![]() ![]() The Smad3 β-hairpin extends beyond the bulk of the MH1 structure to lie within the major groove of the DNA ( 16). The crystal structure of the MH1 region bound to DNA of a highly homologous family member, Smad3, indicates that the protein contacts DNA via an 11-residue β-hairpin motif. The Smad MH1 region was shown to bind to a consensus DNA sequence termed the Smad binding element (SBE) ( 5). Several point mutations map to the MH1 region ( 12– 15), implying that functions of this domain may serve as targets for mutational inactivation ( 9). SMAD4 mutations have been found frequently in pancreatic and colorectal cancers, as well as occasionally in other tumor types ( 11, 12). Mutation or deletion of components of the Smad signaling pathway allows some human tumors to evade the growth suppressive signals of TGF-beta or related ligands. The MH2 region has also been shown to partially interfere with the DNA-binding function of the MH1 region ( 5, 6, 9, 10). The N-terminal MH1 domain is responsible for sequence-specific DNA-binding ( 3– 6), while heteromerization and transactivation functions have been ascribed to the MH2 region ( 7– 9). Smad proteins have two evolutionarily conserved regions, MH1 and MH2 (Mad homology 1 and 2) separated by a linker region. Activated Smads can complex with the common partner, Smad4, translocate to the nucleus and participate in sequence-specific DNA-binding and transcriptional activation through TGF-beta responsive elements ( 1, 2). These receptors then phosphorylate/activate a set of pathway-restricted Smad proteins. Stimulation of a cell by a TGF-beta superfamily member induces autophosphorylation of ligand-specific receptor pairs. These cytokines induce cellular responses such as growth suppression, apoptosis and differentiation by signaling through the Smad family of effector proteins (reviewed in 1, 2). Human Smad4 ( DPC4/MADH4) is a central mediator of signal transduction by the transforming growth factor-beta (TGF-beta) superfamily of cytokines. ![]() Our results demonstrate that the MH1 domain as a whole is very sensitive to changes in overall structure, and that tumorigenic mutations within the region of L43–R135 indeed would target DNA-binding. Previous work demonstrated a β-hairpin protein motif within this region to be responsible for DNA-binding, but suggested that the tumorigenic mutations occurring outside this motif may target a separate function of the MH1 domain. However, mutations in the domain from L43 to R135 caused a dramatic reduction of the ability of Smad4 to bind DNA. Mutation of residues in the less conserved N- and C-terminal areas of the MH1 region had no effect on DNA-binding. We created 20 individual mutations in the MH1 region of human Smad4 and assayed their effect on DNA-binding in vitro. We used alanine scanning mutagenesis and natural mutations to map the subregion of the MH1 domain necessary for that function. Some of these mutations occur in the N-terminal portion of the protein, the Mad homology 1 (MH1) region, which exhibits sequence-specific DNA-binding. SMAD4 is deleted or mutated during tumorigenesis in many human tumors. The transcription factor Smad4 binds DNA in response to a TGF-beta ligand-initiated intracellular signaling cascade. ![]()
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |