The Biotechnology Centre of Oslo, University of Oslo, Gaustadalleen 21, 0349 Oslo, Norway.
J Proteomics. 2013 Oct 8;91:344-57. doi: 10.1016/j.jprot.2013.07.019. Epub 2013 Aug 7.
In order to better understand the cellular responses to the chemotherapeutic drug cisplatin and the mechanisms leading to apoptosis and potential side effects, we performed a SILAC-based quantitative phosphotyrosine analysis of Jurkat T cells exposed to cisplatin. Signaling molecules in the T cell receptor (TCR) pathway were enriched among proteins displaying reduced phosphorylation levels. The results were verified by immunoblotting and/or phospho-flow cytometry for a selected set of proteins, including the tyrosine kinases Lck and Zap70, and downstream targets Itk, Plcγ1 and Erk. In contrast to the effects on the T cell signaling pathways, the dually phosphorylated form of p38α MAPK was increased in treated cells, and activation of this signaling pathway was verified by immunoblot analysis of phosphorylation levels of p38α MAPK and the downstream targets Atf2 and MAPKAPK2. Activation of the p38α MAPK signaling pathway has been suggested to be one of the main mechanisms by which cisplatin induces apoptosis. Our results indicate that cisplatin may reduce the activity of proteins involved in the TCR signaling pathway, which has an important role in regulating proliferation of T cells, and may contribute to explain previous observations where cisplatin has been reported to inhibit proliferation of T cells.
In this study, a quantitative phosphotyrosine analysis was performed to identify changes of the phosphoproteome during exposure of Jurkat T cells by cisplatin. The results of the phosphoproteome analysis were complemented with immunoblotting and temporal phospho-flow analysis. An initial activation of the p38α MAPK signaling pathway was detected at early time points of cisplatin treatment, a response previously suggested to be part of the mechanism by which cisplatin induces apoptosis. Furthermore, reduced phosphorylation levels of proteins involved in signaling downstream of the TCR during apoptosis were found by the phosphotyrosine proteome analysis. Our study can support to elucidate the mechanism behind the previously observed immunosuppressive effect of cisplatin.
为了更好地了解细胞对化疗药物顺铂的反应以及导致细胞凋亡和潜在副作用的机制，我们对暴露于顺铂的 Jurkat T 细胞进行了基于 SILAC 的定量磷酸酪氨酸分析。在磷酸化水平降低的蛋白质中富集了 T 细胞受体 (TCR) 途径中的信号分子。通过免疫印迹和/或磷酸化流式细胞术对一组选定的蛋白质（包括酪氨酸激酶 Lck 和 Zap70 以及下游靶标 Itk、Plcγ1 和 Erk）进行了验证。与对 T 细胞信号通路的影响相反，磷酸化形式的 p38α MAPK 在处理过的细胞中增加，并且通过免疫印迹分析 p38α MAPK 的磷酸化水平和下游靶标 Atf2 和 MAPKAPK2 来验证该信号通路的激活。已经提出 p38α MAPK 信号通路的激活是顺铂诱导细胞凋亡的主要机制之一。我们的结果表明，顺铂可能降低参与 TCR 信号通路的蛋白质的活性，该通路在调节 T 细胞增殖中起着重要作用，并可能有助于解释先前观察到的顺铂抑制 T 细胞增殖的报道。
在这项研究中，进行了定量磷酸酪氨酸分析，以鉴定 Jurkat T 细胞暴露于顺铂时磷酸蛋白质组的变化。磷酸蛋白质组分析的结果通过免疫印迹和时间磷酸化流式分析进行了补充。在顺铂处理的早期时间点检测到 p38α MAPK 信号通路的初始激活，先前认为该反应是顺铂诱导细胞凋亡的机制的一部分。此外，通过磷酸酪氨酸蛋白质组分析发现，在细胞凋亡过程中 TCR 下游信号转导涉及的蛋白质的磷酸化水平降低。我们的研究可以支持阐明先前观察到的顺铂免疫抑制作用背后的机制。
