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.

ACKNOWLEDGMENTS

We are grateful to many colleagues for their help and advice during the course of this work, especially Paul Goldsmith for purified rabbit menin- and RPA2-specific antibodies, George Poy for plasmid sequencing, John Hanover for sharing expertise in confocal microscopy, Richard Baer for providing the HBL-100 cDNA library, and Alexey Bochkarev for the gift of purified RPA2(43-171)-RPA3 for electrophoretic mobility shift assay analysis. We also thank Brian Oliver and Heinz Reiske for critical reading of the manuscript and Aniello Cerrato and Christina Heppner for helpful discussions and technical protocols.