The protein kinase CK2 site (Ser(111/112)) enhances recognition of the Simian virus 40 large T-antigen nuclear localization sequence by importin
The protein kinase CK2 site (Ser(111/112)) enhances recognition of the Simian virus 40 large T-antigen nuclear localization sequence by importin
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DOI：
10.1074/jbc.272.27.17191
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发表时间：
1997-07-04
影响因子：
4.8
通讯作者：
Jans, DA
中科院分区：
文献类型：
--
作者：
Hubner, S;Xiao, CY;Jans, DA
磷酸化调节依赖于核定位序列(NLS)的核蛋白输入的机制在很大程度上尚不清楚。虽然SV40大肿瘤抗原(T-ag)融合蛋白的核积累完全依赖于T-agNLS(氨基酸126-132)，但氨基酸残基111-125，特别是丝氨酸111/112处的蛋白激酶CK2(CK2)的一个位点使核进口速度增加了50倍。由于核蛋白输入的第一步涉及NLS与NLS受体复合体的结合，如Importin 58/97异二聚体，我们建立了一种新的酶联免疫吸附试验来测试NLS的氨基酸末端对NLS和CK2位点的识别是否有影响。我们发现，在含有111-125个氨基酸残基的情况下，Importin 58/97对T-ag NLS的识别能力提高了10倍，并强烈依赖于Importin 97。CK2位点与NLS之间的间隔区减少的T-ag融合蛋白与Importin 58/97的结合减少了30%。该蛋白的最大核积累减少了50%以上，表明正确定位的CK2位点具有重要的生理意义。CR2的磷酸化使T-agNLS与Importin 58/97的结合亲和力进一步提高了40%。我们得出结论，侧翼序列，特别是CK2位点的磷酸化在NLS识别中具有重要的机械意义，并代表着它们增强T-ag核输入的基础。因此，这项研究首次阐明了通过磷酸化调节的NLS中的磷酸化来调节核蛋白输入的机制基础。
The mechanism by which phosphorylation regulates nuclear localization sequence (NLS)-dependent nuclear protein import is largely unclear. Whereas nuclear accumulation of SV40 large tumor antigen (T-ag) fusion proteins is completely dependent on the T-ag NLS (amino acids 126-132), the rate of nuclear import is increased 50-fold by amino acid residues 111-125 and in particular a site for the protein kinase CK2 (CK2) at serine 111/112. Because the first step of nuclear protein import involves the binding of the NLS by an NLS-receptor complex such as the importin 58/97 heterodimer, we established a novel enzyme-linked immunosorbent assay to test whether NLS recognition is influenced by amino acids amino-terminal to the NLS and the CK2 site. We found that recognition of the T-ag NLS by importin 58/97 was enhanced 10-fold in the presence of amino acid residues 111-125 and strongly dependent on importin 97. A T-ag fusion protein in which the spacer between the CK2 site and the NLS was decreased showed 30% reduced binding by importin 58/97. Maximal nuclear accumulation of this protein was reduced by more than 50%, indicating the physiological importance of the correctly positioned CK2 site. Phosphorylation by CR2 increased the T-ag NLS binding affinity for importin 58/97 by a further 40%. We conclude that flanking sequences and in particular phosphorylation at the CK2 site are mechanistically important in NLS recognition and represent the basis of their enhancement of T-ag nuclear import. This study thus represents the first elucidation of the mechanistic basis of the regulation of nuclear protein import through phosphorylation within a phosphorylation-regulated NLS.