Converging genetic, cell biological and biochemical studies have begun to shed some light on to how the mitotic spindle checkpoint components work at a molecular level.
As described, the spindle assembly checkpoint controls both the attachment of microtubules to kinetochores and the tension that is exerted at kinetochores upon bipolar attachment. In the absence of bipolar attachment, the spindle checkpoint proteins have to emit a global signal throughout the cell to inhibit anaphase onset.
How do the Mad/Bub proteins mediate cell cycle arrest and what is the target of the mitotic spindle checkpoint?
It is now clear that one main consequence of spindle checkpoint activation is the inhibition of the APC/C (Anaphase Promoting Complex/Cyclosome), a large multi-subunit E3 ubiquitin-protein ligase (Fig. 5) (Page and Hieter, 1999; King et al., 1996). An E3 ubiquitin ligase transfers ubiquitin to lysine residues in substrate proteins, and proteins modified in such a way are then recognized and degraded by the proteasome (Voges et al., 1999). The APC/C normally becomes active at the metaphase-anaphase transition, and its activity is required for anaphase entry. The
accessory factor Cdc20 and triggers the degradation of an anaphase-inhibiting protein called securin (Yanagida, 2000; Nasmyth et al., 2000). Polyubiquitin chains are added to securin by the APC/C, which leads to its destruction through 26S-proteasome-mediated proteolysis (Yamamoto et al., 1996; Cohen-Fix et al., 1996; Funabiki et al., 1996; Zou et al., 1999). Securin forms a tight complex with an evolutionarily conserved caspase-related protease termed separase (Funabiki et al., 1996; Ciosk et al., 1998; Kumada et al., 1998; Zou et al., 1999; Uhlmann et al., 2000; Waizenegger et al., 2000), thereby inhibiting separase´s activity. Thus, degradation of securin releases separase, which in turn must be phosphorylated, probably directly by cyclin-dependent kinases (Cdk1), in order to efficiently cleave its substrate (Stemmann et al., 2001). The substrate of separase is a subunit of a multiprotein complex termed cohesin, which creates physical links between sister chromatids (Waizenegger et al., 2000; Michaelis et al., 1997). Sister chromatid cohesion is first established during chromosome replication in S phase. Removal of the cohesion complex is regulated by two mechanisms: firstly, in higher eukaryotes the removal of cohesins from the chromosome arms is promoted by phosphorylation of the cohesion complex by Polo-like kinase 1 (Sumara et al., 2002). Residual cohesion at the centromeric region is enough to prevent sister-chromatid separation. Secondly, these remaining complexes must be disrupted through proteolytic cleavage of a cohesin subunit called Scc1 by separase (Uhlmann et al., 1999). This allows the sister chromatids to move poleward along the mitotic spindle, and anaphase is initiated (Fig. 5).
What is the nature of this “wait anaphase” signal that inhibits the activity of the APC/C and therefore prevents sister chromatid separation? The ubiquitin ligase activity of the APC/C towards securin requires association of APC/C with Cdc20, which activates the APC/C by direct binding (Visintin et al., 1997; Hwang et al., 1998; Shirayama et al., 1998; Fang et al., 1998a). Cdc20, the activating protein for the APC/C, is the molecular target of the mitotic spindle checkpoint, and two of the checkpoint proteins, Mad2 and BubR1 have been shown to interact with Cdc20, resulting in APC/C inhibition (Li et al., 1997; Fang et al., 1998b; Hwang et al., 1998;
Kim et al., 1998; Sudakin et al., 2001; Tang et al., 2001; Fang, 2002). In fact, two different checkpoint complexes have been purified from HeLa cells, which are capable of inhibiting the APC/C. One isolated checkpoint complex contains BubR1, Bub3 and substoichiometric amounts of Cdc20 (Tang et al., 2001). In addition, it was shown that recombinant BubR1 directly inhibits the activity of the APC/C and that the
kinase activity of BubR1 is not required for this inhibition (Tang et al., 2001).
Independently, another checkpoint complex has been isolated which is termed mitotic checkpoint complex (MCC). The MCC contains nearly stoichiometric amounts of BubR1, Bub3, Mad2 and Cdc20 (Sudakin et al., 2001), and the MCC is more potent than Mad2 alone at inhibiting the ubiquitin ligase activity of the APC/C. Importantly, the very same complex consisting of Mad3 (BubR1 homolog), Bub3, Mad2 and Cdc20 has been isolated from fission yeast (Millband and Hardwick, 2002) and budding yeast (Fraschini et al., 2001b). Future studies have to resolve the issue of whether Mad2 and BubR1 function independently or synergistically in transducing the anaphase-delaying signal by inhibiting APC/C activity.
Figure 5: The mitotic spindle checkpoint pathway. The checkpoint is activated by lack of microtubule attachment and tension at the kinetochores. The mitotic spindle checkpoint components are recruited to unattached kinetochores and might form several checkpoint protein complexes, e.g. the MCC, which is able to inhibit the APC/C. When all kinetochores achieve bipolar attachment to the mitotic spindle, the checkpoint is inactivated. The active APC/C is activated and ubiquitinates securin. Degradation of securin releases and activates separase, which cleaves Scc1, a subunit of the cohesion complex. Loss of sister chromatid cohesion triggers chromosome segregation and the onset of anaphase. Ub, ubiquitin.
Adopted and modified from (Yu, 2002).
The kinetochore is believed to act as a catalytic site for the production of this
“wait anaphase” signal that diffuses away to block the activity of the APC/C throughout the cell. In fact, it was shown that all of the vertebrate Mad and Bub checkpoint proteins localize to unattached kinetochores (Taylor et al., 2001; Chen et al., 1998; Sharp-Baker and Chen, 2001; Luo et al., 2002), consistent with the proposed role of kinetochores in generating the inhibitory checkpoint signal. In addition, along with the fact that Cdc20 is also enriched at kinetochores, it was suggested that the checkpoint complexes might be assembled at kinetochores (Fang et al., 1998b; Kallio et al., 1998). Furthermore, Cdc20 and several checkpoint proteins, including Mad2 and BubR1, turn over rapidly at the kinetochores in mammalian cells (Yu, 2002; Howell et al., 2000; Kallio et al., 2002a). Therefore, it is possible that unattached kinetochores catalyse the formation of the inhibitory checkpoint protein complexes, which then diffuse away to inhibit the APC/C. The diffusible signal might be the MCC and/or BubR1/Bub3/Cdc20. However, the MCC is present throughout the cell cycle and its formation does not require kinetochores (Sudakin et al., 2001).
Likewise, in yeast, functional kinetochores are not required for the formation of the MCC (Fraschini et al., 2001b). The exact role of the kinetochores in contributing to checkpoint signaling and in the formation of distinct checkpoint protein complexes has to be resolved by future studies.