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 then tested whether MPS1 phosphorylation affects the interactions between MAD1-CTD and MAD2 conformers. We focused on CTD because it is not only easily purified without apparent degradation but also shows functional significance (see below). We did notice that GST-MPS1 could also bind to MAD2 L13A and MAD2 C10 (Fig 4c) . In vitro incubation of MPS1, CTD and MAD2 led to phosphorylation of CTD, as evidenced by the mobility shift and positive signals of phospho-Thr antibody (lane 1 in Fig 4 d and 4e ). However, the association of either MAD2 L13A or MAD2 C10 with CTD did not show obvious changes, when compared to reactions in the absence of ATP or in the presence of MPS1 inhibitor reversine. The interactions between phosphorylated CTD and MPS1, nevertheless, became weaker ( Fig. 4d and 4e). The sites are T8, S22, S62, T323, S598, S610, T624 and T716 (Fig. 5a) . To test the importance of these sites in cells, a mCherry-Mis12-MAD1 8A mutant with all the sites mutated into alanine was expressed and found to be defective in maintaining the mitotic arrest (229±23 min mitotic duration) (Fig. 5b) . Mutating all four NTD sites into alanine showed no effect, but alanine mutants at the CTD four sites (MAD1 CTD4A ) shortened the mitotic duration comparable to MAD1 8A (Fig. 5b) . Further tests found that little CTD 4A is phosphorylated when compared to wild type CTD (CTD WT ) in vitro, supporting that the CTD four sites, S598, S610, T624 and T716, constituted primary phosphorylation sites of MPS1 kinase under our in vitro experimental conditions (Fig. S4a) .