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


CONCLUSIONS
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.
The Biotechnology Centre of Oslo, University of Oslo, Gaustadalleen 21, 0349 Oslo, Norway.
Gro L
GL
Størvold
Johannes
J
Landskron
Margarita
M
Strozynski
Magnus Ø
MØ
Arntzen
Christian J
CJ
Koehler
Maria E
ME
Kalland
Kjetil
K
Taskén
Bernd
B
Thiede
D000970:Antineoplastic Agents / Q000737:chemistry; D017209:Apoptosis; D002470:Cell Survival; D002945:Cisplatin / Q000737:chemistry*; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007166:Immunosuppressive Agents / Q000737:chemistry; D019169:Jurkat Cells; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry; D040901:Proteomics; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000166:cytology; D014443:Tyrosine / Q000737:chemistry*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000970:Antineoplastic Agents; D007166:Immunosuppressive Agents; D011948:Receptors, Antigen, T-Cell; D019000:Phosphotyrosine; D014443:Tyrosine; D048051:p38 Mitogen-Activated Protein Kinases; D002945:Cisplatin
1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-1; 7-AAD; 7-amino-actinomycin D; Apoptosis; Atf2; C-terminal Src kinase; Cisplatin; Csk; Erk; FCB; FSC; Fluorescent cell barcoding; GAPDH; IL-2; ITAM; Itk; Lat; Lck; MAPK; MAPK-activated protein kinase 2; MAPK/ERK kinase; MAPKAPK2; MFI; Mek; Phospho-flow cytometry; Phosphotyrosine; Plcγ1; SH2 domain-containing leukocyte-specific protein of 76kDa; SILAC; SSC; Slp76; T cell receptor; T cell receptor signaling pathway; TCR; Zap70; activating transcription factor 2; extracellular-signal-regulated kinase; fluorescent cell barcoding; forward scatter; glyceraldehyde-3-phosphate dehydrogenase; immunoreceptor tyrosine-based activation motif; interleukin-2; interleukin-2-inducible T-cell kinase; linker for activation of T-cells; median fluorescent intensity; mitogen-activated protein kinase; side scatter; stable isotope labeling with amino acids in cell culture; tyrosine-protein kinase Lck/leukocyte C-terminal SRC kinase; tyrosine-protein kinase ZAP-70/zeta-chain-associated protein of 70kDa