Phosphoinositide and Inositol Phosphate Analysis in Lymphocyte Activation

Karsten Sauer1, Yina Hsing Huang2, Hongying Lin3, Mark Sandberg4, Georg W. Mayr3

1 The Scripps Research Institute, La Jolla, California, 2 Washington University School of Medicine, St. Louis, Missouri, 3 University Medical Center Hamburg‐Eppendorf, Hamburg, Germany, 4 Genomics Institute of the Novartis Research Foundation (GNF), San Diego, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 11.1
DOI:  10.1002/0471142735.im1101s87
Online Posting Date:  November, 2009
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Abstract

Lymphocyte antigen receptor engagement profoundly changes the cellular content of phosphoinositide lipids and soluble inositol phosphates. Among these, the phosphoinositides phosphatidylinositol 4,5‐bisphosphate (PIP2) and phosphatidylinositol 3,4,5‐trisphosphate (PIP3) play key signaling roles by acting as pleckstrin homology (PH) domain ligands that recruit signaling proteins to the plasma membrane. Moreover, PIP2 acts as a precursor for the second messenger molecules diacylglycerol and soluble inositol 1,4,5‐trisphosphate (IP3), essential mediators of PKC, Ras/Erk, and Ca2+ signaling in lymphocytes. IP3 phosphorylation by IP3 3‐kinases generates inositol 1,3,4,5‐tetrakisphosphate (IP4), an essential soluble regulator of PH domain binding to PIP3 in developing T cells. Besides PIP2, PIP3, IP3, and IP4, lymphocytes produce multiple other phosphoinositides and soluble inositol phosphates that could have important physiological functions. To aid their analysis, detailed protocols that allow one to simultaneously measure the levels of multiple different phosphoinositide or inositol phosphate isomers in lymphocytes are provided here. They are based on thin layer, conventional and high‐performance liquid chromatographic separation methods followed by radiolabeling or non‐radioactive metal‐dye detection. Finally, less broadly applicable non‐chromatographic methods for detection of specific phosphoinositide or inositol phosphate isomers are discussed. Support protocols describe how to obtain pure unstimulated CD4+CD8+ thymocyte populations for analyses of inositol phosphate turnover during positive and negative selection, key steps in T cell development. Curr. Protoc. Immunol. 87:11.1.1‐11.1.46. © 2009 by John Wiley & Sons, Inc.

Keywords: lymphocyte; inositol; phosphoinositide; phospholipid; second messenger; T cell; thymocyte; signal transduction; IP3; IP4; IP5; IP6; PIP2; PIP3; HPLC; MDD‐HPLC

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Myo‐[3H] Inositol Labeling and Stimulation of Primary Murine Thymocytes
  • Alternate Protocol 1: Myo‐[3H] Inositol Labeling and Stimulation of Immortalized T Cells
  • Enriching Unstimulated CD4+CD8+ DP Thymocytes
  • Support Protocol 1: MHCI−MHCII− (MHC−) Mice
  • Support Protocol 2: CD53−CD4+CD8+ DP Thymocyte Enrichment by Magnetic Bead Immunoaffinity Cell Sorting (MACS)
  • Basic Protocol 2: [3H] Inositol Phosphate Resolution by HPLC with In‐Line β‐Detector
  • Alternate Protocol 2: [3H]‐Inositol Phosphate Resolution by HPLC without In‐Line β‐Detector
  • Basic Protocol 3: Soluble Inositol Phosphate Resolution by HPLC with Metal Dye Detection (MDD)
  • Alternate Protocol 3: Inositol Phospholipid Separation and Quantification by HPLC with Metal Dye Detection (MDD)
  • Basic Protocol 4: [3H] Inositol Phosphate Resolution by Dowex Anion‐Exchange Chromatography
  • Basic Protocol 5: [3H] Inositol Phospholipid Resolution by Thin Layer Chromatography
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Myo‐[3H] Inositol Labeling and Stimulation of Primary Murine Thymocytes

  Materials
  • PBS and Ca2+‐free PBS ( appendix 2A)
  • Fibronectin (lyophilized human plasma fibronectin; Invitrogen, cat. no. 33016‐015 or equivalent; or fibronectin‐like engineered protein polymer‐plus; Sigma, cat. no. F8141)
  • 1% bovine serum albumin (BSA; Fischer, cat. no. AC61191‐0010 or equivalent) in PBS
  • Mice (6‐week‐old C57BL/6 mice) or purified DP cells
  • CO 2 source
  • M199/FCS/HEPES (see recipe)
  • PharmLyse (BD Biosciences, cat. no. 555899), 1× dilution in double distilled water and filter sterilized, optional
  • Inositol‐free DMEM/2.5% FCS (see recipe)
  • myo‐[3H] inositol (∼2.89 TBq/mmol; GE Healthcare, cat. no. TRK883)
  • Recombinant IL‐7 (R&D Systems, cat. no. 407‐ML)
  • 0.5 mM EDTA in Ca2+‐free PBS
  • HBSS/HEPES (see recipe), ice cold
  • Biotinylated anti‐mouse CD3 antibodies, clones 145‐2C11 or 500A2 (Invitrogen; CALTAG; BD Biosciences)
  • Unconjugated streptavidin (SA; Jackson Immunoresearch, cat. no. 016‐000‐113)
  • Concanavalin A type IV (ConA; Sigma‐Aldrich, cat. no. C5275)
  • 3% perchloric acid (PCA) in water
  • 6‐ and 12‐well tissue culture plastic plates, untreated
  • 37°C incubator with and without 5% CO 2
  • Mouse vivarium with appropriate euthanasia equipment in procedure room
  • 60‐mm petri dishes
  • 40‐µm nylon cell strainer
  • 1‐ to 5‐ml syringes
  • 50‐ml conical tubes
  • Hemacytometer or electronic cell counter
  • 2‐ml microcentrifuge tubes
  • 37°C water bath or heating block
  • Additional reagents and equipment for thymi removal (unit 1.9)

Alternate Protocol 1: Myo‐[3H] Inositol Labeling and Stimulation of Immortalized T Cells

  • Human Jurkat αβT cells (ATCC # TIB‐152) or other cells of interest
  • RPMI/10% FCS/PSG (see recipe)

Support Protocol 1: MHCI−MHCII− (MHC−) Mice

  Materials
  • B6.129‐H2‐Ab1tm1Gru B2mtm1Jae mice [Taconic, cat. no. 004080‐MM‐F (females) or ‐M (males)]
  • PharmLyse (BD Biosciences, cat. no. 555899): dilute to 1× in double distilled water and then filter sterilize
  • PBS containing 2% FCS
  • Fc block: anti‐mouse CD16/32 antibody
  • FACS antibodies: H‐2Kb‐FITC (BD Pharmingen, cat. no. 553569); I‐Ab‐PE (BD Pharmingen, cat. no. 553552); CD4 (L3T4) APC (BD Pharmingen, cat. no. 553051); CD8a (Ly‐2) PE‐Cy7 (BD Pharmingen, cat. no. 552877)
  • Tail digestion buffer (see recipe)
  • 25:24:1 (v/v) phenol/chloroform/isoamyl alcohol (Invitrogen, cat. no. 15593‐031)
  • Genotyping primers: B2m primers [oIMR0160 (mutant): TCTggACgAAgAgCATCAggg; oIMR0184 (common): TATCAgTCTCAgTgggggTg; oIMR0185 (wild type): CTgAgCTCTgTTTTCgTCTg]
  • I‐Ab primers [oIMR5241 (mutant): gTgTTgggTCgTTTgTTCg; oIMR5239 (common): AgggAggTgTgggTCTCC; oIMR5240 (wild type): gTACCAgTTCATgggCgAgT]
  • 96‐well U‐bottom plate
  • Additional reagents and equipment for care and handling of laboratory animals (Chapter 1); flow cytometry (Chapter 5)

