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Genomic tagging of endogenous type IIbeta phosphatidylinositol 5-phosphate 4-kinase in DT40 cells reveals a nuclear localisation.

Abstract：

: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.

journal_name

Cell Signal

journal_title

Cellular signalling

authors

Richardson JP,Wang M,Clarke JH,Patel KJ,Irvine RF

doi

10.1016/j.cellsig.2007.01.010

subject

Has Abstract

pub_date

2007-06-01 00:00:00

pages

1309-14

issue

6

eissn

0898-6568

issn

1873-3913

pii

S0898-6568(07)00027-7

journal_volume

19

pub_type

杂志文章

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