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J. Biol. Chem., Vol. 279, Issue 45, 47222-47232, November 5, 2004
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From the
Departments of Pharmacology and Cancer Biology and of Biochemistry, Howard Hughes Medical Institute, Duke University Medical Center, DUMC 3813, Durham, North Carolina 27710 and Model System Genomics, DCMB Group, Box 91000, Duke University, Durham, North Carolina 27708
Received for publication, July 22, 2004 , and in revised form, August 10, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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or 
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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A molecular basis for the synthesis of higher IPs was first resolved in the budding yeast (Fig. 1A) (7, 8, 16). 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 (8, 17, 18). Ipk1 is a 2-kinase that utilizes primarily I(1,3,4,5,6)P5 but also has activity toward other IPs (7, 19, 20). A third kinase, Kcs1, is an inositol diphosphate synthase capable of phosphorylating IP5 or IP6 to generate PP-IP4 and PP-IP5 (10, 15, 17).
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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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, respectively. PCR reactions were carried out using the Expand High Fidelity PCR System (Roche Applied Science). The following primers were used: dmIP3K: 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. dmIP3K: SmaI and XbaI (for pUNI10 used SmaI and HpaI), dmIpk2: EcoRI and SalI. pGEX-KG plasmids were transformed into DH5-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 ipk2
and ipk2 ipk1 yeast strains using standard yeast transformation techniques.
Bacterial Expression of dmIpk2 and dmIP3K—For recombinant protein expression, transformed Escherichia coli (DH5) were grown at 37 °C to an OD600 of 0.6 and induced with 0.1 mM isopropyl-1-thio--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 x 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 [
-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. (18).
Kinetic Assays—The Km and Vmax of the enzymatic interaction between dmIpk2 and dmIP3K, 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 dmIP3K, 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, dmIP3K, dm5PtaseI, and dmIpk1, respectively. dmIP3K 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), dmIP3K: 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), dmIP3K: 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 x 106 cells/ml and plated into 6-well plates (1x 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 (7).
Construction and Analysis of Fly Lines—Df(2L)BCS16 was obtained from the Bloomington stock center. Nle8 was a kind gift from S. Cohen (27). 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 (28). 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 (29). 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.2 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 (GenBankTM accession number AF177145
[GenBank]
). 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 (dmIP3K, originally annotated as dmIP3K1) was previously identified as an IP3K that confers resistance to oxidative stress when overexpressed in adult flies (31). CG1630 (dmIP3K, 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) (30). 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 synaptojanin ortholog (CG6562), and a putative Type IV family member (CG10426). Based on our analysis, we hypothesized that Drosophila is capable of generating I(1,3,4)P3 through the activities of IP3K and 5-phosphatase. However, it does not appear that the annotated Drosophila genome possess gene products with significant sequence similarity to mammalian I(1,3,4)P3 5-/6-kinases, suggesting that the fly may not synthesize higher IPs through the proposed metazoan route. If this is the case then IP3K-dependent higher IP synthesis in Drosophila may employ a different set of enzymes than previously described.
To identify a Drosophila Ipk1, we searched the genome for putative 2-kinases containing conserved amino acid motifs including EXKPK (7, 19, 20). Using the human IP5 2-kinase (hsIpk1) amino acid sequence, we identified a single ortholog, CG30295 (designated dmIpk1) that translated into a polypeptide sharing 34% similarity and 25% identity to hsIpk1. In summary, our genome analysis indicate that Drosophila have many of the members of both yeast and metazoan pathways required for IP6 synthesis, with the notable exception of a I(1,3,4)P3 5/6-kinase. We therefore postulate that flies synthesize IP5 and IP6 through a pathway that requires the activities of dmIpk2 and dmIpk1.