Support Protocol 2: CD53−CD4+CD8+ DP Thymocyte Enrichment by Magnetic Bead Immunoaffinity Cell Sorting (MACS)

  Materials
  • 6‐week‐old C57BL/6 mice
  • MACS staining buffer (see recipe)
  • Anti‐CD53 Ab (OX‐79) (BD Biosciences, cat. no. 559364)
  • Biotinylated anti‐rat IgG (Jackson Immunoresearch, cat. no. 112‐065‐167)
  • Anti‐biotin microbeads (Miltenyi Biotec, cat. no. 130‐042‐401)
  • MidiMACS separation unit (Miltenyi Biotec, cat. no. 130‐042‐302)
  • Additional reagents and equipment for harvesting thymocytes (see protocol 1); flow cytometry (see Chapter 5)

Basic Protocol 2: [3H] Inositol Phosphate Resolution by HPLC with In‐Line β‐Detector

  Materials
  • HPLC‐grade (NH 4)H 2PO 4
  • Phosphoric acid
  • NaN 3
  • [3H]‐Labeled inositol phosphate standard solutions, e.g.,:
    • D‐myo‐inositol‐1,4,5‐P 3, [inositol‐1‐3H(N)] (Perkin Elmer, cat. no. NET‐911001UC)
    • D‐myo‐inositol‐1,3,4,5‐P 4, [inositol‐1‐3H(N)] (Perkin Elmer, cat. no. NET‐941002UC)
  • Scintillation fluid compatible with high‐salt solutions (Uniscint National Diagnostics, cat. no. LS‐276, or equivalent)
  • HPLC system compatible with aqueous solutions
  • Inline β‐detector (β‐RAM‐RHPLC detector from IN/US with a 500‐µl flow cell or equivalent)
  • Partisphere strong anion exchange (SAX) column (12.5 cm × 4.6–mm; Whatman, cat. no. 4621‐0505 or equivalent) or a 25‐cm column (Whatman, cat. no. 4621‐1507)

Alternate Protocol 2: [3H]‐Inositol Phosphate Resolution by HPLC without In‐Line β‐Detector

  Materials
  • Thymocytes (see protocol 1) or T cell lines (see protocol 2)
  • PBS ( appendix 2A)
  • Lysis buffer (see recipe), ice cold
  • Water‐saturated diethyl ether: prepared by vigorously mixing 1 vol deionized water with 2 vol diethyl ether for at least 2 min
  • 1 M triethanolamine (TEA, p.a., >99% purity; see recipe)
  • Charcoal suspension (see recipe)
  • 0.1 M NaCl ( appendix 2A)
  • Methanol (LiChrosolv)
  • 10% (w/v) trichloroacetic acid (TCA) solution, 4°C
  • 0.2 M EDTA ( appendix 2A)
  • 0.1 M NaF (see recipe)
  • MDD‐HPLC eluent A (see recipe)
  • MDD‐HPLC eluent B (see recipe)
  • Post‐column reagent C (see recipe)
  • HPLC‐injection solution (see recipe)
  • Degassed, filtered HPLC‐grade water
  • 30% analytical‐grade HCl (suprapure)
  • Phytic acid
  • 10 mM 4‐(2‐pyridylazo)‐resorcinol monosodium salt monohydrate (PAR; see recipe)
  • 18 mM yttrium trichloride (YCl 3; see recipe)
  • 0.5 M sodium acetate (see recipe)
  • Cell scraper
  • 12‐ and 14‐ml polypropylene tubes with caps (Greiner Bio‐One, cat. no. 187262)
  • 35°C water bath
  • 2‐ml microcentrifuge tubes
  • SpeedVac
  • Ultra‐Turrax homogenizer
  • 0.22‐µm pore size membrane filters (Millipore, type GV) in a Pyrex glass filtration device
  • Vacuum pump
  • 2‐ml Pyrex glass vials
  • HPLC auto‐sampler with a 1‐ml injection loop and a 2.5‐ml loading syringe (inert valve made from titanium or PEEK; HPLC auto‐sampler 560, Kontron; loading syringe available from Hamilton)
  • MiniQ PC 3.2/3 column (3‐µm bead diameter, GE Healthcare/Pharmacia Biotech, Uppsala)
  • Hand‐made knitted coil from a 40‐cm 1/16‐in. × 0.5‐mm i.d. PTFE capillary with 7 knots (CS‐Chromatographie Service)
  • Two inert HPLC pumps for gradient elution (with titanium or PEEK pump‐head; Pump 422, Kontron)
  • Pump for post‐column dye reagent addition (Shimadzu, cat. no. LC‐10AD)
  • UV/Vis recorder for absorbance recording (with titanium or PEEK flow cell; Shimadzu, cat. no. SPD‐10Avvp)
  • Chromatography data system for control and data processing (e.g., Galaxie chromatography data system, Varian)
  • Graphing/interpolation software (e.g., GraphPad Prism)

Basic Protocol 3: Soluble Inositol Phosphate Resolution by HPLC with Metal Dye Detection (MDD)

  • PIP 2, PIP 3 standards
  • Methanol (LiChrosolv)
  • Chloroform (CHCl 3; LiChrosolv)
  • 0.1 M HCl
  • 3:48:47 (v/v/v) chloroform/methanol/0.6 M HCl
  • n‐Butanol
  • 33% methylamine in ethanol
  • n‐Propanol
  • 20:4:1 (v/v/v) butanol/petroleum‐ether/ethyl formate
  • 53°C water bath or heating block

Alternate Protocol 3: Inositol Phospholipid Separation and Quantification by HPLC with Metal Dye Detection (MDD)

  Materials
  • Cells
  • Medium supplemented with 2 to 20 µCi/ml myo‐[3H] inositol
  • Balanced salt solution (see recipe)
  • Stimulus
  • 10% (w/v) trichloroacetic acid (TCA), ice cold
  • Diethyl ether, water‐saturated
  • 1:100 (v/v) dilution of concentrated ammonia
  • 0.5 g/ml Dowex 1‐X8 resin (100 to 200 mesh; formate form; Bio‐Rad, Sigma‐Aldrich, GFS Chemicals, Serva Electrophoresis; formate form may require custom production), slurry in water
  • 60 mM sodium formate/5 mM disodium tetraborate
  • 0.2 M ammonium formate/0.1 M formic acid
  • 0.4 M ammonium formate/0.1 M formic acid
  • 0.8 M ammonium formate/0.1 M formic acid
  • Scintillation fluor cocktail (compatible with aqueous samples)
  • 13 × 100–mm glass tubes
  • Pasteur pipets
  • 0.6‐cm diameter disposable columns
  • Liquid scintillation counter

