Impaired nuclear translocation, cytoplasmic accumulation of nuclear protein and alteration of cellular physiology

研究了转录因子p53和几种核体形成蛋白cFos的核转运及其对生物功能的调控。年轻的人类成纤维细胞将p53转运到细胞核。然而，在衰老的成人皮肤成纤维细胞中，p53蛋白主要积聚在细胞质中，并定位于波形蛋白细胞骨架上。衰老细胞异位表达p53-GFP，保留在细胞质中。衰老细胞的核转运系统仍然具有功能，因为组蛋白H2B和hnRNP ALF-C1的核输入没有受损。因此，p53的核转运受损似乎是由于锚定在静脉蛋白细胞骨架上。这种锚定需要p53的构象改变，而这种改变可能是由p53的磷酸化引起的。因此，衰老细胞似乎产生了一种假定的p53激酶，它改变了p53的构象。S1-1蛋白是由井上明在1996年发现的。迄今为止，其生物学功能和分子性质仍不清楚。通过本研究，我发现S1-1蛋白家族，p110（全长852AA）和p130（全长929AA）形成了不同的核体。对S1-1和PML（早幼粒细胞白血病）亚型进行初步比对分析，发现了两个高度保守的结构域，分别称为EGKE和Z结构域。Z结构域被确定为S1-1核体形成的负责分子区域。去除z结构域显著地破坏了S1-1和PML4的核体形成。PML4异位表达于cos7细胞细胞质中。这种细胞质保留需要存在于羧基末端74个氨基酸的独特结构域。不同核蛋白的细胞质保留或积累取决于其不同结构域的存在，翻译后修饰和改变的构象。波形蛋白骨架是否与PML4的细胞质保留有关是一个有趣的问题。

Nuclear transport of transcription factor p53 and cFos, a several nuclear body-forming proteins, and their regulation of biological functions were investigated. Young human fibroblasts transport p53 to the nuclei. However, in senescent adult skin fibroblasts, p53 protein accumulated in the cytoplasm dominantly and localized on vimentin cytoskeleton. Ectopically expressed p53-GFP of senescent cells, retained in the cytoplasm. Nuclear transport system of senescent cells is still functional, because the nuclear import of histon H2B and hnRNP ALF-C1 is not impaired. Therefore, the impaired nuclear transport of p53 seems to be due to the anchoring to vimentin cytoskeleton. This anchoring needs the conformational change of p53,which may be induced by phosphorylation of p53. Thus the senescent cells seem to produce a putative p53-kinase which modifies the conformation of p53.S1-1 protein was discovered by Akira Inoue in 1996. So far the biological function and molecular property have been remained unknown. Through this study, I found that S1-1 protein family, p110 (full length 852AA) and p130 (full length 929AA) formed the distinct nuclear body. Primary alignment analysis of S1-1 and PML (promyelocytic leukemia) isoforms revealed the two highly conserved domains, which were termed EGKE and Z domains. Z domain was identified as a responsible molecular region for S1-1 nuclear body formation. Removal of the Z-domain significantly impaired the nuclear body formation of S1-1 and PML4. Ectopically expressed PML4 accumulated in the cytoplasm of cos7 cells. This cytoplasmic retention needs the distinct domain which exists in the carboxyterminal 74 amino acids.Cytoplasmic retention or accumulation of the distinct nuclear proteins depends on the existence of their distinct domains post-tranlational modification, and altered conformation. It is interesting if vimentin cytoskeleton involves with the cytoplasmic retention of PML4.