Global Phosphoproteome Profiling Reveals Unanticipated Networks Responsive to Cisplatin Treatment of Embryonic Stem Cells

Abstract

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.

PIM2 Kinase Is Induced by Cisplatin in Ovarian Cancer Cells and Limits Drug Efficacy

“…24 The upregulation of kinases involved in the MAPK (mitogen activated protein kinase) pathway, such as ERK1/2 and p90RSK, was also expected, as they are critical components of the signaling network activated by CDDP. 5,37 Moreover, our Figure 5. (A, D) PIM2 knockdown to study clonogenic ability of ovarian cancer cells.…”

“…5 Among them, we found H2A.X, which is involved in the initial enzymatic processing step of the DNA damage response. Its phosphorylation on Ser139 is a measure of double-strand breaks (DSBs) 36 and is required for signaling the recruitment of DNA repair proteins to DSBs.…”

Filia Is an ESC-Specific Regulator of DNA Damage Response and Safeguards Genomic Stability

“…Phosphorylation often correlates with a change in protein functions. There is evidence to suggest that S349 residue on Filia is subjected to phosphorylation in response to DNA damage (Pines et al, 2011). Therefore, we investigated if S349 was indeed phosphorylated and whether this modification played a role in modulating Filia’s functions.…”

Human Embryonic Stem Cells Fail to Activate CHK1 and Commit to Apoptosis in Response to DNA Replication Stress

“…However, it is important to point out that mouse ESCs respond very differently to cisplatin than the human ESCs used here. Mouse ESCs show a high level of protein phosphorylation following cisplatin treatment that appears to be driven in part by ATM and/or ATR activation [49]. As with other differences that are well known between human and mouse ESCs, these could reflect the possibility, now widely held, that human ESCs correspond to epistem cells, rather than to late inner cell mass (ICM) as do mouse ESCs (i.e., naïve vs. primed states) [50], but we cannot exclude that they reflect species differences.…”