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• W2029207608 abstract "Metabolism of inositol 1,4,5-trisphosphate (I(1,4,5)P3) results in the production of diverse arrays of inositol polyphosphates (IPs), such as IP4, IP5, IP6, and PP-IP5. Insights into their synthesis in metazoans are reported here through molecular studies in the fruit fly, Drosophila melanogaster. Two I(1,4,5)P3 kinase gene products are implicated in initiating catabolism of these important IP regulators. We find dmIpk2 is a nucleocytoplasmic 6-/3-kinase that converts I(1,4,5)P3 to I(1,3,4,5,6)P5, and harbors 5-kinase activity toward I(1,3,4,6)P4, and dmIP3K is a 3-kinase that converts I(1,4,5)P3 to I(1,3,4,5)P4. To assess their relative roles in the cellular production of IPs we utilized complementation analysis, RNA interference, and overexpression studies. Heterologous expression of dmIpk2, but not dmIP3K, in ipk2 mutant yeast recapitulates phospholipase C-dependent cellular synthesis of IP6. Knockdown of dmIpk2 in Drosophila S2 cells and transgenic flies results in a significant reduction of IP6 levels; whereas depletion of dmIP3K, either α or β isoforms or both, does not decrease IP6 synthesis but instead increases its production, possibly by expanding I(1,4,5)P3 pools. Similarly, knockdown of an I(1,4,5)P3 5-phosphatase results in significant increase in dmIpk2/dmIpk1-dependent IP6 synthesis. IP6 production depends on the I(1,3,4,5,6)P5 2-kinase activity of dmIpk1 and is increased in transgenic flies overexpressing dmIpk2. Our studies reveal that phosphatase and kinase regulation of I(1,4,5)P3 metabolic pools directly impinge on higher IP synthesis, and that the major route of IP6 synthesis depends on the activities of dmIpk2 and dmIpk1, but not dmIP3K, thereby challenging the role of IP3K in the genesis of higher IP messengers. Metabolism of inositol 1,4,5-trisphosphate (I(1,4,5)P3) results in the production of diverse arrays of inositol polyphosphates (IPs), such as IP4, IP5, IP6, and PP-IP5. Insights into their synthesis in metazoans are reported here through molecular studies in the fruit fly, Drosophila melanogaster. Two I(1,4,5)P3 kinase gene products are implicated in initiating catabolism of these important IP regulators. We find dmIpk2 is a nucleocytoplasmic 6-/3-kinase that converts I(1,4,5)P3 to I(1,3,4,5,6)P5, and harbors 5-kinase activity toward I(1,3,4,6)P4, and dmIP3K is a 3-kinase that converts I(1,4,5)P3 to I(1,3,4,5)P4. To assess their relative roles in the cellular production of IPs we utilized complementation analysis, RNA interference, and overexpression studies. Heterologous expression of dmIpk2, but not dmIP3K, in ipk2 mutant yeast recapitulates phospholipase C-dependent cellular synthesis of IP6. Knockdown of dmIpk2 in Drosophila S2 cells and transgenic flies results in a significant reduction of IP6 levels; whereas depletion of dmIP3K, either α or β isoforms or both, does not decrease IP6 synthesis but instead increases its production, possibly by expanding I(1,4,5)P3 pools. Similarly, knockdown of an I(1,4,5)P3 5-phosphatase results in significant increase in dmIpk2/dmIpk1-dependent IP6 synthesis. IP6 production depends on the I(1,3,4,5,6)P5 2-kinase activity of dmIpk1 and is increased in transgenic flies overexpressing dmIpk2. Our studies reveal that phosphatase and kinase regulation of I(1,4,5)P3 metabolic pools directly impinge on higher IP synthesis, and that the major route of IP6 synthesis depends on the activities of dmIpk2 and dmIpk1, but not dmIP3K, thereby challenging the role of IP3K in the genesis of higher IP messengers. Inositol 1,4,5-trisphosphate (I(1,4,5)P3) 1The abbreviations used are: I(1,4,5)P3, inositol 1,4,5-trisphosphate; HPLC, high pressure liquid chromatography; PP-IP4, diphosphoinositol tetrakisphosphate; IP, inositol polyphosphate; IP3 inositol trisphosphate; IP4, inositol tetrakisphosphate; IP5, inositol pentakisphosphate; IP6, inositol