PHD3-dependent hydroxylation of HCLK2 promotes the DNA damage response.
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.
UNC McAllister Heart Institute and Department of Medicine, University of North Carolina, Chapel Hill, NC, USA. pxliang@email.unc.edu
Liang
L
Xie
Xinchun
X
Pi
Ashutosh
A
Mishra
Guohua
G
Fong
Junmin
J
Peng
Cam
C
Patterson
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000378:metabolism; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D049308:Dioxygenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005260:Female; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D011393:Procollagen-Proline Dioxygenase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D018797:Cell Cycle Proteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D049308:Dioxygenases; C522722:PHD3 protein, mouse; D011393:Procollagen-Proline Dioxygenase; C488894:EGLN3 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D011494:Protein Kinases; C066780:Clk dual-specificity kinases; D011505:Protein-Tyrosine Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605869:Chek1 protein, mouse; D017346:Protein Serine-Threonine Kinases