The mitotic checkpoint system delays anaphase until all chromosomes are correctly attached to the mitotic spindle. When the checkpoint is turned on, it promotes the formation of the mitotic checkpoint complex (MCC), which inhibits the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2 bound to the APC/C activator Cdc20. When the checkpoint is satisfied, MCC is disassembled and APC/C becomes active. Previous studies have shown that the Mad2-binding protein p31comet promotes the dissociation of Cdc20 from BubR1 in MCC in a process that requires ATP. We now show that a part of MCC dissociation is blocked by inhibitors of cyclin-dependent kinases (Cdks) and that purified Cdk1–cyclin B stimulates this process. The mutation of all eight potential Cdk phosphorylation sites of Cdc20 partially prevented its release from BubR1. Furthermore, p31comet stimulated Cdk-catalyzed phosphorylation of Cdc20 in MCC. It is suggested that the binding of p31comet to Mad2 in MCC may trigger a conformational change in Cdc20 that facilitates its phosphorylation by Cdk, and that the latter process may promote its dissociation from BubR1.

Cell cycle regulation is characterized by alternating activities of cyclin-dependent kinases (CDKs) and of the ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). During S-phase APC/C is inhibited by early mitotic inhibitor 1 (Emi1) to allow the accumulation of cyclins A and B and to prevent re-replication. Emi1 is degraded at prophase by a Plk1-dependent pathway. Recent studies in which the degradation pathway of Emi1 was disrupted have shown that APC/C is activated at mitotic entry despite stabilization of Emi1. These results suggested the possibility of additional mechanisms other than degradation of Emi1, which release APC/C from inhibition by Emi1 upon entry into mitosis. In this study we report one such mechanism, by which the ability of Emi1 to inhibit APC/C is negatively regulated by CDKs. We show that in Plk1-inhibited cells Emi1 is stabilized and phosphorylated, that Emi1 is phosphorylated by CDKs in mitotic but not S-phase cell extracts, and that Emi1 phosphorylation by mitotic cell extracts or purified CDKs markedly reduces the ability of Emi1 to bind and to inhibit APC/C. Finally, we show that the addition of extracts from S-phase cells to extracts from mitotic cells protects Emi1 from CDK-mediated inactivation.

The mitotic checkpoint system ensures the fidelity of chromosome segregation by preventing the completion of mitosis in the presence of any misaligned chromosome. When activated, it blocks the initiation of anaphase by inhibiting the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). Little is known about the biochemical mechanisms by which this system inhibits APC/C, except for the existence of a mitotic checkpoint complex (MCC) inhibitor of APC/C composed of the APC/C activator Cdc20 associated with the checkpoint proteins Mad2, BubR1, and Bub3. We have been studying the mechanisms of the mitotic checkpoint system in extracts that reproduce its downstream events. We found that inhibitory factors are associated with APC/C in the checkpoint-arrested state, which can be recovered from immunoprecipitates. Only a part of the inhibitory activity was caused by MCC [Braunstein I, Miniowitz S, Moshe Y, Hershko A (2007) Proc Natl Acad Sci USA 104:4870–4875]. Here, we show that during exit from checkpoint, rapid disassembly of MCC takes place while APC/C is still inactive. This observation suggested the possible involvement of multiple factors in the regulation of APC/C by the mitotic checkpoint. We have separated a previously unknown inhibitor of APC/C from MCC. This inhibitor, called mitotic checkpoint factor 2 (MCF2), is associated with APC/C only in the checkpoint-arrested state. The inhibition of APC/C by both MCF2 and MCC was decreased at high concentrations of Cdc20. We propose that both MCF2 and MCC inhibit APC/C by antagonizing Cdc20, possibly by interaction with the Cdc20-binding site of APC/C.

