Functional mapping of the MH1 DNA-binding domain of DPC4/SMAD4

被引:29
作者
Jones, JB
Kern, SE
机构
[1] Johns Hopkins Univ, Predoctoral Program Human Genet, Baltimore, MD USA
[2] Johns Hopkins Univ, Sch Med, Dept Oncol, Baltimore, MD 21205 USA
关键词
D O I
10.1093/nar/28.12.2363
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The transcription factor Smad4 binds DNA in response to a TGF-beta ligand-initiated intracellular signaling cascade. SMAD4 is deleted or mutated during tumorigenesis in many human tumors. Some of these mutations occur in the N-terminal portion of the protein, the Mad homology 1 (MH1) region, which exhibits sequence-specific DNA-binding. We used alanine scanning mutagenesis and natural mutations to map the subregion of the MH1 domain necessary for that function. We created 20 individual mutations in the MH1 region of human Smad4 and assayed their effect on DNA-binding in vitro. Mutation of residues in the less conserved N- and C-terminal areas of the MH1 region had no effect on DNA-binding. However, mutations in the domain from L43 to R135 caused a dramatic reduction of the ability of Smad4 to bind DNA. Previous work demonstrated a beta-hairpin protein motif within this region to be responsible for DNA-binding, but suggested that the tumorigenic mutations occurring outside this motif may target a separate function of the MH1 domain. Our results demonstrate that the MH1 domain as a whole is very sensitive to changes in overall structure, and that tumorigenic mutations within the region of L43-R135 indeed would target DNA-binding.
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收藏
页码:2363 / 2368
页数:6
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