In B cells, phosphatidylinositol 5-phosphate 4-kinase-α synthesizes PI(4,5)P2 to impact mTORC2 and Akt signaling.

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are enigmatic lipid kinases with physiological functions that are incompletely understood, not the least because genetic deletion and cell transfection have led to contradictory data. Here, we used the genetic tractability of DT40 cells to create cell lines in which endogenous PI5P4Kα was removed, either stably by genetic deletion or transiently (within 1 h) by tagging the endogenous protein genomically with the auxin degron. In both cases, removal impacted Akt phosphorylation, and by leaving one PI5P4Kα allele present but mutating it to be kinase-dead or have PI4P 5-kinase activity, we show that all of the effects on Akt phosphorylation were dependent on the ability of PI5P4Kα to synthesize phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] rather than to remove PI5P. Although stable removal of PI5P4Kα resulted in a pronounced decrease in Akt phosphorylation at Thr308 and Ser473, in part because of reduced plasma membrane PIP3, its acute removal led to an increase in Akt phosphorylation only at Ser473. This process invokes activation primarily of mammalian target of rapamycin complex 2 (mTORC2), which was confirmed by increased phosphorylation of other mTORC2 substrates. These findings establish PI5P4Kα as a kinase that synthesizes a physiologically relevant pool of PI(4,5)P2 and as a regulator of mTORC2, and show a phenomenon similar to the "butterfly effect" described for phosphatidylinositol 3-kinase Iα [Hart JR, et al. (2015) Proc Natl Acad Sci USA 112(4):1131-1136], whereby through apparently the same underlying mechanism, the removal of a protein's activity from a cell can have widely divergent effects depending on the time course of that removal.

Nuclear localizations of phosphatidylinositol 5-phosphate 4-kinases α and ß are dynamic and independently regulated during starvation-induced stress.

The chicken B-cell line DT40 has two isoforms of phosphatidylinositol 5-phosphate 4-kinase (PI5P4K), α and ß, which are likely to exist as a mixture of obligate homo- and hetero-dimers. Previous work has led us to speculate that an important role of the ß isoform may be to target the more active PI5P4Kα isoform to the nucleus. In the present study we expand upon that work by genomically tagging the PI5P4Ks with fluorochromes in the presence or absence of stable or acute depletions of PI5P4Kß. Consistent with our original hypothesis we find that PI5P4Kα is predominantly (possible entirely) cytoplasmic when PI5P4Kß is stably deleted from cells. In contrast, when PI5P4Kß is inducibly removed within 1 h PI5P4Kα retains its wild-type distribution of approximately 50:50 between cytoplasm and nucleus even through a number of cell divisions. This leads us to speculate that PI5P4Kα is chromatin-associated. We also find that when cells are in the exponential phase of growth PI5P4Kß is primarily cytoplasmic but translocates to the nucleus upon growth into the stationary phase or upon serum starvation. Once again this is not accompanied by a change in PI5P4Kα localization and we show, using an in vitro model, that this is possible because the dimerization between the two isoforms is dynamic. Given this shift in PI5P4Kß upon nutrient deprivation we explore the phenotype of PI5P4K B-null cells exposed to this stress and find that they can sustain a greater degree of nutrient deprivation than their wild-type counterparts possibly as a result of up-regulation of autophagy.

A short history of inositol lipids.

The diverse family of inositol lipids is now known to be central to many aspects of cell biology. The route from the first discovery of inositol to our present day knowledge of inositol lipids spans more than 150 years and is long and complex. This is a brief account of some of the most important stages along that route.

The function of phosphatidylinositol 5-phosphate 4-kinase γ (PI5P4Kγ) explored using a specific inhibitor that targets the PI5P-binding site.