The activity of cullin-containing ubiquitin protein ligase complexes is stimulated by linkage to cullin of the ubiquitin-like protein Nedd8 (“neddylation”). Neddylation is inhibited by the tight binding of cullins to CAND1 (cullin-associated and neddylation-dissociated 1) protein, and Nedd8 is removed from cullins by specific isopeptidase activity of the COP9/signalosome (CSN) complex. The mechanisms that regulate neddylation and deneddylation of cullins were unknown. We examined this problem for the case of SCF Skp2 , a cullin1 (Cul1)-containing ubiquitin ligase complex that contains the S phase-associated protein Skp2 as the substrate-binding F-box protein subunit. SCF Skp2 targets for degradation the cyclin-dependent kinase (cdk) inhibitor p27 in the G 1 -to-S phase transition, a process that requires its phosphorylation and binding to cdk2-cyclin E. Because levels of Skp2, cyclin E, and the accessory protein Cks1 (cyclin kinase subunit 1) all rise at the end of G 1 phase, it seemed possible that the neddylation of Cul1 in SCF Skp2 is regulated by the availability of the F-box protein and/or the substrate. We found that the supplementation of Skp2–Skp1 and substrate (along with further components necessary for substrate presentation to the ubiquitin ligase) to extracts of HeLa cells synergistically increased levels of neddylated Cul1. Skp2–Skp1 abrogates the inhibitory influence of CAND1 on the neddylation of Cul1 by promoting the dissociation of the cullin–CAND1 complex, whereas substrate, together with substrate-presenting components, prevents the action of CSN to deneddylate cullin. We propose a sequence of events in which the increased availability of Skp2 and substrate in the transition of cells to S phase promotes the neddylation and assembly of the SCF Skp2 ubiquitin ligase complex.

The Cdc25A phosphatase is essential for cell-cycle progression because of its function in dephosphorylating cyclin-dependent kinases. In response to DNA damage or stalled replication, the ATM and ATR protein kinases activate the checkpoint kinases Chk1 and Chk2, which leads to hyperphosphorylation of Cdc25A1,2,3. These events stimulate the ubiquitin-mediated proteolysis of Cdc25A1,4,5 and contribute to delaying cell-cycle progression, thereby preventing genomic instability1,2,3,4,5,6,7. Here we report that β-TrCP is the F-box protein that targets phosphorylated Cdc25A for degradation by the Skp1/Cul1/F-box protein complex. Downregulation of β-TrCP1 and β-TrCP2 expression by short interfering RNAs causes an accumulation of Cdc25A in cells progressing through S phase and prevents the degradation of Cdc25A induced by ionizing radiation, indicating that β-TrCP may function in the intra-S-phase checkpoint. Consistent with this hypothesis, suppression of β-TrCP expression results in radioresistant DNA synthesis in response to DNA damage—a phenotype indicative of a defect in the intra-S-phase checkpoint that is associated with an inability to regulate Cdc25A properly. Our results show that β-TrCP has a crucial role in mediating the response to DNA damage through Cdc25A degradation.

The cyclin-dependent kinase (CDK) inhibitor p27 is degraded in late G1 phase by the ubiquitin pathway1, allowing CDK activity to drive cells into S phase2. Ubiquitinylation of p27 requires its phosphorylation at Thr 187 (refs 3, 4) and subsequent recognition by S-phase kinase associated protein 2 (Skp2; refs 5–8), a member of the F-box family of proteins that associates with Skp1, Cul-1 and ROC1/Rbx1 to form an SCF ubiquitin ligase complex9. However, in vitro ligation of p27 to ubiquitin could not be reconstituted by known purified components of the SCFSkp2 complex. Here we show that the missing factor is CDK subunit 1 (Cks1), which belongs to the highly conserved Suc1/Cks family of proteins that bind to some CDKs and phosphorylated proteins and are essential for cell-cycle progression. Human Cks1, but not other members of the family, reconstitutes ubiquitin ligation of p27 in a completely purified system, binds to Skp2 and greatly increases binding of T187-phosphorylated p27 to Skp2. Our results represent the first evidence that an SCF complex requires an accessory protein for activity as well as for binding to its phosphorylated substrate.