Genomic tagging of endogenous Type IIβ Phosphatidylinositol 5-phosphate 4-kinase in DT40 cells reveals a nuclear localisation

Previous studies from acutely transfected HeLa cells have identified an acidic α-helix in the Type IIβ PtdIns5P 4-kinase (PIPkin IIβ) 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 IIβ physiology are more consistent with an extra-nuclear function. As a means of resolving whether the endogenous PIPkin IIβ 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 IIβ gene. We show that PIPkin IIβ 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.