Basic Protocol 4: [3H] Inositol Phosphate Resolution by Dowex Anion‐Exchange Chromatography

  Materials
  • 1% potassium oxalate/2 mM EDTA
  • Solvent: 60:20:23:18:12 (v/v/v/v/v) chloroform/methanol/acetone/acetic acid/H 2O
  • Cells (see , protocol 1, and protocol 2)
  • Phosphate‐free medium (e.g., GIBCO/BRL) containing 10% heat‐inactivated fetal calf serum (FCS), dialyzed against HBSS or TBS ( appendix 2A) to remove phosphate
  • Carrier‐free [32P] orthophosphate (296 mBq/ml, 8 mCi/ml)
  • Stimulus (see )
  • 50:100:1 (v/v/v) chloroform/methanol/concentrated HCl
  • 100 mM EDTA, pH 7.4 ( appendix 2A)
  • 100 mM KCl
  • Chloroform
  • Nitrogen source
  • 2:1 (v/v) chloroform/methanol
  • PI, PIP, PIP 2, and PA standards (Sigma)
  • Iodine
  • Silica‐gel plates (Baker Si250, J.T. Baker)
  • TLC tanks
  • 100° and 110°C ovens
  • Filter paper
  • 2‐ml microcentrifuge tubes
  • 37°C water bath or heating block
  • Kodak X‐Omat film
  • EN3HANCE spray surface autoradiography enhancer (Perkin Elmer)
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Figures

  •   FigureFigure 11.1.1 Mammalian inositol phosphate metabolism. Simplified scheme of the known inositol phosphate metabolic pathway in mammalian cells. Circled P, phosphate moiety; R, R', fatty acid side chains. The hatched box encloses pathway components for which genetic data suggest relevance in lymphocytes. For more details and discussions of the enzymes involved and of potential cellular inositol phosphate functions, see previously published works (Irvine, , , ; Irvine and Schell, ; Irvine et al., ; Rusten and Stenmark, ; Otto et al., ; Seeds et al., ; Miller et al., ; Alcazar‐Roman and Wente, ; Huang et al., ; Lin et al., ). The membrane phospholipid phosphatidylinositol 4,5‐bisphosphate (PtdIns(4,5)P2/PI(4,5)P2, PIP2) acts as a precursor for the phosphoinositide PI(3,4,5)P3 (PIP3), and for the second messenger molecules diacylglycerol (DAG) and inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3/I(1,4,5)P3/IP3). In mammalian cells, I(1,4,5)P3 acts as a key precursor for multiple higher order, soluble inositol phosphates. An important step in the synthesis of several inositol phosphates is I(1,4,5)P3 phosphorylation into I(1,3,4,5)P4 (IP4) by either one of three IP3 3‐kinases (IP3KA, B or C, also termed ItpkA, B, or C; Pouillon et al., ; Wen et al., ; Huang et al., ) or by IPK2/IPMK (Irvine, ; Irvine et al., ; Otto et al., ). Multiple higher order inositol phosphates have been reported in lymphocytes, including several of those shown here. The levels of some inositol phosphates are modulated after antigen receptor engagement (Imboden and Stobo, ; Stewart et al., , ; Imboden and Pattison, ; Zilberman et al., ; Guse and Emmrich, , ; Guse et al., , ; Pouillon et al., ). Complementing known PIP3, IP3, and DAG functions in lymphocyte development and function (Starr et al., ; Fruman, ; Cante‐Barrett et al., ; Jodi et al., ; Juntilla and Koretzky, ), it has been recently found that IP4 is essential for these processes through novel roles in antigen receptor signaling and myelopoiesis (Pouillon et al., ; Wen et al., ; Huang et al., , ; Jia et al., , ; Marechal et al., ; Miller et al., ). The protocols described here are thus optimized for analyses of IP3 and IP4 isomers.
  •   FigureFigure 11.1.2 Analysis of TCR induced inositol phosphate production in MCHIMHCII thymocytes. (A) HPLC elution profiles of extracts from unstimulated or αCD3‐stimulated MHC murine thymocytes. 2 × 108 cells were labeled overnight with 40 µCi myo‐[3H] inositol, the precursor for all IPs. At 5 min post‐stimulation with medium or 5 µg αCD3 (2C11), cells were lysed in 100 µl of 3% PCA and loaded onto a Whatman cartridge Col SAX PRTSPHR 15‐cm HPLC column. [3H] IP content in the eluates was monitored with an IN/US systems Bram‐4 in‐line β‐detector. IP3 or IP4 retention times were determined by spiking [3H] IP3 or [3H] IP4 into unlabeled cell extracts (not shown). IP3′ represents Ins(1,3,4)P3, an IP3 isomer originating from IP4 metabolism (Pouillon et al., ). IP5 represents a pool of IP5 isomers (Pouillon et al., ). (B) MHC thymocytes contain ≥98% DP cells, shown by FACS analysis of CD4 and CD8 expression on total thymocytes from 6‐week‐old C57BL/6 wild type (wt) or MHCIMHCII (MHC) mice. The two‐dimensional plots indicate CD4 ( y‐axis) or CD8 ( x‐axis) fluorescence intensity for individual cells (dots). The numbers indicate % cells in the respective quadrant.
  •   FigureFigure 11.1.3 Analysis of thymocyte populations pre‐ and post‐anti‐CD53 AB sort. Two‐dimensional plots showing CD4 ( y‐axis) and CD8 ( x‐axis) fluorescence intensities for individual thymocytes (dots) from 6‐week‐old C57BL/6 mice before (pre‐sort) or after (post‐CD53 sort) depletion of CD53+ cells. The numbers indicate percent cells in the respective quadrant.
  •   FigureFigure 11.1.4 A sample trace obtained from Jurkat cells labeled with myo‐[3H] inositol and stimulated for 5 min with OKT3 and αCD28 (1 µg/ml). The inositol phosphate isomers detected are indicated. The peaks corresponding to Ins(1,4,5)P3 and Ins(1,3,4,5)P4 were verified with [3H]‐labeled purified standards (Perkin‐Elmer).
  •   FigureFigure 11.1.5 MDD‐HPLC analysis of phytic acid hydrolysis products. (A) Elution profile. Peak identities were determined by comparison with the retention times for external standards (not shown). Peak 1 (retention time of 6.77 min) contains IP2 isomers, peak 2 (10.9 min) contains I(1,3,4)P3 and I(1,4,5)P3, peak 3 (11.23 min) contains D/L‐I(1,5,6)P3, peak 4 (11.89 min) contains I(4,5,6)P3, peak 5 (13.63 min) contains I(1,2,3,5)P4 and I(1,2,4,6)P4, peak 6 (13.79 min) contains I(1,2,3,4)P4 and I(1,3,4,6)P4, peak 7 (13.91 min) contains I(1,2,4,5)P4 and I(1,3,4,5)P4, peak 8 (14.41 min) contains I(1,2,5,6)P4, peak 9 (14.78 min) contains I(2,4,5,6)P4, peak 10 (15.31 min) contains I(1/3,4,5,6)P4, peak 11 (15.83 min ) contains D/L I(1,2,3,4,6)P5, peak 12 (16.31 min) contains D/L I(1,2,3,4,3)P5, peak 13 (17.04 min) contains D/L I(1,2,4,5,6)P5, peak 14 (17.32 min) contains I(1,3,4,5,6)P5, and peak 15 (18.97 min) contains I(1,2,3,4,5,6)P6 (unhydrolyzed phytic acid). (B) Calibration curve obtained with known amounts of an IP6 external standard.
  •   FigureFigure 11.1.6 MDD‐HPLC analysis of soluble inositol phosphate isomers in Jurkat T cells. The MDD‐HPLC method has been applied in a number of studies to analyze IPs in cells and tissues, including human Jurkat T cells (Guse et al., ). In the example shown here, soluble IPs were extracted from ∼5 × 107 unstimulated or 10 µg/ml OKT3‐stimulated Jurkat T cells. (A) a, separation of an IP standard mixture containing 554 pmol I(1,4,5)P3 (peak 1), 43 pmol I(1,2,3,5)P4 (peak 2), 113 pmol I(1,3,4,6)P4 (peak 3), 217 pmol I(1,3,4,5)P4 (peak 4), 116 pmol I(1,4,5,6)P4 (peak 5), 300 pmol I(1,2,3,4,6)P5 (peak 6), 20 pmol I(1,2,4,5,6)P5 (peak 7), 415 pmol I(1,3,4,5,6)P5 (peak 8), 646 pmol IP6 (peak 9), and 215 pmol PP‐IP5 (peak 10). b‐e, samples from unstimulated (b), 3 (c), 6 (d), or 20 min (e) OKT3‐stimulated Jurkat cells. (B) Quantified amounts of the indicated IPs in the Jurkat cell samples from A. *, p < 0.01, obtained via Student's t‐test.

