Dual-functional significance of ATM-mediated phosphorylation of spindle assembly checkpoint component Bub3 in mitosis and the DNA damage response

Mingming Xiao,Siyue Zhang,Zhuang Liu,Yaqi Mo,Han Wang,Xu Zhao,Xue Yang,Rebecca J Boohaker,Yang Chen,Yamei Han,Hong Liu,Bo Xu

doi:10.1016/j.jbc.2022.101632

Abstract

Both the DNA damage response (DDR) and the mitotic checkpoint are critical for the maintenance of genomic stability. Among proteins involved in these processes, the ataxia–telangiectasia mutated (ATM) kinase is required for both activation of the DDR and the spindle assembly checkpoint (SAC). In mitosis without DNA damage, the enzymatic activity of ATM is enhanced; however, substrates of ATM in mitosis are unknown. Using stable isotope labeling of amino acids in cell culture mass spectrometry analysis, we identified a number of proteins that can potentially be phosphorylated by ATM during mitosis. This list is highly enriched in proteins involved in cell cycle regulation and the DDR. Among them, we further validated that ATM phosphorylated budding uninhibited by benzimidazoles 3 (Bub3), a major component of the SAC, on serine 135 (Ser135) both in vitro and in vivo. During mitosis, this phosphorylation promoted activation of another SAC component, benzimidazoles 1. Mutation of Bub3 Ser135 to alanine led to a defect in SAC activation. Furthermore, we found that ATM-mediated phosphorylation of Bub3 on Ser135 was also induced by ionizing radiation-induced DNA damage. However, this event resulted in independent signaling involving interaction with the Ku70–Ku80–DNA-PKcs sensor/kinase complex, leading to efficient nonhomologous end-joining repair. Taken together, we highlight the functional significance of the crosstalk between the kinetochore-oriented signal and double-strand break repair pathways via ATM phosphorylation of Bub3 on Ser135.

Highlights

The DNA damage response (DDR) has a major impact on preventing genomic instability, which is one of the primary hallmarks of cancer [1, 2]
To achieve a comprehensive understanding of ataxia–telangiectasia mutated (ATM) substrate phosphorylation during mitosis, we employed the SILAC assay in combination with mass spectrometry (Fig. S1A)
These findings provide the first picture of an ATM-mediated global phosphorylation network during mitosis

Summary

Introduction

The DNA damage response (DDR) has a major impact on preventing genomic instability, which is one of the primary hallmarks of cancer [1, 2]. Thereafter, the ataxia–telangiectasia mutated (ATM) kinase, a member of the phosphatidylinositol-3 kinase-like family, is activated to phosphorylate a large number of proteins to execute the optimal DDR, including cell cycle checkpoints and programmed cell death [7, 8]. Thereafter, mediator of DNA damage checkpoint 1 binds to the mitotic checkpoint complex, Mad, and Cdc, and ATM is required for SAC activation. Polo kinase activity facilitates subsequent accumulation of the BubR1–Bub complex at the DSBs, where Bub and BubR1 depend on each other to localize laser-induced DNA lesions [16, 17]. We demonstrate that ATM phosphorylates Bub on serine 135 (Ser135) to activate the SAC and the DNA repair via independent pathways. The dualfunctional role of ATM-mediated Bub Ser135 phosphorylation provides new insights into the interaction between the SAC and the DDR

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Conclusion