Time-resolved dissection of early phosphoproteome and ensuing proteome changes in response to TGF-β

Sci. Signal., 22 July 2014
Vol. 7, Issue 335, p. rs5
DOI: 10.1126/scisignal.2004856

Time-resolved dissection of early phosphoproteome and ensuing proteome changes in response to TGF-β

  1. Rochelle C. J. D’Souza1,*,
  2. Anna M. Knittle2,3,
  3. Nagarjuna Nagaraj1,
  4. Maarten van Dinther2,
  5. Chunaram Choudhary1,4,
  6. Peter ten Dijke2,
  7. Matthias Mann1,4, and
  8. Kirti Sharma1,
  1. 1Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
  2. 2Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands, Leiden University Medical Center, Postbus 9600, 2300 RC Leiden, the Netherlands.
  3. 3Department of Medical Biochemistry and Genetics, and Turku Doctoral Program of Biomedical Sciences, University of Turku, FI-20520 Turku, Finland.
  4. 4Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3b, 2200 Copenhagen, Denmark.
  1. Corresponding author. E-mail: kisharma{at}biochem.mpg.de
  • * Present address: Diabetes and Obesity Program, Garvan Institute of Medical Research, 384 Victoria Road, Darlinghurst, New South Wales 2010, Australia.


Transforming growth factor–β (TGF-β) signaling promotes cell motility by inducing epithelial-to-mesenchymal transitions (EMTs) in normal physiology and development, as well as in pathological conditions, such as cancer. We performed a time-resolved analysis of the proteomic and phosphoproteomic changes of cultured human keratinocytes undergoing EMT and cell cycle arrest in response to stimulation with TGF-β. We quantified significant changes in 2079 proteins and 2892 phosphorylation sites regulated by TGF-β. We identified several proteins known to be involved in TGF-β–induced cellular processes, such as the cytostatic response, extracellular matrix remodeling, and epithelial dedifferentiation. In addition, we identified proteins involved in other cellular functions, such as vesicle trafficking, that were not previously associated with TGF-β signaling. Although many TGF-β responses are mediated by phosphorylation of the transcriptional regulators of the SMAD family by the TGF-β receptor complex, we observed rapid kinetics of changes in protein phosphorylation, indicating that many responses were mediated through SMAD-independent TGF-β signaling. Combined analysis of changes in protein abundance and phosphorylation and knowledge of protein interactions and transcriptional regulation provided a comprehensive representation of the dynamic signaling events underlying TGF-β–induced changes in cell behavior. Our data suggest that in epithelial cells stimulated with TGF-β, early signaling is a mixture of both pro- and antiproliferative signals, whereas later signaling primarily inhibits proliferation.


R. C. J. D’Souza, A. M. Knittle, N. Nagaraj, M. van Dinther, C. Choudhary, P. ten Dijke, M. Mann, and K. Sharma, Time-resolved dissection of early phosphoproteome and ensuing proteome changes in response to TGF-β. Sci. Signal. 7, rs5 (2014).
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