from mitosis, respectively. Anaphase-promoting complex/cyclosome (APC) specifically ubiquitinates Cut2/Pds1 at metaphase–anaphase transition, and ubiquitinates Cyclin B in late mitosis and G1 phase. However, the exact regulatory mechanism of substrate-specific activation of mammalian APC with the right timing remains to be elucidated. We found that not only the binding of the activators Cdc20 and Cdh1 and the inhibitor Mad2 to APC, but also the phosphorylation of Cdc20 and Cdh1 by Cdc2-Cyclin B and that of APC by Polo-like kinase and cAMP-dependent protein kinase, regulate APC activity. The cooperation of the phosphorylation/dephosphorylation and the regulatory factors in regulation of APC activity may thus control the precise progression of mitosis.

Ubiquitin-mediated protein destruction plays a critical role in regulation of cell cycle progression, and is a particularly effective method for promoting unidirectional progression in the cell cycle because of its irreversibility (Murray et al. 1989; King et al. 1994, King et al. 1996; Hershko 1997; Peters 1998; Koepp et al. 1999; Morgan 1999). Proteolysis of the Cut2/Pds1, which triggers sister chromatid separation, is required for the metaphase–anaphase transition and degradation of mitotic cyclin, and Ase1 is essential for exit from mitosis (Glotzer and Murray, 1991; Holloway et al. 1993; Irniger et al. 1995; Cohen-Fix et al. 1996; Funabiki et al. 1996).

Anaphase-promoting complex/cyclosome (APC)1 functions as a cell cycle–regulated ubiquitin ligase that mediates destruction of these cell cycle regulatory factors by the proteasomes during mitosis. It is activated at metaphase–anaphase transition and remains active until late G1 phase (Amon et al. 1994; King et al. 1995; Lahav-Baratz et al. 1995; Sudakin et al. 1995; Tugendreich et al. 1995; Peters et al. 1996; Zachariae et al. 1998a; Yu et al. 1998). The APC is a 20S/36S particle, most of whose core subunits were recently identified (Yu et al. 1998; Zachariae et al. 1998a), but the precise regulatory mechanism of APC activation has remained unresolved.

The APC activity has been thought to be regulated by cell cycle–specific phosphorylation and dephosphorylation (King et al. 1995; Lahav-Baratz et al. 1995; Sudakin et al. 1995; Ishii et al. 1996; Peters et al. 1996; Yamashita et al. 1996; Patra and Dunphy 1998; Yu et al. 1998). It was reported that APC is controlled by Cdc2-Cyclin B (MPF) by reversible phosphorylation (Hershko et al. 1994; Lahav-Baratz et al. 1995; Sudakin et al. 1995; Shteinberg and Hershko 1999; Shteinberg et al. 1999). Recently, the specific kinases that regulate APC activity were identified. Polo-like kinase (Plk) (Glover et al. 1998), a homologue of Drosophila polo and budding yeast Cdc5, phosphorylates at least three APC subunits, APC1, APC3, and APC6, and activates APC (Charles et al. 1998; Descombes and Nigg 1998; Kotani et al. 1998; Shirayama et al. 1998), whereas cAMP-dependent protein kinase (PKA) phosphorylates APC1 and APC3 (Kotani et al. 1998) and suppresses APC activity (Ishii et al. 1996; Yamashita et al. 1996; Yamada et al. 1997; Kotani et al. 1998). Furthermore, PP1 has been found to be required for the activation of APC in metaphase–anaphase transition (Ishii et al. 1996). Therefore, it became clear that phosphorylation and dephosphorylation of APC plays an important role in regulation of APC activity during mitosis.

