Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2.

The mitotic checkpoint prevents cells with unaligned chromosomes from prematurely exiting mitosis by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from targeting key proteins for ubiquitin-mediated proteolysis. We have examined the mechanism by which the checkpoint inhibits the APC/C by purifying an APC/C inhibitory factor from HeLa cells. We call this factor the mitotic checkpoint complex (MCC) as it consists of hBUBR1, hBUB3, CDC20, and MAD2 checkpoint proteins in near equal stoichiometry. MCC inhibitory activity is 3,000-fold greater than that of recombinant MAD2, which has also been shown to inhibit APC/C in vitro. Surprisingly, MCC is not generated from kinetochores, as it is also present and active in interphase cells. However, only APC/C isolated from mitotic cells was sensitive to inhibition by MCC. We found that the majority of the APC/C in mitotic lysates is associated with the MCC, and this likely contributes to the lag in ubiquitin ligase activity. Importantly, chromosomes can suppress the reactivation of APC/C. Chromosomes did not affect the inhibitory activity of MCC or the stimulatory activity of CDC20. We propose that the preformed interphase pool of MCC allows for rapid inhibition of APC/C when cells enter mitosis. Unattached kinetochores then target the APC/C for sustained inhibition by the MCC.

Human BUBR1 is a mitotic checkpoint kinase that monitors CENP-E functions at kinetochores and binds the cyclosome/APC.

Human cells express two kinases that are related to the yeast mitotic checkpoint kinase BUB1. hBUB1 and hBUBR1 bind to kinetochores where they are postulated to be components of the mitotic checkpoint that monitors kinetochore activities to determine if chromosomes have achieved alignment at the spindle equator (Jablonski, S.A., G.K.T. Chan, C.A. Cooke, W.C. Earnshaw, and T.J. Yen. 1998. Chromosoma. 107:386-396). In support of this, hBUB1 and the homologous mouse BUB1 have been shown to be important for the mitotic checkpoint (Cahill, D.P., C. Lengauer, J. Yu, G.J. Riggins, J.K. Willson, S.D. Markowitz, K.W. Kinzler, and B. Vogelstein. 1998. Nature. 392:300-303; Taylor, S.S., and F. McKeon. 1997. Cell. 89:727-735). We now demonstrate that hBUBR1 is also an essential component of the mitotic checkpoint. hBUBR1 is required by cells that are exposed to microtubule inhibitors to arrest in mitosis. Additionally, hBUBR1 is essential for normal mitotic progression as it prevents cells from prematurely entering anaphase. We establish that one of hBUBR1's checkpoint functions is to monitor kinetochore activities that depend on the kinetochore motor CENP-E. hBUBR1 is expressed throughout the cell cycle, but its kinase activity is detected after cells have entered mitosis. hBUBR1 kinase activity was rapidly stimulated when the spindle was disrupted in mitotic cells. Finally, hBUBR1 was associated with the cyclosome/anaphase-promoting complex (APC) in mitotically arrested cells but not in interphase cells. The combined data indicate that hBUBR1 can potentially provide two checkpoint functions by monitoring CENP-E-dependent activities at the kinetochore and regulating cyclosome/APC activity.

The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis.

The ubiquitin-mediated degradation of mitotic cyclins is required for cells to exit from mitosis. Previous work with cell-free systems has revealed four components required for cyclin-ubiquitin ligation and proteolysis: a nonspecific ubiquitin-activating enzyme E1, a soluble fraction containing a ubiquitin carrier protein activity called E2-C, a crude particulate fraction containing a ubiquitin ligase (E3) activity that is activated during M-phase, and a constitutively active 26S proteasome that degrades ubiquitinated proteins. Here, we identify a novel approximately 1500-kDa complex, termed the cyclosome, which contains a cyclin-selective ubiquitin ligase activity, E3-C. E3-C is present but inactive during interphase; it can be activated in vitro by the addition of cdc2, enabling the transfer of ubiquitin from E2-C to cyclin. The kinetics of E3-C activation suggest the existence of one or more intermediates between cdc2 and E3-C. Cyclosome-associated E3-C acts on both cyclin A and B, and requires the presence of wild-type N-terminal destruction box motifs in each cyclin. Ubiquitinated cyclins are then rapidly recognized and degraded by the proteasome. These results identify the cyclosome-associated E3-C as the component of the cyclin destruction machinery whose activity is ultimately regulated by cdc2 and, as such, the element directly responsible for setting mitotic cyclin levels during early embryonic cell cycles.

