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

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.

Download full-text


Available from: Jonathan H Clarke, Sep 28, 2015
10 Reads
  • Source
    • "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
  • Source
    • "We have previously reported that, using specific activities under the same assay conditions [20–22], the three PI5P4K isoforms differ widely in activity, with PI5P4Kα being the most active and PI5P4Kγ being the least active by several orders of magnitude. Enzymatic assays using PI5P4Ks overexpressed in mammalian cells showed no change in activity when the substrate was presented as artificial membranes or as a hexagonal-phase lipid, and no other inositol lipid we tested acted as a better substrate than PtdIns5P (results not shown). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mammals have genes coding for three phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks), and these have different cellular localisations, tissue distributions and lipid kinase activities. We describe here a detailed molecular exploration of human PI5P4Ks α, β and γ, as well as their fly and worm homologs, to understand how and why these differences came to be. The intrinsic ATPase activities of the three isoforms are very similar, and we show that differences in their G-loop regions can account for much of their wide differences in lipid kinase activity. We have also undertaken an extensive in silico evolutionary study of the PI5P4K family, and show experimentally that the single PI5P4K homologs from C. elegans and D. melanogaster are as widely different in activity as the most divergent mammalian isoforms. Finally, we show that the close association of PI5P4Ks α and γ is a true heterodimerisation, and not a higher oligomer association of homodimers. We reveal that structural modeling is consistent with this and with the apparently random heterodimerisation that we had earlier observed between PI5P4Kα and beta (Wang, Bond, Letcher, Richardson, Lilley, Irvine and Clarke, 2010, Biochemical Journal 430, 215-221). Overall the molecular diversity of mammalian PI5P4Ks explains much of their properties and behaviour, but their physiological functionality remains elusive.
    Biochemical Journal 06/2013; 454(Pt 1). DOI:10.1042/BJ20130488 · 4.40 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The beta-isoform of PIP4K (PtdIns5P-4-kinase) regulates the levels of nuclear PtdIns5P, which in turn modulates the acetylation of the tumour suppressor p53. The crystal structure of PIP4Kbeta demonstrated that it can form a homodimer with the two subunits arranged in opposite orientations. Using MS, isoform-specific antibodies against PIP4Ks, RNAi (RNA interference) suppression and overexpression studies, we show that PIP4Kbeta interacts in vitro and in vivo with the PIP4Kalpha isoform. As the two isoforms phosphorylate the same substrate to generate the same product, the interaction could be considered to be functionally redundant. However, contrary to expectation, we find that PIP4Kbeta has 2000-fold less activity towards PtdIns5P compared with PIP4Kalpha, and that the majority of PIP4K activity associated with PIP4Kbeta comes from its interaction with PIP4Kalpha. Furthermore, PIP4Kbeta can modulate the nuclear localization of PIP4Kalpha, and PIP4Kalpha has a role in regulating PIP4Kbeta functions. The results of the present study suggest a rationale for the functional interaction between PIP4Kalpha and PIP4Kbeta and provide insight into how the relative levels of the two enzymes may be important in their physiological and pathological roles.
    Biochemical Journal 09/2010; 430(2):223-35. DOI:10.1042/BJ20100341 · 4.40 Impact Factor
Show more