The Ndc80 complex targets Bod1 to human mitotic kinetochores

Abstract

Regulation of protein phosphatase activity by endogenous protein inhibitors is an important mechanism to control protein phosphorylation in cells. We recently identified Biorientation defective 1 (Bod1) as a small protein inhibitor of protein phosphatase 2A containing the B56 regulatory subunit (PP2A-B56). This phosphatase controls the amount of phosphorylation of several kinetochore proteins and thus the establishment of load-bearing chromosome-spindle attachments in time for accurate separation of sister chromatids in mitosis. Like PP2A-B56, Bod1 directly localizes to mitotic kinetochores and is required for correct segregation of mitotic chromosomes. In this report, we have probed the spatio-temporal regulation of Bod1 during mitotic progression. Kinetochore localization of Bod1 increases from nuclear envelope breakdown until metaphase. Phosphorylation of Bod1 at threonine 95 (T95), which increases Bod1's binding to and inhibition of PP2A-B56, peaks in prometaphase when PP2A-B56 localization to kinetochores is highest. We demonstrate here that kinetochore targeting of Bod1 depends on the outer kinetochore protein Ndc80 and not PP2A-B56. Crucially, Bod1 depletion functionally affects Ndc80 phosphorylation at the N-terminal serine 55 (S55), as well as a number of other phosphorylation sites within the outer kinetochore, including Knl1 at serine 24 and 60 (S24, S60), and threonine T943 and T1155 (T943, T1155). Therefore, Ndc80 recruits a phosphatase inhibitor to kinetochores which directly feeds forward to regulate Ndc80, and Knl1 phosphorylation, including sites that mediate the attachment of microtubules to kinetochores.

The kinetochore–microtubule interface at a glance

“…Auroramediated phosphorylation decreases the affinity of Ndc80 for microtubules (Cheeseman et al, 2006), thereby allowing for correction of aberrant kinetochore-microtubule interactions. Consistent with this, Ndc80 phosphorylation is highest in prometaphase and decreases in metaphase through the combined actions of two phosphatases, PP1 and the PP2A-B56 holoenzyme (Liu et al, 2010;Posch et al, 2010;Schleicher et al, 2017). However, recent work has also suggested that a subset of phosphorylation sites are maintained by Aurora A throughout mitosis to ensure proper microtubule dynamics (DeLuca et al, 2017).…”

Kinase and Phosphatase Cross-Talk at the Kinetochore

“…At least one protein that is thought to regulate PP2A-B56 activity at kinetochores is Bod1. Bod1 localizes to Ndc80 and is able to inhibit BubR1-bound PP2A-B56 (Porter et al, 2013 ; Schleicher et al, 2017 ) (arrow 25, Figure 1 ). PP1-Knl1 activity may also be modulated by various accessory proteins (Posch et al, 2010 ; Eiteneuer et al, 2014 ; Duan et al, 2016 ), however, this regulation is omitted from Figure 1 , because it is thought to occur in the cytoplasm and not at kinetochores.…”

“…Therefore, Cdk1 jointly stimulates both mitotic arrest and mitotic exit, but it hands the control over to microtubules, which can determine whether kinase or phosphatase activities predominate at the kinetochore (as discussed in detail later). Part of this balance may also be controlled by Bod1, which binds to NDC80 and is phosphorylated by Cdk1 to inhibit PP2A-B56 (Porter et al, 2013 ; Schleicher et al, 2017 ; Figure 2A ). It will be interesting to determine whether microtubule attachment or tension can modulate Bod1 localisation or phosphorylation.…”

A homozygous stop gain mutation in BOD1 gene in a Lebanese patient with syndromic intellectual disability

“…BOD1 (Biorientation of chromosomes in cell division 1) is required for chromosomes biorientation during cell division 16 . It is localized at kinetochores from prometaphase until anaphase 16 where it inhibits the phosphatase 2A, thus regulating cell cycle progression 17,18 . A homozygous nonsense mutation in BOD1 (NM_138369: c.334C>T; p.R112*) was previously observed in four female siblings of a consanguineous Iranian family, suffering from mild to moderate ID.…”