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

As a sensitive signaling system, the mitotic checkpoint ensures faithful chromosome segregation by delaying anaphase onset when even a single kinetochore is unattached. The key signal amplification reaction for the checkpoint is the conformational conversion of open MAD2 (O-MAD2) into closed MAD2 (C-MAD2). The reaction was suggested to be catalyzed by an unusual catalyst, a MAD1:C-MAD2 tetramer, but how the catalysis is executed and regulated remains elusive. Here we report that in addition to the well-characterized middle region (MIM), both amino- and carboxyl-terminal domains (NTD and CTD) of MAD1 also contribute to the mitotic checkpoint. In contrast to MIM that stably associates with C-MAD2, MAD1-NTD and CTD surprisingly bind to both O-MAD2 and C-MAD2, suggesting their interactions with substrates and products of the O-C conversion. MAD1-NTD also interacts with CTD. MPS1 kinase interacts with and phosphorylates both NTD and CTD. The phosphorylation reduces the NTD:CTD interaction and CTD interaction with MPS1. Mutating CTD phosphorylation sites including Thr716 compromises MAD2 binding and the checkpoint responses. Ser610 and Tyr634 also contribute to the checkpoint. Our results have uncovered previously unknown interactions of MAD1-NTD and CTD with MAD2 conformers and their regulation by MPS1 kinase, providing novel insights into the mitotic checkpoint signaling.