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

Department of Pharmacology, University of Cambridge, Tennis Court Road, CB2 1QJ, Cambridge, UK.
FEBS Letters (Impact Factor: 3.17). 07/2000; 475(1):57-60. DOI: 10.1016/S0014-5793(00)01625-2
Source: PubMed


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 ¿gamma-(32)PATP and recombinant PIPkin IIalpha, and quantifying the radiolabelled PtdInsP(2) 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.

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    • "c o m / l o c a t e / b b a m c r [12]. Studies revealed that the separate administration of either plateletderived growth factor, vasopressin, prostaglandin, bombesin or EGF evokes Ca 2+ transients and also induces inositol trisphosphate (InsP 3 ) production [13] [14]. In Swiss 3T3 cells, serum-induced Ca 2+ increases are essential, but not sufficient to induce NF-κB activation and subsequent DNA synthesis [15]. "
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    ABSTRACT: Brief changes in the cytosolic and intra-organellar Ca(2+) concentration serve as specific signals for various physiological processes. In mesothelial cells lining the surface of internal organs and the walls of body cavities, a re-entry in the cell cycle (G0-G1 transition) evoked by serum re-administration induces long-lasting Ca(2+) oscillations with a slowly decreasing frequency. Individual mesothelial cells show a wide range of different oscillatory patterns within a single, supposedly homogenous cell population. Changes in the cytoplasmic Ca(2+) concentration (ccyt) show baseline oscillatory patterns i.e., discrete Ca(2+) transients starting from a constant basal ccyt level. The ER Ca(2+) concentration (cER) displays a sawtooth wave at a semi-depleted ER state; the minimum level is reached just briefly after the maximal value for ccyt. These oscillations depend on plasmalemmal Ca(2+) influx and on the inositol trisphosphate concentration [InsP3]; the Ca(2+) influx is a crucial determinant of the oscillation frequency. Partial blocking of SERCA pumps modifies the oscillation frequency in both directions, i.e. increasing it in some cells and lowering it in others. Current mathematical models for Ca(2+) oscillations mostly fail to reproduce two experimentally observed phenomena: the broad range of interspike intervals and constant basal ccyt levels between two Ca(2+) spikes. Here we developed a new model based on -and fitted to- Ca(2+) recordings of ccyt and cER recorded in primary mouse mesothelial cells. The model allowed to explaining many features of experimentally observed Ca(2+) oscillations. We consider this model to be suitable to simulate various types of InsP3 receptor-based baseline Ca(2+) oscillations. Copyright © 2014. Published by Elsevier B.V.
    Full-text · Article · Dec 2014 · Biochimica et Biophysica Acta (BBA) - Molecular Cell Research
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    • "A binding assay and an enzymatic assay that utilized an elaborate liposome-based substrate preparation have been previously reported [17], [18]. Additionally, low-throughput radiolabeled enzymatic thin layer chromatography (TLC) assays were developed where the substrate was prepared in an isotonic KCl solution or as a liposome mixture [20], [21]. The radiometric assay uses γ-32P-ATP and PI5P and measures radiolabeled enzymatic product, PI(4,5)P2 after the separation by TLC [10], [22], [23]. "
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    ABSTRACT: Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z'-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-(32)P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538), was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC(50) of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.
    Full-text · Article · Jan 2013 · PLoS ONE
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    • "However, specific mass assays and imaging using high affinity probes like the plant homeodomain (PHD) of the inhibitor of growth 2 (ING2) lead several groups to suggest that PtdIns5P may act as a second messenger both in the cytoplasm and the nucleus (Gozani et al., 2005; Gozani et al., 2003; Morris et al., 2000). The amount of PtdIns5P can be increased by different stimuli such as thrombin in platelets, insulin in 3T3-L1 cells, T cell receptor engagement, increased tyrosine kinase activity, and stress signals (Grainger et al., 2011; Morris et al., 2000; Pizarro-Cerda and Cossart, 2004; Sbrissa et al., 2001; Sbrissa et al., 2002). In addition, an increase in PtdIns5P was reported during the G1 phase of the cell cycle (Choudhury et al., 2006). "
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