Quantitative phosphoproteome analysis of cisplatin-induced apoptosis in Jurkat T cells

ABSTRACT: Reversible protein phosphorylation is one of the most important posttranslational modifications (PTMs) due to its vital role in cellular functions and signaling pathways. Protein phosphorylation is also known to be involved in the regulation of apoptosis. Previously, we have performed a SILAC-based analysis of phosphotyrosinylation of cisplatin-induced apoptotic Jurkat T cells. To further expand this study, we used the same experimental setup and analyzed the global phosphorylation profile. The different steps of the phosphopeptide enrichment protocol using TiO2 beads were tested and the best approach was applied in combination with LC-MS with different normalized collision energy (NCE) values for HCD fragmentation. In total, more than 2,000 phosphoproteins of more than 4,000 phosphopeptides were identified in the four biological replicates of both states. HCD with NCE 25 accounted for 64% and NCE 35 for 36 % of identified phosphopeptides. 425 phosphopeptides were found to be significantly regulated in cisplatin-treated against control samples. KEGG pathway analysis revealed splicosomal proteins and the MAPK signaling pathway to be upregulated and cell cycle proteins to be downregulated. The transcription factors Atf2 and Jun, which are both involved in the MAPK signaling pathway, were further investigated. For Jun, more than 90 spectral counts were found only in apoptotic cells and covered the well-known stress induced phosphorylation sites at S-63/S-73, as well as S-243 which reduces the ability to bind DNA and is required for GSK3 mediated ubiquitinylation at S-239. For Atf2, more than 60 spectral counts were found in apoptotic cells vs. 8 in control cells. Most significantly upregulated sites for Atf2 have been identified at S-90 and S-112, which are less well studied phosphorylation sites of Atf2 as several other sites. Western blot analyses using non-phosphorylated and phosphorylated antibodies were performed to validate the phosphorylation events of Jun and Atf2. Our findings contribute to the current knowledge of the phosphorylation profiles of apoptotic cells.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell

SUBMITTER: Trung Tran  

LAB HEAD: Bernd Thiede

PROVIDER: PXD004415 | Pride | 2017-05-11

REPOSITORIES: Pride