Molecular and functional consequences of Smad4 C-terminal missense mutations in colorectal tumour cells

• DOI: 10.1042/bj20031886
• Publication Date: 2004-04-13T15:13:59Z
• Authors: Karolien DE BOSSCHER; Caroline S. HILL; Francisco J. NICOLÁS
• Keywords: Cell Nucleus; Models, Molecular; 0301 basic medicine; Macromolecular Substances; Protein Conformation; Active Transport, Cell Nucleus; Mutation, Missense; Forkhead Transcription Factors; Neoplasm Proteins; Protein Structure, Tertiary; 3. Good health; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; 03 medical and health sciences; Amino Acid Substitution; Humans; Point Mutation; ADP-Ribosylation Factor 1; Nerve Growth Factors; Colorectal Neoplasms; Dimerization; Cell Division; Protein Binding

Smad4 is an essential signal transducer of the transforming growth factor β (TGF-β) signalling pathway and has been identified as a tumour suppressor, being mutated in approx. 50% pancreatic cancers 15% colorectal cancers. Two missense mutations C-terminal domain Smad4, D351H (Asp351→His) D537Y (Asp537→Tyr), have described recently human cancer cell lines CACO-2 SW948 respectively [Woodford-Richens, Rowan, Gorman, Halford, Bicknell, Wasan, Roylance, Bodmer Tomlinson (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 9719–9723]. Previous work vitro suggested that only Asp-351 was required for interaction with Smad2 [Wu, Fairman, Penry Shi J. Biol. Chem. 276, 20688–20694]. In present study, we investigate functional consequences these point vivo. We demonstrate neither cells undergo arrest response to TGF-β, which can be explained, at least part, by their inability up-regulate cyclin-dependent kinase inhibitors p21CIP1 or p15INK4b after TGF-β stimulation. Although point-mutated Smad4s are expressed normal levels cells, they cannot interact either TGF-β-induced phosphorylated Smad3. As result, mutants do not accumulate nucleus stimulation, recruited DNA relevant Smad-binding transcription factors generate transcriptionally active DNA-bound complexes. Therefore both completely lack activity owing mutations. Given location three-dimensional structure domain, results also give us significant insights into Smad complex formation.