PNRC1核定位信号序列的鉴定

为鉴定富含脯氨酸核受体辅调节蛋白1(PNRC1)分子的核定位信号序列（nuclear localization signal sequence, NLS）,在生物信息学方法预测的基础上,先构建野生型PNRC1及删除预测NLS的PNRC1突变体的绿色荧光蛋白（GFP）重组表达载体,转染细胞后通过激光共聚焦显微镜观察PNRC1分子在删除预测NLS后细胞内的定位变化.然后,将预测的NLS编码序列直接连到GFP表达载体上,以及将预测的NLS加到胞浆蛋白上构建其GFP重组表达载体,转染细胞,观察预测的NLS能否把构建的重组体都带到细胞核内.结果显示,删除PNRC1中预测的NLS后,其定位从细胞核中变为主要定位在细胞浆中,而预测的NLS能把GFP或胞浆中的蛋白带到细胞核中.研究表明,预测的NLS为PNRC1分子真正的NLS.     

Nuclear translocation of plk1 mediated by its bipartite nuclear localization signal

Polo-like kinase 1 (Plk1), a mammalian ortholog of Drosophila Polo, is a serine-threonine protein kinase implicated in the regulation of multiple aspects of mitosis. The protein level, activity, and localization of Plk1 change during the cell cycle, and its proper subcellular localization is thought to be crucial for its function. Although localization of Plk1 to the centrosome has been established, nuclear localization or nucleocytoplasmic translocation of Plk1 has not been fully addressed. Here we show that Plk1 accumulates in both the nucleus and the cytoplasm in addition to its localization to the centrosome during S and G(2) phases. Our results identify a conserved region in the kinase domain of Plk1 (residues 134-146) as a functional bipartite nuclear localization signal (NLS) sequence that regulates nuclear translocation of Plk1. The identified NLS is necessary and sufficient for directing nuclear localization of Plk1. This bipartite NLS has an unusually short spacer sequence between two clusters of basic amino acids but is sensitive to RanQ69L, a dominant negative form of Ran, similar to ordinary bipartite NLS. Remarkably, the expression of an NLS-disrupted mutant of Plk1 during S phase was found to arrest the cells in G(2) phase. These results suggest that the bipartite NLS-dependent nuclear localization of Plk1 before mitosis is important for ensuring normal cell cycle progression.     

Subcellular translocation signals regulate Geminin activity during embryonic development

BACKGROUND INFORMATION: Geminin (Gem) is a protein with roles in regulating both the fidelity of DNA replication and cell fate during embryonic development. The distribution of Gem is predominantly nuclear in cells undergoing the cell cycle. Previous studies have demonstrated that Gem performs multiple activities in the nucleus and that regulation of Gem activation requires nuclear import in at least one context. In the present study, we defined structural and mechanistic features underlying subcellular localization of Gem and tested whether regulation of the subcellular localization of Gem has an impact on its activity in cell fate specification during embryonic development. RESULTS: We determined that nuclear localization of Gem is dependent on a bipartite NLS (nuclear localization signal) in the N-terminus of Xenopus Gem protein. This bipartite motif mapped to a Gem N-terminal region previously shown to regulate neural cell fate acquisition. Microinjection into Xenopus embryos demonstrated that import-deficient Gem was incapable of modulating ectodermal cell fate, but that this activity was rescued by fusion to a heterologous NLS. Cross-species comparison of Gem protein sequences revealed that the Xenopus bipartite signal is conserved in many non-mammalian vertebrates, but not in mammalian species assessed. Instead, we found that human Gem employs an alternative N-terminal motif to regulate the protein's nuclear localization. Finally, we found that additional mechanisms contributed to regulating the subcellular localization of Gem. These included a link to Crm1-dependent nuclear export and the observation that Cdt1, a protein in the pre-replication complex, could also mediate nuclear import of Gem. CONCLUSIONS: We have defined new structural and regulatory features of Gem, and showed that the activity of Gem in regulating cell fate, in addition to its cell-cycle-regulatory activity, requires control of its subcellular localization. Our data suggest that rather than being constitutively nuclear, Gem may undergo nucleocytoplasmic shuttling through several mechanisms involving distinct protein motifs. The use of multiple mechanisms for modulating Gem subcellular localization is congruent with observations that Gem levels and activity must be stringently controlled during cell-cycle progression and embryonic development.     