On the other hand, recent genetic and biochemical analyses in yeast, Drosophila, and Xenopus have indicated that APC is activated by the WD-repeat proteins, Cdc20/p55CDC/Fizzy (Cdc20) and Cdh1/Hct1/Fizzy-related (Cdh1), in a substrate-specific manner (Visintin et al. 1997; Schwab et al. 1997; Sigrist and Lehner 1997; Kallio et al. 1998; Fang et al. 1998a,Fang et al. 1998b; Kramer et al. 1998; Lorca et al. 1998; Zachariae et al. 1998b), whereas APC is inactivated by a spindle assembly checkpoint through Mad2 (He et al. 1997; Li et al. 1997; Fang et al. 1998b; Gorbsky et al. 1998; Hwang et al. 1998; Kallio et al. 1998; Kim et al. 1998). Very recently, it was reported that whereas Mad1, Mad2, Mad3/Bub1, and Bub3 suppress Cdc20-dependent APC activation, Bub2 localized in the spindle pole body regulates Cdh1-dependent APC activation (Alexandru et al. 1999; Fesquet et al. 1999; Fraschini et al. 1999). Furthermore, it was found that Cdc14, of which activity is regulated by Bub2/Byr4 and RENT complex (Alexandru et al. 1999; Shou et al. 1999; Visintin et al. 1999), dephosphorylates Cdh1/Hct1 and inactivates APC (Visintin et al. 1998). Therefore, APC activity is regulated by at least four distinct mechanisms: activation and inactivation by phosphorylation and dephosphorylation of APC itself; activation by the binding of substrate-specific activators Cdc20 and Cdh1 to APC; suppression of APC activity by the spindle assembly checkpoint through Mad family and Bub family; and regulation of APC activity by Bub2/RENT complex system. However, the precise regulatory mechanism of substrate-specific activation of mammalian APC with the right timing through these complicated mechanisms remains to be elucidated.

We found that not only the binding of the activators Cdc20 and Cdh1 and the inhibitor Mad2 to APC, but also the phosphorylation of Cdc20 and Cdh1 by MPF and that of APC by Plk and PKA, regulate the timing of APC activation. We discuss here the regulatory mechanism of APC activation.

Materials and Methods

cDNA Cloning and Plasmid Constructs

Human Cdc20 and Mad2 cDNA were prepared by reverse transcriptase–PCR. Two human expressed sequence tags homologous to Cdh1/Hct1 were used as probes for the screening of full-length human Cdh1 cDNA in a λgt10 human erythroleukemia K562 cDNA library. The T7- and His6-tagged Cdc20, Cdh1, and Mad2 in pET-23d were expressed in NovaBlue (DE3) in the presence of 0.5 mM isopropyl-β-d-thiogalactopyranoside at 25°C for 20 h. The cell lysates were solubilized (10 mM Tris, pH 8.0, 0.1 M NaH2PO4, and 6 M guanidine isothiocyanate), bound to His-Bind resins, washed (10 mM Tris, pH 8.0, 0.1 M NaH2PO4, and 8 M urea) five times, eluted (10 mM Tris, pH 4.5, 0.1 M NaH2PO4, and 8 M urea), and dialyzed. Rabbit antisera against tagged Cdc20 and Cdh1 were prepared and each specific antibody was purified by antigen affinity chromatography.

In Vitro Kinase Assay

The purified Cdc20, Cdh1, or Mad2 (1 μg) was incubated with 10 μCi of γ-[32P]ATP (3,000 Ci/mmol) and human Cdc2-GST-Cyclin B prepared by baculovirus (0.1 μg) (Kotani et al. 1998) or Plk (0.1 μg) (Kotani et al. 1998) in 50 μl of a kinase buffer (MPF: 10 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mM EGTA, and 0.05% β-mercaptoethanol; Plk: 20 mM Tris, pH 7.4, 10 mM MgCl2, 25 mM NaCl, 0.2 mg/ml BSA, and 0.05% β-mercaptoethanol), at 37°C for 30 min. The labeled proteins were immunoprecipitated with anti-T7 mAb (Novagen), washed, and resolved in 7% SDS-PAGE.

The immunoprecipitates of anti–Cyclin B1-specific antibody in nocodazole-treated K562 cell extracts were incubated with 10 μCi of γ-[32P]ATP in 20 μl of MPF kinase buffer at 30°C for 1 h. 1 μl of 20% SDS was added, mixed, and the sample was centrifuged. The sample was diluted with 500 μl of the kinase buffer and the labeled Cdc20 and Cdh1 were immunoprecipitated with anti–Cdc20- or anti–Cdh1-specific antibody, respectively. The samples were resolved in 7% SDS-PAGE.

Preparation of APC

Mouse NIH3T3 cells were maintained in DME supplemented with 10% FCS. Cells were synchronized at the G1/S boundary by double blocking with 1 μg/ml aphidicolin and harvested 2 h after drug release. APC in the S phase was prepared by Resource Q chromatography and immunoprecipitation with anti-Cdc27 antibody (a gift of Drs. P. Hieter and A. Page, University of British Columbia, Vancouver, Canada) as described (Kotani et al. 1998). Immunoprecipitates were washed three times with a buffer containing 500 mM KCl to remove MPF and Plk, and then washed with the buffer for kinase or ubiquitination reaction.

