Members of the transforming growth factor (TGF)-beta superfamily are keyregulators of lung development and homeostasis, as well as alveolar epithelial cellfunction. TGF-beta signalling involves ligand-dependent phosphorylation of receptorserine/threonine kinases, phosphorylation of pathway-specific transcription factors(Smads), nuclear translocation of Smads, and ultimately, modulation of geneexpression. Although Smad-dependent responses represent the primary signallingsystem activated by TGF-beta receptors, alternative signalling systems have recentlybeen described, which also mediated TGF-beta-induced effects. In this study, aproteomic approach was employed to identify: a) candidate proteins that are subjectedto nuclear-cytoplasmic shuttling in response to TGF-beta stimulation and b) novelproteins phosphorylated within TGF-beta signaling pathway. In order to reducecomplexity of the total proteome subcellular fractionation and phosphoproteomeenrichment of A549 cells had been performed. Cytoplasmic, nuclear, andphosphoprotein enriched fractions were subjected to two dimensional polyacrylamidegel electrophoresis, tryptic digestion, and mass spectrometry in order to identify novelcandidates/mediators of the TGF-beta signaling pathway. In the first part of this study, arapid increase of KHRSP, FUBP1, hnRNP-L, and hnRNP-H1 localization in thecytosol of A549 cells was observed, concomitant with a decrease in their nuclearlocalization after TGF-beta stimulation. Proteomic data were confirmed byimmunofluorescence and immunoblotting analyses. In the second part of this study,25 phosphoproteins were identified after TGF-beta stimulation, 20 of them with differentphosphorylation pattern in 2-DE and 5 of them without changes after TGF-betastimulation. In conclusion, we showed that proteomic approach can be used fordetecting novel intermediate proteins in signalling pathways and that regulatoryfunctions of TGF-beta signalling are broader than previously thought.
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