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

Department of Pharmacology, Tennis Court Road, Cambridge, U.K.
Biochemical Journal (Impact Factor: 4.4). 09/2010; 430(2):215-21. DOI: 10.1042/BJ20100340
Source: PubMed


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.

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Available from: Jonathan H Clarke
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    • "Based on semi-quantitative affinity purifications it is likely that there is less than 1.5 pmole of PI5P4K2α per mg of total cell extract. This corresponds to about 9 × 10 4 molecules of enzyme per cell, and the level of PI5P4K2β is probably at least 5 fold lower [16] [17]. As well as the presence of homodimers, these authors report a new heterodimeric PI5P4K2α: PI5P4K2β association and no other significant interacting proteins as it is likely that binding of this enzyme to the nuclear membrane occurs solely via lipid interactions. "
    Dataset: SILAC-iPAC

    Full-text · Dataset · Feb 2015
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    • "We were able to estimate broadly the actual rate of PI5P4Kα activity in a cell. Data from previous DT40 cell studies give an accurate titre of cellular PI5P4Kα concentration quantified by quantitative MS as 1.5 pmol/mg of protein [20]. Assuming an average DT40 cell size of 10 μm diameter, this number can be converted into a Vmax of approximately 1×103 PtdIns5P molecules phosphorylated per s per cell, although this could be higher depending on the PtdIns5P concentration local to the enzyme. "
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    Preview · Article · Sep 2010 · Biochemical Journal
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