Genomic tagging of endogenous Type IIβ Phosphatidylinositol 5-phosphate 4-kinase in DT40 cells reveals a nuclear localisation
| العنوان: | Genomic tagging of endogenous Type IIβ Phosphatidylinositol 5-phosphate 4-kinase in DT40 cells reveals a nuclear localisation |
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| المؤلفون: | Jonathan H. Clarke, Jonathan P. Richardson, Ketan J. Patel, Robin F. Irvine, Minchuan Wang |
| المصدر: | Cellular Signalling |
| بيانات النشر: | Elsevier Science Ltd, 2007. |
| سنة النشر: | 2007 |
| مصطلحات موضوعية: | Immunoprecipitation, Molecular Sequence Data, Biology, Transfection, Article, Type II PIPkin, Type II Phosphatidylinositol 5-phosphate 4-kinase, Phosphatidylinositol 5-phosphate, 03 medical and health sciences, DT40 cells, medicine, Animals, Humans, Amino Acid Sequence, Peptide sequence, 030304 developmental biology, Cell Nucleus, 0303 health sciences, Genome, Kinase, 030302 biochemistry & molecular biology, Cell Biology, Molecular biology, Transport protein, Phosphatidylinositol 5-phosphate 4-kinase, Cell nucleus, Phosphotransferases (Alcohol Group Acceptor), Protein Transport, medicine.anatomical_structure, Genomic tagging, Homologous recombination, Chickens, Subcellular Fractions |
| الوصف: | 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. |
| اللغة: | English |
| تدمد: | 1873-3913 0898-6568 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::8159a5fdedf4b4e60fb75319ce49476cTest http://europepmc.org/articles/PMC1868964Test |
| حقوق: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....8159a5fdedf4b4e60fb75319ce49476c |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 18733913 08986568 |
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