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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.

Original publication

DOI

10.1016/j.cellsig.2007.01.010

Type

Journal article

Journal

Cell Signal

Publication Date

06/2007

Volume

19

Pages

1309 - 1314

Keywords

Amino Acid Sequence, Animals, Cell Nucleus, Chickens, Genome, Humans, Molecular Sequence Data, Phosphotransferases (Alcohol Group Acceptor), Protein Transport, Subcellular Fractions, Transfection