Dual role of the Smad4/DPC4 tumor suppressor in TGFβ-inducible transcriptional complexes

The transformation growth factor β (TGFβ) family signals a wide variety of biological responses through transcriptional regulation of genes encoding critical determinants of cell fate such as cell-cycle regulators (Pietenpol et al. 1990; Hannon and Beach 1994; Datto et al. 1995; Reynisdottir et al. 1995; Iavarone and Massague 1997), differentiation factors (Zentella and Massague 1992), extracellular matrix proteins (Kerr et al. 1988; Rossi et al. 1988; Keeton et al. 1991; Inagaki et al. 1994) or homeobox gene products (Huang et al. 1995). Clues about the mechanism of transcriptional regulation by the TGFβ family have been provided by the recent discovery of the SMAD proteins as direct substrates of TGFβ family receptors and mediators of receptor signals to the nucleus. The founding member of the SMAD family is the product of the Drosophila gene Mad, which was identified as being required for signaling by the BMP homolog Decapentaplegic (Dpp) (Sekelsky et al. 1995). The discovery of Mad and the identification of nematode and vertebrate Mad-related gene products termed SMADs allowed the elucidation of a signaling pathway in which receptor phosphorylated SMADs move into the nucleus to activate transcription (for review, see Derynck and Zhang 1996; Wrana and Attisano 1996; Massague et al. 1997). The members of the SMAD family contain highly conserved amino- and carboxy-terminal domains (referred to as N and C domains, or MH1 and MH2 domains, respectively), separated by a more divergent linker region. On the basis of structural and functional criteria, the SMAD family can be divided into three subgroups. One group includes those SMADs that are direct receptor substrates. The second group includes co-SMADs, or SMADs that are not direct receptor substrates, but participate in signaling by associating with receptor-activated SMADs (for review, see Derynck and Zhang 1996; Wrana and Attisano 1996; Massague et al. 1997). The third group includes proteins that interfere with SMAD activation and can be referred to as anti-SMADs (Hayashi et al. 1997; Topper et al. 1997). Among the receptor-regulated SMADs, Smad1 and presumably its close homologs Smad5 and Smad9 are bone morphogenetic protein (BMP) receptor substrates and mediators of BMP signals in vertebrates (Graff et al. 1996; Hoodless et al. 1996; Lechleider et al. 1996; Liu et al. 1996; Thomsen 1996; Yingling et al. 1996; Kretzschmar et al. 1997a; Watanabe et al. 1997), whereas Mad in Drosophila (Newfeld et al. 1996; Wiersdorff et al. 1996) and Sma-2 and Sma-3 in Caenorhabditis elegans (Savage et al. 1996) mediate the actions of BMP-like factors in these organisms. Smad2 and Smad3 are substrates and mediators of related TGFβ and activin receptors in vertebrates (Baker and Harland 1996; Eppert et al. 1996; Graff et al. 1996; Macias-Silva et al. 1996; Zhang et al. 1996). Receptor-regulated SMADs are phosphorylated by the receptors at the carboxy-terminal end, which typically is an SSV/MS sequence (Macias-Silva et al. 1996; Kretzschmar et al. 1997a). The N and C domains of these SMADs interact with each other, causing auto inhibition (Baker and Harland 1996; Liu et al. 1996; Hata et al. 1997), and agonist-induced phosphorylation may relieve this inhibition. On phosphorylation of the carboxy-terminal residues, SMADs move into the nucleus (Hoodless et al. 1996; Liu et al. 1996; Nakao et al. 1997a,b) where they participate in agonist-dependent transcriptional activation as originally inferred from studies showing that Smad1 fused to the GAL4 DNA-binding domain has transcriptional activity that is regulated by BMP (Liu et al. 1996). As further evidence for a direct role of SMADs in transcription, activin has been shown to activate the Mix.2 homeobox gene in Xenopus by inducing the association of Smad2 with FAST-1 (Chen et al. 1996), a nuclear protein that belongs to the winged helix transcription factor family and recognizes an activin responsive element (ARE) in the Mix.2 promoter (Huang et al. 1995). This has led to a model in which receptor-activated Smad2 translocates into the nucleus where it associates with a DNA-binding protein forming a transcriptional complex (Chen et al. 1996). Signaling by receptor-regulated SMADs requires the participation of a co-SMAD. The only known member of this group in vertebrates is Smad4. Smad4 has the same overall structure as the receptor-regulated SMADs, but is more divergent and lacks the carboxy-terminal phosphorylation motif. Smad4 was originally identified as the product of the DPC4 tumor suppressor (Hahn et al. 1996) that is mutated or deleted in a high proportion of pancreatic cancers and in a smaller proportion of other cancers (Barrett et al. 1996; Hahn et al. 1996; Kim et al. 1996; Nagatake et al. 1996; Schutte et al. 1996). Inactivating missense mutations have been found both in the N and C domains in different tumor-derived DPC4 alleles. A similar distribution of inactivating mutations has been observed in another tumor suppressor in this family, Smad2 (Eppert et al. 1996; Riggins et al. 1996; Uchida et al. 1996). The C domain of SMADs has effector function in various biological assays (Baker and Harland 1996; Lagna et al. 1996; Liu et al. 1996) and mutations in this domain disrupt its activity (Shi et al. 1997). The crystal structure of the Smad4 C domain reveals that it is a trimer, and certain mutations disrupt the monomer interfaces of this trimer (Shi et al. 1997). Mutations in the N domain of Smad2 and Smad4 augment the autoinhibitory function of this domain (Hata et al. 1997) and may have additional effects (Kim et al. 1997). A general requirement of Smad4 in TGFβ family signaling is suggested not only by the requirement of Smad4 for TGFβ responsiveness in mammalian cells (Lagna et al. 1996; Zhang et al. 1996) but also by its requirement for activin and BMP responses in Xenopus embryo (Lagna et al. 1996, Zhang et al. 1997). Thus, Smad4 is a shared co-Smad, participating in both TGFβ/activin and BMP signaling pathways. Smad4 associates with Smad1 or Smad2 when these SMADs are phosphorylated by specific receptors (Lagna et al. 1996) and is required for their signaling function (Lagna et al. 1996; Zhang et al. 1996, 1997). The interaction between the receptor-regulated SMADs and Smad4 is mediated by their C domains (Hata et al. 1997; Wu et al. 1997). Although Smad4 is recognized as a central mediator for TGFβ signaling, it was not clear which step of the SMAD-signaling pathway requires Smad4 function. In this study we made use of a Smad4-deficient cell line and the FAST-1/Mix.2 system reconstituted in mammalian cells to investigate this problem. We show that Smad4 is not required for nuclear translocation of a receptor-activated SMAD but its association with a receptor-activated SMAD promotes binding of the SMAD complex to DNA and, furthermore, it provides an essential transcriptional activation function.