title: Differential ubiquitination defines the functional status of the tumor suppressor Smad4.
abstract: Smad4 is an essential signal transducer of all transforming growth factor-beta (TGF-beta) superfamily pathways that regulate cell growth and differentiation, and it becomes inactivated in human cancers. Receptor-activated (R-) Smads can be poly-ubiquitinated in the cytoplasm or the nucleus, and this regulates their steady state levels or shutdown of the signaling pathway. Oncogenic mutations in Smad4 and other Smads have been linked to protein destabilization and proteasomal degradation. We analyzed a panel of missense mutants derived from human cancers that map in the N-terminal Mad homology (MH) 1 domain of Smad4 and result in protein instability. We demonstrate that all mutants exhibit enhanced poly-ubiquitination and proteasomal degradation. In contrast, wild type Smad4 is a relatively stable protein that undergoes mono- or oligo-ubiquitination, a modification not linked to protein degradation. Analysis of Smad4 deletion mutants indicated efficient mono- or oligo-ubiquitination of the C-terminal MH2 domain. Mass spectrometric analysis of mono-ubiquitinated Smad4 MH2 domain identified lysine 507 as a major target for ubiquitination. Lysine 507 resides in the conserved L3 loop of Smad4 and participates in R-Smad C-terminal phosphoserine recognition. Mono- or oligo-ubiquitinated Smad4 exhibited enhanced ability to oligomerize with R-Smads, whereas mutagenesis of lysine 507 led to inefficient Smad4/R-Smad hetero-oligomerization and defective transcriptional activity. Finally, overexpression of a mutant ubiquitin that only leads to mono-ubiquitination of Smad4 enhanced Smad transcriptional activity. These data suggest that oligo-ubiquitination positively regulates Smad4 function, whereas poly-ubiquitination primarily occurs in unstable cancer mutants and leads to protein degradation.
authors.affiliation: Ludwig Institute for Cancer Research, Box 595, Biomedical Center, SE-751 24 Uppsala, Sweden.
authors.forename: Anita
authors.initials: A
authors.lastname: Morén
authors.forename: Ulf
authors.initials: U
authors.lastname: Hellman
authors.forename: Yuri
authors.initials: Y
authors.lastname: Inada
authors.forename: Takeshi
authors.initials: T
authors.lastname: Imamura
authors.forename: Carl-Henrik
authors.initials: CH
authors.lastname: Heldin
authors.forename: Aristidis
authors.initials: A
authors.lastname: Moustakas
mesh_terms: D000595:Amino Acid Sequence; D002454:Cell Differentiation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D017124:Conserved Sequence; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020125:Mutation, Missense; D010641:Phenotype; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D051900:Smad3 Protein; D051901:Smad4 Protein; D013997:Time Factors; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D008024:Ligands; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators; D025801:Ubiquitin; D008156:Luciferases; D008239:Lysine