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首页> 外文期刊>Molecular and Cellular Biology >The 32-Kilodalton Subunit of Replication Protein A Interacts with Menin, the Product of the MEN1 Tumor Suppressor Gene
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The 32-Kilodalton Subunit of Replication Protein A Interacts with Menin, the Product of the MEN1 Tumor Suppressor Gene

机译：复制蛋白A的32 Kilodalton亚基与MEN1，MEN1肿瘤抑制基因的产物相互作用。

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摘要

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.
机译：Menin是由 MEN1 编码的70 kDa蛋白，它是在1型多发性内分泌肿瘤中被破坏的抑癌基因。在基于Ras信号重构的酵母双杂交系统中，发现Menin与复制蛋白A（RPA）的32 kDa亚基（RPA2），这是DNA复制，重组和修复所需的异源三聚体蛋白。在常规酵母双杂交系统中以及通过纯化蛋白之间的直接相互作用，证实了menin-RPA2相互作用。 Menin-RPA2结合被多发于1型内分泌肿瘤的个体中的许多menin错义突变所抑制，相互作用区域被定位到menin的N端部分和RPA2的第43至171位氨基酸。 RPA2的此区域包含一个弱的单链DNA结合域，但在体外，menin对RPA-DNA结合没有可检测的作用。 Menin在体外优先与游离RPA2结合，而不是RPA异三聚体或由RPA2与14-kDa亚基（RPA3）结合的亚复合物。但是，70-kDa亚基（RPA1）是通过Menin特异性抗体从HeLa细胞提取物中与RPA2共沉淀的，这表明Menin在体内与RPA异源三聚体或新型RPA1-RPA2复合物结合。这一发现与通过免疫荧光观察到的内源性menin，RPA2和RPA1的核定位模式的广泛重叠是一致的。

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