BRD7核定位信号的结构分析和功能鉴定

目的:对BRD7的核定位信号进行预测、结构分析和功能鉴定,并考察其对BRD7亚细胞定位的影响。方法:通过生物信息学对BRD7的核定位信号进行预测和结构分析,然后利用绿色荧光蛋白(GFP)介导的直接荧光和间接免疫荧光定位方法分别对核定位信号的功能进行鉴定,并考察其对BRD7亚细胞定位的影响。结果:BRD7的65～96位氨基酸残基具有潜在核定位信号(NLS)的结构特征,该核定位信号包含3簇碱性氨基酸残基,可视为由2个紧密相邻、部分重叠的双向核靶序列NLS1和NLS2组成;并发现NLS及其构成上的NLS1和NLS2均具有介导异源蛋白GFP胞核定位的功能,从而证实BRD7的65～96位残基为BRD7功能性核定位信号所在区域,且单簇碱性氨基酸残基的缺失不足以破坏其核定位信号的功能;同时发现野生型BRD7呈胞核分布,而核定位信号缺失型BRD7主要呈胞浆分布。结论:BRD7的65～96位氨基酸残基为BRD7功能性核定位信号所在区域,在BRD7胞核分布模式中发挥了十分重要的作用。     

Identification of novel nuclear localization signal within the ErbB-2 protein

ErbB2, a member of the receptor tyrosine kinase family, is frequently over-expressed in breast cancer. Proteolysis of the extracellular domain of ErbB2 results in constitutive activation of ErbB2 kinase. Recent study reported that ErbB2 is found in the nucleus. Here, we showed that ErbB2 is imported into the nucleus through a nuclear localization signal（NLS）-mediated mechanism. The NLS sequence KRRQQKIRKYTMRR （aa655-668） contains three clusters of basic amino acids and it is sufficient to target GFP into the nucleus. However, mutation in any basic amino acid cluster of this NLS sequence significantly affects its nuclear localization. Furthermore, it was found that this NLS is essential for the nuclear localization of ErbB2 since the intracellular domain of Erb2 lacking NLS completely abrogates its nuclear translocation. Taken together, our study identified a novel nuclear localization signal and reveals a novel mechanism underlying ErbB2 nuclear trafficking and localization.     

Cyclin F regulates the nuclear localization of cyclin B1 through a cyclin-cyclin interaction

The key regulator of G(2)-M transition of the cell cycle is M-phase promoting factor (MPF), a complex composed of cdc2 and a B-type cyclin. Cyclin B1 nuclear localization involves phosphorylation within a region called the cytoplasmic retention signal, which also contains a nuclear export signal. The mechanism of MPF nuclear localization remains unclear since it contains no functional nuclear localization signal (NLS). We exploited the yeast two-hybrid screen to find protein(s) potentially mediating localization of cyclin B1 and identified a novel interaction between cyclin B1 and cyclin F. We found that cdc2, cyclin B1 and cyclin F form a complex that exhibits histone H1 kinase activity. Cyclin B1 and cyclin F also colocalize through immunofluorescence studies. Additionally, deletion analysis revealed that each putative NLS of cyclin F is functional. Taken together, the data suggest that the NLS regions of cyclin F regulate cyclin B1 localization to the nucleus. The interaction between cyclin B1 and cyclin F represents the first example of direct cyclin-cyclin binding, and elucidates a novel mechanism that regulates MPF localization and function.     

