Identification of sequence determinants of human nuclear dUTPase isoform localization.
Identification of sequence determinants of human nuclear dUTPase isoform localization.
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DOI：
10.1016/s0014-4827(03)00048-x
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发表时间：
2003-07
影响因子：
3.7
通讯作者：
Beverly A Tinkelenberg;W. Fazzone;F. Lynch;R. Ladner
中科院分区：
文献类型：
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作者：
Beverly A Tinkelenberg;W. Fazzone;F. Lynch;R. Ladner
dUTP 核苷酸水解酶 (dUTPase) 催化 dUTP 水解为 dUMP 和焦磷酸盐，是细胞 dUTP 池的中心调节因子。该蛋白的核亚型 (DUT-N) 和线粒体亚型 (DUT-M) 已在人类中得到鉴定，并且通过交替使用 5' 外显子而源自同一基因。最近，研究表明这些异构体在一些癌症中异常表达，并且细胞核中 dUTPase 的过度表达与针对胸苷酸生物合成的化疗药物的耐药性相关。在这项研究中，我们使用绿色荧光蛋白融合构建体检查了 dUTPase 同工型定位所需的信号。我们报告说，DUT-N 的 N 端 23 个氨基酸是完整核定位所必需的，但不足以实现。在该区域内，我们鉴定了一小簇碱性残基（K14R15R17），类似于经典的单部分核定位信号（NLS）。这些残基的突变完全消除了核定位。此外，假定的 NLS 附近 Ser11 的磷酸化对 DUT-N 核定位没有影响。通过缺失分析，我们发现当大部分蛋白质序列存在时，DUT-N 到细胞核的排序得到改善。因此，我们得出结论，DUT-N 可能包含位于整个蛋白质中的复杂 NLS。
dUTP nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate and is the central regulator of cellular dUTP pools. Nuclear (DUT-N) and mitochondrial (DUT-M) isoforms of the protein have been identified in humans and arise from the same gene by the alternative use of 5′ exons. Recently, it has been shown that these isoforms are aberrantly expressed in some cancers and overexpression of dUTPase in the nucleus is associated with resistance to chemotherapeutic agents that target thymidylate biosynthesis. In this study, we have examined the signals necessary for dUTPase isoform localization using green fluorescent protein fusion constructs. We report that the N-terminal 23 amino acids of DUT-N are required but not sufficient for complete nuclear localization. Within this region, we identified a small cluster of basic residues (K14R15R17) that resemble a classic monopartite nuclear localization signal (NLS). Mutation of these residues completely abolishes nuclear localization. In addition, phosphorylation of Ser11 near the putative NLS has no affect on DUT-N nuclear localization. Through deletion analysis we show improved sorting of DUT-N to the nucleus when most of the protein sequence is present. Therefore, we conclude that DUT-N may contain a complex NLS that is located throughout the entire protein.