Editing Spindle checkpoint (section)

=== Mitotic checkpoint complex formation ===

The mitotic checkpoint complex is composed of [[BUB3]] together with MAD2 and MAD3 bound to [[Cdc20]]. MAD2 and MAD3 have distinct binding sites on CDC20, and act synergistically to inhibit APC/C. The MAD3 complex is composed of BUB3, which binds to Mad3 and [[BUB1B]] through the [[short linear motif]] known as the GLEBS motif. The exact order of attachments which must take place in order to form the MCC remains unknown. It is possible that Mad2-Cdc20 form a complex at the same time as BUBR1-BUB3-Cdc20 form another complex, and these two subcomplexes are consequently combined to form the mitotic checkpoint complex.<ref name="Mad2"/> In human cells, binding of BUBR1 to CDC20 requires prior binding of MAD2 to CDC20, so it is possible that the MAD2-CDC20 subcomplex acts as an initiator for MCC formation. BUBR1 depletion leads only to a mild reduction in Mad2-Cdc20 levels while Mad2 is required for the binding of BubR1-Bub3 to Cdc20. Nevertheless, BUBR1 is still required for checkpoint activation.<ref name="Nature"/>

The mechanism of formation for the MCC is unclear and there are competing theories for both kinetochore-dependent and kinetochore-independent formation. In support of the kinetochore-independent theory, MCC is detectable in ''[[S. cerevisiae]]'' cells in which core kinetocore assembly proteins have been mutated and cells in which the SAC has been deactivated, which suggests that the MCC could be assembled during mitosis without kinetochore localization. In one model, unattached prometaphase kinetochores can 'sensitize' APC to inhibition of MCC by recruiting the APC to kinetochores via a functioning SAC. Furthermore, depletions of various SAC proteins have revealed that MAD2 and BUBR1 depletions affect the timing of mitosis independently of kinetochores, while depletions of other SAC proteins result in a dysfunctional SAC without altering the duration of mitosis. Thus it is possible that the SAC functions through a two-stage timer where MAD2 and BUBR1 control the duration of mitosis in the first stage, which may be extended in the second stage if there are unattached kinetochores as well as other SAC proteins.<ref name="Nature"/> However, there are lines of evidence which are in disfavor of the kinetochore-independent assembly. MCC has yet to be found during [[interphase]], while MCC does not form from its constituents in ''[[X. laevis]]'' [[meiosis II]] extracts without the addition of sperm of nuclei and [[nocodazole]] to prevent spindle assembly.

The leading model of MCC formation is the "MAD2-template model", which depends on the kinetochore dynamics of MAD2 to create the MCC. MAD1 localizes to unattached kinetochores while binding strongly to MAD2. The localization of MAD2 and BubR1 to the kinetochore may also be dependent on the [[Aurora B kinase]].<ref>{{cite journal | vauthors = Lens SM, Wolthuis RM, Klompmaker R, Kauw J, Agami R, Brummelkamp T, Kops G, Medema RH | title = Survivin is required for a sustained spindle checkpoint arrest in response to lack of tension | journal = The EMBO Journal | volume = 22 | issue = 12 | pages = 2934–47 | date = June 2003 | pmid = 12805209 | pmc = 162159 | doi = 10.1093/emboj/cdg307 }}</ref> Cells lacking Aurora B fail to arrest in metaphase even when chromosomes lack microtubule attachment.<ref>{{cite journal | vauthors = Hauf S, Cole RW, LaTerra S, Zimmer C, Schnapp G, Walter R, Heckel A, van Meel J, Rieder CL, Peters JM | title = The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint | journal = The Journal of Cell Biology | volume = 161 | issue = 2 | pages = 281–94 | date = April 2003 | pmid = 12707311 | pmc = 2172906 | doi = 10.1083/jcb.200208092 }}</ref> Unattached kinetochores first bind to a MAD1-C-MAD2-p31<sup>comet</sup> complex and releases the p31<sup>comet</sup> through unknown mechanisms. The resulting MAD1-C-MAD2 complex recruits the open conformer of Mad2 (O-Mad2) to the kinetochores.  This O-Mad2 changes its conformation to closed Mad2 (C-Mad2) and binds Mad1.  This Mad1/C-Mad2 complex is responsible for the recruitment of more O-Mad2 to the kinetochores, which changes its conformation to C-Mad2 and binds Cdc20 in an auto-amplification reaction. Since MAD1 and CDC20 both contain a similar MAD2-binding motif, the empty O-MAD2 conformation changes to C-MAD2 while binding to CDC20. This [[positive feedback loop]] is negatively regulated by p31<sup>comet</sup>, which competitively binds to C-MAD2 bound to either MAD1 or CDC20 and reduces further O-MAD2 binding to C-MAD2. Further control mechanisms may also exist, considering that p31<sup>comet</sup> is not present in lower eukaryotes.  The 'template model' nomenclature is thus derived from the process where MAD1-C-MAD2 acts as a template for the formation of C-MAD2-CDC20 copies. This sequestration of Cdc20 is essential for maintaining the spindle checkpoint.<ref name="Mad2"/>