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.