Quantitative profiling of tyrosine phosphorylation revealed changes in the activity of the T cell receptor signaling pathway upon cisplatin-induced apoptosis

Abstract

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.