title: Loss of Smad4 function in pancreatic tumors: C-terminal truncation leads to decreased stability.
abstract: At early stages of tumorigenesis, the transforming growth factor-beta (TGF-beta) signaling pathway is thought to have tumor suppressor activity as a result of its ability to arrest the growth of epithelial cells. Smad4 plays a pivotal role in the TGF-beta signaling pathway and has been identified as a tumor suppressor, being mutated or deleted in approximately 50% of pancreatic carcinomas and 15% of colorectal cancers. A nonsense mutation generating a C-terminal truncation of 38 amino acids in the Smad4 protein has been identified in a pancreatic adenocarcinoma (Hahn, S. A., Schutte, M., Hoque, A. T., Moskaluk, C. A., da Costa, L. T., Rozenblum, E., Weinstein, C. L., Fischer, A., Yeo, C. J., Hruban, R. H., and Kern, S. E. (1996) Science 271, 350-353), and here we investigate the functional consequences of this mutation. We demonstrate that the C-terminal truncation prevents Smad4 homomeric complex formation and heteromeric complex formation with activated Smad2. Furthermore, the mutant protein is unable to be recruited to DNA by transcription factors and hence cannot form transcriptionally active DNA-binding complexes. These observations are supported by molecular modeling, which indicates that the truncation removes residues critical for homomeric and heteromeric Smad complex formation. We go on to show that the mutant Smad4 is highly unstable compared with wild type Smad4 and is rapidly degraded through the ubiquitin-proteasome pathway. Consistent with this, we demonstrate that the pancreatic adenocarcinoma harboring this mutated allele, in conjunction with loss of the other allele, expresses no Smad4 protein. Thus we conclude that these tumors completely lack Smad4 activity.
authors.affiliation: Laboratory of Lymphocyte Molecular Biology, London WC2A 3PX, United Kingdom.
authors.forename: D
authors.initials: D
authors.lastname: Maurice
authors.forename: C E
authors.initials: CE
authors.lastname: Pierreux
authors.forename: M
authors.initials: M
authors.lastname: Howell
authors.forename: R E
authors.initials: RE
authors.lastname: Wilentz
authors.forename: M J
authors.initials: MJ
authors.lastname: Owen
authors.forename: C S
authors.initials: CS
authors.lastname: Hill
mesh_terms: D016475:3T3 Cells; D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism; D000483:Alleles; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003062:Codon; D018389:Codon, Nonsense; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D005799:Genes, Dominant; D006801:Humans; D019656:Loss of Heterozygosity; D008648:Mesoderm / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D009414:Nerve Growth Factors; D010190:Pancreatic Neoplasms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D015398:Signal Transduction; D051785:Smad Proteins; D051899:Smad2 Protein; D051901:Smad4 Protein; D013997:Time Factors; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism*; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000378:metabolism; D025801:Ubiquitin / Q000494:pharmacology; D014981:Xenopus; D029867:Xenopus Proteins
chemical_list: D000596:Amino Acids; D003062:Codon; D018389:Codon, Nonsense; D004268:DNA-Binding Proteins; D009414:Nerve Growth Factors; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; C497782:SMAD2 protein, human; C497796:SMAD4 protein, human; D051785:Smad Proteins; D051899:Smad2 Protein; C497785:Smad2 protein, Xenopus; C497783:Smad2 protein, mouse; D051901:Smad4 Protein; C490810:Smad4 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D025801:Ubiquitin; D029867:Xenopus Proteins; C490808:smad4.1 protein, Xenopus; C117263:smad4.2 protein, Xenopus; D003513:Cycloheximide; D012260:Ribonucleases