Genomic tagging reveals a random association of endogenous PtdIns5P 4-kinases IIα and IIβ and a partial nuclear localisation of the IIα isoform
Résumé
Phosphatidylinositol 5-phosphate 4-kinases (PtdIns5P 4-kinases) IIα and IIβ are respectively cytosolic or nuclear when transfected into cells, including DT40 cells (Richardson et al Cellular Signalling 19, 1309-1314; 2007). Here we have genomically tagged both PtdIns5P 4-kinase II isoforms in DT40 cells, and immunoprecipitation of either isoform from tagged cells followed by mass spectrometry showed that they are associated directly with each other, most likely by heterodimerisation. We quantified the cellular levels of the PtdIns5P 4-kinase II mRNAs by real-time quantitative PCR, and the absolute amount of each isoform in immunoprecipitates by mass spectrometry using selective reaction monitoring with 14N13C-labelled internal standard peptides. The data suggest that the dimerisation is complete and random, governed solely by the relative concentrations of the two isoforms. While PtdIns5P 4-kinase IIβ is < 95% nuclear as expected, the distribution of PtdIns4P 4-kinase IIα is 60% cytoplasmic (all bound to membranes) and 40% nuclear. In vitro, PtdIns5P 4-kinase IIα was 2000 times more active as a PtdIns5P 4-kinase than the IIβ isoform. Overall the data suggest a function of PtdIns5P 4-kinase IIβ may be to target the more active IIα isoform into the nucleus.
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