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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: 3.34). 07/2000; 475(1):57-60. DOI: 10.1016/S0014-5793(00)01625-2
Source: PubMed

ABSTRACT 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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    Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 12/2014; 1853(3). DOI:10.1016/j.bbamcr.2014.12.025 · 5.30 Impact Factor
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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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    • "The bisphosphorylated PIs containing PtdIns(3,4)P 2 , PtdIns(4,5)P 2 , and PtdIns(3,5)P 2 moieties play distinct roles in signal prolongation after PtdIns(3,4,5)P 3 inducing stimuli, regulation of the actin cytoskeleton and vesicle transport, respectively [4] [5] [6] [7] [8]. PIs containing phosphoinositide monophosphates were long thought to be mere intermediates in the pathway but are now recognised to possess specific functions by themselves in protein sorting, vesicular trafficking and in osmotic stress response [9] [10] [11] [12]. Analysis of PIs has been achieved in several ways. "
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