Gräf Justus F, Mikicic Ivan, Ping Xiaofei, Scalera Claudia, Mayr Katharina, Stelzl Lukas S, Beli Petra, Wagner Sebastian A
Institute of Molecular Biology (IMB), 55128 Mainz, Germany.
Faculty of Biology, Johannes Gutenberg University, 55128 Mainz, Germany.
iScience. 2022 Aug 9;25(9):104892. doi: 10.1016/j.isci.2022.104892. eCollection 2022 Sep 16.
PPM1D is a p53-regulated protein phosphatase that modulates the DNA damage response (DDR) and is frequently altered in cancer. Here, we employed chemical inhibition of PPM1D and quantitative mass spectrometry-based phosphoproteomics to identify the substrates of PPM1D upon induction of DNA double-strand breaks (DSBs) by etoposide. We identified 73 putative PPM1D substrates that are involved in DNA repair, regulation of transcription, and RNA processing. One-third of DSB-induced S/TQ phosphorylation sites are dephosphorylated by PPM1D, demonstrating that PPM1D only partially counteracts ATM/ATR/DNA-PK signaling. PPM1D-targeted phosphorylation sites are found in a specific amino acid sequence motif that is characterized by glutamic acid residues, high intrinsic disorder, and poor evolutionary conservation. We identified a functionally uncharacterized protein Kanadaptin as ATM and PPM1D substrate upon DSB induction. We propose that PPM1D plays a role during the response to DSBs by regulating the phosphorylation of DNA- and RNA-binding proteins in intrinsically disordered regions.
PPM1D是一种受p53调控的蛋白磷酸酶，可调节DNA损伤反应（DDR），在癌症中经常发生改变。在这里，我们采用化学抑制PPM1D和基于定量质谱的磷酸化蛋白质组学，以鉴定依托泊苷诱导DNA双链断裂（DSB）时PPM1D的底物。我们鉴定出73种推定的PPM1D底物，它们参与DNA修复、转录调控和RNA加工。三分之一的DSB诱导的S/TQ磷酸化位点被PPM1D去磷酸化，表明PPM1D仅部分抵消ATM/ATR/DNA-PK信号传导。在以谷氨酸残基、高内在无序性和低进化保守性为特征的特定氨基酸序列基序中发现了PPM1D靶向的磷酸化位点。我们鉴定出一种功能未明的蛋白Kanadaptin在DSB诱导时作为ATM和PPM1D的底物。我们提出，PPM1D通过调节内在无序区域中DNA和RNA结合蛋白的磷酸化，在对DSB的反应中发挥作用。
