Phosphorylation of Astrin Regulates Its Kinetochore Function

Abstract

The error-free segregation of chromosomes, which requires the precisely timed search and capture of chromosomes by spindles during early mitotic and meiotic cell division, is responsible for genomic stability and is achieved by the spindle assembly checkpoint in the metaphase-anaphase transition. Mitotic kinases orchestrate M phase events, such as the reorganization of cell architecture and kinetochore (KT) composition with the exquisite phosphorylation of mitotic regulators, to ensure timely and temporal progression. However, the molecular mechanisms underlying the changes of KT composition for stable spindle attachment during mitosis are poorly understood. Here, we show that the sequential action of the kinase Cdk1 and the phosphatase Cdc14A control spindle attachment to KTs. During prophase, the mitotic spindle protein Spag5/Astrin is transported into centrosomes by Kinastrin and phosphorylated at Ser-135 and Ser-249 by Cdk1, which, in prometaphase, is loaded onto the spindle and targeted to KTs. We also demonstrate that Cdc14A dephosphorylates Astrin, and therefore the overexpression of Cdc14A sequesters Astrin in the centrosome and results in aberrant chromosome alignment. Mechanistically, Plk1 acts as an upstream kinase for Astrin phosphorylation by Cdk1 and targeting phospho-Astrin to KTs, leading to the recruitment of outer KT components, such as Cenp-E, and the stable attachment of spindles to KTs. These comprehensive findings reveal a regulatory circuit for protein targeting to KTs that controls the KT composition change of stable spindle attachment and chromosome integrity.The maintenance of genome integrity during mitosis is crucial for cell survival and organismal development. To generate two daughter cells with identical genetic information, each sister chromatid must be captured by spindle microtubules (MTs), 4 aligned at the mitotic equator, and segregated toward the spindle poles. Cells build a proteinaceous structure, known as the KT, at the centromere and form a bipolar spindle with an amphitelic spindle attachment to achieve accurate chromosome segregation (1). The KT is made up of protein complexes that control its localization on the chromosome, assembly, attachment to spindle MTs, and chromosome movements, such as congression and segregation (2). KTs lacking essential proteins for KT-MT attachment, such as Knl1, Mis12, and Ndc80 protein subcomplexes, are unable to attach to spindle MTs (3), which in turn results in chromosome loss and concurrent cell death (4).Entry into mitosis is controlled by the concerted action of several mitotic kinases, such as Cyclin-dependent kinase 1 (Cdk1), Polo-like kinase 1 (Plk1), and Aurora A, whose activities are regulated indirectly in a positive feedback loop (5). Plk1 activates Cdk1 by phosphorylating and activating Cdc25, which then removes inhibitory phosphorylation at the Thr-14 and Tyr-15 sites of Cdk1. Aurora A phosphorylates Thr-210 in the active loop of Plk1 with the aid of Bora (6). Cdk1 also activates Plk1 via Bora phosphorylation to promote mit...

The association of Plk1 with the Astrin-Kinastrin complex promotes formation and maintenance of a metaphase plate

“…This idea was corroborated by the analysis of the electrophoretic running properties of Astrin in lysates obtained from cells arrested in mitosis by different treatments (Figure 2A). Confirming previous reports, Astrin was strongly upshifted, indicative of phosphorylation, in nocodazole arrested mitotic cells when compared to thymidine-arrested, interphase cells (Chung et al, 2016). This upshift was further enhanced by arresting cells in STLC, mimicking conditions under which Plk1 and Astrin co-localise at the kinetochore ( Figure 1A).…”

“…In Plk1 immunoprecipitations, both Astrin and Kinastrin were co-precipitated ( Figure S1A). Interestingly, Plk1 only precipitated the slower migrating, mitotically phosphorylated form of Astrin (Chung et al, 2016), suggesting that the interaction between Plk1 and the Astrin complex is phosphorylation-dependent. Plk1 is known to bind to interaction partners via its Cterminal polo-box domain (PBD) to phosphorylated docking sites containing the motif S-pS/pT-P/X ( Figure 1C) (Elia et al, 2003a;Elia et al, 2003b).…”

CDC20B is required for deuterosome-mediated centriole production in multiciliated cells

“…It was shown that SPAG5 inhibits or activates Separase depending on its status of phosphorylation 41 , 42 . As the phosphorylation status of SPAG5 was shown to be controlled by PLK1 44 , our data suggest that the CDC20B/PLK1/SPAG5 complex could control the timing of Separase activation locally in deuterosomes. It is therefore possible that multiple modes of activation of Separase may act in parallel to trigger the release of neo-synthesized centrioles in maturing MCCs.…”

Astrin: A Key Player in Mitosis and Cancer

“…In contrast, the phosphorylase cell division cycle (CDC) 14A dephosphorylates Astrin and negatively regulates Astrin localization on KT ( Figure 1C ). This indicates that the dynamic kinetochore localization of Astrin is also regulated by mitotic kinases ( Chung et al, 2016 ).…”