Abstract
As a sensitive signaling system, the mitotic checkpoint ensures faithful chromosome segregation by delaying anaphase onset even when a single kinetochore is unattached to mitotic spindle microtubules. The key signal amplification reaction for the checkpoint is the conformational conversion of "open" mitotic arrest deficient 2 (O-MAD2) into "closed" MAD2 (C-MAD2). The reaction has been 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 of MAD1 containing the MAD2-interaction motif (MIM), both N- and C-terminal domains (NTD and CTD) of MAD1 also contribute to mitotic checkpoint signaling. Unlike the MIM, which stably associated only with C-MAD2, the NTD and CTD in MAD1 surprisingly bound both O- and C-MAD2, suggesting that these two domains interact with both substrates and products of the O-to-C conversion. MAD1NTD and MAD1CTD also interacted with each other and with the MPS1 protein kinase, which phosphorylated both NTD and CTD. This phosphorylation decreased the NTD:CTD interaction and also CTD's interaction with MPS1. Of note, mutating the phosphorylation sites in the MAD1CTD, including Thr-716, compromised MAD2 binding and the checkpoint responses. We further noted that Ser-610 and Tyr-634 also contribute to the mitotic checkpoint signaling. Our results have uncovered that the MAD1NTD and MAD1CTD directly interact with each other and with MAD2 conformers and are regulated by MPS1 kinase, providing critical insights into mitotic checkpoint signaling.
Highlights
The mitotic checkpoint is a crucial signal transduction pathway that contributes to faithful chromosome segregation [1,2,3,4]
We reasoned that better characterization of the MAD1:closed” mitotic arrest deficient 2 (MAD2) (C-MAD2) catalyst would provide further mechanistic insights into the conversion reaction and the signal amplification step of the mitotic checkpoint
Note no GFP-MAD2L13A was localized at metaphase kinetochores containing mCherry-Mis12-MAD1AA, GFPMAD2L13A did appear at the last few unattached kinetochores, most likely because of presence of endogenous MAD1 there (Fig. S2, compare the second and third columns)
Summary
As a sensitive signaling system, the mitotic checkpoint ensures faithful chromosome segregation by delaying anaphase onset even when a single kinetochore is unattached to mitotic spindle microtubules. We report that in addition to the well-characterized middle region of MAD1 containing the MAD2-interaction motif (MIM), both N- and C-terminal domains (NTD and CTD) of MAD1 contribute to mitotic checkpoint signaling. MAD1NTD and MAD1CTD interacted with each other and with the MPS1 protein kinase, which phosphorylated both NTD and CTD. Our results have uncovered that the MAD1NTD and MAD1CTD directly interact with each other and with MAD2 conformers and are regulated by MPS1 kinase, providing critical insights into mitotic checkpoint signaling. Our nal domain; CTD, C-terminal domain; APC/C, anaphase promoting complex/cyclosome; MCC, mitotic checkpoint complex; MIM, MAD2-interaction motif; TEV, tobacco etch virus. Our work highlights the coordination of different MAD1 domains in efficient mitotic checkpoint signaling and provides further mechanistic insights into the MAD2 O–C conversion reaction
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