| Transforming growth factor-beta I (TGF-beta1) 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 (EMT). TGF-beta1 signals through the TGF-beta type I and type II receptors, and activates the Smad pathway via phosphorylation of Smad2 and Smad3. Evidence suggests that Smad2 and Smad3 signaling is involved in TGF-beta1-induced EMT, cell motility, and increased tumor metastasis, but the contributions of their actions in these phenomena are not fully understood. Furthermore, Smad2 and Smad3 interact with a myriad of proteins and activate a number of different target genes, in response to TGF-beta1 and little is known about their selective activation. We hypothesized that Smad2 and Smad3 can modulate the actin cytoskeleton in a manner distinct from their roles in TGF-beta1-mediated growth inhibition through interactions with novel protein complexes. To test our hypothesis, we screened primary human epithelial cell cultures and established mouse and human cell lines for TGF-beta1 responsiveness including morphological changes, actin cytoskeleton reorganization, cell-cell junction changes, cell proliferation, and Smad phosphorylation. Furthermore, novel Smad2 and Smad3 associated proteins were identified by tandem affinity purification of Myc-His6-Smad2 and Myc-His 6-Smad3 from TGF-beta1 treated HaCaT cells and liquid chromatography (LC) and tandem mass spectrometry (MS/MS) of the resulting eluates. The current study showed that in many cell strains, TGF-beta1 can induce cell cycle arrest and cytoskeleton alterations and that these events are not mutually exclusive. Furthermore, a number of putative novel Smad2 and Smad3 associated proteins were identified. A number of these novel Smad2 and Smad3 interacting proteins are associated with actin cytoskeleton organization and cell motility. One of the proteins identified was Zizimin1, a Cdc42 guanine nucleotide exchange factor. The identification and further analysis of Zizimin1 may provide a link for Smad2 and Smad3 functions in TGF-beta1-mediated cytoskeleton alterations. |
