A novel 16-kilodalton cellular protein physically interacts with and antagonizes the functional activity of c-myc promoter-binding protein 1.
We initially identified c-myc promoter-binding protein 1 (MBP-1) from a human cervical carcinoma cell expression library which negatively regulates c-myc promoter activity. A recent study demonstrated that MBP-1 acts as a general transcriptional repressor (A. K. Ghosh, R. Steele, and R. B. Ray, Mol. Cell. Biol. 19:2880-2886, 1999). In order ... to identify the cellular protein(s) interacting with MBP-1 for transcriptional regulation, a HeLa cell cDNA expression library was screened using a yeast two-hybrid system. An MBP-1-interacting cDNA encoding a polypeptide of 140 amino acid residues with an approximate molecular mass of 16 kDa was identified and named MBP-1 interacting protein-2A (MIP-2A). MIP-2A has a sequence similarity with an unknown mRNA and SEDL. Mutations in the SEDL gene, located at human chromosome Xp22, has recently been implicated with an X-linked genetic disease, although the function of SEDL gene product was not determined (A. K. Gedeon et al., Nat. Genet. 22:400-404, 1999). However, our results suggested the localization of MIP-2A at human chromosome 19. The specificity of interaction between MBP-1 and MIP-2A was verified by an in vitro glutathione S-transferase pulldown experiment, a mammalian two-hybrid analysis, and in vivo coimmunoprecipitation assays. Further analysis revealed that the amino-terminal domain of MBP-1 (amino acids 1 to 95) interacts with MIP-2A. Immunofluorescent staining suggested colocalization of MIP-2A and MBP-1 primarily in the perinuclear membrane of cells. Functional analysis demonstrated that MIP-2A relieves MBP-1 mediated transcriptional repression on c-myc promoter. Additionally, MIP-2A antagonizes cell growth regulatory role of MBP-1. Taken together, these results suggest the functional interaction of MIP-2A and MBP-1 in cell growth regulation.
Mesh Terms:
3T3 Cells, Animals, Binding Sites, Cell Death, Cell Division, Chromosomes, Human, Pair 19, DNA-Binding Proteins, Fluorescent Antibody Technique, Gene Expression Regulation, Genes, Reporter, Genes, myc, Hela Cells, Humans, Membrane Transport Proteins, Mice, Molecular Sequence Data, Molecular Weight, Neoplasm Proteins, Phosphopyruvate Hydratase, Physical Chromosome Mapping, Promoter Regions, Genetic, Protein Binding, Protein Structure, Tertiary, Repressor Proteins, Transcription Factors, Transfection, Tumor Markers, Biological, Tumor Suppressor Proteins, Two-Hybrid System Techniques
3T3 Cells, Animals, Binding Sites, Cell Death, Cell Division, Chromosomes, Human, Pair 19, DNA-Binding Proteins, Fluorescent Antibody Technique, Gene Expression Regulation, Genes, Reporter, Genes, myc, Hela Cells, Humans, Membrane Transport Proteins, Mice, Molecular Sequence Data, Molecular Weight, Neoplasm Proteins, Phosphopyruvate Hydratase, Physical Chromosome Mapping, Promoter Regions, Genetic, Protein Binding, Protein Structure, Tertiary, Repressor Proteins, Transcription Factors, Transfection, Tumor Markers, Biological, Tumor Suppressor Proteins, Two-Hybrid System Techniques
Mol. Cell. Biol.
Date: Jan. 01, 2001
PubMed ID: 11134351
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