Functional cloning of the proto-oncogene brain factor-1 (BF-1) as a Smad-binding antagonist of transforming growth factor-beta signaling.
Using the plasminogen activator inhibitor (PAI) promoter to drive the expression of a reporter gene (mouse CD2), we devised a system to clone negative regulators of the transforming growth factor-beta (TGF-beta) signaling pathway. We infected a TGF-beta-responsive cell line (MvLu1) with a retroviral cDNA library, selecting by fluorescence-activated cell sorter single cells displaying low PAI promoter activity in response to TGF-beta. Using this strategy we cloned the proto-oncogene brain factor-1 (BF-1). BF-1 represses the PAI promoter in part by associating with both unphosphorylated Smad3 (in the cytoplasm) and phosphorylated Smad3 (in the nucleus), thus preventing its binding to DNA. BF-1 also associates with Smad1, -2, and -4; the Smad MH2 domain binds to BF-1, and the C-terminal segment of BF-1 is uniquely and solely required for binding to Smads. Further, BF-1 represses another TGF-beta-induced promoter (p15), it up-regulates a TGF-beta-repressed promoter (Cyclin A), and it reverses the growth arrest caused by TGF-beta. Our results suggest that BF-1 is a general inhibitor of TGF-beta signaling and as such may play a key role during brain development.
Rodriguez
C
C
Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA.
Huang
L J
LJ
Son
J K
JK
McKee
A
A
Xiao
Z
Z
Lodish
H F
HF
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002469:Cell Separation; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors; D005982:Glutathione Transferase / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D015398:Signal Transduction; D051785:Smad Proteins; D051898:Smad1 Protein; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D013936:Thymidine / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D051858:Forkhead Transcription Factors; C489562:Foxd1 protein, mouse; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D051785:Smad Proteins; D051898:Smad1 Protein; C497401:Smad1 protein, mouse; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D051901:Smad4 Protein; C490810:Smad4 protein, mouse; C490811:Smad4 protein, rat; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D004247:DNA; D008156:Luciferases; D005982:Glutathione Transferase; D013936:Thymidine