NIH-12848 (NCGC00012848-02), a putative phosphatidylinositol 5-phosphate 4-kinase γ (PI5P4Kγ) inhibitor, was explored as a tool for investigating this enigmatic, low activity, lipid kinase. PI5P4K assays in vitro showed that NIH-12848 inhibited PI5P4Kγ with an IC50 of approximately 1 µM but did not inhibit the α and ß PI5P4K isoforms at concentrations up to 100 µM. A lack of inhibition of PI5P4Kγ ATPase activity suggested that NIH-12848 does not interact with the enzyme's ATP-binding site and direct exploration of binding using hydrogen-deuterium exchange (HDX)-MS (HDX-MS) revealed the putative PI5P-binding site of PI5P4Kγ to be the likely region of interaction. This was confirmed by a series of mutation experiments which led to the identification of a single PI5P4Kγ amino acid residue that can be mutated to its PI5P4Ks α and ß homologue to render PI5P4Kγ resistant NIH-12848 inhibition. NIH-12848 (10 µM) was applied to cultured mouse principal kidney cortical collecting duct (mpkCCD) cells which, we show, express PI5P4Kγ that increases when the cells grow to confluence and polarize. NIH-12848 inhibited the translocation of Na⁺/K⁺-ATPase to the plasma membrane that occurs when mpkCCD cells grow to confluence and also prevented reversibly their forming of 'domes' on the culture dish. Both these NIH-12848-induced effects were mimicked by specific RNAi knockdown of PI5P4Kγ, but not that of PI5P4Ks α or ß. Overall, the data reveal a probable contribution of PI5P4Kγ to the development and maintenance of epithelial cell functional polarity and show that NIH-12848 is a potentially powerful tool for exploring the cell physiology of PI5P4Ks.

Evolutionarily conserved structural changes in phosphatidylinositol 5-phosphate 4-kinase (PI5P4K) isoforms are responsible for differences in enzyme activity and localization.

Mammals have genes coding for three PI5P4Ks (PtdIns5P 4-kinases), and these have different cellular localizations, tissue distributions and lipid kinase activities. We describe in the present paper a detailed molecular exploration of human PI5P4Ks α, ß and γ, as well as their fly and worm homologues, 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 homologues from Caenorhabditis elegans and Drosophila 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 heterodimerization, and not a higher oligomer association of homodimers. We reveal that structural modelling is consistent with this and with the apparently random heterodimerization that we had earlier observed between PI5P4Kα and PI5P4Kß [Wang, Bond, Letcher, Richardson, Lilley, Irvine and Clarke (2010), Biochem. J. 430, 215-221]. Overall the molecular diversity of mammalian PI5P4Ks explains much of their properties and behaviour, but their physiological functionality remains elusive.

Phosphatidylinositol 4-phosphate 5-kinase Iγ_v6, a new splice variant found in rodents and humans.

Phosphatidylinositol 4-phosphate 5-kinase Iγ (PIP5KIγ) is subject to extensive C-terminal splice variation, with four variants, PIP5KIγ_v1, 2, 4 and 5, described in humans Schill and Anderson (2009) [7]. Here firstly, we report a new rodent splice variant, which includes the exon that was previously unique to the rodent neuron-specific PIP5KIγ93 Giudici et al. (2006) [6], but which omits the C-terminal exon of PIP5KIγ93; this new variant shows a wide tissue expression pattern in mouse. Secondly, we show that in humans there is an alternative splicing site 78 nucleotides from the start of exon 16c, such that humans additionally express both PIP5KIγ93 (which we now call PIP5KIγ_v3) specifically in brain and, again expressed more widely, the new variant described here, which we now name PIP5KIγ_v6.

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

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.

Immunocytochemical techniques reveal multiple, distinct cellular pools of PtdIns4P and PtdIns(4,5)P(2).

PtdIns4P is the major precursor for the synthesis of the multifunctional plasma membrane lipid, PtdIns(4,5)P(2). Yet PtdIns4P also functions as a regulatory lipid in its own right, particularly at the Golgi apparatus. In the present study we define specific conditions that enable preservation of several organellar membranes for the immunocytochemical detection of PtdIns4P. We report distinct pools of this lipid in both Golgi and plasma membranes, which are synthesized by different PI4K (phosphatidylinositol 4-kinase) activities, and also the presence of PtdIns4P in cytoplasmic vesicles, which are not readily identifiable as PI4K containing trafficking intermediates. In addition, we present evidence that the majority of PtdIns4P resides in the plasma membrane, where it is metabolically distinct from the steady-state plasma membrane pool of PtdIns(4,5)P(2).

Do mammals make all their own inositol hexakisphosphate?