hexakisphosphate; PP-IP5, diphosphorylinositol pentakisphosphate; DAPI, 4′,6-diamidino-2-phenylindole; PBS, phosphate-buffered saline; BSA, bovine serum albumin; HPLC, high performance liquid chromatography; GST, glutathione S-transferase; ds, double-stranded; EST, expressed sequence tag.1The abbreviations used are: I(1,4,5)P3, inositol 1,4,5-trisphosphate; HPLC, high pressure liquid chromatography; PP-IP4, diphosphoinositol tetrakisphosphate; IP, inositol polyphosphate; IP3 inositol trisphosphate; IP4, inositol tetrakisphosphate; IP5, inositol pentakisphosphate; IP6, inositol hexakisphosphate; PP-IP5, diphosphorylinositol pentakisphosphate; DAPI, 4′,6-diamidino-2-phenylindole; PBS, phosphate-buffered saline; BSA, bovine serum albumin; HPLC, high performance liquid chromatography; GST, glutathione S-transferase; ds, double-stranded; EST, expressed sequence tag. is a canonical second messenger within the inositol signaling pathway that is produced in response to a wide range of extracellular stimuli and functions to release intracellular calcium through allosteric regulation of an endoplasmic reticulum localized receptor (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6148) Google Scholar, 2Majerus P.W. Annu. Rev. Biochem. 1992; 61: 225-250Crossref PubMed Scopus (348) Google Scholar, 3Mikoshiba K. Curr. Opin. Neurobiol. 1997; 7: 339-345Crossref PubMed Scopus (160) Google Scholar). Another important role of I(1,4,5)P3 production in cells is to serve as a precursor for the synthesis of higher phosphorylated IPs that include inositol tetrakisphosphate (IP4), inositol pentakisphosphate (IP5), inositol hexakisphosphate (IP6), and inositol diphosphates (such as PP-IP4 and PP-IP5) (2Majerus P.W. Annu. Rev. Biochem. 1992; 61: 225-250Crossref PubMed Scopus (348) Google Scholar, 4Irvine R.F. Schell M.J. Nat. Rev. Mol. Cell. Biol. 2001; 2: 327-338Crossref PubMed Scopus (528) Google Scholar, 5Shears S.B. Biochim. Biophys. Acta. 1998; 1436: 49-67Crossref PubMed Scopus (151) Google Scholar). Functional studies of these IPs provide compelling evidence for their roles as messengers that regulate mRNA export, gene expression, chromatin remodeling, DNA break repair, and vesicular trafficking (5Shears S.B. Biochim. Biophys. Acta. 1998; 1436: 49-67Crossref PubMed Scopus (151) Google Scholar, 6York J.D. Bradshaw R.A. Dennis E.A. Handbook of Cell Signaling, Volume 2. 2. Elsevier Science, 2003: 229-232Google Scholar, 7York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (443) Google Scholar, 8Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (342) Google Scholar, 9Hanakahi L.A. Bartlet-Jones M. Chappell C. Pappin D. West S.C. Cell. 2000; 102: 721-729Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 10Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. J. Biol. Chem. 2000; 275: 24686-24692Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 11Shen X. Xiao H. Ranallo R. Wu W.H. Wu C. Science. 2003; 299: 112-114Crossref PubMed Scopus (291) Google Scholar, 12Steger D.J. Haswell E.S. Miller A.L. Wente S.R. O'Shea E.K. Science. 2003; 299: 114-116Crossref PubMed Scopus (313) Google Scholar). PP-IPs contain high energy inositol pyrophosphates that have been implicated in cellular events such as DNA metabolism, chemotaxis and environmental stress responses (13Menniti F.S. Miller R.N. Putney Jr., J.W. Shears S.B. J. Biol. Chem. 1993; 268: 3850-3856Abstract Full Text PDF PubMed Google Scholar, 14Luo H.R. Huang Y.E. Chen J.C. Saiardi A. Iijima M. Ye K. Huang Y. Nagata E. Devreotes P. Snyder S.H. Cell. 2003; 114: 559-572Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 15Dubois E. Scherens B. Vierendeels F. Ho M.M. Messenguy F. Shears S.B. J. Biol. Chem. 2002; 277: 23755-23763Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar).A molecular basis for the synthesis of higher IPs was first resolved in the budding yeast (Fig. 