Davies Rebecca G, Wagstaff Kylie M, McLaughlin Eileen A, Loveland Kate L, Jans David A
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
Priority Research Centre in Chemical Biology and Reproductive Science, University of Newcastle, Callaghan, NSW, Australia.
Biochim Biophys Acta. 2013 Dec;1833(12):3436-3444. doi: 10.1016/j.bbamcr.2013.05.015. Epub 2013 May 23.
Regulation of nuclear protein import is central to many cellular processes such as development, with a key mechanism being factors that retain cargoes in the cytoplasm that normally localize in the nucleus. The breast cancer antigen BRCA1-binding protein BRAP2 has been reported as a novel negative regulator of nuclear import of various nuclear localization signal (NLS)-containing viral and cellular proteins, but although implicated in differentiation pathways and highly expressed in tissues including testis, the gamut of targets for BRAP2 action in a developmental context is unknown. As a first step towards defining the BRAP2 interactome, we performed a yeast-2-hybrid screen to identify binding partners of BRAP2 in human testis. Here we report characterization for the first time of three of these: the high mobility group (HMG)-box-domain-containing chromatin component HMG20A, nuclear mitotic apparatus protein NuMA1 and synaptic nuclear envelope protein SYNE2. Co-immunoprecipitation experiments indicate association of BRAP2 with HMG20A, NuMA1, and SYNE2 in testis, underlining the physiological relevance of the interactions, with immunohistochemistry showing that where BRAP2 is co-expressed with HMG20A and NuMA1, both are present in the cytoplasm, in contrast to their nuclear localization in other testicular cell types. Importantly, quantitative confocal microscopic analysis of cultured cells indicates that ectopic expression of BRAP2 inhibits nuclear localization of HMG20A and NuMA1, and prevents nuclear envelope accumulation of SYNE2, the first report of BRAP2 altering localization of a non-nuclear protein. These results imply for the first time that BRAP2 may have an important role in modulating subcellular localization during testicular development.
核蛋白输入的调控对于许多细胞过程（如发育）至关重要，其中一个关键机制是某些因子将通常定位于细胞核的货物滞留在细胞质中。乳腺癌抗原BRCA1结合蛋白BRAP2已被报道为多种含核定位信号（NLS）的病毒和细胞蛋白核输入的新型负调控因子，但是尽管它参与分化途径且在包括睾丸在内的组织中高表达，但其在发育背景下的作用靶点范围尚不清楚。作为确定BRAP2相互作用组的第一步，我们进行了酵母双杂交筛选以鉴定人睾丸中BRAP2的结合伙伴。在此，我们首次报道了其中三个伙伴的特征：含高迁移率族（HMG）盒结构域的染色质成分HMG20A、核有丝分裂器蛋白NuMA1和突触核被膜蛋白SYNE2。免疫共沉淀实验表明BRAP2与睾丸中的HMG20A、NuMA1和SYNE2存在关联，强调了这些相互作用的生理相关性，免疫组织化学显示，在BRAP2与HMG20A和NuMA1共表达的地方，二者都存在于细胞质中，这与它们在其他睾丸细胞类型中的核定位形成对比。重要的是，对培养细胞的定量共聚焦显微镜分析表明，BRAP2的异位表达抑制了HMG20A和NuMA1的核定位，并阻止了SYNE2在核被膜的积累，这是关于BRAP2改变非核蛋白定位的首次报道。这些结果首次表明，BRAP2可能在睾丸发育过程中调节亚细胞定位方面发挥重要作用。