A highly specific and sensitive mass assay for inositol hexakisphosphate (InsP6) was characterized. This centres around phosphorylating InsP6 with [32P]ATP using a recombinant InsP6 kinase from Giardia lambia, followed by HPLC of the 32P-labelled products with an internal [3H]InsP7 standard. This assay was used to quantify InsP6 levels in a variety of biological samples.Concentrations of InsP6 in rat tissues varied from 10-20 microM (assuming 64% of wet weight of tissue is cytosol water), whereas using the same assumption axenic Dictyostelium discoideum cells contained 352 +/- 11 microM InsP6. HeLa cells were seeded at low density and grown to confluence, at which point they contained InsP6 levels per mg of protein similar to rat tissues. This amounted to 1.952 +/- 0.117 nmol InsP6 per culture dish, despite the cells being grown in serum shown to contain no detectable(less than 20 pmol per dish) InsP6. These results demonstrate that mammalian cells synthesize all their own InsP6. Human blood was analysed, and although the white cell fraction contained InsP6 at a concentration comparable with other tissues, in serum and platelet-free plasma no InsP6 was detected (<1 nM InsP6). Human urine was also examined, and also contained no detectable (<5 nM) InsP6. These results suggest that dietary studies purporting to measure InsP6 at micromolar concentrations in human plasma or urine may not have been quantifying this inositol phosphate. Therefore claims that administrating InsP6 in the diet or applying it topically can produce health benefits by increasing extracellular InsP6 levels may require reassessment.

Nuclear inositide signalling -- expansion, structures and clarification.

The extent and content of this review issue highlights how our understanding of lipid signalling in the nucleus has grown, both in what we actually know, and the breadth of signalling pathways that we now have to consider. Here, a few key issues with regard to nuclear inositide signalling are briefly addressed.

Type II PtdInsP kinases: location, regulation and function.

The regulation of the synthesis of PtdIns(4,5)P2 is emerging as being as complex as we might expect from the multi-functional nature of this lipid. In the present chapter we focus on one aspect of inositide metabolism, which is the functions of the Type II PIPkins (Type II PtdInsP kinases). These are primarily PtdIns5P 4-kinases, although in vitro they will also phosphorylate PtdIns3P to PtdIns(3,4)P2. Thus they have three, not necessarily exclusive, functions: to make PtdIns(4,5)P2 by a quantitatively minor route, to remove PtdIns5P and to make PtdIns(3,4)P2 by a route that does not involve a Class I PtdIns 3-kinase. None of these three possible functions has yet been unambiguously proven or ruled out. Of the three isoforms, alpha and beta are widely expressed, the IIalpha being predominantly cytosolic and the IIbeta primarily nuclear. PIPkin IIgamma has a much more restricted tissue expression pattern, and appears to be localized primarily to intracellular vesicles. Here we introduce in turn each of the three Type II PIPkins, and discuss what we know about their localization, their regulation and their function.

Regulation of type II PIP kinase by PKD phosphorylation.

The type II PIP kinases phosphorylate the poorly understood inositol lipid PtdIns5P, producing the multi-functional lipid product PtdIns(4,5)P(2). To investigate the regulation of these enzymes by phosphorylation, we partially purified a protein kinase from pig platelets that phosphorylated type IIalpha PIP kinase on an activation loop threonine residue, T376. Pharmacological studies suggested this protein kinase was protein kinase D (PKD), and in vitro experiments confirmed this identification. A phospho-specific antibody was developed and used to demonstrate phosphorylation of T376 in living cells, and its enhancement under conditions in which PKD was activated. Although we were unable to determine the effects of phosphorylation on PIP kinase activity directly, mutation of T376 to aspartate significantly inhibited enzyme activity. We conclude that the type II PIP kinases are physiological targets for PKD phosphorylation, and that this modification is likely to regulate inositol lipid turnover by inhibition of these lipid kinases.

Inhibition of PIP4Kγ ameliorates the pathological effects of mutant huntingtin protein.