1A) (7York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (443) Google Scholar, 8Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (342) Google Scholar, 16Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. FEBS Lett. 2000; 468: 28-32Crossref PubMed Scopus (119) Google Scholar). Synthesis of I(1,4,5)P3 from PIP2 via phospholipase C (Plc1) and the subsequent action of the kinases Ipk2 (also known as Arg82) and Ipk1 produce IP6. Ipk2 is a 6-/3-/5-kinase that phosphorylates a variety of I(1,4,5)P3, IP4, and IP5 substrates (8Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (342) Google Scholar, 17Saiardi A. Erdjument-Bromage H. Snowman A.M. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 18Stevenson-Paulik J. Odom A.R. York J.D. J. Biol. Chem. 2002; 277: 42711-42718Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Ipk1 is a 2-kinase that utilizes primarily I(1,3,4,5,6)P5 but also has activity toward other IPs (7York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (443) Google Scholar, 19Ives E.B. Nichols J. Wente S.R. York J.D. J. Biol. Chem. 2000; 275: 36575-36583Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 20Verbsky J.W. Wilson M.P. Kisseleva M.V. Majerus P.W. Wente S.R. J. Biol. Chem. 2002; 277: 31857-31862Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). A third kinase, Kcs1, is an inositol diphosphate synthase capable of phosphorylating IP5 or IP6 to generate PP-IP4 and PP-IP5 (10Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. J. Biol. Chem. 2000; 275: 24686-24692Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 15Dubois E. Scherens B. Vierendeels F. Ho M.M. Messenguy F. Shears S.B. J. Biol. Chem. 2002; 277: 23755-23763Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 17Saiardi A. Erdjument-Bromage H. Snowman A.M. Tempst P. Snyder S.H. Curr. Biol. 1999; 9: 1323-1326Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar).In metazoans, biochemical studies have suggested that IP6 is synthesized from I(1,4,5)P3 via a five-step route of several kinases and a phosphatase as shown in Fig. 1B (2Majerus P.W. Annu. Rev. Biochem. 1992; 61: 225-250Crossref PubMed Scopus (348) Google Scholar, 5Shears S.B. Biochim. Biophys. Acta. 1998; 1436: 49-67Crossref PubMed Scopus (151) Google Scholar, 21Communi D. Vanweyenberg V. Erneux C. Cell Signal. 1995; 7: 643-650Crossref PubMed Scopus (40) Google Scholar). Two key differences between the proposed metazoan and defined yeast pathways are: 1) the initiation step in the synthesis of IP6 occurs through an I(1,4,5)P3 3-kinase (IP3K) and not Ipk2, and 2) the requirement of an I(1,3,4)P3 5/6-kinase, orthologs of which have not yet been found in the budding yeast genome. The cloning of plant and metazoan Ipk2 (also referred to as inositol phosphate “multi-kinase”; IPMK) and Ipk1, both of which were found to possess similar activities as their yeast counterparts, has raised the possibility that synthesis of higher IP messengers in metazoans may occur similarly to that in yeast (18Stevenson-Paulik J. Odom A.R. York J.D. J. Biol. Chem. 2002; 277: 42711-42718Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 20Verbsky J.W. Wilson M.P. Kisseleva M.V. Majerus P.W. Wente S.R. J. Biol. Chem. 2002; 277: 31857-31862Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 22Chang S.C. Miller A.L. Feng Y. Wente S.R. Majerus P.W. J. Biol. Chem. 2002; 277: 43836-43843Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 23Nalaskowski M.M. Deschermeier C. Fanick W. Mayr G.W. Biochem. J. 2002; 366: 549-556Crossref PubMed Scopus (82) Google Scholar, 24Saiardi A. Nagata E. Luo H.R. Sawa A. Luo X. Snowman A.M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2306-2311Crossref PubMed Scopus (93) Google Scholar, 25Shears S.B. Biochem. J. 2004; 377: 265-280Crossref PubMed Scopus (158) Google Scholar, 26Xia H.J. Brearley C. Elge S. Kaplan B. Fromm H. Mueller-Roeber B. Plant Cell. 2003; 15: 449-463Crossref PubMed Scopus (69) Google