Subcellular fractionation of TGF-beta1-stimulated lung epithelial cells: a novel proteomic approach for identifying signaling intermediates

Proteomics. 2009 Mar;9(5):1230-40. doi: 10.1002/pmic.200700604.


Members of the transforming growth factor (TGF)-beta superfamily are key regulators of lung development and homeostasis, in particular by controlling alveolar/bronchial epithelial cell function. TGF-beta signaling involves ligand-dependent activation of receptor serine/threonine kinases, activation and subsequent nuclear translocation of pathway-specific transcription factors (Smads), and ultimately, modulation of gene expression. While Smad-dependent responses represent the primary signaling components activated by TGF-beta receptors, their function is controlled by a variety of cofactors. In addition, alternative signaling systems mediating TGF-beta-induced effects have recently been described such as MAP kinase pathways. To uncover novel proteins that participate in TGF-beta signaling via nuclear/cytoplasmic shuttling in lung epithelial cells, we have analyzed A549 human lung epithelial cells, using subcellular fractionation combined with 2-D PAGE, tryptic digestion, and MS. We identified a rapid increase in the cytosolic localization of KH-type splicing regulatory protein (KHSRP), far upstream element-binding protein (FUBP1), hnRNP-L, and hnRNP-H1, concomitant with a decrease in their nuclear localization in response to TGF-beta1. Proteomic data were confirmed by immunofluorescence and immunoblot analyses. In summary, we represent a powerful novel technology for the identification of previously unknown signaling intermediates.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Differentiation
  • Cell Line
  • Cell Nucleus / chemistry
  • Cell Nucleus / metabolism
  • Cytosol / chemistry
  • Cytosol / metabolism
  • Electrophoresis, Gel, Two-Dimensional
  • Epithelial Cells / cytology*
  • Epithelial Cells / metabolism
  • Humans
  • Lung / cytology*
  • Lung / metabolism
  • Proteome / analysis*
  • Proteome / metabolism
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Subcellular Fractions / chemistry*
  • Subcellular Fractions / metabolism*
  • Transforming Growth Factor beta1 / metabolism*


  • Proteome
  • Transforming Growth Factor beta1