Region between alpha-helices 3 and 4 of the mad homology 2 domain of Smad4: functional roles in oligomer formation and transcriptional activation.
BACKGROUND
Smad4 has a unique region of 35 amino acids between alpha-helices 3 and 4 (termed H3/4 loop) of the Mad homology (MH) 2 domain. In order to elucidate the functional importance of the H3/4 loop, we prepared chimeric constructs of Smad4 containing the region corresponding to the alpha-helix 3, H3/4 loop and alpha-helix 4 of different Smads, including a chimera containing that of Smad2 (Smad4-HL2).


RESULTS
Smad4-HL2 constitutively induced the transcriptional activation of p3TP-Lux, a TGF-beta-responsive reporter construct. However, co-transfection of Smad2 with Smad4-HL2 did not induce a further increase in the activation of p3TP-Lux. Smad4-HL2 did not induce the activation of pAR3-Lux, which contains FAST1-binding sites and is activated by a complex composed of FAST1, Smad2 and Smad4. Smad4-HL2 formed a homo-oligomer more efficiently than wild-type Smad4 in mammalian cells. Moreover, Smad4-HL2 bound to DNA containing the Smad-binding sites with a gretaer affinity than the wild-type Smad4.


CONCLUSIONS
Smad4-HL2 spontaneously forms a homo-oligomer, which may bind to DNA with relatively high affinity and induce transcriptional activation of p3TP-Lux. The H3/4 loop of Smad4 may thus play a role in precluding the spontaneous oligomer formation of Smad4.
Tada
K
K
Department of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research (JFCR), and Research for the Future Program, Japan Society for the Promotion of Science, 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 170-8455, Ja.
Inoue
H
H
Ebisawa
T
T
Makuuchi
M
M
Kawabata
M
M
Imamura
T
T
Miyazono
K
K
D000818:Animals; D019556:COS Cells; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D051899:Smad2 Protein; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D018909:src Homology Domains
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D051899:Smad2 Protein; D015534:Trans-Activators; D008156:Luciferases