Scholar).To further probe the synthesis of higher IPs in metazoans at the molecular level we initiated studies in the fruit fly, Drosophila melanogaster. Using a bioinformatics approach, we identified orthologous kinase and phosphatase genes in the Drosophila that may contribute to higher IP synthesis in metazoans. Detailed biochemical analysis was used to characterize the I(1,4,5)P3 kinases proposed to initiate the first phosphorylation step of higher IP production, and subsequent use of RNA interference in culture cells and transgenic flies provided gene-specific knockdowns of each activity to dissect the IP synthesis pathway. We find that the synthesis of IP5 and IP6 in the fruit fly is dependent on Ipk2 and Ipk1, but not IP3K, 5-phosphatase or I(1,3,4)P3 5/6-kinase activities. Our data provide a novel molecular basis for higher IP production in a metazoan and support a role for Ipk2 as a key gatekeeper for activating this important signaling pathway.MATERIALS AND METHODSPlasmid Construction—Drosophila EST SD14726, SD19941 were obtained from Research Genetics and used as templates for amplification of dmIpk2 and dmIP3Kβ, respectively. PCR reactions were carried out using the Expand High Fidelity PCR System (Roche Applied Science). The following primers were used: dmIP3Kβ: 5′-ATA TCC CCG GGA ATG CCG CGG GAC TAT GGC TAC-3′(forward) and 5′-GCT CTG TTA ACG TCT AGA TTA GGG TTT GCT CTC TTC-3′(reverse), dmIpk2: 5′-ACG TCT GGA ATT CAG ATG GCC AAG AGT GAT CAG GAG-3′ (forward) and 5′-TGA GCT CGA GTC GAC TCA TCG GTG GAG TAT TGA TTG-3′ (reverse). PCR products were then cut with the following enzymes and ligated in-frame with the pUNI10 Lox recombination site or into the pGEX-KG GST expression vector. dmIP3Kβ: SmaI and XbaI (for pUNI10 used SmaI and HpaI), dmIpk2: EcoRI and SalI. pGEX-KG plasmids were transformed into DH5α-competent cells. pUNI10 constructs were recombined into a pRS314 host vector containing Lox site, MYC3, TRP1, CEN, and a copper inducible promoter (CUP1). The recombined plasmid was transformed into ipk2Δ and ipk2Δ ipk1Δ yeast strains using standard yeast transformation techniques.Bacterial Expression of dmIpk2 and dmIP3Kβ—For recombinant protein expression, transformed Escherichia coli (DH5α) were grown at 37 °C to an OD600 of 0.6 and induced with 0.1 mm isopropyl-1-thio-β-d-galactopyranoside for 4 h at 30 °C. Cells were recovered by centrifugation at 4 °C, resuspended in ice-cold 50 mm Tris-HCl pH 7.5, 50 mm KCl, 5 mm dithiothreitol, Complete Mini protease inhibitor mixture (Roche Applied Science) and lysed with four passes through a cell cracker (a high shear fluid processing system for cell rupture, Microfluidics Corp.). Lysates were then cleared by centrifugation at 14,000 × g. The GST fusion proteins were purified over glutathione Sepharose (Amersham Biosciences) according to the manufacturer's instructions. Proteins were eluted from the glutathione Sepharose using 50 mm Tris-HCl pH 8.0, 50 mm NaCl, 5 mm dithiothreitol, and 20 mm glutathione. For antibody production GST-dmIpk2 was cleaved with thrombin protease according to the manufacturer's instructions (Amersham Biosciences). Purified protein was injected into rabbits and antiserum was isolated (BIOSOURCE). dmIpk2 protein was detected using standard Western blotting techniques.Inositol Phosphate Kinase Assay—All unlabeled IPs were purchased from Cell Signals, Inc. Tritiated IPs were purchased from PerkinElmer Life Sciences. I(1,4,5,6)[32P]P4 was generated using 500 ng of recombinant Arabidopsis Ipk2 in buffer containing 50 mm Tris, pH 7.5, 50 mm NaCl, 10 mm MgCl2, 5 μm I(1,4,5)P3, and trace amounts of [32P]ATP. To generate I(1,3,4,6)[32P]P4, unlabeled 5.0 μm I(1,4,5)P3 was incubated at 37 °C in the presence of 7.5 μg dmIP3K and trace amounts of [γ-32P]ATP in buffer containing 50 mm HEPES (pH 7.5), 50 mm NaCl, and 10 mm MgCl2 to create I(1,3,4,5)-[32P]P4. Human Type I 5-phosphatase (900 