Functional consequences of tumorigenic missense mutations in the amino-terminal domain of Smad4

Abstract

Smads, the intracellular e ectors of transforming growth factor-b (TGF-b) family members, are somatically mutated at high frequency in particular types of human cancers. Certain of these mutations a ect the Smad amino-terminal domain, which, in the case of Smad3 and Smad4, binds DNA. We investigated the functional consequences of four missense mutations in the Smad4 amino-terminal domain found in human tumors. The mutant proteins were found to have impaired abilities to bind DNA although they were fully capable of forming complexes with Smad3. All four Smad4 mutants showed decreased protein stability compared to wild-type Smad4. Two of the Smad4 mutants (G65V and P130S) were translocated to the nucleus and were capable of transactivating a Smad-dependent promoter in a liganddependent manner. In contrast, the L43S and R100T mutants were not translocated e ciently to the nucleus and consequently resulted in severely defective transcriptional responses to TGF-b. Moreover, we demonstrate here the critical importance of two basic residues in the b-hairpin loop of Smad3 or Smad4 for DNA binding, consistent with predictions from the Smad3 crystal structure. In addition, our results reveal that in the TGF-b-induced heteromeric signaling complex, loss of DNA binding of Smad4 can be compensated by Smad3, however, both Smad3 and Smad4 are needed for e cient DNA binding and signaling. In conclusion, mutations in the amino-terminal domain of Smad4, that are found in cancer, show loss of multiple functional properties which may contribute to tumorigenesis. Oncogene (2000) 19, 4396 ± 4404.

An extended bipartite nuclear localization signal in Smad4 is required for its nuclear import and transcriptional activity

“…This conclusion is consistent with a previous report (Moren et al, 2000). However, the Smad4 R100T mutation causes destabilization and proteasomal degradation of Smad4 (Moren et al, 2000;Xu and Attisano, 2000), indicating that this mutation may alter the global conformation of the protein. This is also illustrated in Figure 5b.…”

“…As shown by several previous studies, the NLSinhibitory effects of the R100T mutation is probably caused by a general disruption of the overall structural integrity of Smad4 (Shi et al, 1998;Moren et al, 2000;Xu and Attisano, 2000). Thus, we have not assigned a specific role to this residue in nuclear import.…”

Regulating the stability of TGFβ receptors and Smads

“…Deregulation of any of the above-mentioned proteins impacts on either normal embryonic development or, alternatively, on a pathogenic process of human disease. As already discussed above, the first demonstration of ubiquitination as a regulatory mechanism of TGFb pathways came from studies of Smad4 mutants in human tumors [90,91].…”

“…There is more information regarding the poly-ubiquitination and proteasomal degradation of mutant versions of Smad2 and Smad4 that are abundant in various human tumors. Mutations in the N-terminal MH1 domain probably affect the critical folding of this domain, causing general instability of the protein and Ub-mediated proteasomal degradation [90,91]. Such mutations are R133C in Smad2 and L43S, G65V, R100T and P130S in Smad4.…”

To (TGF)β or not to (TGF)β: Fine-tuning of Smad signaling via post-translational modifications

“…In acute myelogenous leukemia a missense mutation in the MH1 domain (P102L) and a frameshift mutation resulting in termination of the MH2 domain, cause rapid SCF βTrCP -mediated proteasomal degradation of Smad4 [87]. Smad4 point mutations identified in pancreatic (L43S and R100T) and colorectal (G65V and P130S) cancers have a higher affinity for the βTrCP F-box protein, and consequently also show enhanced SCF βTrCP -mediated polyubiquitination and degradation as compared to wild-type Smad4 [82,88,89]. Significantly, Liang et al demonstrated that R100T, G65V, and L43S mutations result in massive phosphorylation of Smad4 by JNK/p38 MAPK, causing an increased affinity of mutant Smad4 not only for βTrCP, but also for Skp2 [90].…”