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.
Laboratory of Tumor Biology, Massachusetts General Hospital Cancer Center, Massachusetts General Hospital-East, Charlestown, MA 02129-2060, USA. shioda@helix.mgh.harvard.edu
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T
Shioda
R J
RJ
Lechleider
S L
SL
Dunwoodie
H
H
Li
T
T
Yahata
M P
MP
de Caestecker
M H
MH
Fenner
A B
AB
Roberts
K J
KJ
Isselbacher
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