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Oligopeptide Competition Assay for Phosphorylation Site Determination
Published on: May 18, 2017
Protein phosphorylation profiling identifies potential mechanisms for direct immunotoxicity
Jia Shao1,2,3, Inge Stout1, Peter J M Hendriksen1,3
1a RIKILT-Institute of Food Safety, Wageningen University and Research Centre , Wageningen , the Netherlands .
Abstract:
Signaling networks are essential elements that are involved in diverse cellular processes. One group of fundamental components in various signaling pathways concerns protein tyrosine kinases (PTK). Various toxicants have been demonstrated to exert their toxicity via modulation of tyrosine kinase activity. The present study aimed to identify common cellular signaling pathways that are involved in chemical-induced direct immunotoxicity. To this end, an antibody array-based profiling approach was applied to assess effects of five immunotoxicants, two immunosuppressive drugs and two non-immunotoxic control chemicals on the phosphorylation of 28 receptor tyrosine kinases and 11 crucial signaling nodes in Jurkat T-cells. The phosphorylation of ribosomal protein S6 (RPS6) and of kinases Akt, Src and p44/42 were found to be commonly regulated by immunotoxicants and/or immunosuppressive drugs (at least three compounds), with the largest effect observed upon RPS6. Flow cytometry and Western blotting were used to further examine the effect of the model immunotoxicant TBTO on the components of the mTOR-p70S6K-RPS6 pathway. These analyses revealed that both TBTO and the mTOR inhibitor rapamycin inactivate RPS6, but via different mechanisms. Finally, a comparison of the protein phosphorylation data to previously obtained transcriptome data of TBTO-treated Jurkat cells resulted in a good correlation at the pathway level and indicated that TBTO affects ribosome biogenesis and leukocyte migration. The effect of TBTO on the latter process was confirmed using a CXCL12 chemotaxis assay.
Insights
Chemicals can harm the immune system by altering protein tyrosine kinase (PTK) activity. This study identified common signaling pathways affected by immunotoxicants, focusing on ribosomal protein S6 (RPS6) phosphorylation.
Area of Science:
- Immunotoxicology
- Cellular Signaling
- Molecular Biology
Background:
- Protein tyrosine kinases (PTKs) are crucial in cellular signaling pathways.
- Chemical toxicants can disrupt cellular processes by modulating PTK activity.
- Understanding chemical-induced immunotoxicity requires identifying affected signaling pathways.
Purpose of the Study:
- To identify common cellular signaling pathways involved in chemical-induced direct immunotoxicity.
- To investigate the effects of immunotoxicants and immunosuppressive drugs on tyrosine kinase signaling.
- To elucidate the mechanism of action of specific immunotoxicants on key signaling nodes.
Main Methods:
- Antibody array-based profiling of 28 receptor tyrosine kinases and 11 signaling nodes in Jurkat T-cells.
- Assessment of chemical effects on protein phosphorylation.
- Flow cytometry and Western blotting to analyze the mTOR-p70S6K-RPS6 pathway.
- Comparison of proteomic data with transcriptomic data.
- CXCL12 chemotaxis assay to confirm effects on leukocyte migration.
Main Results:
- Immunotoxicants and immunosuppressive drugs commonly regulated phosphorylation of ribosomal protein S6 (RPS6), Akt, Src, and p44/42 kinases.
- RPS6 phosphorylation showed the most significant regulation.
- The immunotoxicant TBTO and rapamycin inactivated RPS6 through distinct mechanisms.
- Protein phosphorylation data correlated well with transcriptome data at the pathway level.
- TBTO was found to affect ribosome biogenesis and leukocyte migration.
Conclusions:
- Chemicals can induce immunotoxicity by targeting common signaling pathways, particularly those involving RPS6.
- TBTO impacts cellular processes like ribosome biogenesis and leukocyte migration via distinct molecular mechanisms.
- Integrated analysis of proteomic and transcriptomic data provides insights into chemical toxicity mechanisms.
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