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Literature Cited

Literature Cited
   Adelt, S., Plettenburg, O., Stricker, R., Reiser, G., Altenbach, H.J., and Vogel, G. 1999. Enzyme‐assisted total synthesis of the optical antipodes D‐myo‐inositol 3,4,5‐trisphosphate and D‐myo‐inositol 1,5,6‐trisphosphate: Aspects of their structure‐activity relationship to biologically active inositol phosphates. J. Med. Chem. 42:1262‐1273.
   Alcazar‐Roman, A.R. and Wente, S.R. 2008. Inositol polyphosphates: A new frontier for regulating gene expression. Chromosoma 117:1‐13.
   Amaro, R., Escalona, A., and Murillo, M. 2004. HPLC with inductively coupled plasma optical emission spectrometric detection for the analysis of inositol phosphates. J. Chromatogr. Sci. 42:491‐494.
   Andrews, W.V. and Conn, P.M. 1987. Measurement of inositol phospholipid metabolites by one‐dimensional thin‐layer chromatography. Methods Enzymol. 141:156‐168.
   Astoul, E., Edmunds, C., Cantrell, D.A., and Ward, S.G. 2001. PI 3‐K and T‐cell activation: Limitations of T‐leukemic cell lines as signaling models. Trends Immunol. 22:490‐496.
   Azevedo, C. and Saiardi, A. 2006. Extraction and analysis of soluble inositol polyphosphates from yeast. Nat. Protoc. 1:2416‐2422.
   Balla, T. and Varnai, P. 2002. Visualizing cellular phosphoinositide pools with GFP‐fused protein‐modules. Sci. STKE 2002:PL3.
   Barouch‐Bentov, R., Che, J., Lee, C.C., Yang, Y., Herman, A., Jia, Y., Velentza, A., Watson, J., Sternberg, L., Kim, S., Ziaee, N., Miller, A., Jackson, C., Fujimoto, M., Young, M., Batalov, S., Liu, Y., Warmuth, M., Wiltshire, T., Cooke, M.P., and Sauer, K. 2009. A conserved salt bridge in the G loop of multiple protein kinases is important for catalysis and for in vivo lyn function. Mol. Cell 33:43‐52.
   Benjamin, E.R., Haftl, S.L., Xanthos, D.N., Crumley, G., Hachicha, M., and Valenzano, K.J. 2004. A miniaturized column chromatography method for measuring receptor‐mediated inositol phosphate accumulation. J. Biomol. Screen. 9:343‐353.
   Berg, L.J., Finkelstein, L.D., Lucas, J.A., and Schwartzberg, P.L. 2005. Tec family kinases in T lymphocyte development and function. Annu. Rev. Immunol. 23:549‐600.
   Berridge, M.J., Dawson, R.M., Downes, C.P., Heslop, J.P., and Irvine, R.F. 1983. Changes in the levels of inositol phosphates after agonist‐dependent hydrolysis of membrane phosphoinositides. Biochem. J. 212:473‐482.
   Berrie, C.P., Iurisci, C., Piccolo, E., Bagnati, R., and Corda, D. 2007. Analysis of phosphoinositides and their aqueous metabolites. Methods Enzymol. 434:187‐232.
   Boldyreff, B., Rasmussen, T.L., Jensen, H.H., Cloutier, A., Beaudet, L., Roby, P., and Issinger, O.G. 2008. Expression and purification of PI3 kinase {alpha} and development of an ATP depletion and an AlphaScreen PI3 kinase activity assay. J. Biomol. Screen. 13:1035‐1040.
   Brandish, P.E., Hill, L.A., Zheng, W., and Scolnick, E.M. 2003. Scintillation proximity assay of inositol phosphates in cell extracts: High‐throughput measurement of G‐protein‐coupled receptor activation. Anal. Biochem. 313:311‐318.
   Budd, R.C., Winslow, G., Inokuchi, S., and Imboden, J.B. 1990. Intact antigen receptor‐mediated generation of inositol phosphates and increased intracellular calcium in CD4 CD8 T lymphocytes from MRL lpr mice. J. Immunol. 145:2862‐2872.
   Butterfield, S.M., Tran, D.H., Zhang, H., Prestwich, G.D., and Matile, S. 2008. Fluorometric detection of inositol phosphates and the activity of their enzymes with synthetic pores: Discrimination of IP7 and IP6 and phytate sensing in complex matrices. J. Am. Chem. Soc. 130:3270‐3271.
   Cante‐Barrett, K., Gallo, E.M., Winslow, M.M., and Crabtree, G.R. 2006. Thymocyte negative selection is mediated by protein kinase C‐ and Ca2+‐dependent transcriptional induction of bim. J. Immunol. 176:2299‐2306.
   Casals, I., Villar, J.L., and Riera‐Codina, M. 2002. A straightforward method for analysis of highly phosphorylated inositols in blood cells by high‐performance liquid chromatography. Anal. Biochem. 300:69‐76.
   Challiss, R.A., Batty, I.H., and Nahorski, S.R. 1988. Mass measurements of inositol(1,4,5)trisphosphate in rat cerebral cortex slices using a radioreceptor assay: Effects of neurotransmitters and depolarization. Biochem. Biophys. Res. Commun. 157:684‐691.
   Chang, Y.T., Choi, G., Bae, Y.S., Burdett, M., Moon, H.S., Lee, J.W., Gray, N.S., Schultz, P.G., Meijer, L., Chung, S.K., Choi, K.Y., Suh, P.G., and Ryu, S.H. 2002. Purine‐based inhibitors of inositol‐1,4,5‐trisphosphate‐3‐kinase. Chembiochem 3:897‐901.
   Chengalvala, M., Kostek, B., and Frail, D.E. 1999. A multi‐well filtration assay for quantitation of inositol phosphates in biological samples. J. Biochem. Biophys. Methods 38:163‐170.
   Cicchetti, G., Biernacki, M., Farquharson, J., and Allen, P.G. 2004. A ratiometric expressible FRET sensor for phosphoinositides displays a signal change in highly dynamic membrane structures in fibroblasts. Biochemistry 43:1939‐1949.
   Cunha‐Melo, J.R., Dean, N.M., Moyer, J.D., Maeyama, K., and Beaven, M.A. 1987. The kinetics of phosphoinositide hydrolysis in rat basophilic leukemia (RBL‐2H3) cells varies with the type of IgE receptor cross‐linking agent used. J. Biol. Chem. 262:11455‐11463.
   Cunha‐Melo, J.R., Dean, N.M., Ali, H., and Beaven, M.A. 1988. Formation of inositol 1,4,5‐trisphosphate and inositol 1,3,4‐trisphosphate from inositol 1,3,4,5‐tetrakisphosphate and their pathways of degradation in RBL‐2H3 cells. J. Biol. Chem. 263:14245‐14250.
