Premium
Region between α‐helices 3 and 4 of the Mad homology 2 domain of Smad4: functional roles in oligomer formation and transcriptional activation
Author(s) -
Tada Keiichiro,
Inoue Hirofumi,
Ebisawa Takanori,
Makuuchi Masatoshi,
Kawabata Masahiro,
Imamura Takeshi,
Miyazono Kohei
Publication year - 1999
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1046/j.1365-2443.1999.00293.x
Subject(s) - oligomer , biology , smad , transfection , dna , microbiology and biotechnology , biophysics , biochemistry , gene , transforming growth factor , chemistry , organic chemistry
Background Smad4 has a unique region of 35 amino acids between α‐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 α‐helix 3, H3/4 loop and α‐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‐β‐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. Conclusion 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.