Faculty Opinions recommendation of WSTF regulates the H2A.X DNA damage response via a novel tyrosine kinase activity.

Abstract The DNA damage response (DDR) encompasses the cellular response to DNA double-stranded breaks (DSBs), and includes recognition of the DSB, recruitment of numerous factors to the DNA damage site, initiation of signaling cascades, chromatin remodeling, cell-cycle checkpoint activation, and repair of the DSB. Key drivers of the DDR are multiple members of the phosphatidylinositol 3-kinase-related kinase family, including ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). ATM and ATR modulate multiple portions of the DDR, but DNA-PKcs is believed to primarily function in the DSB repair pathway, non-homologous end joining. Utilizing a human cell line in which the kinase domain of DNA-PKcs is inactivated, we show here that DNA-PKcs kinase activity is required for the cellular response to DSBs immediately after their induction. Specifically, DNA-PKcs kinase activity initiates phosphorylation of the chromatin factors H2AX and KAP1 following ionizing radiation exposure and drives local chromatin decondensation near the DSB site. Furthermore, loss of DNA-PKcs kinase activity results in a marked decrease in the recruitment of numerous members of the DDR machinery to DSBs. Collectively, these results provide clear evidence that DNA-PKcs activity is pivotal for the initiation of the DDR.

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Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6

Cells ◽

10.3390/cells9061506 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1506 ◽

Cited By ~ 1

Author(s):

Cecilia Aquino Perez ◽

Matous Palek ◽

Lenka Stolarova ◽

Patrick von Morgen ◽

Libor Macurek

Keyword(s):

Dna Damage ◽

Stress Response ◽

Osmotic Stress ◽

Dna Damage Response ◽

Protein Phosphatase ◽

Gene Editing ◽

Kinase Activity ◽

Cell Cycle Control ◽

Damage Response ◽

Transformed Cell Lines

Polo-like kinases play essential roles in cell cycle control and mitosis. In contrast to other members of this kinase family, PLK3 has been reported to be activated upon cellular stress including DNA damage, hypoxia and osmotic stress. Here we knocked out PLK3 in human non-transformed RPE cells using CRISPR/Cas9-mediated gene editing. Surprisingly, we find that loss of PLK3 does not impair stabilization of HIF1α after hypoxia, phosphorylation of the c-Jun after osmotic stress and dynamics of DNA damage response after exposure to ionizing radiation. Similarly, RNAi-mediated depletion of PLK3 did not impair stress response in human transformed cell lines. Exposure of cells to various forms of stress also did not affect kinase activity of purified EGFP-PLK3. We conclude that PLK3 is largely dispensable for stress response in human cells. Using mass spectrometry, we identify protein phosphatase 6 as a new interacting partner of PLK3. Polo box domain of PLK3 mediates the interaction with the PP6 complex. Finally, we find that PLK3 is phosphorylated at Thr219 in the T-loop and that PP6 constantly dephosphorylates this residue. However, in contrast to PLK1, phosphorylation of Thr219 does not upregulate enzymatic activity of PLK3, suggesting that activation of both kinases is regulated by distinct mechanisms.

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Effect of inhibition of receptor tyrosine kinase AXL by a selective small molecular inhibitor R428 (BGB321) on DNA damage repair response in ovarian cancer cells.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.e15640 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. e15640-e15640

Author(s):

Ruby Yun-Ju Huang ◽

Xun Hui Yeo ◽

Wai Leong Tam

Keyword(s):

Dna Damage ◽

Tyrosine Kinase ◽

Cell Lines ◽

Dna Damage Response ◽

Receptor Tyrosine Kinase ◽

Dna Damage Repair ◽

Damage Repair ◽

Damage Response ◽

Dna Damage Responses

e15640 Background: AXL is a receptor tyrosine kinase that is often overexpressed in many cancers. It contributes to tumor progression, metastasis and drug resistance through activating downstream signaling cascades, making it an emerging therapeutic target. The first-in-class AXL inhibitor R428 (BGB321) was approved by the FDA for the treatment of relapsed or refractory acute myeloid leukemia. R428 (BGB321) was also reported to show selective sensitivity towards ovarian cancers (OC) with a Mesenchymal (Mes) molecular subtype. Recently, a novel role of AXL in the regulation of DNA damage responses has been described. In this study, we explored further the role of AXL in mediating DNA damage responses by using OC as a disease model. Methods: OC cell lines were treated with R428. Accumulation of γH2AX positive foci was assessed for DNA damage response. Western blotting for γH2AX, ATM and ATR levels were performed. Dose response curves of ATR inhibitors were generated by treating OC cells with the fixed dose of R428 (IC20 concentration of each cell line). Results: AXL inhibition by using R428 resulted in the increase of DNA damage foci in Mes OC cells SKOV3 and HeyA8. This occurred concurrently with the up-regulation of classic DNA damage response signaling molecules such as γH2AX, ATM and ATR. The IC50 of the ATR inhibitor significantly decreased for 2-3 folds in all OC cell lines tested. AXL inhibitor R428 sensitized both BRCA-mutated and non-BRCA-mutated OC cells to a potent and highly selective ATR inhibitor. Conclusions: Our results showed that AXL inhibition rendered cells more sensitive to the inhibition of ATR, a crucial mediator for replication stress, paving ways to the rationale for potential combinatory use of AXL and DNA damage repair inhibitors.

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