Quantitative phosphoproteome analysis of a mouse liver cell line reveals specificity of phosphatase inhibitors

Proteomics. 2008 Nov;8(21):4534-46. doi: 10.1002/pmic.200800105.

Abstract

The liver is a central organ involved in many aspects of physiology and disease. Signaling properties of hepatocytes, the main liver cell type, are of special interest in metabolic diseases and in regeneration. For this reason we investigated the phosphoproteome of the mouse liver cell line Hepa1-6 by stable isotope labeling by amino acids in cell culture (SILAC) and high resolution MS. Using stringent statistical evaluation criteria, we obtained 5433 phosphorylation sites on 1808 proteins. The phosphoproteome encompasses all major protein classes, including a large number of transcription factors. We compared control and phosphatase inhibitor treated cells by SILAC. This enabled ready identification of in vivo phosphorylation sites by sequencing the more abundant, inhibitor induced version of the peptide while still observing the endogenous version. We employed a mixture of pervanadate for blocking protein tyrosine phosphatases (PTPs) and calyculin A and deltamethrin for blocking the activities of serine/threonine phosphatases. Interestingly, these commonly used inhibitors in standard concentrations affected only 28% of the phosphopeptides by at least two-fold. The unaffected sites may be substrates of phosphatases that are not efficiently inhibited, have slow kinetic or sites that are almost stoichiometric in normally growing cells. Finally, we devised a triple labeling strategy comprising control cells, stimulated cells, and phosphatase treated cells to derive an upper bound on phosphorylation occupancy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Chromatography, Liquid
  • Computational Biology
  • Hepatocytes / drug effects
  • Hepatocytes / metabolism*
  • Insulin / pharmacology
  • Liver / cytology
  • Mice
  • Nitriles / pharmacology
  • Oxazoles / pharmacology
  • Phosphoprotein Phosphatases / antagonists & inhibitors*
  • Phosphoproteins / analysis*
  • Phosphorylation
  • Protein Tyrosine Phosphatases / antagonists & inhibitors*
  • Proteome / analysis*
  • Pyrethrins / pharmacology
  • Substrate Specificity
  • Tandem Mass Spectrometry
  • Transcription Factors / analysis
  • Vanadates / pharmacology

Substances

  • Insulin
  • Nitriles
  • Oxazoles
  • Phosphoproteins
  • Proteome
  • Pyrethrins
  • Transcription Factors
  • pervanadate
  • decamethrin
  • Vanadates
  • calyculin A
  • Phosphoprotein Phosphatases
  • Protein Tyrosine Phosphatases