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...

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Bibliographic Details
Published in:The Journal of biological chemistry 2001-08, Vol.276 (32), p.30224-30230
Main Authors: Rodriguez, C, Huang, L J, Son, J K, McKee, A, Xiao, Z, Lodish, H F
Format: Article
Language:English
Subjects:
3T3 Cells
Animals
BF-1 gene
Binding Sites
Brain - metabolism
brain factor 1
CD2 gene
Cell Separation
Cloning, Molecular
DNA - metabolism
DNA, Complementary - metabolism
DNA-Binding Proteins - metabolism
Flow Cytometry
Forkhead Transcription Factors
Glutathione Transferase - metabolism
Luciferases - metabolism
Mice
Microscopy, Fluorescence
Mutation
Nerve Tissue Proteins - metabolism
Phosphorylation
Promoter Regions, Genetic
Protein Binding
Protein Structure, Tertiary
Recombinant Proteins - metabolism
Retroviridae - genetics
Signal Transduction
Smad protein
Smad Proteins
Smad1 Protein
Smad2 Protein
Smad3 Protein
Smad4 Protein
Thymidine - metabolism
Trans-Activators - metabolism
Transfection
Transforming Growth Factor beta - metabolism
Tumor Cells, Cultured
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M102759200