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

• Published in: Proteomics (Weinheim) 2011-08, Vol.11 (16), p.3390-3401
• Main Authors: Ali, Naveid A., Molloy, Mark P.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Cancer; Cell biology; Cell Line, Tumor; Colonic Neoplasms - genetics; Colonic Neoplasms - metabolism; Fundamental and applied biological sciences. Psychology; Gastroenterology. Liver. Pancreas. Abdomen; Humans; Isotope Labeling; Mass Spectrometry; Medical sciences; Miscellaneous; Molecular Sequence Data; Peptide Fragments - metabolism; Phosphoproteins - analysis; Phosphoproteins - chemistry; Phosphoproteins - metabolism; Phosphorylation; Protein-Serine-Threonine Kinases - metabolism; Proteins; Proteome - analysis; Proteome - chemistry; Proteome - metabolism; Proteomics - methods; Signal Transduction - physiology; Smad4; Smad4 Protein - biosynthesis; Smad4 Protein - genetics; Smad4 Protein - metabolism; Stomach. Duodenum. Small intestine. Colon. Rectum. Anus; Titanium - chemistry; TNF Receptor-Associated Factor 2 - metabolism; Transforming Growth Factor beta - metabolism; Transforming growth factor-β; Tumors
• ISSN: 1615-9853; 1615-9861
• 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.