Ubiquitination Assay

Cdc20 and Cdh1 (1 μg) were phosphorylated by incubating with 0.1 μg of Cdc2-GST-Cyclin B and 0.1 mM ATP in 10 μl of MPF kinase buffer at 37°C for 30 min. The phosphorylated Cdc20 and Cdh1 were bound to His-Bind resin, washed with the kinase buffer containing 0.5 M KCl three times to remove MPF, and washed with the ubiquitination buffer. In some cases, the purified APC was phosphorylated by PLK phosphorylated by MPF (pPlk) and/or PKA in 50 μl of Plk kinase buffer containing 1 mM ATP, 0.1 μg of human Cdc2-GST-Cyclin B prepared by baculovirus (Kotani et al. 1998) and 0.1 μg of Plk, or in PKA buffer (10 mM Tris, pH 7.4, 10 mM MgCl2, and 0.1 mM EGTA) with 0.5 μg of bovine PKA catalytic subunit, and washed twice with 5 mM Tris, pH 7.6, and 0.5 mM MgCl2. The phosphorylated or untreated APC was incubated with 2 μg of GST-Cut2 (a gift of Dr. M. Yanagida, Kyoto University, Kyoto, Japan) (Funabiki et al. 1997) or 2 μg of human Cdc2-GST-Cyclin B (a gift of Dr. N. Watanabe, RIKEN, Japan) (Kotani et al. 1998) in the presence of various amounts (0–1 μg) of phosphorylated or unphosphorylated forms of Cdc20 and Cdh1 in 30 μl of 5 mM Tris, pH 7.6, 0.5 mM MgCl2, 2 mM ATP, 2 mM DTT, 2 mM creatine phosphate, 1 μg/ml creatine phosphokinase, 0.2 mg/ml bovine ubiquitin (Sigma Chemical Co.), 40 μg/ml mouse recombinant E1 and 50 μg/ml human recombinant hE2-C (Kotani et al. 1998), and then incubated at 25°C for 30 min or the indicated time. The samples were applied to 7% SDS-PAGE, and the ubiquitinated GST-Cyclin B or GST-Cut2 was detected by immunoblotting with anti–Cyclin B1 antibody and anti–GST antibody, respectively, and visualized by the enhanced chemiluminescence reaction.

Binding Assay

The purified APC in the S phase, which was immunoprecipitated with anti–Cdc27 antibody, was incubated with or without pPlk or PKA as described. The pretreated APC was incubated with T7-tagged human Cdc20, pCdc20, Cdh1, or Cdh1-phosphorylated MPF (pCdh1) in the ubiquitination buffer at 25°C for 30 min, washed with the buffer five times, and the proteins bound to the APC were resolved by 7% SDS-PAGE. The bound proteins were detected by immunoblotting with anti-T7 antibody.

The purified S phase APC was incubated with Cdc2-GST-Cyclin B and γ-[32P]ATP in the presence of Cdc20 or Cdh1 in MPF kinase buffer at 37°C for 30 min. The 32P-labeled proteins were immunoprecipitated by anti–Cdc27 antibody and were resolved in 7% SDS-PAGE.

Results

MPF Phosphorylates Cdc20 and Cdh1

The T7-tagged human Cdc20, Cdh1, and Mad2 were prepared in Escherichia coli, and their possible kinases, human Cdc2-GST-Cyclin B (MPF) prepared by baculovirus and His-tagged Plk produced in E. coli, were homogeneously purified as shown in Fig. 1 A. The in vitro phosphorylation was performed by incubating these recombinant Cdc20, Cdh1, and Mad2 with recombinant MPF or Plk in the presence of γ-[32P]ATP. Both Cdc20 and Cdh1 could be phosphorylated by MPF (Fig. 1 B, lanes 1 and 3) but not by Plk (Fig. 1 B, lanes 2 and 4) in vitro. Mad2 was not phosphorylated by these kinases (data not shown).