Cloning and Characterization of Drosophila I(1,4,5)P3 Ki nases—Of the three I(1,4,5)P3 kinase gene products found, we tested if dmIpk2 and dmIP3K translated into functional I(1,4,5)P3-specific kinases. GST fusion proteins of each kinase were purified from E. coli and incubated with I(1,4,5)P3 plus ATP, and the reaction products were analyzed by HPLC. dmIP3K phosphorylated I(1,4,5)P3 to IP4 (Fig. 2, A and B); whereas dmIpk2 phosphorylated I(1,4,5)P3 to generate IP5 (Fig. 3, A and B). To confirm that the product of the dmIP3K reaction is I(1,3,4,5)P4, we tested it for sensitivity to purified human type I 5-phosphatase, a I(1,3,4,5)P4 5-phosphatase. Treatment completely converted the product to I(1,3,4)P3, demonstrating that dmIP3K functions as an I(1,4,5)P3 3-kinase (Fig. 2C). We determined the kinetic properties of dmIP3K and found that the kinase has a high affinity for I(1,4,5)P3 (Km = 72 nM), transfers the phosphate to the D3 position at a rate of 25 nmol/min/mg and has a catalytic efficiency of 2.47 x 105 s-1 M-1 (Table I).
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dmIpk2 Rescues IP6 Production in Yeast Ipk2-deleted Cells— Given the similar substrate specificity of dmIpk2 and yeast Ipk2, we performed complementation analysis of ipk2 mutant yeast. Yeast cells lacking Ipk2 fail to generate IP6 and accumulate I(1,4,5)P3 (Fig. 5A, top trace). We observed complete restoration of IP6 synthesis when we heterologously expressed dmIpk2 in yeast ipk2 (Fig. 5A, bottom trace). Deletion of Ipk1 in yeast results in the accumulation of its substrate, I(1,3,4,5,6)P5, and a diphosphoryl species PP-IP4 that is generated from IP5. Expression of dmIpk2 in yeast ipk1 ipk2 recapitulated the appearance of an ipk1 mutant as IP5 and PP-IP4 accumulated in these cells (Fig. 5B, bottom). These data provide evidence that dmIpk2 acts as a dual specificity IP4/IP5 kinase in vivo and can employ an Ipk1-dependent mechanism to generate IP6. Conversely, IP6 synthesis was not restored when dmIP3K was heterologously expressed in ipk2-null cells. This result is not surprising because yeast lack several enzymes that are needed to generate IP6 from I(1,3,4,5)P4, including I(1,3,4,5)P4 5-phosphatase and I(1,3,4)P3 5-/6-kinases. Taken together, our data suggest that dmIpk2, but not dmIP3K, can cooperate with an IP5 2-kinase to generate IP6 in dividing cells.
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, dmIpk2, and dm5PtaseI mRNA levels were reduced by at least 80%, 61%, and 82% respectively. Of note, the 61% dmIpk2 mRNA reduction may be an underestimate as degraded RNA interfered with our measurement. To this end, a Western blot analysis with a dmIpk2-specific polyclonal antibody revealed a 90% reduction in protein levels in cells treated with dmIpk2 dsRNA compared with untreated cells (Fig. 6B).
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and dmIpk2 toward the phosphorylation I(1,4,5)P3, we carried out kinase reactions from dsRNA-treated S2 extracts. Competing kinase and phosphatase reactions in the assays made individual specific activity measurements difficult to determine using conventional time course assays. Therefore, we analyzed reaction products at early time points (and less than 5% substrate conversion) by HPLC under conditions that separated IP molecules ranging from IP1 to IP5 (for example Fig. 6C). In vitro phosphorylation of I(1,4,5)P3 to IP4 and IP5 was significantly reduced in extracts made from either dmIP3K- or dmIpk2-depleted cells indicating 1) that each kinase was functional in extracts and 2) that the RNAi worked for both (data not shown). When dsRNA to both dmIP3K and dmIpk2 were simultaneously added to cells, extracts had little observable I(1,4,5)P3 phosphorylation as compared with an equal amount of control treated extract (Fig. 6C), suggesting that dmIpk2 and dmIP3K comprise nearly all the I(1,4,5)P3 kinase activities in S2 cells. Kinase activity derived from dmIP3K may not be detectable in appreciable amounts in S2 cells. We further quantified the effect of dmIpk2 knockdown on I(1,4,5,6)P4 kinase activity (Fig 7A) and found that RNAi treatment reduced activity over 95%.
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Molecular Analysis of Higher IP Production in Drosophila Schneider (S2) Cells—To determine the roles of the kinases and phosphatase in the cellular production of higher IPs, we steady state-labeled various RNAi-depleted S2 cells with [3H]inositol. HPLC analysis of the extracted radiolabeled IPs from control-treated S2 cells revealed two major peaks corresponding to IP5 and IP6, whereas I(1,4,5)P3 and IP4 peaks are relatively minor (Fig. 8A). The IP5 isomer detected co-elutes with both I(2,3,4,5,6)P5 and I(1,2,4,5,6)P5 standards, which under these conditions are not separable. We report that relative distribution of radioactivity into the Ins, IP6, and IP5 peaks is 
92, 3.0, and 0.8% of the total tritiated sample recovered from the soluble extract.
