title: Quantitative phosphoproteomics of transforming growth factor-β signaling in colon cancer cells.
abstract: 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.
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia.
authors.forename: Naveid A
authors.initials: NA
authors.lastname: Ali
authors.forename: Mark P
authors.initials: MP
authors.lastname: Molloy
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism*; D000081410:Germinal Center Kinases; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D020543:Proteome / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction / Q000502:physiology*; D051901:Smad4 Protein / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D047992:TNF Receptor-Associated Factor 2 / Q000378:metabolism; D014025:Titanium / Q000737:chemistry; D016212:Transforming Growth Factor beta / Q000378:metabolism*
chemical_list: D000081410:Germinal Center Kinases; D010446:Peptide Fragments; D010750:Phosphoproteins; D020543:Proteome; C497796:SMAD4 protein, human; D051901:Smad4 Protein; D047992:TNF Receptor-Associated Factor 2; D016212:Transforming Growth Factor beta; C009495:titanium dioxide; D014025:Titanium; D017346:Protein Serine-Threonine Kinases