Coordination between discrete Mitotic Arrest Deficient 1 (MAD1) domains is required for efficient mitotic checkpoint signaling

Wenbin Ji; Yibo Luo; Ejaz Ahmad; Song-Tao Liu

We have demonstrated that MPS1 interacts with and phosphorylates MAD1-NTD and CTD (Fig. 4) . Phospho-resistant mutants at in vitro MPS1 phosphorylation sites in MAD1-CTD especially the T716A mutation compromised the mitotic checkpoint responses in cells and showed reduced interaction with MAD2 in vitro (Fig 5) . No increase in MAD2 interactions with MAD1-CTD was detected either using phospho-mimic T716E mutant or after direct in vitro phosphorylation by MPS1 (Fig. 4, Fig. 5 ). We cannot exclude the possibility that in cells and in the context of full length MAD1, CTD phosphorylation show differential binding towards either conformer of MAD2. Phosphorylation by MPS1 did reduce its own interaction with CTD and the interaction between NTD and CTD (Fig. 4, Fig. 6 ). We propose that the NTD:CTD interaction occurs in interphase cells and represents an inactive state of MAD1 even though its MIM associates with C-MAD2 in a cell cycle independent manner (18,24). The interaction between MPS1 and MAD1 might contribute to the MAD1 recruitment to kinetochores. Once concentrated at unattached kinetochores, MPS1 kinase becomes activated and phosphorylates BUB1 to stably anchor MAD1 (30, 31, 57) . Activated MPS1 also phosphorylates MAD1-CTD, most likely at T716, relieving itself and the NTD from CTD. The now exposed and phosphorylated CTD could bind to O-MAD2 and C-MAD2 and also facilitate MAD2 O-C conversion through increased association with CDC20 (30) or other more direct mechanisms as discussed in last section.