Quantitative phosphoproteomics reveal cellular responses from caffeine, coumarin and quercetin in treated HepG2 cells

Toxicol Appl Pharmacol. 2022 Aug 15:449:116110. doi: 10.1016/j.taap.2022.116110. Epub 2022 Jun 7.


Protein phosphorylation is the most common type of post-translational modification where serine, threonine or tyrosine are reversibly bound to the phosphate group of ATP in a reaction catalyzed by protein kinases. Phosphorylation plays an important role in regulation of cell homeostasis, including but not limited to signal perception and transduction, gene expression and function of proteins. Protein phosphorylation happens on a fast time scale and represents an energy-efficient way for the cell to adapt to exposure to chemical stressors. To understand the cascade of cellular signaling induced by exposure to chemicals, we have exposed HepG2 cells to three chemicals with different modes of action, namely, caffeine, coumarin, and quercetin in a concentration and time response manner. Significantly upregulated and downregulated phosphosites were screened to analyze the activation/deactivation of signaling pathways by protein kinases. In total, 69, 44 and 12 signaling pathways were found enriched in caffeine, coumarin and quercetin treated cells, respectively, of which 9 pathways were co-enriched with 11 jointly responded kinases. Among identified co-responded kinases, CDK1, MAPK1 and MAPK3 play important roles in cell cycle and insulin signaling pathways. Quantitative phosphoproteomics can sensitively distinguish the effects of different chemicals on cells, allowing the assessment of chemical safety through changes in substrates and metabolic pathways at the cellular level, which is important for the development of non-animal approaches for chemical safety assessment.

Keywords: Cell cycle; Insulin signaling pathway; Kinase; Phosphoproteomics; Phosphorylation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Caffeine* / pharmacology
  • Coumarins* / pharmacology
  • Hep G2 Cells
  • Humans
  • Phosphorylation
  • Protein Kinases / metabolism
  • Proteomics
  • Quercetin* / pharmacology


  • Coumarins
  • Caffeine
  • Quercetin
  • Protein Kinases