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or dmIpk2 by labeling cells that were RNAi-depleted for either gene. No significant changes in IP5 or IP6 synthesis were found in dmIP3K-depleted cells after six or fifteen days of knockdown (Fig. 8, B and F). Similarly, cells treated with dsRNA for dmIP3K or dmIP3K and dmIP3K together did not affect higher IP synthesis (data not shown). In contrast, IP5 and IP6 levels were significantly decreased in dmIpk2-depleted cells (Fig. 8, C and F). We found in repeated experiments that IP5 levels were often reduced below the limit of detection on our HPLC apparatus, whereas IP6 levels where decreased by an average of 75%. These data demonstrate that dmIpk2 is required for IP5 and IP6 synthesis in Drosophila S2 cells.
Our data indicate that dmIpk2 contributes to IP6 synthesis in S2 cells by converting I(1,4,5)P3 to I(1,3,4,5,6)P5, which may then be phosphorylated to IP6 by the 2-kinase dmIpk1. Analysis of steady-state radiolabeled dmIpk1-depleted S2 cells show that IP6 levels were reduced, as measured from four independent experiments, by an average of 44% (Fig. 8, D and F). RNAi of dmIpk1 also caused levels of I(1,2,4,5,6)P5 or I(2,3,4,5,6)P5 to decrease by up to 54% suggesting that the isomer is a product of the kinase via 2-phosphorylation of I(1,4,5,6)P4 or I(3,4,5,6)P4. It is also possible that the D2-phosphorylated IP5 peak arises from dephosphorylation of IP6 at the 3- or 1-position, in which case depletion of IP6 may also reduce this IP5 peak by mass action. Additionally, in the dmIpk1-depleted labeled cells, we observed the accumulation of a new IP5 isomer that co-elutes with an I(1,3,4,5,6)P5 standard. A similar buildup of I(1,3,4,5,6)P5 occurs in yeast ipk1, supporting the hypothesis that dmIpk1 uses I(1,3,4,5,6)P5 as a substrate in vivo (7). These data indicate that dmIpk1 contributes to IP6 synthesis in Drosophila S2 cells by phosphorylating I(1,3,4,5,6)P5. To further determine if IP6 synthesis occurs through a single pathway of phosphorylation by dmIpk2 and dmIpk1, we simultaneously treated cells with dsRNA for both kinases. IP levels from the double-depleted cells were indistinguishable from those seen when dmIpk2 was depleted alone (Fig. 8F). This suggests that dmIpk2 and dmIpk1 are epistatic in S2 cellular production of IP5 and IP6.
We also tested whether the Drosophila IP6 synthesis pathway requires a type I 5-phosphatase as predicted for step three in the metazoan model (Fig. 1A). This would be surprising in light of our results indicating that RNAi depletion of dmIP3K and dmIP3K (step 1 of that model) did not disrupt IP6 synthesis. Nonetheless, we treated cells with dsRNA targeting dm5PtaseI, radiolabeled with inositol and analyzed extracts by HPLC. Interestingly, rather than a depletion, we noticed a significant increase in IP5 and IP6 levels as compared with control cells (Fig. 8, E and F). Likewise, co-depletion of dm5PtaseI and dmIpk2 led to an elevation of IP5 and IP6 as compared with dmIpk2-depleted cells alone (Fig. 8, F and G). One possible explanation for this is that depletion of dm5PtaseI leads to an increase in an I(1,4,5)P3 pool that is shared with the dmIpk2-dependent pathway. Similar increases in higher IPs were measured when dmIP3K was depleted together with dmIpk2 or dmIpk1 suggesting that the kinase may also draw from the same I(1,4,5)P3 pool (Fig. 8, F and G).
