Department of Toxicogenetics, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.
Mol Cell Biol. 2011 Dec;31(24):4964-77. doi: 10.1128/MCB.05258-11. Epub 2011 Oct 17.
Cellular responses to DNA-damaging agents involve the activation of various DNA damage signaling and transduction pathways. Using quantitative and high-resolution tandem mass spectrometry, we determined global changes in protein level and phosphorylation site profiles following treatment of SILAC (stable isotope labeling by amino acids in cell culture)-labeled murine embryonic stem cells with the anticancer drug cisplatin. Network and pathway analyses indicated that processes related to the DNA damage response and cytoskeleton organization were significantly affected. Although the ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) consensus sequence (S/T-Q motif) was significantly overrepresented among hyperphosphorylated peptides, about half of the >2-fold-upregulated phosphorylation sites based on the consensus sequence were not direct substrates of ATM and ATR. Eleven protein kinases mainly belonging to the mitogen-activated protein kinase (MAPK) family were identified as being regulated in their kinase domain activation loop. The biological importance of three of these kinases (cyclin-dependent kinase 7 [CDK7], Plk1, and KPCD1) in the protection against cisplatin-induced cytotoxicity was demonstrated by small interfering RNA (siRNA)-mediated knockdown. Our results indicate that the cellular response to cisplatin involves a variety of kinases and phosphatases not only acting in the nucleus but also regulating cytoplasmic targets, resulting in extensive cytoskeletal rearrangements. Integration of transcriptomic and proteomic data revealed a poor correlation between changes in the relative levels of transcripts and their corresponding proteins, but a large overlap in affected pathways at the levels of mRNA, protein, and phosphoprotein. This study provides an integrated view of pathways activated by genotoxic stress and deciphers kinases that play a pivotal role in regulating cellular processes other than the DNA damage response.
细胞对 DNA 损伤剂的反应涉及各种 DNA 损伤信号转导途径的激活。使用定量和高分辨率串联质谱法，我们在使用抗癌药物顺铂处理 SILAC（稳定同位素标记的细胞培养中的氨基酸）标记的鼠胚胎干细胞后，测定了蛋白质水平和磷酸化位点谱的全局变化。网络和途径分析表明，与 DNA 损伤反应和细胞骨架组织相关的过程受到显著影响。尽管 ATM（共济失调毛细血管扩张症突变）和 ATR（ATM 和 Rad3 相关）的共有序列（S/T-Q 基序）在高磷酸化肽中明显过表达，但根据共有序列，超过 2 倍上调的磷酸化位点中约有一半不是 ATM 和 ATR 的直接底物。鉴定出 11 种主要属于丝裂原活化蛋白激酶（MAPK）家族的蛋白激酶，其激酶结构域激活环受到调节。通过小干扰 RNA（siRNA）介导的敲低，证明了这三种激酶（细胞周期蛋白依赖性激酶 7 [CDK7]、Plk1 和 KPCD1）在保护细胞免受顺铂诱导的细胞毒性方面的生物学重要性。我们的研究结果表明，细胞对顺铂的反应不仅涉及作用于细胞核的多种激酶和磷酸酶，还涉及调节细胞质靶标，导致广泛的细胞骨架重排。转录组学和蛋白质组学数据的整合表明，相对转录物水平的变化与其相应蛋白质水平之间相关性较差，但在 mRNA、蛋白质和磷酸化蛋白质水平上，受影响途径的重叠很大。该研究提供了对基因组毒性应激激活途径的综合观点，并阐明了在调节除 DNA 损伤反应以外的细胞过程中发挥关键作用的激酶。
