Kinetic characterization of the human retinoblastoma protein bipartite nuclear localization sequence (NLS) in vivo and in vitro - A comparison with the SV40 large T-antigen NLS
Kinetic characterization of the human retinoblastoma protein bipartite nuclear localization sequence (NLS) in vivo and in vitro - A comparison with the SV40 large T-antigen NLS
复制标题
DOI：
10.1074/jbc.272.35.22134
复制
发表时间：
1997-08-29
影响因子：
4.8
通讯作者：
Jans, DA
中科院分区：
文献类型：
--
作者：
Efthymiadis, A;Shao, HM;Jans, DA
视网膜母细胞瘤（RE）肿瘤抑制因子是对细胞生长控制重要的核磷蛋白，并且能够特异性结合病毒癌蛋白如SV 40大肿瘤抗原（T-ag）。人RE具有由氨基酸860-877内的两簇碱性氨基酸组成的二分核定位序列（NLS），其也存在于小鼠和爪蟾同源物中，其类似于核质蛋白，T-ag NLS代表不同类型的NLS，仅由一段碱性氨基酸组成。为了比较RE的二分NLS赋予的核输入动力学与T-ag NLS赋予的核输入动力学，我们使用含有RE或T-ag的NLS的β-半乳糖苷酶融合蛋白，RE NLS能够在体内将β-半乳糖苷酶靶向细胞核，（在HTC大鼠肝癌系的显微注射细胞中）和体外（在机械穿孔的HTC细胞中），NLS的近端碱性残基的突变取代消除了核靶向活性，证实了其二分特征，RE融合蛋白的核积累在约8分钟内达到半最大值，体内最大水平为细胞质中的3-4倍，小于T-ag融合蛋白达到的最大水平的50%，而RE蛋白的核输入的初始速率也小于T-ag的一半，两种NLS在体外赋予的核输入依赖于胞质溶胶和ATP，并被不可水解的GTP类似物GTP γ S抑制。使用基于ELISA的结合测定，我们确定与T-ag NLS相比，RE二分NLS对高亲和力异源二聚体NLS结合蛋白复合物importin 58/97的亲和力严重降低，这种差异可能代表体内最大核积累和输入速率降低的基础，这些结果支持了NLS结合蛋白识别NLS的亲和力在决定核蛋白输入动力学方面至关重要的假设。
The retinoblastoma (RE) tumor suppressor is a nuclear phosphoprotein important for cell growth control and able to bind specifically to viral oncoproteins such as the SV40 large tumor antigen (T-ag), Human RE possesses a bipartite nuclear localization sequence (NLS) consisting of two clusters of basic amino acids within amino acids 860-877, also present in mouse and Xenopus homologs, which resembles that of nucleoplasmin, The T-ag NLS represents a different type of NLS, consisting of only one stretch of basic amino acids, To compare the nuclear import kinetics conferred by the bipartite NLS of RE to those conferred by the T-ag NLS, we used beta-galactosidase fusion proteins containing the NLSs of either RE or T-ag, The RE NLS was able to target beta-galactosidase to the nucleus both in vivo (in microinjected cells of the HTC rat hepatoma line) and in vitro (in mechanically perforated HTC cells), Mutational substitution of the proximal basic residues of the NLS abolished nuclear targeting activity, confirming its bipartite character, Nuclear accumulation of the RE fusion protein was half-maximal within about 8 min in vivo maximal levels being between 3-4-fold those in the cytoplasm, which was less than 50% of the maximal levels attained by the T-ag fusion protein, while the initial rate of nuclear import of the RE protein was also less than half that of T-ag, Nuclear import conferred by both NLSs in vitro was dependent on cytosol and ATP and inhibited by the nonhydrolyzable GTP analog GTP gamma S. Using an ELISA-based binding assay, we determined that the RE bipartite NLS had severely reduced affinity, compared with the T-ag NLS, for the high affinity heterodimeric NLS-binding protein complex importin 58/97, this difference presumably representing the basis of the reduced maximal nuclear accumulation and import rate in vivo, The results support the hypothesis that the affinity of NLS recognition by NLS-binding proteins is critical in determining the kinetics of nuclear protein import.