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

Abstract

Local phosphatase regulation is critical for determining when phosphorylation signals are activated or deactivated. A typical example is the spindle assembly checkpoint (SAC) during mitosis, which regulates kinetochore PP1 and PP2A-B56 activities to switch-off signalling events at the correct time. In this case, kinetochore phosphatase activation dephosphorylates MELT motifs on KNL1 to remove SAC proteins, including the BUB complex. We show here that, surprisingly, neither PP1 or PP2A are required to dephosphorylate the MELT motifs. Instead, they remove polo-like kinase 1 (PLK1) from the BUB complex, which can otherwise maintain MELT phosphorylation in an autocatalytic manner. This is their principle role in the SAC, because both phosphatases become redundant if PLK1 is inhibited or BUB-PLK1 interaction is prevented. Therefore, phosphatase regulation is critical for the SAC, but primarily to restrain and extinguish autonomous kinase activity. We propose that these circuits have evolved to generate a semi-autonomous SAC signal that can be synchronously silenced following kinetochore-microtubule tension.

BUB1 and CENP-U, Primed by CDK1, Are the Main PLK1 Kinetochore Receptors in Mitosis

“…Without priming kinases, addition of Mg 2+ -ATP did not promote binding to MBP-PLK1 PBD , indicating that despite considerable auto-phosphorylation BUB1 kinase activity is not required for binding to MBP-PLK1 PBD . These observations are in line with previous work on the role of BUB1 and BUBR1 as PLK1 receptors (Cordeiro et al, 2020;Elowe et al, 2007;Qi et al, 2006) and show that CDK1 and PLK1 prime BUB1 and BUBR1 to become direct binding partners of PLK1 PBD . Aurora B and BUB1 Contribute Indirectly to PLK1 Recruitment MPS1 phosphorylates KNL1 to generate binding sites for BUB1 (and, indirectly, through the latter for BUBR1) (Krenn et al, 2014;Overlack et al, 2015;Vleugel et al, 2013 Article (Santaguida et al, 2010) led to a strong decrease of BUB1 kinetochore levels (Figures S4A and S4B), concomitant with the previously reported increase of MPS1 kinetochore levels (Santaguida et al, 2010) (Figures S4C and S4D).…”

“…). Conversely, depletion of BUBR1 led to an only marginal co-depletion of kinetochore PLK1, in agreement with previous studies (Cordeiro et al, 2020;Elowe et al, 2007;Ikeda and Tanaka, 2017). We conclude that BUB1 and CENP-U represent the two main kinetochore recruitment axes for PLK1 in nocodazole-arrested cells.…”

“…Another Kinetochore Binding Site for PLK1 BUB1 is required for kinetochore localization of BUBR1 (Overlack et al, 2015), possibly explaining why BUBR1 depletion has no major consequences on kinetochore PLK1 (Cordeiro et al, 2020;Elowe et al, 2007). BUB1 also contributes to a positive feedback loop that promotes localized accumulation and activation of Aurora B (Baron et al, 2016;Caldas et al, 2013;Hindriksen et al, 2017;Liu et al, 2013;van der Waal et al, 2012;Wang et al, 2011).…”

“…Dynamic regulation of kinase-phosphatase switches within kinetochores, including those involving PLK1, is crucial for kinetochore-microtubule attachment and spindle assembly checkpoint but remains very poorly understood (Cordeiro et al, 2020;Elowe et al, 2007Elowe et al, , 2010Ikeda and Tanaka, 2017;Jia et al, 2016;Kaisari et al, 2019;Lé ná rt et al, 2007;Liu et al, 2012;Maia et al, 2012;Moutinho-Santos et al, 2012;O'Connor et al, 2015;Peters et al, 2006;von Schubert et al, 2015). High PLK1 activity at kinetochores in prometaphase stabilizes kinetochore microtubules and balances a destabilizing activity attributed to Aurora B (Foley et al, 2011;Liu et al, 2012).…”

“…High PLK1 activity at kinetochores in prometaphase stabilizes kinetochore microtubules and balances a destabilizing activity attributed to Aurora B (Foley et al, 2011;Liu et al, 2012). PLK1 has been previously implicated in the control of PP2A activity (Joukov and De Nicolo, 2018;Saurin, 2018) through the creation of docking sites for the B56 regulatory subunit on BUB1 and BUBR1 (Cordeiro et al, 2020;Elowe et al, 2007;Hertz et al, 2016;Kruse et al, 2013;Suijkerbuijk et al, 2012b). MYPT1, a PP1 regulator and a CDK1:cyclin A substrate, has instead emerged as a potential negative regulator of PLK1 (Dumitru et al, 2017;Yamashiro et al, 2008).…”