Sternsdorf T, Jensen K, Will H
Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, D-20251 Hamburg, Federal Republic of Germany.
J Cell Biol. 1997 Dec 29;139(7):1621-34. doi: 10.1083/jcb.139.7.1621.
PML and Sp100 proteins are associated with nuclear domains, known as nuclear dots (NDs). They were discovered in the context of leukemic transformation and as an autoantigen in primary biliary cirrhosis, respectively. Both proteins are expressed in the form of many COOH-terminally spliced variants, and their expression is enhanced by interferons (IFN). The recent finding that PIC1/SUMO-1, a small ubiquitin-like protein, is covalently linked to the RanGAP1 protein of the nuclear pore complex and also binds PML in yeast cells led us to determine whether PML is covalently modified by PIC1/SUMO-1 and whether the same is true for Sp100. We found an immune reaction of PML and Sp100 proteins with a PIC1/SUMO-1-specific monoclonal antibody by immunoblotting when using cell extracts prepared from stably transfected cells inducibly expressing one isoform of each protein as well as from nontransfected cells. In contrast, both proteins did not react when synthesized in vitro. Immunofluorescence staining showed that PIC1/SUMO-1 colocalized with Sp100 and PML in NDs except in mitotic cells, in which PML and Sp100 are dissociated. Cell fractionation and immunoblotting demonstrated that PIC1/SUMO-1 immunoreactive Sp100 in IFN-treated and untreated cells was exclusively nuclear, whereas nonmodified Sp100 was also found in the cytoplasm. Taken together, these data strongly suggest covalent modification of specific nuclear isoforms of Sp100 and PML by PIC1/SUMO-1. This modification may play a regulatory role in ND structure, composition, and function.
早幼粒细胞白血病（PML）蛋白和Sp100蛋白与核结构域相关，即所谓的核点（NDs）。它们分别是在白血病转化过程中以及作为原发性胆汁性肝硬化中的自身抗原被发现的。这两种蛋白均以多种COOH末端剪接变体的形式表达，并且它们的表达可被干扰素（IFN）增强。最近发现，一种小的泛素样蛋白PIC1/SUMO-1与核孔复合体的RanGAP1蛋白共价连接，并且在酵母细胞中也与PML结合，这促使我们确定PML是否被PIC1/SUMO-1共价修饰，以及Sp100是否也是如此。当使用从稳定转染的细胞制备的细胞提取物时，我们通过免疫印迹发现PML和Sp100蛋白与PIC1/SUMO-1特异性单克隆抗体发生免疫反应，这些稳定转染的细胞可诱导表达每种蛋白的一种异构体，同时也使用了未转染细胞的提取物。相比之下，这两种蛋白在体外合成时不发生反应。免疫荧光染色显示，除了在有丝分裂细胞中PML和Sp100解离外，PIC1/SUMO-1与Sp100和PML在核点中共定位。细胞分级分离和免疫印迹表明，在IFN处理和未处理的细胞中，PIC1/SUMO-1免疫反应性Sp100仅存在于细胞核中，而未修饰的Sp100也存在于细胞质中。综上所述，这些数据强烈表明PIC1/SUMO-1对Sp100和PML的特定核异构体进行了共价修饰。这种修饰可能在核点的结构、组成和功能中发挥调节作用。