The discovery of the causative gene for Huntington's disease (HD) has promoted numerous efforts to uncover cellular pathways that lower levels of mutant huntingtin protein (mHtt) and potentially forestall the appearance of HD-related neurological defects. Using a cell-based model of pathogenic huntingtin expression, we identified a class of compounds that protect cells through selective inhibition of a lipid kinase, PIP4Kγ. Pharmacological inhibition or knock-down of PIP4Kγ modulates the equilibrium between phosphatidylinositide (PI) species within the cell and increases basal autophagy, reducing the total amount of mHtt protein in human patient fibroblasts and aggregates in neurons. In two Drosophila models of Huntington's disease, genetic knockdown of PIP4K ameliorated neuronal dysfunction and degeneration as assessed using motor performance and retinal degeneration assays respectively. Together, these results suggest that PIP4Kγ is a druggable target whose inhibition enhances productive autophagy and mHtt proteolysis, revealing a useful pharmacological point of intervention for the treatment of Huntington's disease, and potentially for other neurodegenerative disorders.

The intracellular localisation and mobility of Type Igamma phosphatidylinositol 4P 5-kinase splice variants.

There are three known splice variants of Type Igamma phosphatidylinositol 4-phosphate 5-kinase (PIPkin Igamma): PIPkins Igamma87, Igamma90, and the most recently cloned (Giudici, M.L., Emson, P.C. and Irvine, R.F. (2004) A novel neuronal-specific splice variant of Type I phosphatidylinositol 4-phosphate 5-kinase isoform gamma. Biochem. J. 379, 489-496) PIPkin IgammaC (here called PIPkin Igamma93). Here, we have explored the subcellular localisation and mobility of Type I PIPkins in transfected cells by confocal microscopy and flourescence recovery after photobleaching. The unique behaviour shown by PIPkin Igamma93 is consistent with its suggested distinct function. Moreover, the markedly different localisation and mobility of active versus inactive PIPkin Igamma93 provide insights into the factors that dictate cellular targeting of Type Igamma PIPkins.

The behaviour of myo-inositol hexakisphosphate in the presence of magnesium(II) and calcium(II): protein-free soluble InsP6 is limited to 49 microM under cytosolic/nuclear conditions.

Progress in the biology of myo-inositol hexakisphosphate (InsP(6)) has been delayed by the lack of a quantitative description of its multiple interactions with divalent cations. Our recent initial description of these [J. Torres, S. Dominguez, M.F. Cerda, G. Obal, A. Mederos, R.F. Irvine, A. Diaz, C. Kremer, J. Inorg. Biochem. 99 (2005) 828-840] predicted that under cytosolic/nuclear conditions, protein-free soluble InsP(6) occurs as Mg(5)(H(2)L), a neutral complex that exists thanks to a significant, but undefined, window of solubility displayed by solid Mg(5)(H(2)L).22H(2)O (L is fully deprotonated InsP(6)). Here we complete the description of the InsP(6)-Mg(2+)-Ca(2+) system, defining the solubilities of the Mg(2+) and Ca(2+) (Ca(5)(H(2)L).16H(2)O) solids in terms of K(s0)=[M(2+)](5)[H(2)L(10-)], with pK(s0)=32.93 for M=Mg and pK(s0)=39.3 for M=Ca. The concentration of soluble Mg(5)(H(2)L) at 37 degrees C and I=0.15M NaClO(4) is limited to 49muM, yet InsP(6) in mammalian cells may reach 100muM. Any cytosolic/nuclear InsP(6) in excess of 49muM must be protein- or membrane-bound, or as solid Mg(5)(H(2)L).22H(2)O, and any extracellular InsP(6) (e.g. in plasma) is surely protein-bound.

Agonist-induced regulation of mitochondrial and endoplasmic reticulum motility.

Using fluorescently tagged markers for organelles in conjunction with confocal microscopy, we have studied the effects of agonist-induced Ca2+ signals on the motility of mitochondria and the ER (endoplasmic reticulum). We observed that the muscarinic agonist carbachol produced a rapid, simultaneous and reversible cessation of the movements of both organelles, which was dependent on a rise in cytosolic Ca2+. This rise in Ca2+ was shown to cause a fall in cellular ATP levels, and the effect of carbachol on organelle movement could be mimicked by depleting ATP with metabolic inhibitors in the absence of any such rise in Ca2+. However, a Ca2+-sensing process independent of ATP appears also to be involved, because we identified conditions where the ATP depletion was blocked (by inhibitors of the Ca2+ pumps), but the organelle movements still ceased following a rise in cytosolic Ca2+. We conclude that the co-ordinated cessation of mitochondria and ER motility is a process regulated by the cytosolic concentration of both Ca2+ and ATP, and that these two parameters are likely to synergize to regulate the localization of the two organelles, and to facilitate the transfer of Ca2+ between them.

