Subcellular fractionation of TGF-beta1-stimulated lung epithelial cells: a novel proteomic approach for identifying signaling intermediates.
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.
Milosevic
Jadranka
J
Department of Medicine, University of Giessen Lung Center, Giessen, Germany.
Bulau
Patrick
P
Mortz
Ejvind
E
Eickelberg
Oliver
O
D002454:Cell Differentiation; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000166:cytology* / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions / Q000737:chemistry* / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*
D020543:Proteome; D053773:Transforming Growth Factor beta1