title: Molecular and functional consequences of Smad4 C-terminal missense mutations in colorectal tumour cells.
abstract: Smad4 is an essential signal transducer of the transforming growth factor beta (TGF-beta) signalling pathway and has been identified as a tumour suppressor, being mutated in approx. 50% of pancreatic cancers and approx. 15% of colorectal cancers. Two missense mutations in the C-terminal domain of Smad4, D351H (Asp351-->His) and D537Y (Asp537-->Tyr), have been described recently in the human colorectal cancer cell lines CACO-2 and SW948 respectively [Woodford-Richens, Rowan, Gorman, Halford, Bicknell, Wasan, Roylance, Bodmer and Tomlinson (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 9719-9723]. Previous work in vitro suggested that only Asp-351 was required for interaction with Smad2 [Wu, Fairman, Penry and Shi (2001) J. Biol. Chem. 276, 20688-20694]. In the present study, we investigate the functional consequences of these point mutations in vivo. We demonstrate that neither of these colorectal cancer cells undergo growth arrest in response to TGF-beta, which can be explained, at least in part, by their inability to up-regulate cyclin-dependent kinase inhibitors p21 (CIP1 ) or p15 ( INK4b) after TGF-beta stimulation. Although the point-mutated Smad4s are expressed at normal levels in these colorectal cancer cells, they cannot interact with either TGF-beta-induced phosphorylated Smad2 or Smad3. As a result, these Smad4 mutants do not accumulate in the nucleus after TGF-beta stimulation, are not recruited to DNA by relevant Smad-binding transcription factors and cannot generate transcriptionally active DNA-bound complexes. Therefore both these colorectal tumour cells completely lack functional Smad4 activity owing to the missense mutations. Given the location of these mutations in the three-dimensional structure of the Smad4 C-terminal domain, the results also give us significant insights into Smad complex formation.
authors.affiliation: Laboratory of Developmental Signalling, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.
authors.forename: Karolien
authors.initials: K
authors.lastname: De Bosscher
authors.forename: Caroline S
authors.initials: CS
authors.lastname: Hill
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Nicolás
mesh_terms: D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism; D021581:Active Transport, Cell Nucleus; D019943:Amino Acid Substitution; D002455:Cell Division / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism; D015179:Colorectal Neoplasms / Q000378:metabolism* / Q000473:pathology; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D019281:Dimerization; D051858:Forkhead Transcription Factors; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D046911:Macromolecular Substances; D008958:Models, Molecular; D020125:Mutation, Missense; D009363:Neoplasm Proteins / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D009414:Nerve Growth Factors; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D051785:Smad Proteins; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D029867:Xenopus Proteins
chemical_list: D004268:DNA-Binding Proteins; C510350:FOXH1 protein, human; D051858:Forkhead Transcription Factors; C491117:Foxh1 protein, mouse; D046911:Macromolecular Substances; D009363:Neoplasm Proteins; D009414:Nerve Growth Factors; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; D051785:Smad Proteins; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D029867:Xenopus Proteins; C490808:smad4.1 protein, Xenopus; C117263:smad4.2 protein, Xenopus; D020823:ADP-Ribosylation Factor 1