Transcriptional activating activity of Smad4: roles of SMAD hetero-oligomerization and enhancement by an associating transactivator.
Smad4 plays a pivotal role in signal transduction of the transforming growth factor beta superfamily cytokines by mediating transcriptional activation of target genes. Hetero-oligomerization of Smad4 with the pathway-restricted SMAD proteins is essential for Smad4-mediated transcription. We provide evidence that SMAD hetero-oligomerization is directly required for the Smad4 C-terminal domain [Smad4(C)] to show its transcriptional transactivating activity; this requirement obtains even when Smad4(C) is recruited to promoters by heterologous DNA-binding domains and in the absence of the inhibitory Smad4 N-terminal domain. Defined mutations of GAL4 DNA-binding domain fusion of Smad4(C) that disrupt SMAD hetero-oligomerization suppressed transcriptional activation. Importantly, we found that an orphan transcriptional activator MSG1, a nuclear protein that has strong transactivating activity but apparently lacks DNA-binding activity, functionally interacted with Smad4 and enhanced transcription mediated by GAL4 DNA-binding domain-Smad4(C) and full-length Smad4. Transcriptional enhancement by MSG1 depended on transforming growth factor beta signaling and was suppressed by Smad4(C) mutations disrupting SMAD hetero-oligomerization or by the presence of Smad4 N-terminal domain. Furthermore, Smad4(C) did not show any detectable transactivating activity in yeast when fused to heterologous DNA-binding domains. These results demonstrate additional roles of SMAD hetero-oligomerization in Smad4-mediated transcriptional activation. They also suggest that the transcriptional-activating activity observed in the presence of Smad4 in mammalian cells may be derived, at least in part, from endogenously expressed separate transcriptional activators, such as MSG1.
Shioda
T
T
Laboratory of Tumor Biology, Massachusetts General Hospital Cancer Center, Massachusetts General Hospital-East, Charlestown, MA 02129-2060, USA. shioda@helix.mgh.harvard.edu
Lechleider
R J
RJ
Dunwoodie
S L
SL
Li
H
H
Yahata
T
T
de Caestecker
M P
MP
Fenner
M H
MH
Roberts
A B
AB
Isselbacher
K J
KJ
D016475:3T3 Cells; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D001665:Binding Sites; D004268:DNA-Binding Proteins; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D051901:Smad4 Protein; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D051017:Apoptosis Regulatory Proteins; C512688:CITED1 protein, human; C102208:Cited1 protein, mouse; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; C497796:SMAD4 protein, human; D029701:Saccharomyces cerevisiae Proteins; D051901:Smad4 Protein; C490810:Smad4 protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta