Genomic tagging of endogenous Type IIβ phosphatidylinositol 5-phosphate 4-kinase in DT40 cells reveals a nuclear localisation
Genomic tagging of endogenous Type IIβ phosphatidylinositol 5-phosphate 4-kinase in DT40 cells reveals a nuclear localisation
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DOI：
10.1016/j.cellsig.2007.01.010
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发表时间：
2007-06-01
影响因子：
4.8
通讯作者：
Irvine, R. F.
中科院分区：
文献类型：
--
作者：
Richardson, Jonathan P.;Wang, Minchuan;Irvine, R. F.
先前对急性转染的HeLa细胞的研究已经鉴定了II型β PtdIns 5 P 4-激酶（PIPkin II β）中的酸性α-螺旋作为推定的新的核定位序列（Ciruela等人，Biochem.J.364，587-5912000）。然而，总是观察到细胞定位的一些异质性，并且PIPkin II β生理学的其他已发表方面与核外功能更一致。作为解决内源性PIPkin II β是否是核的手段，我们已经使用DT 40细胞的高同源重组频率敲除表位标签（Mosedale et al.，12，763-771 2005）转化到DT 40 PIPkin II β基因的等位基因之一中。我们表明，PIPkin II β是作为一个标记的蛋白质表达，是活跃的免疫沉淀和酶测定所揭示的，细胞分级显示，它确实是核。DT 40细胞内源性蛋白质的基因组标记是一种在研究内源性信号蛋白方面具有独特优势的技术。(c)2007爱思唯尔公司All rights reserved.
Previous studies from acutely transfected HeLa cells have identified an acidic alpha-helix in the Type II beta PtdIns5P 4-kinase (PIPkin II beta) as a putative novel nuclear localisation sequence (Ciruela et al. Biochem. J. 364, 587-5912000). However, some heterogeneity in cellular localisation was always observed, and other published aspects of PIPkin II beta physiology are more consistent with an extra-nuclear function. As a means of resolving whether the endogenous PIPkin II beta is nuclear, we have used the high homologous recombination frequency of DT40 cells to knock an epitope tag (Mosedale et al., Nat Struct Biol. 12, 763-771 2005) into one of the alleles of the DT40 PIPkin II beta gene. We show that PIPkin II beta is expressed as a tagged protein, is active as revealed by immunoprecipitation and enzyme assay, and that cellular fractionation reveals that it is indeed nuclear. Genomic tagging of endogenous proteins in DT40 cells is a technique that offers unique advantages in studying endogenous signalling proteins. (c) 2007 Elsevier Inc. All rights reserved.