title: Ribonucleic acid polymerase II binding subunit 3 (Rpb3), a potential nuclear target of insulin-like growth factor binding protein-3.
abstract: IGF-binding protein (IGFBP)-3 has intrinsic antiproliferative and proapoptotic functions that are independent of IGF binding and may involve nuclear localization. We determined that exogenous IGFBP-3 rapidly translocates to myoblast nuclei and that a 22-residue peptide containing the metal binding domain (MBD) and nuclear localization sequence (NLS) can similarly direct chimeric GFP into myoblast nuclei. Furthermore, a non-IGF-binding IGFBP-3 mutant inhibited myoblast proliferation without stimulating apoptosis. These results suggest that IGFBP-3 inhibits muscle cell growth in an IGF-independent manner that may be influenced by its rapid nuclear localization. We therefore identified IGFBP-3 interacting proteins by screening a rat L6 myoblast cDNA library using the yeast two-hybrid assay and two N-terminal deletion mutants as bait: BP3/231 (231 residues, L61 to K291) and BP3/111 (K181-K291). Proteins previously known to interact with IGFBP-3 as well as several novel proteins were identified, including RNA polymerase II binding subunit 3 (Rpb3). The domain necessary for Rpb3 binding was subsequently identified using different IGFBP-3 deletion mutants and was localized to the MBD/NLS epitope. Rpb3/IGFBP-3 binding was confirmed by coimmunoprecipitation assays with specific antisera, whereas a NLS mutant IGFBP-3 did not associate with Rpb3, suggesting that a functional NLS is required. Rpb3 facilitates recruitment of the polymerase complex to specific transcription factors and is necessary for the transactivation of many genes. Its association with IGFBP-3 provides a functional role for IGFBP-3 in the direct modulation of gene transcription.
authors.affiliation: Department of Animal Sciences, Washington State University, Pullman, 99164-6351, USA.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Oufattole
authors.forename: Sally Wan-Jung
authors.initials: SW
authors.lastname: Lin
authors.forename: Bingrong
authors.initials: B
authors.lastname: Liu
authors.forename: Desmond
authors.initials: D
authors.lastname: Mascarenhas
authors.forename: Pinchas
authors.initials: P
authors.lastname: Cohen
authors.forename: Buel D
authors.initials: BD
authors.lastname: Rodgers
mesh_terms: D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism*; D049109:Cell Proliferation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D000939:Epitopes / Q000737:chemistry; D017353:Gene Deletion; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012319:RNA Polymerase II / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013347:Subcellular Fractions; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques
chemical_list: D000939:Epitopes; D018972:Insulin-Like Growth Factor Binding Protein 3; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; C491783:POLR2C protein, human; D012319:RNA Polymerase II