CDK2 and PKA Mediated-Sequential Phosphorylation Is Critical for p19INK4d Function in the DNA Damage Response

DNA damage triggers a phosphorylation-based signaling cascade known as the DNA damage response. p19INK4d, a member of the INK4 family of CDK4/6 inhibitors, has been reported to participate in the DNA damage response promoting DNA repair and cell survival. Here, we provide mechanistic insight into the activation mechanism of p19INK4d linked to the response to DNA damage. Results showed that p19INK4d becomes phosphorylated following UV radiation, β-amyloid peptide and cisplatin treatments. ATM-Chk2/ATR-Chk1 signaling pathways were found to be differentially involved in p19INK4d phosphorylation depending on the type of DNA damage. Two sequential phosphorylation events at serine 76 and threonine 141 were identified using p19INK4d single-point mutants in metabolic labeling assays with 32P-orthophosphate. CDK2 and PKA were found to participate in p19INK4d phosphorylation process and that they would mediate serine 76 and threonine 141 modifications respectively. Nuclear translocation of p19INK4d induced by DNA damage was shown to be dependent on serine 76 phosphorylation. Most importantly, both phosphorylation sites were found to be crucial for p19INK4d function in DNA repair and cell survival. In contrast, serine 76 and threonine 141 were dispensable for CDK4/6 inhibition highlighting the independence of p19INK4d functions, in agreement with our previous findings. These results constitute the first description of the activation mechanism of p19INK4d in response to genotoxic stress and demonstrate the functional relevance of this activation following DNA damage.

DNA损伤可触发一类以磷酸化为核心的信号级联反应，即DNA损伤应答(DNA damage response)。p19INK4d作为INK4家族的CDK4/6抑制剂(CDK4/6 inhibitors)成员，已有研究表明其可参与DNA损伤应答，促进DNA修复与细胞存活。本研究旨在阐明p19INK4d在DNA损伤应答中的激活机制。实验结果显示，在紫外线辐射、β-淀粉样肽和顺铂处理后，p19INK4d会发生磷酸化修饰。ATM-Chk2/ATR-Chk1信号通路会根据DNA损伤类型的不同，差异化参与p19INK4d的磷酸化过程。通过采用32P-正磷酸进行代谢标记实验，并结合p19INK4d单点突变体，研究人员鉴定出p19INK4d上的两个连续磷酸化位点：丝氨酸76与苏氨酸141。研究证实，CDK2与蛋白激酶A(PKA)分别参与了p19INK4d的磷酸化过程，并分别介导上述两个位点的修饰。DNA损伤诱导的p19INK4d核转位过程，依赖于丝氨酸76位点的磷酸化修饰。最为关键的是，这两个磷酸化位点均对p19INK4d在DNA修复与细胞存活中的功能至关重要。与之相反，丝氨酸76与苏氨酸141位点对于p19INK4d的CDK4/6抑制活性并非必需，这表明p19INK4d的不同功能相互独立，与我们此前的研究结果一致。本研究首次阐明了p19INK4d在应对遗传毒性应激时的激活机制，并证实了该激活过程在DNA损伤应答中的功能相关性。