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Plk1 mitotic phosphorylation in the N-terminal region of the BRCA2 protein : identification, characterization and role in protein interactions

Abstract : BRCA2 is an oncoprotein frequently mutated in hereditary breast cancers. To improve the diagnosis of these cancers, several molecular studies have identified BRCA2 key positions and characterized mutations in these regions causing a BRCA2 loss of function. However, these studies mainly focused on the C-terminal globular domain of BRCA2. Here, we characterized the N-terminal region of BRCA2 from aa 48 to aa 284 (BRCA2₄₈₋₂₈₄). This well-conserved region is disordered, i.e. it lacks stable secondary structure (Julien et al. 2020 Biomol. NMR Assign). It is also highly phosphorylated by the kinase Plk1 at the entry into mitosis. However, previous studies using mass spectrometry didn’t allow to precisely identify all the phosphorylation sites, limiting their characterization. To circumvent this problem, we used real-time NMR to monitor phosphorylations of the N-terminal region of BRCA2, at the residue level. We developed 2 protocols for disordered regions allowing phosphorylation in a large range of temperatures and pHs (Julien et al. 2020 Methods Mol. Biol; Alik et al. 2020 An- gew. Chem.). Then, we identified that Plk1 phosphorylates BRCA2 at 2 conserved positions: pS193 and pT207. We further searched for the function of these phosphoresidues. First, we identified by biophysical methods that BRCA2pT207 creates a docking site for the regulatory domain of Plk1. In collaboration with the group of Dr. Aura Carreira, we showed that this interaction contributes to the assembly of a quaternary complex involving BRCA2, Plk1, BubR1 and PP2A that regulates chromosome alignment in mitosis (Ehlen et al. 2020. Nat Commun.). We also demonstrated that breast cancer variants impact the phosphorylation of BRCA2 and the formation of the complex in vitro and in cell. Then, we per- formed proteomics experiments to identify new mitotic partners specific to phospho-BRCA2, and found Plk1 as well as other proteins involved in mitosis. I started the characterization of two new BRCA2 partners: Ki2C and Chk2. We also explored the role of Cdk1 phosphorylation of BRCA2T77 on further Plk1 phosphorylation. We found that early BRCA2T77 phosphorylation increases the phosphoryla- tion rate of BRCA2S193 by Plk1. Finally, we initiated a project about the Plk1 kinase, an interesting cancer-target as it is often overexpressed in several cancers, including breast cancers. Here, I produced several constructs of the kinase for further structural characterization.
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Submitted on : Friday, September 10, 2021 - 6:18:11 PM
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  • HAL Id : tel-03341485, version 1

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Manon Julien. Plk1 mitotic phosphorylation in the N-terminal region of the BRCA2 protein : identification, characterization and role in protein interactions. Structural Biology [q-bio.BM]. Université Paris-Saclay, 2021. English. ⟨NNT : 2021UPASQ011⟩. ⟨tel-03341485⟩

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