TGF-β1 responsive Smad2 and Smad3 associated proteins identified by tandem affinity purification and mass spectrometry

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Transforming growth factor-beta 1 (TGF-β1) is proposed to inhibit the growth of epithelial cells in early tumorigenesis, and promote tumor cell motility and invasion in the later stages of carcinogenesis through the induction of an epithelial to mesenchymal transition. TGF-β1 signals through the TGF-β type I and type II receptors, and activates the Smad pathway via phosphorylation of Smad2 and Smad3. Little is known about the selective activation of Smad2 versus Smad3. The Smad2 and Smad3 knockout mouse phenotypes and studies comparing Smad2 and Smad3 activation of TGF-β1 target genes, suggest that Smad2 and Smad3 may have distinct roles in TGF-β signaling. Furthermore, the observation that TGF-β1 inhibits proliferation of Smad3 null mammary gland epithelial cells, whereas Smad3 deficient fibroblasts are only partially growth inhibited, suggests that the role of Smad3 in epithelial cells may be different than its role in fibroblasts. We hypothesize that Smad2 and Smad3 play distinct roles in TGF-β1 signaling in epithelial cells through interactions with a unique subset of proteins. To test our hypothesis, human keratinocytes (HaCaT) were transduced with Myc-His6-Smad2 or Myc-His6-Smad3-expressing retrovirus and were treated with TGF-β1. Myc-His6-Smad2 or Myc-His6-Smad3 was purified by tandem affinity purification and eluates were subject to SDS-PAGE and Colloidal Blue staining. Select protein bands were digested with trypsin. The resulting tryptic peptides were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS/MS) and the SEQUEST algorithm was employed to identify proteins in the bands. A number of proteins that are known to interact with Smad2 and Smad3 were identified by LC-MS/MS. These proteins include Erbin, MAN1, Nedd-4, SKI, SKIP, Smad4, SMURF2, and SNO. In addition, a number of putative novel Smad2 and Smad3 associated proteins were identified. Fourteen proteins were identified that uniquely associate with Smad2, twenty proteins were identified that uniquely associate with Smad3, and eighteen proteins were identified that associate with both Smad2 and Smad3. Efforts are underway to confirm the interaction between Smad2, Smad3, and the novel associated proteins and to understand the biological significance of these interactions. These novel Smad2 and Smad3 associated proteins will be presented.