Faculty Bibliography

The mitotic (or spindle assembly) checkpoint system ensures accurate chromosome segregation by preventing anaphase initiation until all chromosomes are correctly attached to the mitotic spindle. It affects the activity of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets inhibitors of anaphase initiation for degradation. The mechanisms by which this system regulates APC/C remain obscure. Some models propose that the system promotes sequestration of the APC/C activator Cdc20 by binding to the checkpoint proteins Mad2 and BubR1. A different model suggests that a mitotic checkpoint complex (MCC) composed of BubR1, Bub3, Cdc20, and Mad2 inhibits APC/C in mitotic checkpoint [Sudakin V, Chan GKT, Yen TJ (2001) J Cell Biol 154:925-936]. We examined this problem by using extracts from nocodazole-arrested cells that reproduce some downstream events of the mitotic checkpoint system, such as lag kinetics of the degradation of APC/C substrate. Incubation of extracts with adenosine-5'-(gamma-thio)triphosphate (ATP[gammaS]) stabilized the checkpoint-arrested state, apparently by stable thiophosphorylation of some proteins. By immunoprecipitation of APC/C from stably checkpoint-arrested extracts, followed by elution with increased salt concentration, we isolated inhibitory factors associated with APC/C. A part of the inhibitory material consists of Cdc20 associated with BubR1 and Mad2, and is thus similar to MCC. Contrary to the original MCC hypothesis, we find that MCC disassembles upon exit from the mitotic checkpoint. Thus, the requirement of the mitotic checkpoint system for the binding of Mad2 and BubR1 to Cdc20 may be for the assembly of the inhibitory complex rather than for Cdc20 sequestration

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 anaphase-promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin-protein ligase that targets for degradation cell-cycle regulatory proteins during exit from mitosis and in the G1 phase of the cell cycle. The activity of APC/C in mitosis and in G1 requires interaction with the activator proteins Cdc20 and Cdh1, respectively. Substrates of APC/C-Cdc20 contain a recognition motif called the 'destruction box' (D-box). The mode of the action of APC/C activators and their possible role in substrate binding remain poorly understood. Several investigators suggested that Cdc20 and Cdh1 mediate substrate recognition, whereas others proposed that substrates bind to APC/C or to APC/C-activator complexes. All these studies used binding assays, which do not necessarily indicate that substrate binding is functional and leads to product formation. In the present investigation we examined this problem by an 'isotope-trapping' approach that directly demonstrates productive substrate binding. With this method we found that the simultaneous presence of both APC/C and Cdc20 is required for functional substrate binding. By contrast, with conventional binding assays we found that either Cdc20 or APC/C can bind substrate by itself, but only at low affinity and relaxed selectivity for D-box. Our results are consistent with models in which interaction of substrate with specific binding sites on both APC/C and Cdc20 is involved in selective and productive substrate binding

A procedure is described for the affinity purification of the mitotic form of anaphase-promoting complex/cyclosome (APC/C) from HeLa cells. It is based on the binding of mitotically phosphorylated APC/C to the phosphate-binding site of p13(suc1), followed by specific elution with a phosphate-containing compound. The procedure is rapid, simple, and yields 50- to 70-fold purification of soluble APC/C, with a approximately 30% recovery of activity

Early mitotic inhibitor 1 (Emi1) inhibits the activity of the anaphase promoting complex/cyclosome (APC/C), which is a multisubunit ubiquitin ligase that targets mitotic regulators for degradation in exit from mitosis. Levels of Emi1 oscillate in the cell cycle: it accumulates in the S phase and is rapidly degraded in prometaphase. The degradation of Emi1 in early mitosis is necessary for the activation of APC/C in late mitosis. Previous studies have shown that Emi1 is targeted for degradation in mitosis by a Skp1-Cullin1 F-box protein (SCF) ubiquitin ligase complex that contains the F-box protein beta-TrCP. As with other substrates of SCF(beta-TrCP), the phosphorylation of Emi1 on a DSGxxS sequence is required for this process. However, the protein kinase(s) involved has not been identified. We find that Polo-like kinase 1 (Plk1), a protein kinase that accumulates in mitosis, markedly stimulates the ligation of Emi1 to ubiquitin by purified SCF(beta-TrCP). Cdk1-cyclin B, another major mitotic protein kinase, has no influence on this process by itself but stimulates the action of Plk1 at low, physiological concentrations. Plk1 phosphorylates serine residues in the DSGxxS sequence of Emi1, as suggested by the reduced phosphorylation of a derivative in which the two serines were mutated to nonphosphorylatable amino acids. Transfection with an small interfering RNA duplex directed against Plk1 caused the accumulation of Emi1 in mitotically arrested HeLa cells. It is suggested that phosphorylation of Emi1 by Plk1 is involved in its degradation in mitosis

We have recently demonstrated that regulation of Cdc25A protein abundance during S phase and in response to DNA damage is mediated by SCF(betaTrCP) activity. Based on sequence homology of known betaTrCP substrates, we found that Cdc25A contains a conserved motif (DSG), phosphorylation of which is required for interaction with betaTrCP.1 Here, we show that phosphorylation at Ser 82 within the DSG motif anchors Cdc25A to betaTrCP and that Chk1-dependent phosphorylation at Ser 76 affects this interaction as well as betaTrCP-dependent degradation. We propose that a hierarchical order of phosphorylation events commits Cdc25A to betaTrCP-dependent degradation. According to our model, phosphorylation at Ser 76 is a 'priming' step required for Ser 82 phosphorylation, which in turn allows recruitment of Cdc25A by betaTrCP and subsequent betaTrCP-dependent degradation

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 Cdc25A. These events stimulate the ubiquitin-mediated proteolysis of Cdc25A and contribute to delaying cell-cycle progression, thereby preventing genomic instability. Here we report that beta-TrCP is the F-box protein that targets phosphorylated Cdc25A for degradation by the Skp1/Cul1/F-box protein complex. Downregulation of beta-TrCP1 and beta-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 beta-TrCP may function in the intra-S-phase checkpoint. Consistent with this hypothesis, suppression of beta-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 beta-TrCP has a crucial role in mediating the response to DNA damage through Cdc25A degradation

The cyclin-dependent kinase inhibitor p21Cip1 has important roles in the control of cell proliferation, differentiation, senescence, and apoptosis. It has been observed that p21 is a highly unstable protein, but the mechanisms of its degradation remained unknown. We show here that p21 is a good substrate for an SCF (Skp1-Cullin1-F-box protein) ubiquitin ligase complex, which contains the F-box protein Skp2 (S phase kinase-associated protein 2) and the accessory protein Cks1 (cyclin kinase subunit 1). A similar ubiquitin ligase complex has been previously shown to be involved in the degradation of a related cyclin-dependent kinase inhibitor, p27Kip1. The levels of Skp2 oscillate in the cell cycle, reaching a maximum in S phase. The ubiquitylation of p21 in vitro required the supplementation of all components of the SCF complex as well as of Cks1 and Cdk2-cyclin E. The protein kinase Cdk2-cyclin E acts both by the phosphorylation of p21 on Ser-130 and by the formation of a complex with p21, which is required for its presentation to the ubiquitin ligase. As opposed to the case of p27, the phosphorylation of p21 stimulates its ubiquitylation but is not absolutely required for this process. Levels of p21 are higher in Skp2-/- mouse embryo fibroblasts than in wild-type fibroblasts in the S phase, and the rates of the degradation of p21 are slower in cells that lack Skp2. It is suggested that SCFSkp2 participates in the degradation of p21 in the S phase