ng) was then added to this reaction and incubated at 37 °C to produce I(1,3,4)[32P]P3. The I(1,3,4)[32P]P3 product was incubated at 37 °C in a reaction containing 2 mm ATP, 10 mm MgCl2, 14 mm phosphocreatine, phosphocreatine kinase, 0.1 mg/ml BSA, 600 ng of human type I 5-phosphatase and 200 ng of 5/6 kinase (a kind gift from Steve Shears) to yield I(1,3,4,6)[32P]P4. Kinase assays were carried out essentially as described by Stevenson-Paulik et al. (18Stevenson-Paulik J. Odom A.R. York J.D. J. Biol. Chem. 2002; 277: 42711-42718Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar).Kinetic Assays—The Km and Vmax of the enzymatic interaction between dmIpk2 and dmIP3Kβ, and various substrates were determined. The following reaction mixture was prepared: 250 mm HEPES (pH 7.5), 250 mm NaCl, 10% glycerol, 0.1% BSA, 2 mm ATP, 100 mm MgCl2, 80 cpm/μl radiolabeled substrate, 10 ng dmIpk2 or dmIP3Kβ, and various concentrations of unlabeled substrate in a 20-μl reaction volume. The reaction was stopped by the addition of HPLC buffer or 1.0 m HCl. 10 mm NH4H2PO4 (pH 3.5) was then added to the reaction and analyzed by Partisphere strong anion exchange HPLC. The kinase kinetics was calculated from measured changes in the substrate area under the curve (AUC) peak.RNAi in S2 Cultured Cells—Drosophila ESTs SD14726, SD19941, RE1770, and GH07317 were obtained from Research Genetics and used as templates for amplification of dmIpk2, dmIP3Kβ, dm5PtaseI, and dmIpk1, respectively. dmIP3Kα template was amplified using genomic DNA prepared from S2 cells. FTZ (dsRNA control) template was amplified from a cDNA library provided by Rick Fehon. Templates for dsRNA synthesis were made by PCR using the EST clones as templates and the following primers. dmIpk2: 5′-TTAAT ACGAC TCACT ATAGG GAGAA CAGGT TGCGG GTCAC ACATT-3′ (forward) and 5′-TTAAT ACGAC TCACT ATAGG GAGAT GCAGC TGGCG GAGTA CTTCC-3′ (reverse), dmIP3Kβ: 5′-TTAAT ACGAC TCACT ATAGG GAGAA CCGGC AAGAA GCAGA GCTCC-3′(forward) and 5′-TTAAT ACGAC TCACT ATAGG GAGAC GCTCT TCTTG ATGCC CTCGA-3′ (reverse), dm5Ptase: 5′-TTAAT ACGAC TCACT ATAGG GAGAG GATGT GTTTC TGGTC ACGGC-3′ (forward) and 5′-TTAAT ACGAC TCACT ATAGG GAGAG GTATG AACTA GGGCT CTTCT G-3′ (reverse), (dsRNA control) Ftz: 5′-TTAAT ACGAC TCACT ATAGG GAGAG CCGAC AACAT GAACA TGTAC-3′ (forward) and 5′-TTAAT ACGAC TCACT ATAGG GAGAC CATTC TTCAG CTTCT GCGTC-3′ (reverse), dmIpk1: 5′-TTAAT ACGAC TCACT ATAGG GAGAG GAGCA GCGAG GAGTG GTGGA G-3′ (forward) and 5′-TTAAT ACGAC TCACT ATAGG GAGAC GTTTA TCCAG AATCT ATCGT C-3′ (reverse), dmIP3Kβ: 5′-TTAAT ACGAC TCACT ATAGG GAGAG ACTCA AGCAG CTATG GAAGC-3′ (forward) and 5′-TTAAT ACGAC TCACT ATAGG GAGAG CTGCA CTTGA CACTT GAAAC G-3′ (reverse). Primers all contain T7 promoters on their 5′-ends. Transcription reactions were carried out using the DNA templates with the MEGAscript T7 transcription kit (Ambion). The RNA was purified by phenol:chloroform extraction and isopropyl alcohol precipitation and resuspended in DEPC-treated H2O to a final concentration of 3 μg/μl. RNA strands were then annealed by heating to 70 °C for 30 min and cooling to room temperature overnight.S2 cells were propagated in Schneider's Drosophila media (Invitrogen) containing 10% fetal calf serum dialyzed and heat inactivated (Hyclone) and 10 μg/ml penicillin and streptomycin. The cells were pelleted and resuspended in Drosophila-SFM (Invitrogen) to a density of 1 × 106 cells/ml and plated into 6-well plates (1× 106 cells/well). 15 μg of dsRNA was added to each well and incubated at room temperature for 1 h. The cells were then supplemented with 2 ml of Schneider's Drosophila media/fetal bovine serum/Penn/Strep and incubated for 4–6 days at 25 °C. In the case that RNAi knockdowns were extended over a period of 15 days, dsRNA-treated cells were counted after 8 days, split, and again treated with dsRNA as described above.Northern Analysis of S2 Cells—Probes for Northern analysis were made using DNA templates for dsRNA as described above. DNA templates were then incubated with 32P-labeled dCTP and the random-primed DNA labeling kit (Amersham Biosciences) according to the manufacturer's instructions. S2 cells were treated with dsRNA for 6 days as described above. 