Binding of activated cyclosome to p13(suc1). Use for affinity purification.

Previous studies have indicated that a approximately 1,500-kDa complex, designated the cyclosome or anaphase-promoting complex, has a regulated cyclin-ubiquitin ligase activity that targets cyclin B for degradation at the end of mitosis. The cyclosome is inactive in the interphase of the embryonic cell cycle and is converted to the active form in late mitosis in a phosphorylation-dependent process initiated by protein kinase Cdc2-cyclin B. We show here that the active, phosphorylated form of the cyclosome from clam oocytes binds to p13(suc1), a protein known to associate with Cdc2. The following evidence indicates that the binding of the cyclosome to p13(suc1) is not mediated via the Cdc2-cyclin B complex: (a) activated cyclosome binds to p13(suc1)-Sepharose following its separation from Cdc2-cyclin B by gel filtration chromatography; (b) cyclosome from interphase extracts, activated by a kinase in which cyclin B has been replaced by an N-terminally truncated derivative fused to glutathione S-transferase, binds well to p13(suc1)-Sepharose but not to glutathione-agarose. An alternative possibility, that the phosphorylated cyclosome binds directly to a phosphate-binding site of p13(suc1), is supported by the observation that the cyclosome is efficiently eluted from p13(suc1)-Sepharose by phosphate-containing compounds. This information was utilized to develop a procedure for the affinity purification of the cyclosome. A factor abundant in the fraction not adsorbed to p13(suc1)-Sepharose stimulates the activity of purified cyclosome. It is suggested that binding of Suc1 may have a role in the regulation of cyclosome activity.

Reversible phosphorylation controls the activity of cyclosome-associated cyclin-ubiquitin ligase.

Cyclin B/cdc2 is responsible both for driving cells into mitosis and for activating the ubiquitin-dependent degradation of mitotic cyclins near the end of mitosis, an event required for the completion of mitosis and entry into interphase of the next cell cycle. Previous work with cell-free extracts of rapidly dividing clam embryos has identified two specific components required for the ubiquitination of mitotic cyclins: E2-C, a cyclin-selective ubiquitin carrier protein that is constitutively active during the cell cycle, and E3-C, a cyclin-selective ubiquitin ligase that purifies as part of a approximately 1500-kDa complex, termed the cyclosome, and which is active only near the end of mitosis. Here, we have separated the cyclosome from its ultimate upstream activator, cdc2. The mitotic, active form of the cyclosome can be inactivated by incubation with a partially purified, endogenous okadaic acid-sensitive phosphatase; addition of cdc2 restores activity to the cyclosome after a lag that reproduces that seen previously in intact cells and in crude extracts. These results demonstrate that activity of cyclin-ubiquitin ligase is controlled by reversible phosphorylation of the cyclosome complex.

Components of a system that ligates cyclin to ubiquitin and their regulation by the protein kinase cdc2.

Cyclin B, a positive regulatory subunit of the cdc2 protein kinase complex, is synthesized across the cell cycle and then rapidly degraded at the end of mitosis. Degradation of cyclin B is triggered by increased levels of active cdc2 and is required for exit from mitosis. It was shown previously that cyclin degradation is carried out by the ubiquitin system, but the components responsible for the specificity and regulation of cyclin-ubiquitin ligation have not been identified. The formation of ubiquitin-protein conjugates usually requires the sequential action of three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-carrier protein (E2), and a ubiquitin-protein ligase (E3). In this work we employed a fractionation approach to identify the components of a clam oocyte system responsible for specific ubiquitination of cyclin and to determine which components are regulated by cdc2. Experimental conditions were established under which a fusion protein containing an amino-terminal fragment of cyclin B is ligated to ubiquitin only in extracts from M-phase but not from interphase cells. Fractionation of M-phase extracts by DEAE-cellulose and high speed centrifugation yielded three fractions that were all required for cell cycle stage-specific cyclin-ubiquitin ligation. Only one of these fractions could be replaced by a previously known enzyme of the ubiquitin system, E1. A second fraction contained a novel species of E2, termed E2-C, which acts in the ligation of ubiquitin to cyclin but not to other endogenous proteins. A third component is associated with particulate material. Whereas E2-C from either M-phase or interphase extracts is active, the particulate component is active only in M-phase. Incubation of the particulate fraction from interphase cells with the protein kinase cdc2 activates it for cyclin-ubiquitin ligation, after a lag of about 30 min. These findings suggest that the particulate fraction may contain an E3 enzyme that acts on cyclin, as well as additional factors activated by cdc2.