PHD3-dependent hydroxylation of HCLK2 promotes the DNA damage response
Liang Xie, Xinchun Pi, Ashutosh Mishra, Guohua Fong, Junmin Peng, Cam Patterson
Liang Xie, Xinchun Pi, Ashutosh Mishra, Guohua Fong, Junmin Peng, Cam Patterson
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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 5

PHD3 hydroxylation of HCLK2 mediates the interaction between HCLK2 and ATR.

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PHD3 hydroxylation of HCLK2 mediates the interaction between HCLK2 and A...
(A) In vitro hydroxylation assays using 2-oxo-glutarate [1-14C] as the cosubstrate were performed in the presence of GST-HCLK2340–530 or PHD3 as indicated. The released 14CO2 (mean ± SEM) was trapped and measured. n = 3; *P < 0.01 versus PHD3 alone. (B) Flag-HCLK2340–530 immunoprecipitated from HeLa cells was trypsin digested and analyzed by LC-MS/MS. Modified peptide sequence AVLIC57LAQLGEP#ELR (C57 indicating mass shift of 57.0215 Da by iodoacetamide alkylation on the Cys residue; # indicating proline hydroxylation) was identified based on the high-resolution MS/MS survey scan comparison between the unmodified form (dashed lines) and the hydroxylated form (solid lines) as well as the MS/MS scan of the hydroxylated peptide. The unmodified form was detected as a doubly charged monoisotopic ion of 841.4690 m/z, whereas the modified form was identified as 849.4674 m/z. The modification was further validated by multiple product ions (b and y ions) in the MS/MS scan by collision-induced dissociation. (C) GST or GST-HCLK2340–530 immobilized on GSH beads were incubated with Flag-ATR purified from HEK293 cells, either directly or after 60 minutes of incubation with PHD3 in the presence or absence of DMOG. The pull-down products were analyzed by Western blot analysis. (D) HeLa cells transfected with pcDNA3, Flag-HCLK2, or Flag-HCLK2P374A/P419A/P422A were treated with DMOG for 6 hours. Coimmunoprecipitation and Western blot analysis were then performed with the indicated antibodies. In C and D, representative blots from 2 similar experiments are shown.