Andrew J. Letcher

Thrombin stimulation of platelets causes an increase in phosphatidylinositol 5-phosphate revealed by mass assay

Phosphatidylinositol 5‐phosphate (PtdIns5P), a novel inositol lipid, has been shown to be the major substrate for the type II PtdInsP kinases (PIPkins) [Rameh et al. (1997) Nature 390, 192–196]. A PtdInsP fraction was prepared from cell extracts by neomycin chromatography, using a protocol devised to eliminate the interaction of acidic solvents with plasticware, since this was found to inhibit the enzyme. The PtdIns5P in this fraction was measured by incubating with [γ‐32P]ATP and recombinant PIPkin IIα, and quantifying the radiolabelled PtdInsP2 formed. This assay was used on platelets to show that during 10 min stimulation with thrombin, the mass level of PtdIns5P increases, implying the existence of an agonist‐stimulated synthetic mechanism.

PiUS (Pi uptake stimulator) is an inositol hexakisphosphate kinase.

A cDNA cloned from its ability to stimulate inorganic phosphate uptake in Xenopus oocytes (phosphate uptake stimulator (PiUS)) shows significant similarity with inositol 1,4,5‐trisphosphate 3‐kinase. However, the expressed PiUS protein showed no detectable activity against inositol 1,4,5‐trisphosphate, nor the 1,3,4,5‐ or 3,4,5,6‐isomers of inositol tetrakisphosphate, whereas it was very active in converting inositol hexakisphosphate (InsP6) to inositol heptakisphosphate (InsP7). PiUS is a member of a family of enzymes found in many eukaryotes and we discuss the implications of this for the functions of InsP7 and for the evolution of inositol phosphate kinases.

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 μM (assuming 64% of wet weight of tissue is cytosol water), whereas using the same assumption axenic Dictyostelium discoideum cells contained 352±11 μM 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.

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 IIα PtdInsP kinase, or transient overexpression of Type Iα or IIβ 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.

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

PtdIns5P 4-kinases IIα and IIβ 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 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-fold more active as a PtdIns5P 4-kinase than the IIβ isoform. Overall the results suggest a function of PtdIns5P 4-kinase IIβ may be to target the more active IIα isoform into the nucleus.

Type IIalpha phosphatidylinositol phosphate kinase associates with the plasma membrane via interaction with type I isoforms.

The phosphatidylinositol phosphate kinases (PIPkins) are a family of enzymes involved in regulating levels of several functionally important inositol phospholipids within cells. The PIPkin family is subdivided into three on the basis of substrate specificity, each subtype presumably regulating levels of different subsets of the inositol lipids. The physiological function of the type II isoforms, which exhibit a preference for phosphatidylinositol 5-phosphate, a lipid about which very little is known, is particularly poorly understood. In the present study, we demonstrate interaction between, and co-immunoprecipitation of, type IIalpha PIPkin with the related, but biochemically and immunologically distinct, type I PIPkin isoforms. Type IIalpha PIPkin interacts with all three known type I PIPkins (alpha, beta and gamma), and in each case co-expression of the type I isoform with type IIalpha results in recruitment of the latter from the cytosol to the plasma membrane of the cell. This change in subcellular localization could result in improved access of the type IIalpha PIPkin to its lipid substrates.

Regulation of type IIalpha phosphatidylinositol phosphate kinase localisation by the protein kinase CK2.

Inositol lipid synthesis is regulated by several distinct families of enzymes [1]. Members of one of these families, the type II phosphatidylinositol phosphate kinases (PIP kinases), are 4-kinases and are thought to catalyse a minor route of synthesis of the multifunctional phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) from the inositide PI(5)P [2]. Here, we demonstrate the partial purification of a protein kinase that phosphorylates the type IIalpha PIP kinase at a single site unique to that isoform - Ser304. This kinase was identified as protein kinase CK2 (formerly casein kinase 2). Mutation of Ser304 to aspartate to mimic its phosphorylation had no effect on PIP kinase activity, but promoted both redistribution of the green fluorescent protein (GFP)-tagged enzyme in HeLa cells from the cytosol to the plasma membrane, and membrane ruffling. This effect was mimicked by mutation of Ser304 to alanine, although not to threonine, suggesting a mechanism involving the unmasking of a latent membrane localisation sequence in response to phosphorylation.

The effect of inositol 1,3,4,5-tetrakisphosphate on inositol trisphosphate-induced Ca2+ mobilization in freshly isolated and cultured mouse lacrimal acinar cells.

Earlier reports have shown a remarkable synergism between InsP(4) and InsP(3) [either Ins(1,4,5)P(3) or Ins(2,4,5)P(3)] in activating Ca(2+)-dependent K(+) and Cl(-) currents in mouse lacrimal cells [Changya, Gallacher, Irvine, Potter and Petersen (1989) J. Membr. Biol. 109, 85-93; Smith (1992) Biochem. J. 283, 27-30]. However, Bird, Rossier, Hughes, Shears, Armstrong and Putney [(1991) Nature (London) 352, 162-165] reported that they could see no such synergism in the same cell type. A major experimental difference between the two laboratories lies in whether or not the cells were maintained in primary culture before use. Here we have compared directly the responses to inositol polyphosphates in freshly isolated cells versus cells cultured for 6-72 h. In the cultured cells, Ins(2,4,5)P(3) at 100 microM produced a robust stimulation of K(+) and Cl(-) currents, as much as an order of magnitude greater than that observed in the freshly isolated cells. However, the freshly isolated cells could be restored to a sensitivity similar to cultured cells by the addition of InsP(4) at a concentration two orders of magnitude lower than that of Ins(2,4,5)P(3). We discuss the implications of this with respect to the actions of InsP(4), including the possibility that disruption of the cellular structure during the isolation of the cells exposes an extreme manifestation of a possible physiological role for InsP(4) in controlling calcium-store integrity.

An inositol 1,4,5-trisphosphate-6-kinase activity in pea roots

A soluble extract from pea (Pisum sativum L.) roots, when incubated with ATP and inositol 1,4,5-trisphosphate, produced an inositol tetrakisphosphate. The chromatographic properties of this inositol tetrakisphosphate, and of the products formed by its chemical degradation, identify it as inositol 1,4,5,6-tetrakisphosphate. No evidence was obtained for a 3-phosphorylation of inositol 1,4,5-trisphosphate. The importance of these observations with respect to inositol phosphates and calcium signalling in higher plants, is discussed.

One-dimensional thin-layer chromatographic separation of the lipids involved in arachidonic acid metabolism.

A one-dimensional thin-layer chromatographic separation technique is described that separates the phospholipids and neutral lipids that are principally involved in arachidonic acid metabolism from each other, and from arachidonic acid and its eicosanoid derivatives. The technique is useful in studies on changes in arachidonic acid metabolism following cell stimulation. Balance sheets may be drawn up between the loss of radioactivity from phospholipids, and the gain in free fatty acid, eicosanoids, and esterified lipids of the phosphatidylinositol cycle.