   Dean, N.M. and Beaven, M.A. 1989. Methods for the analysis of inositol phosphates. Anal. Biochem. 183:199‐209.
   Donie, F. and Reiser, G. 1989. A novel, specific binding protein assay for quantitation of intracellular inositol 1,3,4,5‐tetrakisphosphate (InsP4) using a high‐affinity InsP4 receptor from cerebellum. FEBS Lett. 254:155‐158.
   Drees, B.E., Weipert, A., Hudson, H., Ferguson, C.G., Chakravarty, L., and Prestwich, G.D. 2003. Competitive fluorescence polarization assays for the detection of phosphoinositide kinase and phosphatase activity. Comb. Chem. High Throughput Screen. 6:321‐330.
   Esty, A. 1991. Receptor‐specific serum‐free cell attachment using a highly stable engineered protein polymer. Am. Biotechnol. Lab. 9:44.
   Feske, S. 2007. Calcium signaling in lymphocyte activation and disease. Nat. Rev. Immunol. 7:690‐702.
   Folch, J. 1949. Complete fractionation of brain cephalin; isolation from it of phosphatidyl serine, phosphatidyl ethanolamine, and diphosphoinositide. J. Biol. Chem. 177:497‐504.
   Fox, C.J., Hammerman, P.S., and Thompson, C.B. 2005. Fuel feeds function: Energy metabolism and the T‐cell response. Nat. Rev. Immunol. 5:844‐852.
   Freeburn, R.W., Wright, K.L., Burgess, S.J., Astoul, E., Cantrell, D.A., and Ward, S.G. 2002. Evidence that SHIP‐1 contributes to phosphatidylinositol 3,4,5‐trisphosphate metabolism in T lymphocytes and can regulate novel phosphoinositide 3‐kinase effectors. J. Immunol. 169:5441‐5450.
   Fruman, D.A. 2004. Phosphoinositide 3‐kinase and its targets in B‐cell and T‐cell signaling. Curr. Opin. Immunol. 16:314‐320.
   Gray, A., Olsson, H., Batty, I.H., Priganica, L., and Peter Downes, C. 2003. Nonradioactive methods for the assay of phosphoinositide 3‐kinases and phosphoinositide phosphatases and selective detection of signaling lipids in cell and tissue extracts. Anal. Biochem. 313:234‐245.
   Grusby, M.J., Auchincloss, H. Jr., Lee, R., Johnson, R.S., Spencer, J.P., Zijlstra, M., Jaenisch, R., Papaioannou, V.E., and Glimcher, L.H. 1993. Mice lacking major histocompatibility complex class I and class II molecules. Proc. Natl. Acad. Sci. U.S.A. 90:3913‐3917.
   Guillou, H., Lecureuil, C., Anderson, K.E., Suire, S., Ferguson, G.J., Ellson, C.D., Gray, A., Divecha, N., Hawkins, P.T., and Stephens, L.R. 2007a. Use of the GRP1 PH domain as a tool to measure the relative levels of PtdIns(3,4,5)P3 through a protein‐lipid overlay approach. J. Lipid Res. 48:726‐732.
   Guillou, H., Stephens, L.R., and Hawkins, P.T. 2007b. Quantitative measurement of phosphatidylinositol 3,4,5‐trisphosphate. Methods Enzymol. 434:117‐130.
   Guse, A.H. and Emmrich, F. 1991. T cell receptor–mediated metabolism of inositol polyphosphates in Jurkat T‐lymphocytes. Identification of a D‐myo‐inositol 1,2,3,4,6‐pentakisphosphate‐2‐phosphomonoesterase activity, a D‐myo‐inositol 1,3,4,5,6‐pentakisphosphate‐1/3‐phosphatase activity and a D/L‐myo‐inositol 1,2,4,5,6‐pentakisphosphate‐1/3‐kinase activity. J. Biol. Chem. 266:24498‐24502.
   Guse, A.H. and Emmrich, F. 1992. Determination of inositol polyphosphates from human T‐lymphocyte cell lines by anion‐exchange high‐performance liquid chromatography and post‐column derivatization. J. Chromatogr. 593:157‐163.
   Guse, A.H., Roth, E., Broker, B.M., and Emmrich, F. 1992. Complex inositol polyphosphate response induced by co‐cross‐linking of CD4 and Fc gamma receptors in the human monocytoid cell line U937. J. Immunol. 149:2452‐2458.
   Guse, A.H., Greiner, E., Emmrich, F., and Brand, K. 1993. Mass changes of inositol 1,3,4,5,6‐pentakisphosphate and inositol hexakisphosphate during cell cycle progression in rat thymocytes. J. Biol. Chem. 268:7129‐7133.
   Guse, A.H., da Silva, C.P., Emmrich, F., Ashamu, G.A., Potter, B.V., and Mayr, G.W. 1995a. Characterization of cyclic adenosine diphosphate‐ribose‐induced Ca2+ release in T lymphocyte cell lines. J. Immunol. 155:3353‐3359.
   Guse, A.H., Goldwich, A., Weber, K., and Mayr, G.W. 1995b. Non‐radioactive, isomer‐specific inositol phosphate mass determinations: High‐performance liquid chromatography‐micro‐metal‐dye detection strongly improves speed and sensitivity of analyses from cells and micro‐enzyme assays. J. Chromatogr. B Biomed. Appl. 672:189‐198.
   Ham, R.G. and McKeehan, W.L. 1979. Media and growth requirements. Methods Enzymol. 58:44‐93.
   Hatzack, F. and Rasmussen, S.K. 1999. High‐performance thin‐layer chromatography method for inositol phosphate analysis. J. Chromatogr. B Biomed. Sci. Appl. 736:221‐229.
   Holz, R.W., Hlubek, M.D., Sorensen, S.D., Fisher, S.K., Balla, T., Ozaki, S., Prestwich, G.D., Stuenkel, E.L., and Bittner, M.A. 2000. A pleckstrin homology domain specific for phosphatidylinositol 4, 5‐bisphosphate (PtdIns‐4,5‐P2) and fused to green fluorescent protein identifies plasma membrane PtdIns‐4,5‐P2 as being important in exocytosis. J. Biol. Chem. 275:17878‐17885.
   Horn, S., Endl, E., Fehse, B., Weck, M.M., Mayr, G.W., and Jucker, M. 2004. Restoration of SHIP activity in a human leukemia cell line downregulates constitutively activated phosphatidylinositol 3‐kinase/Akt/GSK‐3b signaling and leads to an increased transit time through the G1 phase of the cell cycle. Leukemia. 18:1839‐1849.
   Huang, Y.H., Grasis, J.A., Miller, A.T., Xu, R., Soonthornvacharin, S., Andreotti, A.H., Tsoukas, C.D., Cooke, M.P., and Sauer, K. 2007. Positive regulation of Itk PH domain function by soluble IP4. Science 316:886‐889.
