In situ SUMOylation analysis reveals a modulatory role of RanBP2 in the nuclear rim and PML bodies
In situ SUMOylation analysis reveals a modulatory role of RanBP2 in the nuclear rim and PML bodies
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DOI：
10.1016/j.yexcr.2006.01.013
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发表时间：
2006-05-01
影响因子：
3.7
通讯作者：
Nakao, M
中科院分区：
文献类型：
--
作者：
Saitoh, N;Uchimura, Y;Nakao, M
SUMO修饰在许多细胞功能中起关键作用，包括核质转运、基因表达、细胞周期和亚核结构如早幼粒细胞白血病（PML）体的形成。为了鉴定细胞中发生SUMO化的位点，我们开发了使用半完整细胞系统的原位SUMO化测定，随后将其与基于siRNA的敲低核孔蛋白RanBP 2（也称为Nup 358，其是已知的SUMO E3蛋白之一）相结合。通过原位SUMO化分析，我们发现除了有丝分裂器外，核边缘和PML小体都是SUMO化的主要靶点。分析可能的SUMO缀合位点的能力将是研究细胞中SUMO E3样活性和/或SUMO底物存在的有价值的工具。RanBP 2的特异性敲除完全废除了沿着核边缘的SUMO化，并使RanGAP 1从核孔复合物中脱位。有趣的是，与其它正常出现的核区室（包括核纤层、核仁和染色质）相比，RanBP 2的缺失显著减少了PML体的数量，这表明RanBP 2和PML体之间存在新的联系。由RanBP 2在核边缘促进的SUMO化可能是形成特定亚核组织的关键步骤。我们的数据表明，SUMO E3蛋白，如RanBP 2促进时空SUMO化的某些核结构和功能。(c)2006年爱思唯尔公司All rights reserved.
SUMO modification plays a critical role in a number of cellular functions including nucleocytoplasmic transport, gene expression, cell cycle and formation of subnuclear structures such as promyelocytic leukemia (PML) bodies. In order to identify the sites where SUMOylation takes place in the cell, we developed an in situ SUMOylation assay using a semi-intact cell system and subsequently combined it with siRNA-based knockdown of nucleoporin RanBP2, also known as Nup358, which is one of the known SUMO E3 proteins. With the in situ SUMOylation assay, we found that both nuclear rim and PML bodies, besides mitotic apparatuses, are major targets for active SUMOylation. The ability to analyze possible SUMO conjugation sites would be a valuable tool to investigate where SUMO E3-like activities and/or SUMO substrates exist in the cell. Specific knockdown of RanBP2 completely abolished SUMOylation along the nuclear rim and dislocated RanGAP1 from the nuclear pore complexes. interestingly, the loss of RanBP2 markedly reduced the number of PML bodies, in contrast to other, normal-appearing nuclear compartments including the nuclear lamina, nucleolus and chromatin, suggesting a novel link between RanBP2 and PML bodies. SUMOylation facilitated by RanBP2 at the nuclear rim may be a key step for the formation of a particular subnuclear organization. Our data imply that SUMO E3 proteins like RanBP2 facilitate spatio-temporal SUMOylation for certain nuclear structure and function. (c) 2006 Elsevier Inc. All rights reserved.