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

Since MAD1-MIM, CTD and NTD are all required for an effective mitotic checkpoint (Fig. 1) , we next investigated whether there is coordination between these domains. The presumed non-coiled coil domains along the length of MAD1 might provide certain bends and turns to the molecule, and an earlier model did suggested that MAD1 CTD folds back to the proximity of the catalytic core consisting of MIM and associated C-MAD2 (11) . We examined and found that NTD directly interacts with CTD (Fig. 6a , lane 1). CTD interacts with itself (lane 3), agreeing with published crystal structure and acting as a positive control in this binding assay (33) . MIM does not directly interact with CTD nor NTD (Fig. 6a , lane 2 and Fig. 6b ). Addition of MPS1 kinase reduced the interactions between MAD1 NTD and MAD1 CTD (Fig. 6c) . The effect depends on the MPS1 kinase activity as omitting ATP from the reactions or adding MPS1 inhibitors (reversine or AZ3146) reversed the MPS1 effect on NTD:CTD interaction (compare His signals in lane 2 and those in lanes [3] [4] [5] .