   Huang, Y.H., Hoebe, K., and Sauer, K. 2008. New therapeutic targets in immune disorders: ItpkB, Orai1 and UNC93B. Expert Opin. Ther. Targets 12:391‐413.
   Imai, A. and Gershengorn, M.C. 1987. Measurement of lipid turnover in response to thyrotropin‐releasing hormone. Methods Enzymol. 141:100‐101.
   Imboden, J.B. and Pattison, G. 1987. Regulation of inositol 1,4,5‐trisphosphate kinase activity after stimulation of human T cell antigen receptor. J. Clin. Invest. 79:1538‐1541.
   Imboden, J.B. and Stobo, J.D. 1985. Transmembrane signaling by the T cell antigen receptor. Perturbation of the T3‐antigen receptor complex generates inositol phosphates and releases calcium ions from intracellular stores. J. Exp. Med. 161:446‐456.
   Imboden, J., Weyand, C., and Goronzy, J. 1987. Antigen recognition by a human T cell clone leads to increases in inositol trisphosphate. J. Immunol. 138:1322‐1324.
   Inokuchi, S. and Imboden, J.B. 1990. Antigen receptor‐mediated regulation of sustained polyphosphoinositide turnover in a human T cell line. Evidence for a receptor‐regulated pathway for production of phosphatidylinositol 4,5‐bisphosphate. J. Biol. Chem. 265:5983‐5989.
   Irvine, R.F. 1986. The structure, metabolism, and analysis of inositol lipids and inositol phosphates. In Receptor Biochemistry and Methodology. pp. 89‐108. (J.W. Putney, ed.). Wiley‐Liss, New York.
   Irvine, R.F. 1990. Methods in Inositide Research. Raven Press, New York.
   Irvine, R.F. 2001. Inositol phosphates: Does IP(4) run a protection racket? Curr. Biol. 11:R172‐R174.
   Irvine, R.F. 2003. Nuclear lipid signaling. Nat. Rev. Mol. Cell Biol. 4:349‐360.
   Irvine, R.F. 2005. Inositide evolution—Towards turtle domination? J. Physiol. 566:295‐300.
   Irvine, R.F. 2006. Nuclear inositide signaling—Expansion, structures and clarification. Biochim. Biophys. Acta 1761:505‐508.
   Irvine, R.F. 2007. Cell signaling. The art of the soluble. Science 316:845‐846.
   Irvine, R.F. and Schell, M.J. 2001. Back in the water: The return of the inositol phosphates. Nat. Rev. Mol. Cell Biol. 2:327‐338.
   Irvine, R.F., Letcher, A.J., Heslop, J.P., and Berridge, M.J. 1986. The inositol tris/tetrakisphosphate pathway—Demonstration of Ins(1,4,5)P3 3‐kinase activity in animal tissues. Nature 320:631‐634.
   Irvine, R.F., Lloyd‐Burton, S.M., Yu, J.C., Letcher, A.J., and Schell, M.J. 2006. The regulation and function of inositol 1,4,5‐trisphosphate 3‐kinases. Adv. Enzyme Regul. 46:314‐323.
   Jenkinson, S. 1995. Separation of labeled inositol phosphate isomers by high‐pressure liquid chromatography (HPLC). Methods Mol. Biol. 41:151‐165.
   Jia, Y., Subramanian, K.K., Erneux, C., Pouillon, V., Hattori, H., Jo, H., You, J., Zhu, D., Schurmans, S., and Luo, H.R. 2007. Inositol 1,3,4,5‐tetrakisphosphate negatively regulates phosphatidylinositol‐3,4,5‐trisphosphate signaling in neutrophils. Immunity 27:453‐467.
   Jia, Y., Loison, F., Hattori, H., Li, Y., Erneux, C., Park, S.Y., Gao, C., Chai, L., Silberstein, L.E., Schurmans, S., and Luo, H.R. 2008. Inositol trisphosphate 3‐kinase B (InsP3KB) as a physiological modulator of myelopoiesis. Proc. Natl. Acad. Sci. U.S.A. 105:4739‐4744.
   Jodi, L., Buckler, X.L., Laurence, A., and Turka, L.A. 2008. Regulation of T cell responses by PTEN. Immunol. Rev. 224:239‐248.
   Johnson, C.M., Chichili, G.R., and Rodgers, W. 2008. Compartmentalization of phosphatidylinositol 4,5‐bisphosphate signaling evidenced using targeted phosphatases. J. Biol. Chem. 283:29920‐29928.
   Jones, R.G. and Thompson, C.B. 2007. Revving the engine: Signal transduction fuels T cell activation. Immunity 27:173‐178.
   Juntilla, M.M. and Koretzky, G.A. 2008. Critical roles of the PI3K/Akt signaling pathway in T cell development. Immunol. Lett. 116:104‐110.
   Krauss, S., Brand, M.D., and Buttgereit, F. 2001. Signaling takes a breath—New quantitative perspectives on bioenergetics and signal transduction. Immunity 15:497‐502.
   Kuksis, A. 2003. Inositol Phospholipid Metabolism and Phosphatidyl Inositol Kinases, vol. 30. Elsevier, New York.
   Lapetina, E.G. and Siess, W. 1987. Measurement of inositol phospholipid turnover in platelets. Methods Enzymol. 141:176‐192.
   Lin, H., Fridy, P.C., Ribeiro, A.A., Choi, J.H., Barma, D.K., Vogel, G., Falck, J.R., Shears, S.B., York, J.D., and Mayr, G.W. 2009. Structural analysis and detection of biological inositol pyrophosphates reveal that the family of VIP/diphosphoinositol pentakisphosphate kinases are 1/3‐kinases. J. Biol. Chem. 284:1863‐1872.
   Liu, J.J., Hartman, D.S., and Bostwick, J.R. 2003. An immobilized metal ion affinity adsorption and scintillation proximity assay for receptor‐stimulated phosphoinositide hydrolysis. Anal. Biochem. 318:91‐99.
   Lorke, D.E., Gustke, H., and Mayr, G.W. 2004. An optimized fixation and extraction technique for high resolution of inositol phosphate signals in rodent brain. Neurochem. Res. 29:1887‐1896.
   Majerus, P.W., Zou, J., Marjanovic, J., Kisseleva, M.V., and Wilson, M.P. 2008. The role of inositol signaling in the control of apoptosis. Adv. Enzyme Regul. 48:10‐17.
   Marechal, Y., Pesesse, X., Jia, Y., Pouillon, V., Perez‐Morga, D., Daniel, J., Izui, S., Cullen, P.J., Leo, O., Luo, H.R., Erneux, C., and Schurmans, S. 2007. Inositol 1,3,4,5‐tetrakisphosphate controls proapoptotic Bim gene expression and survival in B cells. Proc. Natl. Acad. Sci. U.S.A. 104:13978‐13983.
   Mayr, G.W. 1988. A novel metal‐dye detection system permits picomolar‐range HPLC analysis of inositol polyphosphates from non‐radioactively labeled cell or tissue specimens. Biochem. J. 254:585‐591.
