Summary
Members of the transforming growth factor (TGF)-b superfamily are key regulators of lung development and homeostasis, in particular by controlling alveolar/bronchial epithelial cell function. TGF-b 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-b receptors, their function is controlled by a variety of cofactors. In addition, alternative signaling systems mediating TGF-binduced effects have recently been described such as MAP kinase pathways. To uncover novel proteins that participate in TGF-b 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-b1. Proteomic data were confirmed by immunofluorescence and immunoblot analyses.

Figure 3. 2-DE separation of cytoplasmic and nuclear fractions of A549 cells. Proteins were separated by 2-DE (first dimension, 11 cm 3¡V10 IPG strips, second dimension 12% SDS-PAGE) and visualized by staining with Sypro Ruby. The 18 annotated spots from the cytoplasmic fraction (Fig. 3A) and 19 spots from the nuclear fraction (Fig. 3B) are consecutively identified by MS and MS/MS in Table 1 (Fig. 3A) and Table 2 (Fig. 3B). Gels are representative for three independent experiments each, comprising at least 15 independent protein extractions from control and TGF-b1-stimulated A549 cells.

Reference:
Milosevic J. et al. ¡§Subcellular fractionation of TGF-ƒÒ1-stimulated lung epithelial cells: A novel proteomic approach for identifying signaling intermediates¡¨, Proteomics, 9, p.1230-1240, 2009.