Quantitative phosphoproteomics of transforming growth factor-β signaling in colon cancer cells

Proteomics. 2011 Aug;11(16):3390-401. doi: 10.1002/pmic.201100036.


The transforming growth factor-β (TGF-β) signaling pathway progresses through a series of protein phosphorylation regulated steps. Smad4 is a key mediator of the classical TGF-β signaling pathway; however, reports suggest that TGF-β can activate other cellular pathways independent of Smad4. By investigating the TGF-β-regulated phosphoproteome, we aimed to uncover new functions controlled by TGF-β. We applied titanium dioxide to enrich phosphopeptides from stable isotope labeling with amino acids in cell culture (SILAC)-labeled SW480 cells stably expressing Smad4 and profiled them by mass spectrometry. TGF-β stimulation for 30 min resulted in the induction of 17 phosphopeptides and the repression of 8 from a total of 149 unique phosphopeptides. Proteins previously not known to be phosphorylated by TGF-β including programmed cell death protein 4, nuclear ubiquitous casein and cyclin-dependent kinases substrate, hepatoma-derived growth factor and cell division kinases amongst others were induced following TGF-β stimulation, while the phosphorylation of TRAF2 and NCK-interacting protein kinase are examples of proteins whose phosphorylation status was repressed. This phosphoproteomic screen has identified new TGF-β-modulated phosphorylation responses in colon carcinoma cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cell Line, Tumor
  • Colonic Neoplasms / genetics
  • Colonic Neoplasms / metabolism*
  • Germinal Center Kinases
  • Humans
  • Isotope Labeling
  • Mass Spectrometry
  • Molecular Sequence Data
  • Peptide Fragments / metabolism
  • Phosphoproteins / analysis
  • Phosphoproteins / chemistry
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Protein Serine-Threonine Kinases / metabolism
  • Proteome / analysis
  • Proteome / chemistry
  • Proteome / metabolism
  • Proteomics / methods*
  • Signal Transduction / physiology*
  • Smad4 Protein / biosynthesis
  • Smad4 Protein / genetics
  • Smad4 Protein / metabolism
  • TNF Receptor-Associated Factor 2 / metabolism
  • Titanium / chemistry
  • Transforming Growth Factor beta / metabolism*


  • Germinal Center Kinases
  • Peptide Fragments
  • Phosphoproteins
  • Proteome
  • SMAD4 protein, human
  • Smad4 Protein
  • TNF Receptor-Associated Factor 2
  • Transforming Growth Factor beta
  • titanium dioxide
  • Titanium
  • Protein Serine-Threonine Kinases