3 ml of cells were then recovered by centrifugation at 4 °C. Total RNA was then isolated using the Qiagen RNA purification system according to the manufacturer's instructions. RNA was then separated on a 2% agarose gel and transferred to hybond nitrocellulose paper (Amersham Biosciences). The DNA probe was then hybridized in ExpressHyb hybridization solution according to the man ufacturer's instructions (Clontech).Kinase Assays on S2 Cell Extracts—dsRNA-treated cells (3 ml) were recovered by centrifugation at 4 °C. Pellets were resuspended in 200 μl of ice-cold 50 mm Tris, pH 7.5, 3 mm MgCl2, 2.5 mm EGTA, 0.5 mm EDTA, 1 mm dithiothreitol, and Complete Mini protease inhibitor mixture. The cells were lysed with ten half-second pulses, three times on a setting of 3 using a Branson sonifier and diluted to a stock concentration of 1 mg/ml. Activity assays were carried out in 20-μl volumes with 5 μg of S2 extract, 10 mm HEPES pH 7.5, 10 mm NaCl, 10 mm MgCl2, 2 mm ATP, 0.25 units/μl creatine phosphokinase, 10 mm phosphocreatine, 5 μm inositol polyphosphate, and 10,000–20,000 cpm labeled inositol polyphosphate. Labeled inositol polyphosphates were [3H]I(1,4,5)P3, I(1,4,5,6)[32P]P4, and 32P-I(1,3,4,5,6)P5. Reactions were incubated at 37 °C for various times from 5 to 200 min. Reactions were stopped with the addition of 0.5 n HCl and analyzed by HPLC or TLC.In Vivo Labeling of S2 Cells and Saccharomyces cerevisiae—S2 cells were treated with dsRNA as described above or no dsRNA as a control. On day four or thirteen, [3H]inositol (American Radiolabeled Chemicals) was added to the media to a final concentration of 80 μCi/ml. Cells were then incubated for 2 days at 25 °C. On day 6 or 15, the cells were recovered by centrifugation and washed once in Dulbecco's phosphate-buffered saline (Invitrogen Life Technologies, Inc.). Yeast cultures were incubated in minimal media lacking tryptophan and 150 μm CuSO4 for 2 days at 30 °C for 2 days. [3H]Inositol was added to a final concentration of 80 μCi/ml. Soluble inositol polyphosphates were harvested and analyzed by HPLC using a Partisphere SAX strong-anion exchange column as described previously (7York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (443) Google Scholar).Construction and Analysis of Fly Lines—Df(2L)BCS16 was obtained from the Bloomington stock center. NleΔ8 was a kind gift from S. Cohen (27Royet J. Bouwmeester T. Cohen S.M. EMBO J. 1998; 17: 7351-7360Crossref PubMed Scopus (57) Google Scholar). For dmIpk2 RNAi, we obtained the pWIZ vector from Richard Carthew to construct a transgene containing inverted repeats of a 200-bp region of dmIpk2 separated by a 74-bp intron spacer (28Lee Y.S. Carthew R.W. Methods. 2003; 30: 322-329Crossref PubMed Scopus (306) Google Scholar). The following primers were used to generate the dmIpk2 region containing XbaI restriction sites on both ends: 5′-CTAGT CTAGA ACGAC TATTC AAGAC TGGCT G-3′ (forward) and 5′-CTACT CTAGA TCATC ATCGG TGGAG TATTG-3′ (reverse). The PCR product was then subcloned into the pWIZ vector using AvrII and NheI sites and checked for proper orientation. For expression of FLAG-dmIpk2 we cloned the dmIpk2 coding region into pBSIISK with sequence for an N-terminal FLAG epitope inserted (a kind gift from Rick Fehon). The following primers were used to PCR dmIpk2 from the EST containing SmaI and HindIII restriction sites: 5′-TCTGG ACCCG GGATG GCCAA GAGTG ATCAGG AG-3′ (forward) and 5′-TGAGC T CGA A AGCTT TCATC GGTGG AGTAT TGATT G-3′ (reverse). The FLAG epitope and dmIpk2 were then subcloned into pP[UAST]. pWIZ-Ipk2 and pP[UAST]-FLAG-dmIpk2 constructs and a P-element transposase plasmid were injected in w1118 embryos and germ line transformants were selected with the w+ marker (29Beall E.L. Mahoney M.B. Rio D.C. Genetics. 