Abstract
ABSTRACTErrors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic chromosomes must align at the metaphase plate with stable amphitelic attachments to microtubules emanating from opposing spindle poles. The astrin–kinastrin (astrin is also known as SPAG5 and kinastrin as SKAP) complex, also containing DYNLL1 and MYCBP, is a spindle and kinetochore protein complex with important roles in bipolar spindle formation, chromosome alignment and microtubule–kinetochore attachment. However, the molecular mechanisms by which astrin–kinastrin fulfils these diverse roles are not fully understood. Here, we characterise a direct interaction between astrin and the mitotic kinase Plk1. We identify the Plk1-binding site on astrin as well as four Plk1 phosphorylation sites on astrin. Regulation of astrin by Plk1 is dispensable for bipolar spindle formation and bulk chromosome congression, but promotes stable microtubule–kinetochore attachments and metaphase plate maintenance. It is known that Plk1 activity is required for effective microtubule–kinetochore attachment formation, and we suggest that astrin phosphorylation by Plk1 contributes to this process.
Highlights
During animal cell division the efficient end-on attachment of kinetochores to microtubules is a prerequisite for successful chromosome segregation
To understand when and where during mitosis astrin and Pololike kinase 1 (Plk1) interact, untreated HeLa cells at prometaphase or metaphase, or HeLa cells arrested in mitosis by treatment with the microtubule-depolymerising drug nocodazole or the Eg5 kinesin inhibitor S-trityl-L-cysteine (STLC) (Skoufias et al, 2006), were stained for astrin and Plk1
Plk1 kinetochore staining was strongest on unattached kinetochores but still present at attached kinetochores at metaphase plates or in STLC-arrested cells
Summary
During animal cell division the efficient end-on attachment of kinetochores to microtubules is a prerequisite for successful chromosome segregation. Analysis of HeLa cells with an antibody raised against the doubly phosphorylated pS157/S159 peptide showed prominent staining on attached kinetochores, which was dependent on the presence of both astrin and Plk1 activity, as well as the phosphorylatable residues (Fig. 2D,E,G; Fig. S2E), and was not diminished by inhibition of other mitotic kinases (Fig. S2B,C).
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