title: The 32-kilodalton subunit of replication protein A interacts with menin, the product of the MEN1 tumor suppressor gene.
abstract: Menin is a 70-kDa protein encoded by MEN1, the tumor suppressor gene disrupted in multiple endocrine neoplasia type 1. In a yeast two-hybrid system based on reconstitution of Ras signaling, menin was found to interact with the 32-kDa subunit (RPA2) of replication protein A (RPA), a heterotrimeric protein required for DNA replication, recombination, and repair. The menin-RPA2 interaction was confirmed in a conventional yeast two-hybrid system and by direct interaction between purified proteins. Menin-RPA2 binding was inhibited by a number of menin missense mutations found in individuals with multiple endocrine neoplasia type 1, and the interacting regions were mapped to the N-terminal portion of menin and amino acids 43 to 171 of RPA2. This region of RPA2 contains a weak single-stranded DNA-binding domain, but menin had no detectable effect on RPA-DNA binding in vitro. Menin bound preferentially in vitro to free RPA2 rather than the RPA heterotrimer or a subcomplex consisting of RPA2 bound to the 14-kDa subunit (RPA3). However, the 70-kDa subunit (RPA1) was coprecipitated from HeLa cell extracts along with RPA2 by menin-specific antibodies, suggesting that menin binds to the RPA heterotrimer or a novel RPA1-RPA2-containing complex in vivo. This finding was consistent with the extensive overlap in the nuclear localization patterns of endogenous menin, RPA2, and RPA1 observed by immunofluorescence.
authors.affiliation: Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-1802, USA. KarenS@intra.niddk.nih.gov
authors.forename: Karen E
authors.initials: KE
authors.lastname: Sukhodolets
authors.forename: Alison B
authors.initials: AB
authors.lastname: Hickman
authors.forename: Sunita K
authors.initials: SK
authors.lastname: Agarwal
authors.forename: Maxim V
authors.initials: MV
authors.lastname: Sukhodolets
authors.forename: Victor H
authors.initials: VH
authors.lastname: Obungu
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Novotny
authors.forename: Judy S
authors.initials: JS
authors.lastname: Crabtree
authors.forename: Settara C
authors.initials: SC
authors.lastname: Chandrasekharappa
authors.forename: Francis S
authors.initials: FS
authors.lastname: Collins
authors.forename: Allen M
authors.initials: AM
authors.lastname: Spiegel
authors.forename: A Lee
authors.initials: AL
authors.lastname: Burns
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Marx
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016193:G1 Phase; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D009377:Multiple Endocrine Neoplasia / Q000235:genetics; D020125:Mutation, Missense; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D051716:Replication Protein A; D016196:S Phase; D014162:Transfection; D020798:Two-Hybrid System Techniques
chemical_list: D001426:Bacterial Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C105373:MEN1 protein, human; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; C497167:RPA1 protein, human; D011994:Recombinant Proteins; C048817:RepB protein, Bacteria; D051716:Replication Protein A; C497168:Rpa1 protein, mouse; D004247:DNA; D005982:Glutathione Transferase