Chromosome segregation errors: a double-edged sword - TI Pharma

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years before this experimental evidence was published, some of the components that are responsible
for this mitotic checkpoint signal were identified by two independent screens for budding yeast
mutants that were unable to delay mitosis in the presence of microtubule destabilizing drugs 71,72 .
These identified genes included Bub1, Bub3 71 , MAD1, MAD2 and MAD3 72 and were later found to be
conserved in higher eukaryotes 73-76 . All checkpoint components are recruited to outer kinetochores
in prometaphase, where they monitor MT attachment status and delay anaphase onset through
inhibition of the E3 ubiquitin ligase activity of the Anaphase Promoting Complex or Cyclosome
(APC/C). The APC/C is a 1.5-MDa complex which ubiquitinates various substrates important for cell
cycle progression (reviewed in 77 ) by collaborating with three E2 enzymes, UbcH5, UbcH10 and Ube2S
78-81 . The unattached kinetochore is thought to function as a catalytic platform for the generation of
the mitotic checkpoint complex (MCC) 82 , consisting of Mad2, Bub3 and the pseudo-kinase BubR1 83
(Mad3 in budding yeast), which directly binds and sequesters the APC/C co-activator Cdc20 84-94 . By
binding Cdc20, the MCC inhibits the ubiquitination of two APC/C substrates, Securin and Cyclin B1,
whose degradation by the 26S proteasome is necessary for cells to progress into anaphase 95-99 (Fig.4).
The protease Separase is required to separate sister chromatids in anaphase by cleaving the cohesin
complex present in between both DNA strands 99,100 and is kept inactive by its chaperone Securin 101 .
Therefore, degradation of Securin in anaphase is required to allow cohesin cleavage and subsequent
separation of sister chromatids to opposite sides of the cell98-100 . Degradation of Cyclin B1 results in
inactivation of the major mitotic regulator Cyclin B1- Cdk1, an event that is essential for mitotic exit96,97 .
Unattached kinetochores catalyze MCC formation through the presence of a hetero-tetrameric complex
consisting of MAD2 and MAD1 102,103 (Fig.4). MAD2 homodimerization at this kinetochore-bound
complex induces a conformational change in the inactive cytoplasmic form of MAD2, called open- or
O-MAD2, to the active form, termed closed- or C-MAD2 104,105 . This closed form of MAD2 is capable
of forming the MCC complex with BubR1, Bub3 and Cdc20 and therefore to inhibit APC/C activity 106 .
BubR1 binds to the MCC through its conserved N-terminal KEN-box 107-110 , which is a motif specifically
recognized by Cdc20 111 . Another KEN-Box motif in BubR1, more centrally located, can also directly
bind the APC/C and is thought to act as a pseudosubstrate 107,108,110 . The final component of the MCC is
Bub3, which is, besides MCC binding 91 , required for kinetochore localization of both BubR1 and Bub1 76 .
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MAD1 C-MAD2
Bub1
Bub3
Mps1
BubR1
BubR1
Bub3
O-MAD2
Cdc20
C-MAD2
APC/C
Cdc20
Ub
Ub
Ub
Ub
Cyclin B1
Ub
Ub
Ub
Ub
Securin
Figure 4. Mitotic checkpoint signaling
Mitotic checkpoint components are recruited to unattached kinetochores, thereby creating a platform for the formation of the
Mitotic Checkpoint Complex (MCC) consisting of MAD2, BubR1 and Bub3, which inhibits APC/C dependent Cyclin B1 and Securin
degradation through sequestration of Cdc20.