Identification of novel Smad2 and Smad3 associated proteins in response to TGF‐β1

Transforming growth factor-beta 1 (TGF-β1) is an important growth regulator of many cell types to aid in maintaining tissue homeostasis. TGF-β1 can inhibit epithelial cell proliferation while promoting the growth of some fibroblasts. The activity of TGF-β1 occurs by signaling through the TGF-β type I (TβRI) and TGF-β type II (TβRII) transmembrane serine/threonine protein kinase receptors, and activates the Smad pathway through C-terminal phosphorylation of Smad2 and Smad3 on serine residues [Macias-Silva et al., 1996]. Activated Smad2 and Smad3 oligomerize with Smad4 and translocate to the nucleus where they interact with transcription factors, transcriptional co-repressors, or transcriptional co-activators to repress or activate a host of TGF-β1 responsive genes [Abdollah et al., 1997; de Caestecker et al., 2000; Heldin et al., 1997; Kloos et al., 2002; Massague, 2000; Massague and Wotton, 2000; Nakao et al., 1997; Souchelnytskyi et al., 1997; Ten Dijke et al., 2002; Wu et al., 1997]. Resistance to the antiproliferative effects of TGF-β1 is observed in many cancers including head and neck [Chen et al., 2001b; Garrigue-Antar et al., 1995; Munoz-Antonia et al., 1996], lung [Hougaard et al., 1999], gastric [Kang et al., 1999; Myeroff et al., 1995; Park et al., 1994], colon [Grady et al., 1999; Markowitz, 2000; Markowitz et al., 1995; Myeroff et al., 1995], pancreatic [Goggins et al., 1998], ovarian [Chen et al., 2001a; Wang et al., 2000], and some recurrent breast cancers [Chen et al., 1998; Lucke et al., 2001]. This loss of sensitivity to TGF-β1 is often due to mutations in the genes that encode Smad2 and Smad4, mothers against decapentaplegic homologue (MADH) 2 and MADH4 respectively [Eppert et al., 1996; Hahn et al., 1996; Salovaara et al., 2002; Yanagisawa et al., 2000]. Smad2 and Smad3 have an amino acid sequence identity of 91%; therefore it is often thought that Smad2 and Smad3 have redundant roles in TGF-β1 signaling. In spite of this similarity, data from Smad2 and Smad3 null mice and Smad2 and Smad3 silencing in epithelial cells, suggest that Smad2 and Smad3 have distinct roles, in addition to compensatory roles (reviewed in [Brown et al., 2007]). Microarray analysis of Smad2 and Smad3 null mouse embryonic fibroblasts (MEFs) and of epithelial cells in which Smad2 or Smad3 is silenced show an activation or repression of different and overlapping subsets of genes [Kretschmer et al., 2003; Yang et al., 2003]. Interestingly, silencing of Smad3, but not silencing of Smad2, blocks the growth inhibitory response of TGF-β1 in HaCaT cells indicating that Smad3 may have a more important role in TGF-β1-mediated cell cycle arrest than Smad2 [Kretschmer et al., 2003]. The cytostatic function of Smad3 over Smad2 in TGF-β1 signaling is also revealed in a study where Smad3 silencing results in inhibition of TGF-β1-mediated cell cycle arrest in a number of TGF-β1 sensitive cell lines [Kim et al., 2005]. Furthermore, raising the relative endogenous ratio of Smad3 to Smad2 by depleting Smad2 enhances the TGF-β1 cytostatic response and Smad3 activation and transcriptional activity of TGF-β1-treated cells [Kim et al., 2005]. In contrast, Yang et al. observe TGF-β1-mediated growth inhibition in Smad3 deficient mammary gland epithelial cells in culture [Yang et al., 2002]. Compensatory changes in protein levels or phosphorylation of Smad2 were not seen in the Smad3 deficient epithelium [Yang et al., 2002]. From these Smad2 or Smad3 depletion studies it is clear that in some cell types the roles of Smad2 and Smad3 are not simply redundant but are distinct. Overexpression of Smad2 and Smad3 in human cells further elucidates the roles of Smad2 versus Smad3. Primary hepatic stellate cells that overexpress Smad3 have increased secretion of fibronectin and type I collagen, increased chemotaxis, decreased proliferation, more focal adhesions, and increased α-smooth muscle actin organization in actin stress fibers, compared to cells overexpressing Smad2 [Uemura et al., 2005]. In HepG2 human hepatoma cells, overexpression of Smad3/4 results in higher levels of transcriptional activation of the p21 promoter than overexpression of Smad2/4 [Moustakas and Kardassis, 1998]. In addition, overexpression of Smad3 induces apoptosis in lung epithelial cells, whereas overexpression of Smad2 does not induce apoptosis to the same extent [Yanagisawa et al., 1998]. These overexpression data also suggest that Smad2 and Smad3 have distinct functions. The regulation of Smad2 and Smad3 is complex and can occur at the level of interaction with the TGF-β receptors, nuclear import and export, and/or at the transcriptional level. This regulation occurs through the interaction of Smad2 and Smad3 with a myriad of proteins in response to TGF-β1. However, little is known about the selective activation of Smad2 versus Smad3. Collectively, the studies described above indicate that Smad2 and Smad3 may play distinct roles in TGF-β1-mediated cytoskeleton rearrangement and cell cycle control. Therefore, we further investigated the roles and regulation of Smad2 and Smad3 in TGF-β1 signaling by identifying novel Smad2 and Smad3 associated proteins by affinity purification and liquid chromatography (LC) and tandem mass spectrometry (MS/MS). A number of putative novel Smad2 and Smad3 associated proteins were identified in addition to proteins that are known to interact with Smad2 and Smad3. Specifically, a Smad2 and Smad3 association with a novel interacting protein identified by LC-MS/MS, Zizimin1, was confirmed by co-immunoprecipitation experiments. These novel associated proteins have functions in cell proliferation, apoptosis, actin cytoskeleton regulation, cell motility, transcription, and Ras or insulin signaling and may reveal how Smad2 and Smad3 are regulated and/or uncover new functions of Smad2 and Smad3 in TGF-β1 signaling.