Regulation of the subcellular shuttling of Sgo1 between centromeres and chromosome arms by Aurora B-mediated phosphorylation

https://doi.org/10.1016/j.bbrc.2014.10.103Get rights and content

Highlights

  • Aurora B kinase interacts with and phosphorylates Sgo1.

  • Aurora B-mediated phosphorylation of Sgo1 regulates the distribution of Sgo1 between centromeres and chromosome arms.

  • Aurora B-mediated phosphorylation of Sgo1 facilitates the accurate separation of mitotic chromosomes.

Abstract

A minor fraction of cohesin complexes at chromosome arms is not removed by the prophase pathway, and maintained until metaphase and enriched at centromeres. Sgo1 localizes to chromosome arms from prophase to metaphase, and is indispensable for removing cohesin complexes from chromosome arms. However, it has not been established how the chromosome arm localization of Sgo1 leads to the establishment of cohesion on chromosomes. Here, we report that Aurora B kinase interacts with and phosphorylates Sgo1 in vitro and in vivo. Furthermore, the phosphorylation of Sgol by Aurora B kinase regulated the distribution of Sgo1 between centromeres and chromosome arms, and the expression of Aurora B kinase-dead mutants of Sgo1 caused mislocalization from centromeres to chromosome arms. These results suggest Aurora B kinase directly regulates the subcellular distribution of Sgo1 to facilitate the accurate separation of mitotic chromosomes

Introduction

Sister chromatid cohesion is mediated by a multi-subunit complex called cohesin, and the cohesin complex consists of four subunits, that is, two structural proteins, Smc1 and Smc3, and two regulatory proteins, Scc1/Rad21 and Scc3/SA (SA1 and SA2 in vertebrates). Recent reports suggest that cohesin forms a ring-like structure which topologically appears to embrace two sister chromatids [1], [2], [3], [4]. In animal cells during early mitosis, most cohesin dissociates from chromosome arms in a process known as “prophase pathway” removal [5], which is partially dependent on the phosphorylation of cohesin by mitotic kinases, that is, Aurora B and Plk1 [6]. However, cohesin at centromeres is retained until metaphase to anaphase transition due to the function of shugoshin (Sgo) proteins [7], [8]. In particular, Sgo1 associates with serine and threonine protein phosphatase 2A (PP2A) to protect cohesin subunit proteins from phosphorylation [7], [9]. If Sgo1 is inactivated, cohesin dissociates prematurely from centromeres in the absence of separase activity. Therefore, Sgo1 is believed to play an essential role by protecting centromeric cohesion from the prophase removal pathway and to maintain cohesion until separase becomes active in metaphase. At metaphase, chromosomes are attached to spindle microtubule via their kinetochores, and this bipolar attachment is stabilized by the tension generated by the force pulling spindle microtubules and counteracting cohesive forces at centromeres. Thus, centromeric cohesion, which is largely protected by Sgo1, is essential for establishing bipolar attachment [10], [11].

The recruitment of Sgo1 to centromeres depends on Bub1 mitotic checkpoint kinase and HP1a [12], [13]. Loss of Bub1, like Sgo1 depletion, leads to separation of sister chromatids. Interestingly, inhibition of Aurora B kinase affects Sgo1 localization by causing its redistribution to chromosome arms during mitosis and meiosis [14], [15], [16]. Although this phenotype resulting from Aurora B inhibition might be related to the observed requirement for Aurora B in the prophase pathway, the role of Aurora B-mediated phosphorylation of Sgo1 in centromeric localization seems distinct. However, it has not been established whether the relocalization of Sgo1 to chromosome arms is also regulated by Aurora B-mediated phosphorylation. It is interesting that cohesion between chromosome arms is never completely dissolved during unperturbed mitosis [5], [11]. The complete removal of arm cohesion likely appears in cells that remain in prometaphase for prolonged periods of times, and therefore, is probably due to continuous activity of the prophase pathway [3], [4], [5]. However, it has been recently shown that cleavage of Scc1 by small amounts of separase activity is also required for cohesin removal signaling [11]. Importantly, the requirement for separase during chromosome arm opening is abolished if Sgo1 is depleted, which implies that small amounts of Sgo1 also protect cohesion on chromosome arms during the early stage of mitosis [11], [17]. Nevertheless, the molecular link responsible for Sgo1 shuttling between centromeres and chromosome arms and its dependence on Aurora B remains elusive.

Recent evidence suggests that when cohesin release from chromatid arms is impaired, a substantial amount of Sgo1 is also concentrated on a discrete axis-like structure along the entire length of chromosomes where cohesin is also enriched [18]. In addition, if Sgo1 is depleted centromeric staining for Sgo1 is reduced, and Sgo1 id delocalized alienating chromosome arms [11]. These observed strongly indicate Sgo1 is essential for the regulation of sister chromatid cohesion and dissociation at centromeres and at chromosome arms. However, loss of Sgo1 from sister chromatid arms appears to be insufficient to promote cohesin release and arm resolution, and that instead Sgo1 plays an important role in stabilizing cohesion along chromosome arms [18]. Although the role of Sgo1 in the stabilization of centromeric cohesion during mitosis and meiosis has been extensively characterized, its potential contribution to stabilizing arm cohesion remains largely unrecognized. Therefore, we sought to unveil how in Aurora B depleted or inactivated cells, Sgo1 redistributes to chromosome arms from centromeres, and how Aurora B inactivation causes this redistribution to chromosome arms.

Section snippets

Generations of plasmids, shRNAs, and siRNAs

Sgo1 S38A, S303A, S307A, S314A, and T319A alleles were generated by site-directed mutagenesis. cDNAs for Sgo1 WT and Sgo1 mutants were subcloned into GFP epitope-encoding vector to generate GFP-Sgo1 WT and mutants. For shRNA synthesis, the following gene-specific sequences were generated using pSuper vector (Oligoengine); Aurora B shRNA, 5′-GCAGA AGAGC TGCAC ATTT-3′; luciferase shRNA, 5′-CTACG CGGAA TACTT CGA-3′; Sgo1 shRNA, 5′-GTCTA CTGAT AATGT CTTA-3′. Gene-specific sequences for siRNA

Aurora B interacted with and phosphorylated Sgo1 in vitro and in vivo

To determine whether Sgo1 interacts functionally with Aurora B, endogenous Sgo1 was immunoprecipitated from asynchronized HeLa cells and immunoblotted with Aurora B antibody (Fig. 1A). In addition, Sgo1 was immunoprecipitated from HeLa cells inducibly expressing GFP-Sgo1 WT treated with thymidine (Thy), doxorubicin (Doxo), or nocodazole (Noco) to synchronize cells at the G1-S boundary, G2, or mitosis, respectively, and immunoblotted with Aurora B antibody (Fig. 1B). These assays revealed that

Discussion

Since Sgo1 protects cohesin complexes, the localization and regulation of Sgo1 are important aspects of faithful chromosome segregation. Despite the importance of chromosome segregation, little is known of the mechanisms that regulate the localization of Sgo1 in vertebrate cells. In this study, Sgo1 was found to interact directly with Aurora B and act as a key substrate. In addition, it was observed that the phosphorylation of Sgo1 is required for dissociating Sgo1 from chromosome arms, thereby

Acknowledgments

We would like to thank the Samsung Biomedical Research Institute for supplying the equipment, technical assistance, and financial support. This study was supported by a National Research Foundation grant funded by the Korean government (MEST) (2011-0030833 and 2012-R1A1A2041656).

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