Mpf with but Not without Suc1/Cks1 Phosphorylates APC

Next, we performed in vitro phosphorylation of APC by MPF in the presence or absence of recombinant purified human Suc1/Cks1 (CksHs-1 or CksHs-2). There exist two human Suc1/Cks1 homologues, CksHs-1 and CksHs-2. As shown in Fig. 1 C, none of the APC core subunits could be phosphorylated by MPF alone (Fig. 1 C, lane 1). In the presence of CksHs-1 (Fig. 1 C, lane 3) or CksHs-2 (Fig. 1 C, lane 4), however, MPF could clearly phosphorylate APC1/Tsg24 and APC3/Cdc27 but not APC6/Cdc16, which is consistent with the results of Patra and Dunphy 1998. The MPF-activated Plk could phosphorylate APC1/Tsg24, APC3/Cdc27, APC6/Cdc16, and an unidentified protein of 85 kD (Fig. 1 C, lane 2) as we previously showed (Kotani et al. 1998). In the presence of all three components (MPF, CksHs-1 or CksHs-2, and Plk), these APC core subunits were most efficiently phosphorylated (Fig. 1 C, lanes 5 and 6). These results indicate that none of the APC core subunits can be phosphorylated by MPF alone, but in the presence of Suc1/Cks1, two APC core subunits, APC1/Tsg24 and APC3/Cdc27, can be phosphorylated by MPF.

pCdc20 but Not Cdc20 Activates Ubiquitination of Cut2 and Cyclin B

In vitro reconstituted ubiquitination assay was performed to assess the substrate- and time-specific activation or inactivation of mammalian APC, and the effects of phosphorylation of the regulatory factors as well as APC on the ubiquitination activity were determined. Fig. 2A and Fig. B, show that the purified APC in the S phase had no activity to ubiquitinate GST-Cut2 (Fig. 2 A, lane 1) or GST-Cyclin B (Fig. 2 B, lane 1), even in the presence of Cdc20 (Fig. 2A and Fig. B, lanes 2 and 3).

Since we found that Cdc20 could be phosphorylated by MPF, we next examined the effect of Cdc20 phosphorylated by MPF (pCdc20) on APC activity. The Cdc20 was phosphorylated by MPF, bound to His-Bind resin, and washed with 0.5 M KCl to remove MPF. It was confirmed by the immunoblot and MPF assay that the pCdc20 prepared in this way was free of MPF (data not shown). Interestingly, when the APC was incubated with pCdc20, both GST-Cut2 and GST-Cyclin B were ubiquitinated in a dose-dependent manner (Fig. 2A and Fig. B, lanes 4–6). Furthermore, Mad2 inhibited these ubiquitination activities in a dose-dependent manner (Fig. 2A and Fig. B, lanes 7 and 8). These results indicate that pCdc20 but not Cdc20 activates ubiquitination of both Cut2/Pds1 and Cyclin B, and the pCdc20-dependent APC activation can be suppressed by Mad2.

Cdh1 but Not pCdh1 Activates Ubiquitination of Cyclin B but Not Cut2

No GST-Cut2 was ubiquitinated even in the presence of a large excess of Cdh1 (Fig. 3 A, lanes 2 and 3) or Cdh1 phosphorylated by MPF (pCdh1) (Fig. 3 A, lanes 4 and 5). The pCdh1 was prepared by the same procedure as pCdc20, and it contained no MPF activity. The ubiquitination of GST-Cyclin B was activated by Cdh1 in a dose-dependent manner (Fig. 3 B, lanes 1–4), and the activation was not blocked by the addition of a large excess of Mad2 (Fig. 3 B, lane 5). In contrast, even sufficiently large amounts of pCdh1 could not activate the ubiquitination of GST-Cyclin B (Fig. 3 B, lanes 6 and 7). These results indicate that APC in the presence of Cdh1 but not pCdh1, effectively and specifically ubiquitinates Cyclin B (but not Cut2/Pds1) at least in vitro, and that Mad2 suppresses only pCdc20-dependent APC activation.

Positive and Negative Effects of APC Phosphorylation by pPlk and PKA on Ubiquitination Activity in the Presence of pCdc20 or Cdh1

Next, we examined the effect of phosphorylation of APC itself on APC activity in the presence of the activated form of the regulatory factors, pCdc20 and Cdh1. As previously reported, the pPlk-activated APC (pAPC) ubiquitinated GST-Cyclin B (Fig. 4, lower panel, lane 1), but its activation was suppressed by phosphorylation with PKA (Fig. 4, lower panel, lane 3). However, pAPC could not ubiquitinate GST-Cut2 (Fig. 4, upper panel, lane 1). Furthermore, if the APC or pAPC was once phosphorylated by PKA, the GST-Cyclin B could not be ubiquitinated even in the presence of the active regulatory factors, pCdc20 or Cdh1 (Fig. 4, lower panel, lanes 4–7). These results indicate that the ubiquitination of Cyclin B is suppressed by PKA as well as by Mad2, and that pPlk specifically stimulates the ubiquitination of Cyclin B but not Cut2/Pds1.