   Miller, A.T., Sandberg, M., Huang, Y.H., Young, M., Sutton, S., Sauer, K., and Cooke, M.P. 2007. Production of Ins(1,3,4,5)P(4) mediated by the kinase Itpkb inhibits store‐operated calcium channels and regulates B cell selection and activation. Nat. Immunol. 8:514‐521.
   Miller, A.T., Chamberlain, P.P., and Cooke, M.P. 2008. Beyond IP3: Roles for higher order inositol phosphates in immune cell signaling. Cell Cycle 7:463‐467.
   Mueller, P., Massner, J., Jayachandran, R., Combaluzier, B., Albrecht, I., Gatfield, J., Blum, C., Ceredig, R., Rodewald, H.R., Rolink, A.G., and Pieters, J. 2008. Regulation of T cell survival through coronin‐1‐mediated generation of inositol‐1,4,5‐trisphosphate and calcium mobilization after T cell receptor triggering. Nat. Immunol. 9:424‐431.
   Mustelin, T., Poso, H., Iivanainen, A., and Andersson, L.C. 1986. myo‐Inositol reverses Li+‐induced inhibition of phosphoinositide turnover and ornithine decarboxylase induction during early lymphocyte activation. Eur. J. Immunol. 16:859‐861.
   Nasuhoglu, C., Feng, S., Mao, J., Yamamoto, M., Yin, H.L., Earnest, S., Barylko, B., Albanesi, J.P., and Hilgemann, D.W. 2002. Nonradioactive analysis of phosphatidylinositides and other anionic phospholipids by anion‐exchange high‐performance liquid chromatography with suppressed conductivity detection. Anal. Biochem. 301:243‐254.
   Oatey, P.B., Venkateswarlu, K., Williams, A.G., Fletcher, L.M., Foulstone, E.J., Cullen, P.J., and Tavare, J.M. 1999. Confocal imaging of the subcellular distribution of phosphatidylinositol 3,4,5‐trisphosphate in insulin‐ and PDGF‐stimulated 3T3‐L1 adipocytes. Biochem. J. 344:511‐518.
   Otto, J.C., Mulugu, S., Fridy, P.C., Chiou, S.T., Armbruster, B.N., Ribeiro, A.A., and York, J.D. 2007. Biochemical analysis of inositol phosphate kinases. Methods Enzymol. 434:171‐185.
   Palmer, S. and Wakelam, M.J. 1989. Mass measurement of inositol phosphates. Biochim. Biophys. Acta 1014:239‐246.
   Park, T.J., Song, S.K., and Kim, K.T. 1997. A2A adenosine receptors inhibit ATP‐induced Ca2+ influx in PC12 cells by involving protein kinase A. J. Neurochem. 68:2177‐2185.
   Parmryd, I., Adler, J., Patel, R., and Magee, A.I. 2003. Imaging metabolism of phosphatidylinositol 4,5‐bisphosphate in T cell GM1‐enriched domains containing Ras proteins. Exp. Cell Res. 285:27‐38.
   Pettitt, T.R., Dove, S.K., Lubben, A., Calaminus, S.D., and Wakelam, M.J. 2006. Analysis of intact phosphoinositides in biological samples. J. Lipid Res. 47:1588‐1596.
   Phillippy, B.Q., White, K.D., Johnston, M.R., Tao, S.H., and Fox, M.R. 1987. Preparation of inositol phosphates from sodium phytate by enzymatic and nonenzymatic hydrolysis. Anal. Biochem. 162:115‐121.
   Pouillon, V., Hascakova‐Bartova, R., Pajak, B., Adam, E., Bex, F., Dewaste, V., Van Lint, C., Leo, O., Erneux, C., and Schurmans, S. 2003. Inositol 1,3,4,5‐tetrakisphosphate is essential for T lymphocyte development. Nat. Immunol. 4:1136‐1143.
   Prestwich, G.D. 2004. Phosphoinositide signaling: From affinity probes to pharmaceutical targets. Chem. Biol. 11:619‐637.
   Prestwich, G.D. 2005. Visualization and perturbation of phosphoinositide and phospholipid signaling. Prostaglandins Other Lipid Mediat. 77:168‐178.
   Puls, K.L., Hogquist, K.A., Reilly, N., and Wright, M.D. 2002. CD53, a thymocyte selection marker whose induction requires a lower affinity TCR‐MHC interaction than CD69, but is up‐regulated with slower kinetics. Int. Immunol. 14:249‐258.
   Rathmell, J.C., Vander Heiden, M.G., Harris, M.H., Frauwirth, K.A., and Thompson, C.B. 2000. In the absence of extrinsic signals, nutrient utilization by lymphocytes is insufficient to maintain either cell size or viability. Mol. Cell 6:683‐692.
   Resnick, A.C. and Saiardi, A. 2008. Inositol polyphosphate multikinase: Metabolic architect of nuclear inositides. Front. Biosci. 13:856‐866.
   Rusten, T.E. and Stenmark, H. 2006. Analyzing phosphoinositides and their interacting proteins. Nat. Methods 3:251‐258.
   Saiardi, A., Bhandari, R., Resnick, A.C., Snowman, A.M., and Snyder, S.H. 2004. Phosphorylation of proteins by inositol pyrophosphates. Science 306:2101‐2105.
   Sakaguchi, R., Endoh, T., Yamamoto, S., Tainaka, K., Sugimoto, K., Fujieda, N., Kiyonaka, S., Mori, Y., and Morii, T. 2009. A single circularly permuted GFP sensor for inositol‐1,3,4,5‐tetrakisphosphate based on a split PH domain. Bioorg. Med. Chem. 17:7381‐7386.
   Sato, M., Ueda, Y., Takagi, T., and Umezawa, Y. 2003. Production of PtdInsP3 at endomembranes is triggered by receptor endocytosis. Nat. Cell Biol. 5:1016‐1022.
   Schenk, U., Westendorf, A.M., Radaelli, E., Casati, A., Ferro, M., Fumagalli, M., Verderio, C., Buer, J., Scanziani, E., and Grassi, F. 2008. Purinergic control of T cell activation by ATP released through pannexin‐1 hemichannels. Sci. Signal 1:ra6.
   Schwartzberg, P.L., Finkelstein, L.D., and Readinger, J.A. 2005. TEC‐family kinases: Regulators of T‐helper‐cell differentiation. Nat. Rev. Immunol. 5:284‐295.
   Seeds, A.M. and York, J.D. 2007. Inositol polyphosphate kinases: Regulators of nuclear function. Biochem. Soc. Symp. 2007:183‐197.
   Seeds, A.M., Frederick, J.P., Tsui, M.M., and York, J.D. 2007. Roles for inositol polyphosphate kinases in the regulation of nuclear processes and developmental biology. Adv. Enzyme Regul. 47:10‐25.
   Sergeant, S. and McPhail, L.C. 2007. Measurement of phospholipid metabolism in intact neutrophils. Methods Mol. Biol. 412:69‐83.
