Enrichment of Aurora B kinase at the inner kinetochore controls outer kinetochore assembly

Abstract

Outer kinetochore assembly enables chromosome attachment to microtubules and spindle assembly checkpoint (SAC) signaling in mitosis. Aurora B kinase controls kinetochore assembly by phosphorylating the Mis12 complex (Mis12C) subunit Dsn1. Current models propose Dsn1 phosphorylation relieves autoinhibition, allowing Mis12C binding to inner kinetochore component CENP-C. Using Xenopus laevis egg extracts and biochemical reconstitution, we found that autoinhibition of the Mis12C by Dsn1 impedes its phosphorylation by Aurora B. Our data indicate that the INCENP central region increases Dsn1 phosphorylation by enriching Aurora B at inner kinetochores, close to CENP-C. Furthermore, centromere-bound CENP-C does not exchange in mitosis, and CENP-C binding to the Mis12C dramatically increases Dsn1 phosphorylation by Aurora B. We propose that the coincidence of Aurora B and CENP-C at inner kinetochores ensures the fidelity of kinetochore assembly. We also found that the central region is required for the SAC beyond its role in kinetochore assembly, suggesting that kinetochore enrichment of Aurora B promotes the phosphorylation of other kinetochore substrates.

Oocyte spindle assembly depends on multiple interactions between HP1 and the CPC

“…Our results are similar to studies in Xenopus, where centromeric CPC localization is not required for kinetochore assembly (Haase et al, 2017). Correct spatial regulation and specific interactions with inner kinetochore or centromere proteins are required for the CPC to promote outer kinetochore assembly (Bonner et al, 2019), which ensures kinetochore assembly at the correct location and time. Some of our mutants (HP1:Incenp and Incenp ΔCEN ), were able to support limited kinetochore assembly but less K-fibers, suggesting that low levels of CPC are sufficient for kinetochore assembly, but higher levels and/or specific localization are required for spindle assembly.…”

“…We suggest that, in order to build K-fibers, Borealin needs to be recruited to the centromeric regions to cluster the CPC and function more efficiently. A notable difference compared to Xenopus (Bonner et al, 2019), however, is that in the Drosophila oocytes the SAH domain is not required for kinetochore assembly.…”

The proteomic landscape of centromeric chromatin reveals an essential role for the Ctf19CCANcomplex in meiotic kinetochore assembly

“…We hypothesised that the inability to recruit Ipl1 AURORA B to centromeres results in a failure to incorporate Mtw1c MIS12c into the kinetochore, and lethality. To test this idea, we used the auxin-inducible degron to degrade Bir1-aid upon release of otherwise wild-type and mcm21D cells from a G1 arrest, and Although several studies have implicated Ipl1 AURORA B in stabilizing interactions between the inner and outer layers of the kinetochore (Akiyoshi et al, 2009b;Bonner et al, 2019;Kim and Yu, 2015), paradoxically, its activity does not appear to be essential for outer kinetochore assembly, at least in otherwise wild-type yeast cells (e.g. (Akiyoshi et al, 2013a;Marco et al, 2013;Meyer et al, 2015a;Pinsky et al, 2006).…”

“…How kinetochore assembly is regulated remains unclear; however, in budding and fission yeast, Xenopus egg extracts, chicken and human cells, Aurora B kinase facilitates kinetochore assembly by phosphorylating two conserved serines on the Dsn1 DSN1 component of the Mtw1c MIS12c (Akiyoshi et al, 2013a;Bonner et al, 2019;Hara et al, 2018;Kim and Yu, 2015;Rago et al, 2015;Zhou et al, 2017). This displaces an autoinhibitory fragment of Dsn1 DSN1 , exposing a binding site on Mtw1c MIS12c for the inner kinetochore Mif2 CENP-C and, in yeast, Ame1 CENP-U proteins (Dimitrova et al, 2016;Petrovic et al, 2016).…”

“…In yeast, Ipl1 AURORA B may further stabilise the kinetochore through phosphorylation of Cse4 CENP-A (Boeckmann et al, 2013). However, given that depletion of Aurora B does not cause a profound kinetochore assembly defect in multiple organisms, with the exception of Xenopus egg extracts, Aurora B cannot be critical for kinetochore assembly, suggesting the existence of additional mechanisms (Bonner et al, 2019;Emanuele et al, 2008;Haase et al, 2017).…”