pCdc20 or Cdh1 Acts Synergistically with pPlk on Cyclin B Ubiquitination

Time course experiments of Cyclin B ubiquitination with pAPC demonstrated that the kinetics of the ubiquitination reaction became much faster in the presence of the active regulatory factors, pCdc20 (Fig. 5 A) or Cdh1 (Fig. 5 B), i.e., a shorter incubation time (10 min) was enough to reach the saturation level of GST-Cyclin B ubiquitination (Fig. 5A and Fig. B, lane 8), indicating that pCdc20 or Cdh1 acts synergistically with pPlk on Cyclin B ubiquitination. It was also found that Mad2 does not inhibit pAPC activity (Fig. 5 A, lane 10); thus, Mad2 inhibits only pCdc20-dependent APC activation, whereas PKA suppresses both pCdc20- and pPlk-dependent APC activation.

Binding of Cdc20, pCdc20, and Cdh1 to APC and pAPC

The binding assay of the regulatory factors to APC demonstrated that both Cdc20 and pCdc20 constitutively bound to APC (Fig. 6 A, lanes 1 and 2) and to pAPC (Fig. 6 A, lanes 7 and 8) but not to APC phosphorylated by PKA (pAPC(PKA)) (Fig. 6 A, lanes 13 and 14). These results indicate that pCdc20-induced APC activation is dependent on the phosphorylation of Cdc20 with MPF rather than on the binding preference of pCdc20 to APC. Furthermore, the binding of Cdc20 and pCdc20 to APC or to pAPC was not affected by the addition of Mad2 (Fig. 6 A, lanes 3, 4, 9, and 10), suggesting that Mad2 inhibition of pCdc20-induced APC activation is not due to the binding inhibition of pCdc20 to APC, but due to the direct functional inhibition of pCdc20 by Mad2. In contrast, Cdh1 could bind to either APC (Fig. 6 A, lane 5) or pAPC (Fig. 6 A, lane 11) but not to pAPC(PKA) (Fig. 6 A, lane 15), whereas pCdh1 did not bind to any forms of APC (Fig. 6 A, lanes 6, 12, and 16). This indicates that Cdh1-induced APC activation totally depends on the binding of Cdh1 to APC or pAPC. These results suggest that Cdh1 (but not pCdh1) specifically binds to and activates APC, and that the PKA-induced inhibition of APC activity is due to the inhibition of the binding of these active regulatory factors to APC.

To confirm these binding data, the purified S phase APC was incubated with Cdc2-GST-Cyclin B and γ-[32P]ATP in the presence of Cdc20 or Cdh1, and the 32P-labeled proteins were immunoprecipitated by anti–Cdc27 antibody. The results showed that MPF directly phosphorylated Cdc20 but none of the components of the purified APC (Fig. 6 B, lane 1), and that pCdc20 actually bound to APC (Fig. 6 B, lane 1) but pCdh1 could not bind to APC (Fig. 6 B, lane 2). These results also confirmed that there is no need to consider the effect of phosphorylation of APC by MPF on ubiquitination activity.

MPF Phosphorylates Cdc20 and Cdh1 during Mitosis In Vivo

To demonstrate that MPF phosphorylates Cdc20 and Cdh1 during mitosis in vivo, the immunoprecipitates of anti–Cyclin B-specific antibody from mitotic K562 cell extracts were incubated with γ-[32P]ATP in MPF kinase buffer. The sample was washed with 1% SDS, diluted 25-fold with the kinase buffer, and the labeled Cdc20 and Cdh1 were immunoprecipitated with anti–Cdc20- or anti–Cdh1-specific antibody, respectively. The 32P-labeled Cdc20 and Cdh1 can be clearly seen (Fig. 7, lanes 2 and 3). Fig. 7, lane 1, shows the total 32P-labeled proteins without immunoprecipitation by anti–Cdc20 or anti–Cdh1 antibody, and the most prominent band corresponded to the 32P-labeled Cyclin B as shown. The preimmune antibody could immunoprecipitate none of these proteins (Fig. 7, lane 4). The phosphorylation could not be observed in cell extracts prepared from cells in S phase (data not shown). These results strongly suggest that MPF phosphorylates Cdc20 and Cdh1 during mitosis in vivo, while it cannot be completely ruled out the possibility that another kinase coimmunoprecipitated with anti–Cyclin B antibody phosphorylates these proteins.