   Shan, X., Czar, M.J., Bunnell, S.C., Liu, P., Liu, Y., Schwartzberg, P.L., and Wange, R.L. 2000. Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation. Mol. Cell Biol. 20:6945‐6957.
   Shears, S.B. 2007. Understanding the biological significance of diphosphoinositol polyphosphates (‘inositol pyrophosphates’). Biochem. Soc. Symp. 2007:211‐221.
   Singh, A.K. and Jiang, Y. 1995. Quantitative chromatographic analysis of inositol phospholipids and related compounds. J. Chromatogr. B Biomed. Appl. 671:255‐280.
   Skippen, A., Swigart, P., and Cockcroft, S. 2006. Measurement of phospholipase C by monitoring inositol phosphates using [3H]‐inositol‐labeling protocols in permeabilized cells. Methods Mol. Biol. 312:183‐193.
   Sommers, C.L., Samelson, L.E., and Love, P.E. 2004. LAT: A T lymphocyte adapter protein that couples the antigen receptor to downstream signaling pathways. Bioessays 26:61‐67.
   Starr, T.K., Jameson, S.C., and Hogquist, K.A. 2003. Positive and negative selection of T cells. Annu. Rev. Immunol. 21:139‐176.
   Stevenson‐Paulik, J., Chiou, S.T., Frederick, J.P., dela Cruz, J., Seeds, A.M., Otto, J.C., and York, J.D. 2006. Inositol phosphate metabolomics: Merging genetic perturbation with modernized radiolabeling methods. Methods 39:112‐121.
   Stewart, S.J., Prpic, V., Powers, F.S., Bocckino, S.B., Isaacks, R.E., and Exton, J.H. 1986. Perturbation of the human T cell antigen receptor‐T3 complex leads to the production of inositol tetrakisphosphate: Evidence for conversion from inositol trisphosphate. Proc. Natl. Acad. Sci. U.S.A. 83:6098‐6102.
   Stewart, S.J., Kelley, L.L., and Powers, F.S. 1987. Production of inositol pentakisphosphate in a human T lymphocyte cell line. Biochem. Biophys. Res. Commun. 145:895‐902.
   Stokes, A.J., Shimoda, L.M., Lee, J.W., Rillero, C., Chang, Y.T., and Turner, H. 2006. Fcepsilon RI control of Ras via inositol (1,4,5) trisphosphate 3‐kinase and inositol tetrakisphosphate. Cell Signal 18:640‐651.
   Takazawa, K., Lemos, M., Delvaux, A., Lejeune, C., Dumont, J.E., and Erneux, C. 1990. Rat brain inositol 1,4,5‐trisphosphate 3‐kinase. Ca2(+)‐sensitivity, purification and antibody production. Biochem. J. 268:213‐217.
   Ullman, E.F., Kirakossian, H., Switchenko, A.C., Ishkanian, J., Ericson, M., Wartchow, C.A., Pirio, M., Pease, J., Irvin, B.R., Singh, S., Singh, R., Patel, R., Dafforn, A., Davalian, D., Skold, C., Kurn, N., and Wagner, D.B. 1996. Luminescent oxygen channeling assay (LOCI): Sensitive, broadly applicable homogeneous immunoassay method. Clin. Chem. 42:1518‐1526.
   van der Kaay, J., Batty, I.H., Cross, D.A., Watt, P.W., and Downes, C.P. 1997. A novel, rapid, and highly sensitive mass assay for phosphatidylinositol 3,4,5‐trisphosphate (PtdIns(3,4,5)P3) and its application to measure insulin‐stimulated PtdIns(3,4,5)P3 production in rat skeletal muscle in vivo. J. Biol. Chem. 272:5477‐5481.
   van der Kaay, J., Cullen, P.J., and Downes, C.P. 1998. Phosphatidylinositol(3,4,5)trisphosphate (Ptdins(3,4,5)P3) mass measurement using a radioligand displacement assay. Methods Mol. Biol. 105:109‐125.
   Vats, P., Bhushan, B., Chakarborti, A.K., and Banerjee, U.C. 2008. Separation and identification of enzymatically prepared dephosphorylated products of myo‐inositolhexakisphosphate using LC‐MS. J. Sep. Sci. 31:3829‐3833.
   Verhoeven, A.J., Tysnes, O.B., Horvli, O., Cook, C.A., and Holmsen, H. 1987. Stimulation of phosphate uptake in human platelets by thrombin and collagen. Changes in specific 32P labeling of metabolic ATP and polyphosphoinositides. J. Biol. Chem. 262:7047‐7052.
   Weernink, P.A.O., Schulte, P., Guo, Y., Wetzel, J., Amano, M., Kaibuchi, K., Haverland, S., Vo, M., Schmidt, M., Mayr, G.W., and Jakobs, K.H. 2000. Stimulation of phosphatidylinositol‐4‐phosphate 5‐kinase by rho‐kinase. J. Biol. Chem. 275:10168‐10174.
   Wen, B.G., Pletcher, M.T., Warashina, M., Choe, S.H., Ziaee, N., Wiltshire, T., Sauer, K., and Cooke, M.P. 2004. Inositol (1,4,5) trisphosphate 3 kinase B controls positive selection of T cells and modulates Erk activity. Proc. Natl. Acad. Sci. U.S.A. 101:5604‐5609.
   Wenk, M.R., Lucast, L., Di Paolo, G., Romanelli, A.J., Suchy, S.F., Nussbaum, R.L., Cline, G.W., Shulman, G.I., McMurray, W., and De Camilli, P. 2003. Phosphoinositide profiling in complex lipid mixtures using electrospray ionization mass spectrometry. Nat. Biotechnol. 21:813‐817.
   Williams, A. and Frasca, V. 1998. Ion‐exchange chromatography. Curr. Protoc. Mol. Biol. 44:10.10.1‐10.10.30.
   Wreggett, K.A., Howe, L.R., Moore, J.P., and Irvine, R.F. 1987. Extraction and recovery of inositol phosphates from tissues. Biochem. J. 245:933‐934.
   Wreggett, K.A., Lander, D.J., and Irvine, R.F. 1990. Two‐stage analysis of radiolabeled inositol phosphate isomers. Methods Enzymol. 191:707‐718.
   Zeidman, R., Lofgren, B., Pahlman, S., and Larsson, C. 1999. PKCepsilon, via its regulatory domain and independently of its catalytic domain, induces neurite‐like processes in neuroblastoma cells. J. Cell Biol. 145:713‐726.
   Zheng, W., Brandish, P.E., Kolodin, D.G., Scolnick, E.M., and Strulovici, B. 2004. High‐throughput cell‐based screening using scintillation proximity assay for the discovery of inositol phosphatase inhibitors. J. Biomol. Screen. 9:132‐140.
   Zilberman, Y., Howe, L.R., Moore, J.P., Hesketh, T.R., and Metcalfe, J.C. 1987. Calcium regulates inositol 1,3,4,5‐tetrakisphosphate production in lysed thymocytes and in intact cells stimulated with concanavalin A. Embo J. 6:957‐962.
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