Plk1 bound to Bub1 contributes to spindle assembly checkpoint activity during mitosis

Abstract

For faithful chromosome segregation, the formation of stable kinetochore–microtubule attachment and its monitoring by the spindle assembly checkpoint (SAC) are coordinately regulated by mechanisms that are currently ill-defined. Here, we show that polo-like kinase 1 (Plk1), which is instrumental in forming stable kinetochore–microtubule attachments, is also involved in the maintenance of SAC activity by binding to Bub1, but not by binding to CLASP2 or CLIP-170. The effect of Plk1 on the SAC was found to be mediated through phosphorylation of Mps1, an essential kinase for the SAC, as well as through phosphorylation of the MELT repeats in Knl1. Bub1 acts as a platform for assembling other SAC components on the phosphorylated MELT repeats. We propose that Bub1-bound Plk1 is important for the maintenance of SAC activity by supporting Bub1 localization to kinetochores in prometaphase, a time when the kinetochore Mps1 level is reduced, until the formation of stable kinetochore-microtubule attachment is completed. Our study reveals an intricate mechanism for coordinating the formation of stable kinetochore–microtubule attachment and SAC activity.

Kinetochore phosphatases suppress autonomous kinase activity to control the spindle assembly checkpoint

“…The kinase, in this case, is PLK1 which is recruited via the BUB complex to the SAC scaffold KNL1. PLK1 is able to phosphorylate KNL1 on MELT motifs to recruit further BUB complexes, which, directly or indirectly, recruits all other proteins needed to generate the SAC signal 1,2,[25][26][27] . Therefore, this positive feedback loop helps to maintain the SAC in a semi-autonomous manner (pMELT→BUB:PLK1→pMELT, Figure 4B, P1).…”

“…Therefore, this creates over a thousand active MELT repeats per kinetochore, which the BUB-PLK1 module can use to rapidly amplifying SAC signalling downstream of MPS1. Furthermore, this may be reinforced by an additional positive feedback loop to MPS1 itself, since PLK1 can phosphorylate the activation loop of MPS1 to stimulate its kinase activity 26,27,37 (MPS1→BUB:PLK1→MPS1, Figure 4B, P2). This may help to explain the recent observation that this 'autoactivation' site remains phosphorylated when MPS1 is inhibited in BUBR1 PP2A cells 38 .…”

“…PLK1 is able to support SAC signalling by phosphorylating the MELT motifs directly [25][26][27] . Therefore, we hypothesised that the increased BUB-PLK1 levels in KNL1 PP1 and BUBR1 PP2A cells could help to sustain MELT phosphorylation and the SAC in the absence of MPS1 activity.…”

“…PLK1 interacts via its polo-box domains (PBDs) to phospho-epitopes on various different kinetochore complexes, including to two CDK1 phosphorylation sites on the BUB complex (BUB1-pT609 and BUBR1-pT620) [20][21][22] . PLK1 has similar substrates preferences to MPS1 23,24 and it shares at least two key substrates that are critical for SAC signalling: the KNL1-MELT motifs and MPS1 itself, including key sites in the MPS1 activation loop [25][26][27] . PLK1 can therefore enhance MPS1 kinase activity and also directly phosphorylate the MELT motifs to support SAC signalling, perhaps from its localised binding site on BUB1 27 .…”

“…PLK1 has similar substrates preferences to MPS1 23,24 and it shares at least two key substrates that are critical for SAC signalling: the KNL1-MELT motifs and MPS1 itself, including key sites in the MPS1 activation loop [25][26][27] . PLK1 can therefore enhance MPS1 kinase activity and also directly phosphorylate the MELT motifs to support SAC signalling, perhaps from its localised binding site on BUB1 27 . It is unclear why PLK1 is needed to cooperate with MPS1 in SAC signalling and, importantly, what inhibits PLK1 signalling to allow MELT dephosphorylation and SAC silencing upon microtubule attachment.…”