PHD3-dependent hydroxylation of HCLK2 promotes the DNA damage response
Liang Xie, … , Junmin Peng, Cam Patterson
Liang Xie, … , Junmin Peng, Cam Patterson
Published July 17, 2012
Citation Information: J Clin Invest. 2012;122(8):2827-2836. https://doi.org/10.1172/JCI62374.
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Research Article Oncology

PHD3-dependent hydroxylation of HCLK2 promotes the DNA damage response

Abstract

The DNA damage response (DDR) is a complex regulatory network that is critical for maintaining genome integrity. Posttranslational modifications are widely used to ensure strict spatiotemporal control of signal flow, but how the DDR responds to environmental cues, such as changes in ambient oxygen tension, remains poorly understood. We found that an essential component of the ATR/CHK1 signaling pathway, the human homolog of the Caenorhabditis elegans biological clock protein CLK-2 (HCLK2), associated with and was hydroxylated by prolyl hydroxylase domain protein 3 (PHD3). HCLK2 hydroxylation was necessary for its interaction with ATR and the subsequent activation of ATR/CHK1/p53. Inhibiting PHD3, either with the pan-hydroxylase inhibitor dimethyloxaloylglycine (DMOG) or through hypoxia, prevented activation of the ATR/CHK1/p53 pathway and decreased apoptosis induced by DNA damage. Consistent with these observations, we found that mice lacking PHD3 were resistant to the effects of ionizing radiation and had decreased thymic apoptosis, a biomarker of genomic integrity. Our identification of HCLK2 as a substrate of PHD3 reveals the mechanism through which hypoxia inhibits the DDR, suggesting hydroxylation of HCLK2 is a potential therapeutic target for regulating the ATR/CHK1/p53 pathway.

Authors

Liang Xie, Xinchun Pi, Ashutosh Mishra, Guohua Fong, Junmin Peng, Cam Patterson

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Figure 2

Prolyl hydroxylase inhibitor DMOG specifically inhibits the ATR/CHK1/p53 pathway following DNA damage.

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Prolyl hydroxylase inhibitor DMOG specifically inhibits the ATR/CHK1/p53...
(A) A simplified schematic of the signaling pathways induced by DNA damage. (B) HeLa cells were pretreated with DMOG for 4 hours, followed by NCS treatment as indicated. Western blots were performed with indicated antibodies. (C) HeLa cells were pretreated with DMOG for 4 hours followed by UV treatment at different doses. Cells were harvested after 2 hours, and Western blots were performed with the indicated antibodies. (D–F) HeLa cells were pretreated with DMOG for 4 hours, followed by UV treatment (250 j/m2), and harvested at different time points as indicated. (D) Western blots were performed with the indicated antibodies. TUNEL staining was performed at 8 hours after UV treatment. Representative images of cells pretreated with PBS or DMOG are shown in E. Scale bars: 20 μm. Original magnification, ×100. (F) TUNEL-positive cells (mean ± SEM) were quantified. n = 3; *P < 0.01. In B–D, representative blots from 3 similar experiments are shown.