Ku70 Serine 155 mediates Aurora B inhibition and activation of the DNA damage response

Yutoku

The Journal of Veterinary Medical Science

2017

Understanding the molecular mechanisms of DNA double-strand break (DSB) repair machinery, specifically non-homologous DNA-end joining (NHEJ), is crucial for developing next-generation radiotherapies and common chemotherapeutics for human and animal cancers. The localization, protein-protein interactions and post-translational modifications of core NHEJ factors, might play vital roles for regulation of NHEJ activity. The human Ku heterodimer (Ku70/Ku80) is a core NHEJ factor in the NHEJ pathway and is involved in sensing of DSBs. Companion animals, such as canines, have been proposed to be an excellent model for cancer research, including development of chemotherapeutics. However, the post-translational modifications, localization and complex formation of canine Ku70 have not been clarified. Here, we show that canine Ku70 localizes in the nuclei of interphase cells and that it is recruited quickly at laser-microirradiated DSB sites. Structurally, two DNA-PK phosphorylation sites (S6 and S51), an ubiquitination site (K114), two canonical sumoylation consensus motifs, a CDK phosphorylation motif, and a nuclear localization signal (NLS) in the human Ku70 are evolutionarily conserved in canine and mouse species, while the acetylation sites in human Ku70 are partially conserved. Intriguingly, the primary candidate nucleophile (K31) required for 5’dRP/AP lyase activity of human and mouse Ku70 is not conserved in canines, suggesting that canine Ku does not possess this activity. Our findings provide insights into the molecular mechanisms of Ku-dependent NHEJ in a canine model and form a platform for the development of next-generation common chemotherapeutics for human and animal cancers.

Section: Discussionsupporting

confidence: 64%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Cloning, localization and focus formation at DNA damage sites of canine Ku70

Yutoku

The Journal of Veterinary Medical Science

2017

“…Duolink II (Sigma-Aldrich Inc., St. Louis, MO, USA) in situ proximity ligation assay (PLA) was performed as previously described [ 48 ]. Cover slips were mounted onto glass slides with Prolong Gold antifade reagent with DAPI (Molecular Probes by Life Technologies, Burlington, ON, Canada) and were analyzed with an Olympus BX51 microscope (Olympus America Inc., Center Valley, PA, USA).…”

Section: Methodsmentioning

confidence: 99%

Regulation of c-Raf Stability through the CTLH Complex

McTavish

Bérubé-Janzen

Wang

et al. 2019

IJMS

Self Cite

c-Raf is a central component of the extracellular signal-regulated kinase (ERK) pathway which is implicated in the development of many cancer types. RanBPM (Ran-Binding Protein M) was previously shown to inhibit c-Raf expression, but how this is achieved remains unclear. RanBPM is part of a recently identified E3 ubiquitin ligase complex, the CTLH (C-terminal to LisH) complex. Here, we show that the CTLH complex regulates c-Raf expression through a control of its degradation. Several domains of RanBPM were found necessary to regulate c-Raf levels, but only the C-terminal CRA (CT11-RanBPM) domain showed direct interaction with c-Raf. c-Raf ubiquitination and degradation is promoted by the CTLH complex. Furthermore, A-Raf and B-Raf protein levels are also regulated by the CTLH complex, indicating a common regulation of Raf family members. Finally, depletion of CTLH subunits RMND5A (required for meiotic nuclear division 5A) and RanBPM resulted in enhanced proliferation and loss of RanBPM promoted tumour growth in a mouse model. This study uncovers a new mode of control of c-Raf expression through regulation of its degradation by the CTLH complex. These findings also uncover a novel target of the CTLH complex, and suggest that the CTLH complex has activities that suppress cell transformation and tumour formation.

“…AurB is also involved in the NHEJ pathway to repair DNA damage. Notably, the Ku heterodimer (Ku70/Ku80), a scaffolding protein of the NHEJ complex, represses AurB kinase activity after irradiation [ 114 ]. In response to DNA damage, PARP1 (poly(ADP-ribose) polymerase 1), a chromatin-associated DNA repair enzyme, also antagonizes AurB to block mitosis and histone H3 phosphorylation [ 115 ].…”

Section: Somatic Cellsmentioning

confidence: 99%

The functional diversity of Aurora kinases: a comprehensive review

et al. 2018

Aurora kinases are serine/threonine kinases essential for the onset and progression of mitosis. Aurora members share a similar protein structure and kinase activity, but exhibit distinct cellular and subcellular localization. AurA favors the G2/M transition by promoting centrosome maturation and mitotic spindle assembly. AurB and AurC are chromosome-passenger complex proteins, crucial for chromosome binding to kinetochores and segregation of chromosomes. Cellular distribution of AurB is ubiquitous, while AurC expression is mainly restricted to meiotically-active germ cells. In human tumors, all Aurora kinase members play oncogenic roles related to their mitotic activity and promote cancer cell survival and proliferation. Furthermore, AurA plays tumor-promoting roles unrelated to mitosis, including tumor stemness, epithelial-to-mesenchymal transition and invasion. In this review, we aim to understand the functional interplay of Aurora kinases in various types of human cells, including tumor cells. The understanding of the functional diversity of Aurora kinases could help to evaluate their relevance as potential therapeutic targets in cancer.