Richardson Jonathan P, Wang Minchuan, Clarke Jonathan H, Patel Ketan J, Irvine R F
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK.
Cell Signal. 2007 Jun;19(6):1309-14. doi: 10.1016/j.cellsig.2007.01.010. Epub 2007 Jan 20.
Previous studies from acutely transfected HeLa cells have identified an acidic alpha-helix in the Type IIbeta PtdIns5P 4-kinase (PIPkin IIbeta) as a putative novel nuclear localisation sequence (Ciruela et al. Biochem. J. 364, 587-591 2000). However, some heterogeneity in cellular localisation was always observed, and other published aspects of PIPkin IIbeta physiology are more consistent with an extra-nuclear function. As a means of resolving whether the endogenous PIPkin IIbeta 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 IIbeta gene. We show that PIPkin IIbeta 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.
此前对急性转染的HeLa细胞进行的研究已确定，IIβ型磷脂酰肌醇5-磷酸4-激酶（PIPkin IIβ）中的一个酸性α螺旋是一种假定的新型核定位序列（Ciruela等人，《生物化学杂志》364, 587 - 591, 2000年）。然而，总是观察到细胞定位存在一些异质性，并且PIPkin IIβ生理学的其他已发表方面与核外功能更为一致。作为解决内源性PIPkin IIβ是否定位于细胞核的一种方法，我们利用DT40细胞的高同源重组频率，将一个表位标签（Mosedale等人，《自然结构生物学》12, 763 - 771, 2005年）敲入DT40 PIPkin IIβ基因的一个等位基因中。我们发现PIPkin IIβ以带标签的蛋白质形式表达，通过免疫沉淀和酶活性测定显示其具有活性，并且细胞分级分离表明它确实定位于细胞核。在DT40细胞中对内源性蛋白质进行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。
