Subcellular fractionation of TGF-β1-stimulated lung epithelial cells: A novel proteomic approach for identifying signaling intermediates

Autor: Oliver Eickelberg, Ejvind Mørtz, Jadranka Milosevic, Patrick Bulau
Rok vydání: 2009
Předmět:
Zdroj: PROTEOMICS. 9:1230-1240
ISSN: 1615-9861
1615-9853
Popis: 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.
Databáze: OpenAIRE
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