2002; 162: 217-227PubMed Google Scholar). Homozygous w+ lines were generated with standard balancer chromosomes. For fly labeling, 3–6-day-old adult males were fed a 5% sucrose solution containing 500 μCi/ml [3H]inositol for 4 days. 2B. Elliot and V. Bankaitis, personal communication. Soluble IPs were extracted as described above.Salivary Gland Stains—FLAG-dmIpk2 expression was induced by crossing p[FLAG-dmIpk2]/cyo to Gal4 expressing flies under the actin promoter (P[FLAG-dmIpk2]/Actin). Salivary glands were dissected from 3rd instar larvae and fixed in PBS + 0.1% Triton X-100 (PBS-T) and 3.7% paraformaldehyde for fifteen minutes. Glands were then washed in PBS-T and blocked overnight in PBS-T + 5% goat serum. Anti-FLAG M2 monoclonal antibody (Sigma) was added to the blocking buffer and incubated for one hour with agitation. Salivary glands were washed three times with PBS-T. An anti-mouse cy3 secondary antibody (Jackson ImmunoResearch) was incubated with the salivary glands for 1 h. The glands were washed, stained with DAPI (Sigma), and mounted with ProLong Antifade (Molecular Probes). Glands were visualized with a Zeiss Axioscope equipped with Metamorph software.RESULTSIdentification of Drosophila Genes Involved in Inositol Polyphosphate Synthesis—In this study, we utilize the tractable fly model system to study mechanisms of higher IP synthesis in metazoans. Initially, our analysis focused on two I(1,4,5)P3 kinase-dependent IP synthesis models, one derived from genetic yeast studies and one proposed from biochemical characterization of pathways metazoans (Fig. 1A). The initiation steps for each model are distinct and require I(1,4,5)P3 kinase activities encoded by separate gene products. Ipk2 functions in yeast as a dual specificity kinase that converts I(1,4,5)P3 to I(1,3,4,5,6)P5 and in metazoans IP3K phosphorylates I(1,4,5)P3 to I(1,3,4,5)P4. Of interest, the two I(1,4,5)P3 kinases are members of a small family of IP kinases that share an evolutionary ancestor and common amino acid motifs, but possess divergent substrate specificities (6York J.D. Bradshaw R.A. Dennis E.A. Handbook of Cell Signaling, Volume 2. 2. Elsevier Science, 2003: 229-232Google Scholar, 8Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (342) Google Scholar). The second and third steps of the pathway proposed in metazoans require inositol polyphosphate 5-phosphatase, which converts I(1,3,4,5)P4 to I(1,3,4)P3, and I(1,3,4)P3 5/6-kinase that produces I(1,3,4,6)P4 (30Majerus P.W. Kisseleva M.V. Norris F.A. J. Biol. Chem. 1999; 274: 10669-10672Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). The fourth step in the metazoan pathway is proposed to be accomplished through the 5-kinase activity of Ipk2, which produces I(1,3,4,5,6)P5 from the I(1,3,4,6)P4 substrate. The last step for IP6 synthesis in both yeast and metazoans requires Ipk1, an I(1,3,4,5,6)P5 2-kinase.In order to identify various kinase and phosphatase members of these pathways, we performed in silico searches (www.ncbi.nlm.nih.gov/BLAST/and www.flybase.net/blast/) of the Drosophila genome using hallmark motifs for each gene product. Four predicted gene products were found that harbor the IP kinase motif PXXXDXKXG: CG13688 (PcvmDvKmG), CG4026 (PcvmDiKmG), CG1630 (PcvmDcKvG), and CG10082 (PcilDlKmG). Sequence analysis of CG13688 (designated here as dmIpk2) revealed that it is most similar to Ipk2 homologs from other species (Fig. 1B). dmIpk2 is a 310 amino acid polypeptide encoded by a single exon with 37% similarity and 24% identity to the human Ipk2. CG10082 (designated dmIP6K) shared the highest degree of homology with mammalian IP6 kinases (also referred to as diphosphoryl inositol synthetases). IP6Ks use IP5 and IP6 as substrates to generate inositol pyrophosphate species. The