Phosphorylation propels p31^comet for mitotic exit

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Phosphorylation of Xenopus p31comet potentiates mitotic checkpoint exit

The spindle checkpoint ensures accurate chromosome segregation and prevents aneuploidy. Unattached kinetochores during mitosis activate the checkpoint, which inhibits the anaphase-promoting complex or cyclosome (APC/C) and delays chromosome segregation.¹ The critical checkpoint protein Mad2 has multiple conformers, including the inactive open Mad2 (O-Mad2) and the active closed Mad2 (C-Mad2). At unattached kinetochores, the Mad1–C-Mad2 complex recruits cytosolic O-Mad2 and converts it to intermediate Mad2 (I-Mad2) (Fig. 1). I-Mad2 binds Cdc20 (the mitotic activator of APC/C) to form the Cdc20–C-Mad2 complex. Cdc20–C-Mad2 then associates with BubR1–Bub3 to form the mitotic checkpoint complex (MCC), which is an effective inhibitor of APC/C.

Figure 1. Activating p31^comet through phosphorylation in Xenopus egg extracts. During checkpoint activation, Mad1–C-Mad2 at unattached kinetochores recruit O-Mad2 and converts it to I-Mad2, which binds Cdc20. Cdc20–C-Mad2 associates with BubR1–Bub3 to form MCC, which inhibits APC/C. During checkpoint inactivation, IKKβ phosphorylates p31^comet and promotes its binding to C-Mad2, thereby blocking Mad2 activation and triggering MCC disassembly.

The Mad2-binding protein p31^comet stimulates spindle checkpoint silencing and controls timely chromosome segregation.¹ It specifically binds to C-Mad2, but not to O-Mad2, and inhibits C-Mad2 in 2 ways.² First, p31^comet binds Mad1–C-Mad2 and prevents conformational activation of O-Mad2. Second, p31^comet weakens MCC by disrupting the BubR1–C-Mad2 interaction, and collaborates with the ATPase Trip13 to disassemble the Cdc20–C-Mad2 complex, leading to MCC disassembly and APC/C activation. Despite the functional importance of p31^comet, how it is regulated during mitosis is poorly understood. The study of Mo et al. in this issue provides new insights into this important question.³

Proteomic analysis revealed that p31^comet is a phospho-protein during mitosis in human cells.⁴ An earlier report showed that S102 phosphorylation of human p31^comet inhibits its function and weakens its interaction with Mad2.⁵ Using Xenopus egg extracts as the model system, Mo et al. have now demonstrated that the kinase IKKβ phosphorylates and activates p31^comet during mitotic exit.³

The Xenopus egg extract is a powerful cell-free system that recapitulates many mitotic events. Mature Xenopus eggs are arrested at metaphase by the cytostatic factor (CSF) Emi2/xErp1, an inhibitor of APC/C^Cdc20. The mitotic CSF extracts can turn added sperm nuclei into discrete condensed chromosomes, which nucleate the formation of bipolar spindles. Addition of Ca²⁺ triggers Emi2 degradation and mitotic exit. Microtubule depolymerization by nocodazole followed by Ca²⁺ addition to these extracts creates a mitotic arrest dependent on the spindle checkpoint.

Mo et al. first showed that p31^comet promoted Ca²⁺-triggered mitotic exit in CSF extracts supplemented with sperm nuclei, as evidenced by delayed cyclin B degradation in p31^comet-depleted extracts.³ Co-depletion of Mad2 rescued the mitotic delay caused by p31^comet depletion, indicating that p31^comet promotes checkpoint inactivation through antagonizing Mad2 in these extracts, rather than through Emi2-dependent mechanisms. This finding further suggests that, in the absence of nocodazole, the spindle checkpoint is engaged in these reconstituted extracts permissible for spindle formation.

Mo et al. then showed that the level of p31^comet phosphorylation was high during mitosis and decreased after mitotic exit.³ They identified IKKβ as a kinase of p31^comet. Depletion or chemical inhibition of IKKβ delayed mitotic exit. IKKβ phosphorylated S4, T6, and T179 of Xenopus p31^comet. Recombinant purified phospho-mimicking p31^comet EEE mutant bound more tightly to Mad2 in vitro. The phosphorylation-deficient p31^comet AAA mutant was less efficient in promoting mitotic exit whereas p31^comet EEE was more effective. Strikingly, p31^comet EEE greatly reduced the amount of Mad2 bound to chromosomes, suggesting that p31^comet phosphorylation was particularly important for blocking Mad2 activation at kinetochores. Thus, phosphorylation-enhanced binding of p31^comet to Mad2 blocks Mad2 activation at kinetochores and promotes MCC disassembly in the cytosol, leading to APC/C activation and mitotic exit (Fig. 1).

S4 and T6 are located in the flexible N-terminal region of p31^comet dispensable for Mad2 binding. It is not apparent how their phosphorylation might enhance p31^comet binding to Mad2. On the other hand, our structural modeling reveals that T179 of p31^comet is located in a loop close to the Mad2–p31^comet interface. Intriguingly, 2 conserved arginines of Mad2, R129 and R133, lie in the vicinity of T179, and could conceivably engage in favorable electrostatic interactions with phospho-T179. T179 phosphorylation of Xenopus p31^comet might underlie the increased affinity toward Mad2.

Unfortunately, T179 is not conserved in other vertebrate p31^comet proteins.³ Moreover, there are conflicting reports on the mitotic role of IKKβ in human cells.^6,7 Additional studies are needed to test whether IKKβ-mediated phosphorylation regulates p31^comet in other species and during the somatic cell cycle. Regardless, the Mo et al. study provides the first evidence that phosphorylation can boost the function of p31^comet during mitotic exit. It also raises the exciting possibility that upstream signals in the canonical, pro-survival NF-κB pathway might have non-canonical roles in regulating the spindle checkpoint and mitotic progression.