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

Biochem J. 2004 Apr 1;379(Pt 1):209-16. doi: 10.1042/BJ20031886.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADP-Ribosylation Factor 1 / metabolism
  • Active Transport, Cell Nucleus
  • Amino Acid Substitution
  • Cell Division / drug effects
  • Cell Nucleus / metabolism
  • Colorectal Neoplasms / metabolism*
  • Colorectal Neoplasms / pathology
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / deficiency
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • DNA-Binding Proteins / physiology*
  • Dimerization
  • Forkhead Transcription Factors
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Macromolecular Substances
  • Models, Molecular
  • Mutation, Missense*
  • Neoplasm Proteins / chemistry
  • Neoplasm Proteins / deficiency
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / physiology*
  • Nerve Growth Factors
  • Point Mutation
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Tertiary
  • Signal Transduction
  • Smad Proteins
  • Smad2 Protein
  • Smad3 Protein
  • Smad4 Protein
  • Structure-Activity Relationship
  • Trans-Activators / chemistry
  • Trans-Activators / deficiency
  • Trans-Activators / genetics
  • Trans-Activators / metabolism
  • Trans-Activators / physiology*
  • Transcription Factors / metabolism
  • Transcription, Genetic
  • Transforming Growth Factor beta / pharmacology
  • Xenopus Proteins*

Substances

  • DNA-Binding Proteins
  • FOXH1 protein, human
  • Forkhead Transcription Factors
  • Foxh1 protein, mouse
  • Macromolecular Substances
  • Neoplasm Proteins
  • Nerve Growth Factors
  • SMAD2 protein, human
  • SMAD3 protein, human
  • SMAD4 protein, human
  • Smad Proteins
  • Smad2 Protein
  • Smad3 Protein
  • Smad4 Protein
  • Trans-Activators
  • Transcription Factors
  • Transforming Growth Factor beta
  • Xenopus Proteins
  • smad4.1 protein, Xenopus
  • smad4.2 protein, Xenopus
  • ADP-Ribosylation Factor 1