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

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 wellcharacterized middle region (MIM), both aminoand 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. _______________________________________ The mitotic checkpoint is a crucial signal transduction pathway that contributes to faithful chromosome segregation (1) (2) (3) (4) . A single unattached kinetochore delays anaphase onset, underscoring the importance of signal amplification for the mitotic checkpoint (5) . The conversion of MAD2 from open (O-MAD2) to closed (C-MAD2) conformation is a well-recognized signal amplification mechanism for the mitotic checkpoint (6, 7) . O-MAD2 is the predominant conformer in interphase cells (8, 9) . During prometaphase intracellular C-MAD2 concentration is increased to promote formation of the mitotic checkpoint complex (MCC), which binds and inhibits the anaphase promoting complex/cyclosome (APC/C) (1) (2) (3) (4) . In the current model, the MAD2 O-C conversion is catalyzed by a 2:2 MAD1:C-MAD2 tetramer localized at unattached kinetochores, whereby cytoplasmic O-MAD2 hetero-dimerizes with the C-MAD2 moiety in the catalyst and morphs into C-MAD2 through unknown mechanism but possibly involving some intermediate folding states (I-MAD2) (6, 7, 10) .