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.
Ghosh
A K
AK
Departments of Pathology, Saint Louis University, St. Louis, Missouri 63104, USA.
Majumder
M
M
Steele
R
R
White
R A
RA
Ray
R B
RB
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D014408:Biomarkers, Tumor; D016923:Cell Death; D002455:Cell Division; D002888:Chromosomes, Human, Pair 19 / Q000235:genetics; D004268:DNA-Binding Proteins; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D016259:Genes, myc / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D026901:Membrane Transport Proteins; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase; D020161:Physical Chromosome Mapping; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D025521:Tumor Suppressor Proteins; D020798:Two-Hybrid System Techniques
D014408:Biomarkers, Tumor; D004268:DNA-Binding Proteins; D026901:Membrane Transport Proteins; D009363:Neoplasm Proteins; D012097:Repressor Proteins; C120759:TRAPPC2 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C095157:ENO1 protein, human; D010751:Phosphopyruvate Hydratase