A novel smad nuclear interacting protein, SNIP1, suppresses p300-dependent TGF-beta signal transduction.

TGF-β is the prototypic member of a large family of structurally related cytokines including the TGF-βs, activins, and bone morphogenetic proteins (BMPs), which regulate cell fate and extracellular matrix deposition through the transcriptional regulation of diverse gene targets. These ligands initiate intracellular signals by associating with two classes of interacting transmembrane receptor serine-threonine kinases (Derynck and Feng 1997; Massague 1998). Smad proteins, which serve as direct targets of phosphorylation by the activated type I receptors, have been demonstrated to be downstream mediators of specific TGF-β/BMP-signaling pathways from the receptors to the nucleus. Three classes of Smads have been identified and are referred to as the receptor-activated Smads, co-Smads, and inhibitory Smads. Receptor-activated Smads (R-Smads), Smad2, and Smad3 are specific mediators of TGF-β and activin signaling pathways, whereas Smad1, Smad5, and Smad8 are involved in BMP responses (Massague 1998). Smad4 is a co-Smad and is not regulated by phosphorylation, but acts as a common and essential mediator of TGF-β, activin, and BMP-signaling responses (Candia et al. 1997; de Caestecker et al. 1997). A third class of Smads includes Smad6 and Smad7, and act as an inhibitor of this pathway by binding to the activated receptors or to R-Smads, thereby sequestering the transcriptionally active complex from the promoter. Like many transcription factors, the R-Smad proteins interact with the paralogous transcriptional coactivators CBP and p300 through their MH2 domains (Feng et al. 1998; Janknecht et al. 1998; Pouponnot et al. 1998; Topper et al. 1998). Although these two proteins were characterized by different means, they are now considered to be orthologous and to act as a crucial scaffold to bring together transcription factors and basal factors in the transcriptional initiation complex. They are essential coactivators for a wide variety of transcriptional factors including nuclear hormone receptors and NF-κB (Mannervik et al. 1999). Besides possessing intrinsic histone acetyltransferase (HAT) activity, these proteins are also able to interact with other HATs such as p/CAF and steroid receptor coactivators (SRCs) (Torchia et al. 1998). Although recent publications have concentrated on the HAT activity of CBP/p300, these proteins also contain three highly conserved cysteine-histidine-rich domains (C/H1, C/H2, and C/H3). Among these, the C/H1 domain was shown to interact with the Smad activation domain (SAD) of Smad4, which is necessary for transcriptional activating activity of the Smads (de Caestecker et al. 2000), whereas the C/H3 domain is the locus of interaction with R-Smads (Feng et al. 1998; Janknecht et al. 1998; Nishihara et al. 1998; Shen et al. 1999). Many recent investigations have focused on identification of proteins that interact with Smad proteins to modulate both their trafficking and stability in the cytoplasm and their transcriptional activating activity in the nucleus. Cytoplasmic interactors include Smurf1, which was found to modulate the BMP signal-transduction pathway by regulating the level of Smad1 and Smad5 in the cell (Zhu et al. 1999), and SARA, which interacts directly with Smad2 and Smad3 and functions to recruit Smad2 to the TGF-β receptor (Tsukazaki et al. 1998). An increasing number of nuclear activators including the AP-1 complex, vitamin D receptor, and TFE3 transcriptional factor (Hua et al. 1998; Zhang et al. 1998; Yanagi et al. 1999; Yanagisawa et al. 1999) have been shown to interact specifically with Smad proteins. In addition, a number of transcriptional inhibitors have been shown to interact with Smad proteins and to repress their transcriptional activating activity. These include the oncoproteins Evi-1 (Kurokawa et al. 1998), Ski and SnoN, (Luo et al. 1999), TGIF, which recruits the histone deacetylase HDAC (Wotton et al. 1999), and the δEF1/Zfh-1 family member SIP1 (Verschueren et al. 1999). In this study, we characterize a novel nuclear protein, termed SNIP1 for Smad nuclear interacting protein 1, which also serves to suppress the TGF-β-signaling pathway. We demonstrate that, whereas this inhibitor can interact with the MH2 domain of Smad4 in a TGF-β dependent manner, its principal mechanism of action appears to be through inhibition of transcription by binding to CBP/p300 and interfering with the ability of these coactivators to interact with Smad4.