Identification and characterization of the nuclear import and export signals of the mammalian Ste20-like protein kinase 3

Mst3, a human Ste20-like protein kinase, has been recently demonstrated to undergo a caspase-mediated cleavage during apoptosis. The proteolytic cleavage of the C-terminus of Mst3 caused nuclear translocation of its kinase domain. This work provides evidence that Mst3 may contain a bipartite-like nuclear localization sequence (NLS) at the C-terminus of its kinase domain (residues 278-292). The removal of NLS from the kinase domain of Mst3 led to the cytoplasmic accumulation of EGFP-Mst3(Delta277). The presence of nuclear exporting signals in the Mst3 was also demonstrated by leptomycin B-treatment and serial deletion of the C-terminal regulatory domain of Mst3. A nuclear export signal was also postulated to be in the regions of amino acids 335-386. In conclusion, Mst3 contains both NLS and NES signals, which may cooperate to control the subcellular distribution of Mst3.     

FAK nuclear export signal sequences

Ubiquitously expressed focal adhesion kinase (FAK), a critical component in transducing signals from sites of cell contacts with extracellular matrix, was named after its typical localization in focal adhesions. A nuclear localization of FAK has been also reported and its scaffolding role in nucleus and requirement for p53 ubiquitination were only recently described. Whereas FAK nuclear localization signal (NLS) was found in F2 lobe of FERM domain, nuclear export signal (NES) sequences have not been yet determined. Here we demonstrate that FAK has two NES sequences, NES1 in F1 lobe of FERM domain and NES2 in kinase domain. Although, both NES1 and NES2 are evolutionary conserved, and present as well in FAK-related protein kinase Pyk2, only NES2 demonstrates full biological nuclear export activity.     

A ligand-activated nuclear localization signal in cellular retinoic acid binding protein-II

Primary sequences of proteins often contain motifs that serve as "signatures" for subcellular targeting, such as a nuclear localization signal (NLS). However, many nuclear proteins do not harbor a recognizable NLS, and the pathways that mediate their nuclear translocation are unknown. This work focuses on CRABP-II, a cytosolic protein that moves to the nucleus upon binding of retinoic acid. While CRABP-II does not contain an NLS in its primary sequence, such a motif could be recognized in the protein's tertiary structure. We map the retinoic acid-induced structural rearrangements that result in the presence of this NLS in holo- but not apo-CRABP-II. The signal, whose three-dimensional configuration aligns strikingly well with a "classical" NLS, mediates ligand-induced association of CRABP-II with importin alpha and is critical for nuclear localization of the protein. The ligand-controlled NLS "switch" of CRABP-II may represent a general mechanism for posttranslational regulation of the subcellular distribution of a protein.     

Regulation of subcellular localization of the antiproliferative protein Tob by its nuclear export signal and bipartite nuclear localization signal sequences

Tob, a member of the Tob and BTG antiproliferative protein family, plays an important role in many cellular processes including cell proliferation. In this study, we have addressed molecular mechanisms regulating subcellular localization of Tob. Treatment with leptomycin B, an inhibitor of nuclear export signal (NES) receptor, resulted in a change in subcellular distribution of Tob from its pan-cellular distribution to nuclear accumulation, indicating the existence of NES in Tob. Our results have then identified an N-terminal region (residues 2-14) of Tob as a functional NES. They have also shown that Tob has a functional, bipartite nuclear localization signal (NLS) in residues 18-40. Thus, Tob is shuttling between the nucleus and the cytoplasm by its NES and NLS. To examine a possible relationship between subcellular distribution of Tob and its function, we exogenously added a strong NLS sequence or a strong NES sequence or both to Tob. The obtained results have demonstrated that the strong NLS-added Tob has a much weaker activity to inhibit cell cycle progression from G0/G1 to S phase. These results suggest that cytoplasmic localization or nucleocytoplasmic shuttling is important for the antiproliferative function of Tob.