Genomic tagging reveals a random association of endogenous PtdIns5P 4-kinases IIalpha and IIbeta and a partial nuclear localization of the IIalpha isoform

Abstract

PtdIns5P 4-kinases IIalpha and IIbeta are cytosolic and nuclear respectively when transfected into cells, including DT40 cells [Richardson, Wang, Clarke, Patel and Irvine (2007) Cell. Signalling 19, 1309-1314]. In the present study we have genomically tagged both type II PtdIns5P 4-kinase isoforms in DT40 cells. Immunoprecipitation of either isoform from tagged cells, followed by MS, revealed that they are associated directly with each other, probably by heterodimerization. We quantified the cellular levels of the type II PtdIns5P 4-kinase mRNAs by real-time quantitative PCR and the absolute amount of each isoform in immunoprecipitates by MS using selective reaction monitoring with 14N,13C-labelled internal standard peptides. The results suggest that the dimerization is complete and random, governed solely by the relative concentrations of the two isoforms. Whereas PtdIns5P 4-kinase IIbeta is >95% nuclear, as expected, the distribution of PtdIns4P 4-kinase IIalpha is 60% cytoplasmic (all bound to membranes) and 40% nuclear. In vitro, PtdIns5P 4-kinase IIalpha was 2000-fold more active as a PtdIns5P 4-kinase than the IIbeta isoform. Overall the results suggest a function of PtdIns5P 4-kinase IIbeta may be to target the more active IIalpha isoform into the nucleus.

Figure 1. Differential expression of PtdIns5P 4-kinase II isoforms from endogenously tagged alleles

Expression from tagged and untagged PIP5K2A and PIP5K2B alleles determined by qRT-PCR using the comparative threshold cycle (C_t) method. Normalized expression is presented as the relative fold increase above untagged PIP5K2B in WT cells (n=9). PCR was performed on cDNA made from RNA extracts of WT, JPR3 and MW2 cells as described in the Materials and methods section.

Figure 2. Detection of AQUA and analyte peptides by MS

Integrated signals derived from two selected transitions (see Supplementary Table S1 at http://www.BiochemJ.org/bj/430/bj4300215add.htm) for (A) the internal AQUA standard peptide (570.38>770.87, top panel; 570.38>699.89, lower panel) and (B) the corresponding analyte peptide (565.38>760.87, top panel; 565.38>689.99, lower panel) for quantification of PtdIns5P 4-kinase IIα in a whole cell lysate by SRM.

Figure 3. Fractionation and Western blotting of DT40 cell lysates

(A) Purity of isolated nuclei were assessed by adhering whole cells or nuclei (prepared as described in the Materials and methods section) to coverslips using cell-tak and decorating with a combination of wheat germ agglutinin plasma membrane stain (conjugated to Alexa Fluor® 555) and DAPI nuclear stain. Scale bar=5 μm. Representative Western blot for fractionation of (B) JPR3 or (C) MW2 cell lysates. The same proportion of each cell lysate fraction (see Supplementary Figure S1 at http://www.BiochemJ.org/bj/430/bj4300215add.htm) was either Western blotted for actin or histone H1, or used for immunoprecipitation and detection of FLAG-tagged protein as described in the Materials and methods section. (D) Quantification of PtdIns5P 4-kinase IIα, actin and histone H1 levels in Western blots of MW2 cell lysate fractionations [the cytosolic fractions (C+P) were combined]. Results are means±S.E.M. (n=4 for PtdIns5P 4-kinase IIα; n=3 for actin and histone H1). C+P, cytosol; M, cytoplasmic membranes; S, sucrose cushion; N, extracted nuclei.