Abstract
Local phosphatase regulation is needed at kinetochores to silence the mitotic checkpoint (a.k.a. spindle assembly checkpoint [SAC]). A key event in this regard is the dephosphorylation of MELT repeats on KNL1, which removes SAC proteins from the kinetochore, including the BUB complex. We show here that PP1 and PP2A-B56 phosphatases are primarily required to remove Polo-like kinase 1 (PLK1) from the BUB complex, which can otherwise maintain MELT phosphorylation in an autocatalytic manner. This appears to be their principal role in the SAC because both phosphatases become redundant if PLK1 is inhibited or BUB-PLK1 interaction is prevented. Surprisingly, MELT dephosphorylation can occur normally under these conditions even when the levels or activities of PP1 and PP2A are strongly inhibited at kinetochores. Therefore, these data imply that kinetochore phosphatase regulation is critical for the SAC, but primarily to restrain and extinguish autonomous PLK1 activity. This is likely a conserved feature of the metazoan SAC, since the relevant PLK1 and PP2A-B56 binding motifs have coevolved in the same region on MADBUB homologues.
Highlights
The mitotic checkpoint, known as the spindle assembly checkpoint (SAC), prevents mitotic exit until chromosomes have attached to microtubules via the kinetochore (Corbett, 2017; Saurin, 2018)
PP1-KNL1 and PP2A-B56 antagonize Polo-like kinase 1 (PLK1) recruitment to the BUB complex Inhibition of PP1-KNL1 or knockdown of PP2A-B56 both enhance PLK1 recruitment to kinetochores (Foley et al, 2011; Liu et al, 2012). To test whether this was due to localized phosphatase inhibition at the BUB complex, we inhibited the recruitment of PP2A-B56 to BUBR1 (BUBR1ΔPP2A) and compared this to a PP1KNL1 mutant (KNL1ΔPP1), as used previously (Liu et al, 2012; Nijenhuis et al, 2014; note that, in these and all subsequent experiments, siRNA-mediated gene knockdown was used in combination with doxycycline-inducible replacement of the WT or mutant gene from an FRT locus; see Materials and methods)
PLK1 binds via its Polo-box domain (PBD) to CDK1 phosphorylation sites on BUB1 and BUBR1; we raised antibodies to these sites and validated their specificity in cells (Fig. S2, A–C)
Summary
The mitotic checkpoint, known as the spindle assembly checkpoint (SAC), prevents mitotic exit until chromosomes have attached to microtubules via the kinetochore (Corbett, 2017; Saurin, 2018). PLK1 can enhance MPS1 kinase activity and directly phosphorylate the MELT motifs to support SAC signaling, perhaps from its localized binding site on BUB1 (Ikeda and Tanaka, 2017).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