pCdc20 but Not pCdh1 Binds to APC during Mitosis In Vivo

Next, we examined in vivo phosphorylation of Cdc20 and Cdh1 during mitosis and the interaction of these phosphorylated factors with APC in vivo. K562 cells were labeled with [32P]orthophosphate during mitosis in vivo and the APC was purified by immunoprecipitation with anti–Cdc27 antibody. The 32P-labeled Cdc20 could be immunoprecipitated with anti–Cdc20-specific antibody from this purified APC (Fig. 8, lane 1) and from the total cell lysates (Fig. 8, lane 2), indicating that Cdc20 can indeed be phosphorylated and binds to APC during mitosis in vivo. In contrast, the 32P-labeled Cdh1 could not be immunoprecipitated with its specific antibody (Fig. 8, lane 3) from the purified APC, whereas the 32P-labeled Cdh1 could be immunoprecipitated from the total cell lysates (Fig. 8, lane 4), demonstrating that Cdh1 can actually be phosphorylated but pCdh1 cannot interact with APC during mitosis in vivo. These results perfectly agree with the findings shown in the in vitro reconstituted system (Fig. 1,Fig. 2,Fig. 3,Fig. 4,Fig. 5,Fig. 6).

Discussion

Taken together with the findings described above and other observations recently reported, the scheme of regulation of APC activity is depicted in Fig. 9. We showed in this paper that phosphorylation of Cdc20 is required for Cdc20-dependent APC activation at least in vitro (Fig. 2). It has been reported that Cdc20 is expressed during G2 phase and mitosis (Fang et al. 1998a; Kramer et al. 1998) and binds to APC (Fig. 6 A and 8) in early mitotic stages (Fang et al. 1998b). Whereas, Fang et al. 1998a described that the in vitro translated Cdc20, which might be phosphorylated during preparation, activated APC in vitro and that the phosphatase treatment of Cdc20 had no effect on APC activity, it was recently demonstrated that Cdc20 is clearly phosphorylated during mitosis in HeLa cells (Kramer et al. 1998), and that Cdc20 is phosphorylated by MPF in Xenopus embryos (Lorca et al. 1998). Actually, the possible Cdc2 phosphorylation site is conserved in budding yeast Cdc20, fission yeast Slp1, and mammalian Cdc20. Therefore, it is most likely that Cdc20 is indeed phosphorylated during mitosis in vivo. Taken together, we concluded that Cdc20 activates APC when it is phosphorylated by MPF.

The activation of APC can be blocked by the binding of Mad2 to pCdc20 (Fig. 2A and Fig. B; He et al. 1997; Li et al. 1997; Fang et al., 1998; Gorbsky et al. 1998; Hwang et al. 1998; Kallio et al. 1998; Kim et al. 1998). Therefore, at least two events may be required to activate APC at metaphase–anaphase transition: release of Mad2 from pCdc20 after spindle assembly checkpoint is released, and phosphorylation of Cdc20 bound to APC by MPF or binding of pCdc20 to APC. Furthermore, we found that at least in vitro APC activation can be suppressed by PKA (Fig. 4 and Fig. 6 A; Ishii et al. 1996), which phosphorylates two APC subunits, APC1 and APC3 (Kotani et al. 1998). Thus, dephosphorylation of PKA phosphorylation sites on APC by a specific phosphatase yet unidentified, which may be PP1 as previously suggested (Ishii et al. 1996), might also be required for the onset of anaphase.

Mad2 cannot inhibit Cdh1-induced APC activation (Fig. 3 B), and pAPC can ubiquitinate Cyclin B even in the presence of Mad2 (Fig. 5 A). Thus Mad2 inhibits APC activation only through Cdc20. Further, the binding of Cdc20 or pCdc20 to APC or pAPC was not affected by Mad2 (Fig. 6 A), indicating that Mad2 inhibits the function of pCdc20 but not the binding of pCdc20 to APC. Very recently, it was reported that whereas Mad1, Mad2, Mad3/Bub1, and Bub3 suppress Cdc20-dependent APC activation, Bub2 localized in the spindle pole body regulates Cdh1-dependent APC activation (Alexandru et al. 1999; Fesquet et al. 1999; Fraschini et al. 1999). Further detailed functional analyses of the Mad family and Bub family are required to clarify the molecular mechanisms of spindle assembly checkpoint and of APC regulation.

