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A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening
Published on: May 12, 2017
Global phosphoproteome profiling reveals unanticipated networks responsive to cisplatin treatment of embryonic stem
Alex Pines1, Christian D Kelstrup, Mischa G Vrouwe
1Department of Toxicogenetics, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.
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.
Insights
This study reveals that cisplatin triggers extensive cellular responses, affecting DNA damage repair and cytoskeleton organization. Key protein kinases, beyond ATM/ATR, regulate these processes, impacting cell survival.
Area of Science:
- Proteomics and Systems Biology
- Molecular Cell Biology
- Cancer Research
Background:
- Cellular responses to DNA damage are crucial for maintaining genomic stability.
- Anticancer drugs like cisplatin induce DNA damage, activating complex signaling pathways.
- Understanding these pathways is key to developing more effective cancer therapies.
Purpose of the Study:
- To comprehensively map protein and phosphoprotein changes in response to cisplatin treatment.
- To identify key kinases and signaling pathways involved in the cellular response to genotoxic stress.
- To investigate the role of identified kinases in cisplatin-induced cytotoxicity.
Main Methods:
- Quantitative proteomics using SILAC-labeled murine embryonic stem cells.
- High-resolution tandem mass spectrometry to profile protein and phosphorylation changes.
- Bioinformatic analyses including network and pathway analysis, and siRNA-mediated knockdown.
Main Results:
- Cisplatin treatment significantly altered DNA damage response and cytoskeleton organization pathways.
- While ATM/ATR motifs were enriched, many hyperphosphorylated sites were not direct substrates.
- Eleven protein kinases, including MAPK family members, showed regulated activation loops; CDK7, Plk1, and KPCD1 knockdown protected against cisplatin toxicity.
- Transcriptomic and proteomic data showed poor correlation in individual gene/protein levels but pathway overlap.
Conclusions:
- The cellular response to cisplatin involves diverse kinases and phosphatases in both nuclear and cytoplasmic compartments.
- Extensive cytoskeletal rearrangements are a significant outcome of cisplatin-induced genotoxic stress.
- This study deciphers kinases regulating processes beyond DNA damage response, offering insights into broader cellular functions.
