Intracellular localization of human Ins(1,3,4,5,6)P5 2-kinase
Intracellular localization of human Ins(1,3,4,5,6)P5 2-kinase
复制标题
DOI：
10.1042/bj20070382
复制
发表时间：
2007-12-15
影响因子：
4.1
通讯作者：
Mayr, Georg W.
中科院分区：
文献类型：
--
作者：
Brehm, Maria A.;Schenk, Tobias M. H.;Mayr, Georg W.
InSP 6是由IP 5 K [Ins（1，3，4，5，6）P-5 2-kinase]合成的具有多种功能的细胞内信号分子。在本研究中，我们在NRK（正常大鼠肾脏），COS 7和H1299细胞中过表达增强型绿色荧光蛋白（EGFP）-IP 5 K融合蛋白。结果表明，有空间微异质性的IP 5 K的细胞内定位，也可以证实为内源性酶。这可能有助于InSP 6水平在其作用部位的变化。例如，过表达的IP 5 K在细胞核内显示出结构化的组织。该激酶优先定位于常染色质和核仁，并与mRNA共定位。在细胞质中，过表达的IP 5 K在离散病灶中显示局部高浓度。以mRNA、PABP [poly（A）-binding protein]和TIAR（TIA-1-related protein）为标志物，将后者归因于应激颗粒。应激颗粒的发生率，其中IP 5 K保持高度集中，进一步增加嘌呤霉素治疗。使用FRAP（光漂白后的荧光恢复），我们确定IP 5 K被主动转运到细胞核中。通过定点突变，我们确定了一个核输入信号和一个肽段介导的IP 5 K的核输出。
InSP6 is an intracellular signal with several proposed functions that is synthesized by IP5K [Ins(1,3,4,5,6)P-5 2-kinase]. In the present study, we overexpressed EGFP (enhanced green fluorescent protein)-IP5K fusion proteins in NRK (normal rat kidney), COS7 and H1299 cells. The results indicate that there is spatial microheterogeneity in the intracellular localization of IP5K that could also be confirmed for the endogenous enzyme. This may facilitate changes in InSP6 levels at its sites of action. For example, overexpressed IP5K showed a structured organization within the nucleus. The kinase was preferentially localized in euchromatin and nucleoli, and co-localized with mRNA. In the cytoplasm, the overexpressed IP5K showed locally high concentrations in discrete foci. The latter were attributed to stress granules by using mRNA, PABP [poly(A)-binding protein] and TIAR (TIA-1-related protein) as markers. The incidence of stress granules, in which IP5K remained highly concentrated, was further increased by puromycin treatment. Using FRAP (fluorescence recovery after photobleaching) we established that IP5K was actively transported into the nucleus. By site-directed mutagenesis we identified a nuclear import signal and a peptide segment mediating the nuclear export of IP5K.