Proper chromosome alignment depends on BRCA2 phosphorylation by PLK1

Åsa Ehlén,Sophie Zinn-Justin,Virginie Ropars,Ahmed El Marjou,Aura Carreira,Simona Miron,Manon Julien,Romane Beaurepere,Patricia Duchambon,Charlotte Martin,Gaetana Sessa,François-Xavier Theillet,Virginie Boucherit

doi:10.1038/s41467-020-15689-9

Abstract

The BRCA2 tumor suppressor protein is involved in the maintenance of genome integrity through its role in homologous recombination. In mitosis, BRCA2 is phosphorylated by Polo-like kinase 1 (PLK1). Here we describe how this phosphorylation contributes to the control of mitosis. We identify a conserved phosphorylation site at T207 of BRCA2 that constitutes a bona fide docking site for PLK1 and is phosphorylated in mitotic cells. We show that BRCA2 bound to PLK1 forms a complex with the phosphatase PP2A and phosphorylated-BUBR1. Reducing BRCA2 binding to PLK1, as observed in BRCA2 breast cancer variants S206C and T207A, alters the tetrameric complex resulting in unstable kinetochore-microtubule interactions, misaligned chromosomes, faulty chromosome segregation and aneuploidy. We thus reveal a role of BRCA2 in the alignment of chromosomes, distinct from its DNA repair function, with important consequences on chromosome stability. These findings may explain in part the aneuploidy observed in BRCA2-mutated tumors.

Highlights

The BRCA2 tumor suppressor protein is involved in the maintenance of genome integrity through its role in homologous recombination
Several missense variants of uncertain significance (VUS) identified in BRCA2 in breast cancer patients are located in the N-terminal region predicted to be phosphorylated by Pololike kinase 1 (PLK1) (Breast information core (BIC)[26] and BRCAShare27), summarized in Supplementary Table 1
To find out if any of these variants affected PLK1 phosphorylation in this region, we purified fragments comprising amino acids 1 to 250 of BRCA2 from human embryonic kidney cells (HEK293T) and used an in vitro kinase assay to assess the phosphorylation by PLK1 of the fragments containing either the WT sequence, the different BRCA2 variants M192T, S196N, S206C, and T207A, or the mutant S193A, previously reported to reduce the phosphorylation of BRCA2 by PLK114

Summary

Introduction

The BRCA2 tumor suppressor protein is involved in the maintenance of genome integrity through its role in homologous recombination. PLK1 directly binds and phosphorylates BUBR1 at several residues including the two tension-sensitive sites S67618 and T68019 in prometaphase allowing the formation of stable kinetochore–microtubule attachments This activity needs to be tightly regulated to ensure proper alignment of the chromosomes at the metaphase plate[8,9,18]. The kinase activity of Aurora B is essential to destabilize erroneous kinetochore–microtubule interactions[20] whereas the phosphatase PP2A protects initial kinetochore–microtubule interactions from excessive destabilization by Aurora B21 This function is achieved through the interaction of PP2A-B56 subunit with BUBR1 phosphorylated at the Kinetochore Attachment and Regulatory Domain (KARD) motif (including residues S670, S676, and T680)[19]. A defect in this function of BRCA2 is manifested in chromosome misalignment, chromosome segregation errors, mitotic delay and aneuploidy, leading to chromosomal instability

Methods

Results

Conclusion