Alpha-helix 2 in the amino-terminal mad homology 1 domain is responsible for specific DNA binding of Smad3.
Smads, signal transducers of the transforming growth factor-beta (TGF-beta) superfamily proteins, directly bind to DNA and regulate transcription of target genes. Smad3 binds to CAGA box, whereas Smad1 and Smad5 preferentially bind to GC-rich sequences. The beta-hairpin loop in the amino-terminal Mad homology 1 (MH1) domain is the direct DNA-binding site of Smad3; however, the amino acid sequences of the beta-hairpin loop of Smad3 and Smad1/5 are identical, suggesting that other regions may be responsible for the differential DNA binding of Smad3 and Smad1/5. To identify regions other than the beta-hairpin loop responsible for specific DNA binding of Smad3, we generated chimeras containing various regions of Smad3 and Smad1. Luciferase assays using a TGF-beta-responsive reporter (CAGA)9-MLP-Luc and gel-mobility shift assays using 3xCAGA as a probe revealed that alpha-helix 2 (H2) in the amino-terminal part of the MH1 domain plays an important role in specific DNA binding and transcriptional activation of Smad3. Luciferase assays using natural TGF-beta-responsive reporters also revealed the functional importance of H2 in the Smad3 MH1 domain in direct DNA binding. Smad3 thus binds to DNA directly through the beta-hairpin loop, and H2 supports specific DNA binding of Smad3.
Kusanagi
K
K
Department of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research, 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 170-8455, Japan.
Kawabata
M
M
Mishima
H K
HK
Miyazono
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008907:Mink; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D051900:Smad3 Protein; D015534:Trans-Activators / Q000737:chemistry*; D015533:Transcriptional Activation; D016212:Transforming Growth Factor beta / Q000378:metabolism
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; C497790:SMAD3 protein, human; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D004247:DNA; D008156:Luciferases