Regulation of the localization and activity of inositol 1,4,5-trisphosphate 3-kinase B in intact cells by proteolysis.

IP3K (inositol 1,4,5-trisphosphate 3-kinase) catalyses the Ca2+-regulated phosphorylation of the second messenger Ins(1,4,5)P3, thereby inactivating the signal to release Ca2+ and generating Ins(1,3,4,5)P4. Here we have investigated the localization and activity of IP3KB and its modulation by proteolysis. We found that the N- and C-termini (either side of residue 262) of IP3KB localized predominantly to the actin cytoskeleton and ER (endoplasmic reticulum) respectively, both in COS-7 cells and in primary astrocytes. The functional relevance of this was demonstrated by showing that full-length (actin-localized) IP3KB abolished the histamine-induced Ca2+ response in HeLa cells more effectively than truncated constructs localized to the ER or cytosol. The superior efficacy of full-length IP3KB was also attenuated by disruption of the actin cytoskeleton. By transfecting COS-7 cells with double-tagged IP3KB, we show that the translocation from actin to ER may be a physiologically regulated process caused by Ca2+-modulated constitutive proteolysis in intact cells.

Phosphatidylinositol 5-phosphate 4-kinase γ (PI5P4Kγ), a lipid signalling enigma.

The phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are an important family of enzymes, whose physiological roles are being teased out by a variety of means. Phosphatidylinositol-5-phosphate 4-kinase γ (PI5P4Kγ) is especially intriguing as its in vitro activity is very low. Here we review what is known about this enzyme and discuss some recent advances towards an understanding of its physiology. Additionally, the effects of the ATP-competitive inhibitor I-OMe Tyrphostin AG-538 on all three mammalian PI5P4Ks was explored, including two PI5P4Kγ mutants with altered ATP- or PI5P-binding sites. The results suggest a strategy for targeting non-ATP binding sites on inositol lipid kinases.

The structural integrity of the endoplasmic reticulum, and its possible regulation by inositol 1,3,4,5-tetrakisphosphate.

The endoplasmic reticulum (ER) is a dynamic organelle thought to consist of a single interconnected network of membranes. Using fluorescence recovery after photobleaching (FRAP) of HEK-293 cells dually transfected with soluble fluorescent proteins targeted to the ER (GFP) and mitochondria (DsRed), we have confirmed this continuity, which contrasts that of the mitochondria, which behave as a population of discrete organelles. The degree of ER integrity (interconnected versus fragmented) has been suggested to be regulated in some cells by inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). In HEK-293 and freshly isolated murine lacrimal acinar cells, we manipulated ER structure by disrupting cellular Ca(2+) homeostasis with the Ca(2+) ionophore ionomycin, and by permeabilisation of the plasma membrane, protocols known to cause ER fragmentation. However, we were subsequently unable to detect by FRAP any significant effect of Ins(1,3,4,5)P(4) on ER integrity.

Effects of lipid kinase expression and cellular stimuli on phosphatidylinositol 5-phosphate levels in mammalian cell lines.

Phosphatidylinositol 5-phosphate (PtdIns5P) is a relatively recently discovered inositol lipid whose metabolism and functions are not yet clearly understood. We have transfected cells with a number of enzymes that are potentially implicated in the synthesis or metabolism of PtdIns5P, or subjected cells to a variety of stimuli, and then measured cellular PtdIns5P levels by a specific mass assay. Stable or transient overexpression of Type IIalpha PtdInsP kinase, or transient overexpression of Type Ialpha or IIbeta PtdInsP kinases caused no significant change in cellular PtdIns5P levels. Similarly, subjecting cells to oxidative stress or EGF stimulation had no significant effect on PtdIns5P, but stimulation of HeLa cells with a phosphoinositide-specific PLC-coupled agonist, histamine, caused a 40% decrease within 1 min. Our data question the degree to which inositide kinases regulate PtdIns5P levels in cells, and we discuss the possibility that a significant part of both the synthesis and removal of this lipid may be regulated by phosphatases and possibly phospholipases.