putative dmIP6K open reading frame codes for an 893 amino acid protein and contains 36% similarity to human IP6K2 (GenBank™ accession number AF177145). Further studies will be required to elucidate whether this putative gene translates into a functional IP6 kinase as it will not be discussed here. The other two PXXXDXKXG-containing proteins (CG4026 and CG1630) share 59% similarity and are highly homologous to mammalian IP3K (Fig. 1B). Three different isoforms of IP3K (A–C) are described in higher eukaryotes that differ in sequence, tissue and subcellular localization. CG4026 (dmIP3Kα, originally annotated as dmIP3K1) was previously identified as an IP3K that confers resistance to oxidative stress when overexpressed in adult flies (31Monnier V. Girardot F. Audin W. Tricoire H. Free Radic. Biol. Med. 2002; 33: 1250-1259Crossref PubMed Scopus (32) Google Scholar). CG1630 (dmIP3Kβ, originally annotated as dmIP3K2) is most similar to the human IP3K B (45% similar and 37% identical). The putative IP3Ks do not contain calmodulin-binding sites at their N terminus as are found in mammalian I(1,4,5)P3 3-kinases.Type I enzymes in the 5-phosphatase family have high catalytic efficiency as D5 specific phosphatases against I(1,4,5)P3 and I(1,3,4,5)P4, and contain a highly conserved motif (r/n)-XP(s/a)(w/y)(c/t)DR(i/v)(l/i) (30Majerus P.W. Kisseleva M.V. Norris F.A. J. Biol. Chem. 1999; 274: 10669-10672Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). This pattern motif was used to search Drosophila genome and identified a predicted gene product, CG31107 (designated dm5PtaseI), of 400 amino acids having 58% similarity and 43% identity to the human type I 5-phosphatase identified. Although no other putative type I 5-phosphatases were identified in the annotated genome we found that Drosophila contain open reading frames that encode two type II 5-phosphatase isozymes (CG6805 and CG9784), a synapto" @default.
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• W2029207608 title "A Molecular Basis for Inositol Polyphosphate Synthesis in Drosophila melanogaster" @default.
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• W2029207608 cites W1953537738 @default.
• W2029207608 cites W1966677529 @default.
• W2029207608 cites W1971517152 @default.
• W2029207608 cites W1972877825 @default.
• W2029207608 cites W1973389973 @default.
• W2029207608 cites W1977870999 @default.
• W2029207608 cites W1980166477 @default.
• W2029207608 cites W1983536335 @default.
• W2029207608 cites W1990668649 @default.
• W2029207608 cites W1994071091 @default.
• W2029207608 cites W1994473838 @default.
• W2029207608 cites W1994974292 @default.
• W2029207608 cites W1997739014 @default.
• W2029207608 cites W2002312465 @default.
• W2029207608 cites W2006499135 @default.
• W2029207608 cites W2020238791 @default.
• W2029207608 cites W2021198651 @default.
• W2029207608 cites W2023543654 @default.
• W2029207608 cites W2028957094 @default.
• W2029207608 cites W2034654184 @default.
• W2029207608 cites W2046122733 @default.
• W2029207608 cites W2059141024 @default.
• W2029207608 cites W2063761685 @default.
• W2029207608 cites W2066432020 @default.
• W2029207608 cites W2069340514 @default.
• W2029207608 cites W2070433129 @default.
• W2029207608 cites W2080209900 @default.
• W2029207608 cites W2080419891 @default.
• W2029207608 cites W2087358641 @default.
• W2029207608 cites W2105933335 @default.
• W2029207608 cites W2107025263 @default.
• W2029207608 cites W2111888383 @default.
• W2029207608 cites W2112038940 @default.
• W2029207608 cites W2112919363 @default.
• W2029207608 cites W2124864193 @default.
• W2029207608 cites W2136120616 @default.
• W2029207608 cites W2146718650 @default.
• W2029207608 cites W2154052554 @default.
• W2029207608 cites W2159070856 @default.
• W2029207608 cites W2164577098 @default.
• W2029207608 cites W2395442271 @default.
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