Aurora B Phosphorylates Spatially Distinct Targets to Differentially Regulate the Kinetochore-Microtubule Interface

Abstract

Summary Accurate chromosome segregation requires carefully regulated interactions between kinetochores and microtubules, but how plasticity is achieved to correct diverse attachment defects remains unclear. Here, we demonstrate that Aurora B kinase phosphorylates three spatially distinct targets within the conserved outer kinetochore KNL1/Mis12 complex/Ndc80 complex (KMN) network, the key player in kinetochore-microtubule attachments. The combinatorial phosphorylation of the KMN network generates graded levels of microtubule binding activity, with full phosphorylation severely compromising microtubule binding. Altering the phosphorylation state of each protein causes corresponding chromosome segregation defects. Importantly, the spatial distribution of these targets along the kinetochore axis leads to their differential phosphorylation in response to changes in tension and attachment state. In total, rather than generating exclusively binary changes in microtubule binding, our results suggest a mechanism for the tension-dependent fine tuning of kinetochore-microtubule interactions.

Temporal changes in Hec1 phosphorylation control kinetochore–microtubule attachment stability during mitosis

“…This group further reported that phosphorylation of KNL1 on sites Ser24 and Ser60 by Aurora B kinase negatively regulates PP1 binding to KNL1 as a phosphomimetic version of KNL1 bound to PP1 in vitro with much less affinity than did wild-type KNL1 . A later study demonstrated that KNL1 Ser24 and Ser60 are phosphorylated at kinetochores in vivo in an Aurora-B-kinasedependent manner and, as mentioned above, using phosphorylationspecific antibodies, these authors measured a 30-50% decrease in phosphorylated KNL1 on kinetochores from prometaphase to metaphase (Welburn et al, 2010). Presumably, this reduction of phosphorylation on KNL1 at kinetochores is sufficient to increase the affinity of KNL1 for PP1 so that kinetochore targets can be dephosphorylated.…”

“…Based on the findings that Hec1 N-terminal phosphorylation is significantly reduced after chromosome bi-orientation, it is probable that other mechanisms for inducing kinetochore-MT detachment and error correction are in place during late mitosis (Bakhoum et al, 2009). Recently, it has been demonstrated that both KNL1 and DSN1 (a member of the Mis12 complex) are phosphorylated in an Aurora-B-kinase-dependent manner in vivo, and phosphorylation of these proteins results in decreased affinity of the KMN complex for MTs in vitro (Welburn et al, 2010). Phosphorylated forms of both KNL1 and DSN1 at kinetochores decrease only modestly from prometaphase to metaphase (~33-55% reduction for KNL1-P and ~20% reduction for DSN1-P), thus it is possible that phosphorylation of these members of the KMN complex by Aurora B in late mitosis is sufficient to induce some level of kinetochore-MT turnover even in the absence of high levels of Hec1 phosphorylation.…”

“…Under this experimental condition, active Aurora B kinase remains at the outer kinetochore, indicating that although the kinase is spatially positioned to rephosphorylate Hec1, it does not. Because it is probable that other outer kinetochore targets are regulated by spatial positioning of the kinase (Liu et al, 2009;Liu et al, 2010;Welburn et al, 2010), the mechanism controlling Aurora-Bdependent phosphorylation cycles might differ between kinetochore targets.…”

A Redundant Function for the N-Terminal Tail of Ndc80 in Kinetochore–Microtubule Interaction in Saccharomyces cerevisiae

“…The N-terminal tail is unstructured and not present in the crystal structure of Ndc80 complexes or in cryo-electron microscopy images of the Ndc80 complex bound to MTs. The N-terminal tail of Ndc80 is enriched for Ipl1/AuroraB phosphorylation sites, and MT binding can be regulated by the kinase (Guimaraes et al 2008;Welburn et al 2010;Lampson and Cheeseman 2011;. MT-binding affinity of yeast and human Ndc80 complexes is decreased when the Ndc80 N-terminal tail is deleted (Wei et al 2005;Ciferri et al 2008).…”

The Spindle Assembly Checkpoint: Clock or Domino?

“…This protein kinase, which is located at the centromeres between the two sister-kinetochores, plays an essential role in the correction of erroneous kinetochore-microtubule attachment Nezi and Musacchio 2009). Specifically, it phosphorylates the microtubule-binding KMN network at kinetochores in a proximity-dependent manner if kinetochores are not stretched apart through a bipolar attachment Welburn et al 2010). But it has also been implicated directly in the spindle checkpoint signaling, as it contributes to the loading of Mad2 to kinetochores in human cells and is essential for the spindle checkpoint in S. pombe (Johnson et al 2004;Vanoosthuyse and Hardwick 2009).…”