It was clearly shown that pAPC can ubiquitinate Cyclin B but not Cut2 in vitro, but it remains unclear whether free pAPC actually exists during mitosis in vivo. It is most likely that the majority of pAPC forms a complex with either pCdc20 or Cdh1 that ubiquitinates Cyclin B in vivo. Furthermore, phosphorylation of APC by pPlk may not be essential for APC activation at the metaphase–anaphase transition, but our results suggest that it is required for complete Cyclin B ubiquitination in later stages of mitosis.

We showed here that pCdc20 and Cdh1, but neither Cdc20 nor pCdh1 activates APC in vitro, and the following switching mechanism from pCdc20 to Cdh1 could be speculated. When the cells enter anaphase after Cut2/Pds1 is entirely degraded, the pAPC–pCdc20 complex steadily ubiquitinates Cyclin B and, consequently, MPF activity decreases by the end of mitosis. The pCdc20 can be dephosphorylated by a specific phosphatase and released from pAPC or it may be degraded as previously suggested (Fang et al. 1998a; Shirayama et al. 1998). When MPF activity is high, Cdh1 is phosphorylated and remains inactive, but when Cdc14 is activated, Cdh1 is dephosphorylated by Cdc14 as suggested (Visintin et al. 1998, Visintin et al. 1999; Shou et al. 1999) and binds to and activates APC. Consequently, the pAPC–pCdc20 complex is replaced by the pAPC–Cdh1 (or APC–Cdh1) complex in late mitosis. The pAPC–Cdh1 or APC–Cdh1 complex further ubiquitinates Cyclin B in late mitosis and G1 phase. Thus, the switch mechanism from pCdc20 to Cdh1 may be dependent upon the MPF activity during mitosis, and the MPF activity itself is controlled by the pCdc20- and Cdh1-dependent APC activity.

Cdc20 has been reported to activate ubiquitination of the factors regulating sister chromatid separation and Cdh1 promotes ubiquitination of mitotic cyclins (Visintin et al. 1997; Shirayama et al. 1998). However, it was recently found that in Xenopus early embryos, Cdh1 is not expressed (Lorca et al. 1998), which is consistent with the observation that Cdh1 is not expressed before stage 13 in Drosophila embryos (Sigrist and Lehner 1997). It was also described that Cdc20 regulates ubiquitination of both Cut2/Pds1 and Cyclin B in the early embryonic cell cycle (Lorca et al. 1998). Very recently, Clute and Pines 1999 reported that ubiquitin-dependent proteolysis of Cyclin B begins at metaphase–anaphase transition in HeLa cells, which is consistent with the results obtained with clam embryo (Hunt et al. 1992). These findings are consistent with our observation that pCdc20 can activate ubiquitination not only of Cut2/Pds1, but also of Cyclin B. Thus, pCdc20 alone may be enough for cells to go through mitosis without Cdh1. However, in the somatic cell cycle, Cdh1 in addition to pCdc20, may be required for effective and complete ubiquitination of Cyclin B in later stages of mitosis and G1 phase.

We demonstrated in this paper that Cdh1 can be phosphorylated by MPF (Fig. 1 B and 7), and Cdh1 but not pCdh1 binds to and activates APC only after dephosphorylation (Fig. 3 B, 6, A and B, and 8). These in vitro results are consistent with in vivo data in budding yeast that the dephosphorylated form of Cdh1/Hct1 activates APC (Zachariae et al. 1998b). It was recently described that Cdh1 is constantly expressed throughout the cell cycle (Fang et al. 1998a; Kramer et al. 1998), binds to APC in late mitosis and G1 phase (Fang et al. 1998b), and is phosphorylated during mitosis in HeLa cells (Kramer et al. 1998). It was also reported that Cdh1/Hct1 binding to APC is regulated by cyclin-dependent kinases (Zachariae et al. 1998b). Very recently, it was demonstrated that Cdc14 dephosphorylates Cdh1/Hct1 and inactivates APC (Visintin et al. 1998). Further, it was found that the activation of Cdc14 is regulated by Bub2/Byr4 and RENT complex (Alexandru et al. 1999; Shou et al. 1999; Visintin et al. 1999), although it remains unresolved whether the regulation by RENT complex works in the mammalian system. All these results in vivo agree well with our results in vitro.

