Aurora B phosphorylates Bub1 to promote spindle assembly checkpoint signaling

Abstract

SummaryAccurate chromosome segregation during cell division requires amphitelic attachment of each chromosome to the spindle apparatus. This is ensured by the Spindle Assembly Checkpoint (SAC) [1], which delays anaphase onset in response to unattached chromosomes, and an error correction mechanism, which eliminates syntelic chromosome attachments [2]. The SAC is activated by the Mps1 kinase. Mps1 sequentially phosphorylates the kinetochore protein Spc105/KNL1 to license the recruitment of several signaling proteins including Bub1. These proteins produce the Mitotic Checkpoint Complex (MCC), which delays anaphase onset [3-8]. The error correction mechanism is regulated by the Aurora B kinase, which phosphorylates the microtubule-binding interface of the kinetochore. Aurora B is also known to promote SAC signaling indirectly [9-12]. Here we present evidence that Aurora B kinase activity directly promotes MCC production in budding yeast and human cells. Using the ectopic SAC activation (eSAC) system, we find that the conditional dimerization of Aurora B (or an Aurora B recruitment domain) with either Bub1 or Mad1, but not the ‘MELT’ motifs in Spc105/KNL1, leads to a SAC-mediated mitotic arrest [13-16]. Importantly, ectopic MCC production driven by Aurora B requires the ability of Bub1 to bind both Mad1 and Cdc20. These and other data show that Aurora B cooperates with Bub1 to promote MCC production only after Mps1 licenses Bub1 recruitment to the kinetochore. This direct involvement of Aurora B in SAC signaling is likely important for syntelically attached sister kinetochores that must delay anaphase onset in spite of reduced Mps1 activity due to their end-on microtubule attachment.

Signaling protein abundance modulates the strength of the Spindle Assembly Checkpoint

“…To ensure accurate quantification and comparative analysis, we used genome-edited HeLa cells that express either mNeonGreen-Bub1, mNeonGreen-BubR1, or Mad1-mNeonGreen [“mNeonGreen” is abbreviated as “mNG” henceforth; (Shaner et al, 2013)]. Because the fluorescent tags are separated from the tagged proteins by flexible linkers, and we have previously found that N-terminally tagged BubR1 and C-terminally tagged Mad1 are functional in budding yeast (Aravamudhan et al, 2016; Roy et al, 2022). Because of the similarity of the domain organizations of Bub1 and BubR1, we expect that Bub1 function will also be unaffected by the N-terminal tag.…”

BubR1 recruitment to the kinetochore via Bub1 enhances Spindle Assembly Checkpoint signaling

“…In fission yeast, BubR1 binding to Bub1 is essential for SAC activity (Leontiou et al, 2019). However, BubR1 is unlikely to be recruited to budding yeast kinetochores (Roy et al, 2022; Tromer et al, 2016), and in nocodazole-treated HeLa cells, the disruption of Bub1-mediated BubR1 recruitment slightly strengthens the SAC (Overlack et al, 2015; Zhang et al, 2016) because the disruption of BubR1 recruitment also disrupted BubR1-mediated PP2A recruitment (Elowe et al, 2007; Suijkerbuijk et al, 2012). Therefore, to isolate and quantitatively define the effect of Bub1-BubR1 heterodimerization on Bub1-mediated MCC assembly, we used the ectopic SAC activation system or the eSAC.…”

“…In HeLa cells, induced dimerization of the central domain of Bub1 (Bub1 231-620 -mNG-2xFkbp12, diagram in Figure 1B) with the Mps1 kinase domain (Frb-mCherry-Mps1 500-817 ) produces a dosage-dependent increase in the duration of mitosis with a maximal duration of ∼ 145 minutes (Figure 1C replotted for comparison from (Chen et al, 2019)). Importantly, the extended mitosis was due to increased Mad2-Cdc20 and MCC formation in HeLa cells and fission yeast (Leontiou et al, 2019; Roy et al, 2022). To assess the contribution of Bub1-BubR1 heterodimerization to Bub1-mediated MCC assembly, we created a truncated Bub1 phosphodomain lacking the BubR1 heterodimerization domain (Bub1 444-620 -mNG-2xFkbp12) (Overlack et al, 2015; Zhang et al, 2016).…”

“…Rapamycin-induced dimerization of this phosphodomain with Frb-mCherry-Mps1 500- 817 elicited a significantly weaker eSAC activity, with a maximal mitotic duration of 105 ± 6 minutes (Figure 1C, estimated from a fit with the 4-parameter Hill equation, the range indicates 95% confidence intervals, see Methods for details). The absence of the Bub3-binding GLEBS domain in Bub1 444-620 -mNG-2xFkbp12 cannot explain the observed decrease in eSAC activity (Roy et al, 2022). Thus, Bub1-BubR1 heterodimerization promotes MCC mediated by the Bub1 phosphodomain.…”

“…We find that Bub1-BubR1 heterodimerization elevates Bub1-mediated MCC generation. On the other hand, the recruitment of Bub1 and BubR1 via the 'KI' motifs in the kinetochore protein Knl1 does not contribute to MCC generation mediated by the MELT motifs within the Knl1 phosphodomain (Bolanos-Garcia et al, mitotic delay induced by the eSAC activity (Roy et al, 2021). Thus, Bub1-BubR1 heterodimerization contributes to the ability of the Bub1 phosphodomain to generate MCC.…”