In our scheme, it is possible that Cdh1–APC complex activity is maintained until late G1 phase, while it might be diminished by the phosphorylation of Cdh1 with Cdk2, Cdk4, or another unidentified specific kinase that is active at G1/S transition (Amon et al. 1994). It has been reported that the low level of Cyclin B is translated even in G1 phase (Brandeis and Hunt 1996). In the G1 phase, APC–Cdh1 complex may, thus, effectively ubiquitinate this newly translated Cyclin B to avoid activation of MPF. The possible involvement of G1/S Cdks or other specific kinases in Cdh1-dependent APC inactivation must be further studied.

Patra and Dunphy 1998 recently reported that Cdc2-Cyclin B phosphorylates APC3/Cdc27 and APC1/BIME of Xenopus APC in the presence of the Xenopus Suc1/Cks1 protein, Xe-p9. We confirmed that in the presence of human Suc1/Cks1 (CksHs-1 or CksHs-2), MPF could phosphorylate APC1/Tsg24 and APC3/Cdc27 but not APC6/Cdc16, whereas none of the APC core subunits could be phosphorylated by MPF alone (Fig. 1 C). The MPF-activated Plk could phosphorylate APC1/Tsg24, APC3/Cdc27, APC6/Cdc16, and one more additional unidentified protein as we previously showed (Kotani et al. 1998). It has been reported that in Xenopus the phosphorylated APC components during mitosis are at least APC1/Tsg24, APC3/Cdc27, APC6/Cdc16, and APC8/Cdc23 (Peters et al. 1996). Therefore, the kinase that phosphorylates Cdc23/APC8 and may affect APC activity has not yet been identified. Furthermore, Patra and Dunphy 1998 have not demonstrated that phosphorylation with Cdk1-Suc1/Cks1 actually activates ubiquitination of cyclin B. Very recently, however, Shteinberg and Hershko 1999 demonstrated that in early embryonic cell cycle Cdk1 activated Xenopus APC in the presence of Suc1/Cks1. Further, Shteinberg et al. 1999 described that phosphorylation of APC by MPF is required for its stimulation by Cdc20. In contrast, Kaiser et al. 1999 very recently reported that Cks1 neither bound to nor activated APC, but that it did regulate proteasome activity in yeast. We also found that the APC was not activated only by phosphorylation with MPF. These data support the notion that MPF can phosphorylate APC in the presence of Suc1/Cks1, but itself may not be enough to completely activate APC, and that other kinases like Plk and/or factors such as Cdc20 and Cdh1 are required for full APC activation.

Our findings in vitro and in vivo are consistent with the other in vivo observations (Fang et al. 1998a,Fang et al. 1998b; Kramer et al. 1998; Lorca et al. 1998), and strongly support the notion that pCdc20 but not Cdc20 activates APC, and that Cdh1 but not pCdh1 binds to and activates APC. Therefore, phosphorylation and dephosphorylation of APC regulatory factors by MPF are critical for their binding to APC and/or APC activation. The MPF activity itself is regulated by the pCdc20- and Cdh1-dependent APC activity, and this feedback control precisely regulates APC activity. Taken together, the APC activity is regulated by the phosphorylation and dephosphorylation of APC and of the regulatory factors, Cdc20 and Cdh1, by MPF, Plk, PKA, and PP1, as well as by the binding of positive and negative regulatory factors, Cdc20, Cdh1, and Mad2, to APC. These elaborate regulatory mechanisms might control the precise progression of mitosis.

Acknowledgments

We thank P. Hieter and A. Page for anti–Cdc27 antibody, M. Yanagida for Cut2, and N. Watanabe for MPF. We also thank M. Peters, E.R. Kramer, K. Nasmyth, W. Zachariae, M. Shirayama (Research Institute of Molecular Pathology, Vienna, Austria), and T. Oyamatsu (RIKEN, Japan) for valuable discussions throughout this work.

This work was supported in part by a Special Grant for Promotion of Research from the Institute of Physical and Chemical Research (RIKEN) and grants from the Ministry of Education, Science and Culture of Japan and from the Science Technology Agency of Japan.

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1.used in this paper: APC, anaphase promoting complex/cyclosome; MPF, Cdc2-Cyclin B or Cdc2-GST-Cyclin B; pAPC, APC phosphorylated by pPlk or APlk-activated APC; pCdc20, Cdc20 phosphorylated by MPF; pCdh1, Cdh1 phosphorylated MPF; PKA, cAMP-dependent protein kinase; Plk, Polo-like kinase; pPlk, Plk phosphorylated by MPF