Ipk2-mediated Higher IP Production in Adult Flies—We next tested whether higher IP synthesis in the adult fly depends on dmIpk2 through loss and gain of function analysis in transgenic insects. Wild-type (w1118) male flies were in vivo labeled by feeding them a 5% sucrose solution containing [3H]inositol for 4 days. Distribution of radioactive metabolites observed by HPLC analysis of the whole fly extracts revealed an IP profile similar to S2 cells (compare Fig. 8A to Fig. 9A, top panel). To test whether the IP synthesis depends on dmIpk2, we examined IP6 levels in fly lines that were deficient in expression of the kinase. Df(2L)BCS16 is a deficiency line that fails to complement Ush and Gsc that reside on either side of dmIpk2 at 21E2 (Kevin Cook-Bloomington stock center). Another deficiency line was obtained that is derived from a P-element excision from Nle, a neighboring gene to dmIpk2 (Nle8) (27). We speculated that the break points of the Nle8 deficiency include the dmIpk2 open reading frame. Additionally, we generated a transgenic line that expresses an inverted repeat of dmIpk2-specific sequence under the Gal4 promoter for RNAi (p[WIZ-Ipk2]) (28). Western blot analysis of flies from each line revealed that dmIpk2 levels were reduced in all three deficient lines as compared with wild type (Fig. 9B). Antigen levels of dmIpk2 in both Df(2L)BCS16 and Nle8 were reduced by 50%, consistent with them being heterozygous for dmIpk2 (Fig. 9B). Ubiquitous expression of the dmIpk2 inverted repeat using an Actin-Gal4 promoter caused dmIpk2 levels to decrease 81% (Fig. 9B).
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8 flies had IP6 levels that were reduced 20–30% as compared with w1118 flies whereas levels in the p[WIZ-Ipk2]/Actin-Gal4 flies were reduced by an average of 60% (Fig. 9C). The dose-dependent reduction in IP6 levels observed in the fly mutants, suggests that dmIpk2 activity is an important rate-limiting step in the synthesis pathway. Transgenic flies overexpressing epitope-tagged dmIpk2 under control of the actin promoter had a 5-fold increase in higher IP production as compared with wild-type flies (Fig. 9A, bottom trace). Interestingly, the levels of several different IP species increased in these flies, including IP2 and IP3 uncovering a potential catabolic component of the pathway. Collectively, our results demonstrate that regulation of higher IP production in adult flies requires a dmIpk2-dependent synthesis pathway.
The Cellular Localization of dmIpk2—Given the role and localization of yeast Ipk2 in the nucleus, we analyzed the subcellular localization of dmIpk2 in cultured cells and whole fly tissue. We observed that FLAG-tagged dmIpk2 localizes to the nucleus when expressed in Drosophila S2 cells, suggesting that Ipk2-dependent IP synthesis in the fly is nuclear (data not shown). In yeast, Ipk2 initiates a nuclear IP synthesis pathway whose products regulate events such as transcription and chromatin remodeling, raising the possibility that Ipk2 associates with active zones of chromatin DNA (7, 8, 11, 12). The Drosophila polytene chromosomes present in giant cells of the salivary glands derived from 3rd instar larvae represent a unique system to observe nuclear proteins. FLAG-tagged dmIpk2 was expressed in larvae under control of the actin promoter and immunofluorescence studies demonstrate that the majority of protein appears localized within the nucleus (Fig. 10, top panel). Closer examination of the nuclear localization and concomitant staining of the chromosomal DNA with DAPI revealed that the bulk of dmIpk2 does not appear to be bound to chromatin DNA itself, rather it appears enriched in the non-DAPI nucleosol (Fig. 10, bottom panel). The same distribution was observed for the endogenous protein using an anti-dmIpk2 antibody, indicating the FLAG-dmIpk2 localization was not a consequence of overexpression (data not shown). These data provide new insights into a possible nuclear role for Ipk2 and its products in D. melanogaster.
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or dm5PtaseI depletion causes an increase in IP levels is through expansion of an I(1,4,5)P3 pool. Both of these enzymes are known to modulate I(1,4,5)P3-stimulated calcium signaling through the phosphorylation or dephosphorylation of this second messenger (4). Depletion of either of the two enzymes could lead to increases in the I(1,4,5)P3 pool that is available for feeding into the Ipk2/Ipk1 pathway. Alternatively, separable IP synthesis pathways may exist in Drosophila that can impinge on each other when they are disrupted or down-regulated. Speed et al. (33) reported similar increases in inositol polyphosphate production in mammalian cells by depleting a human Type I 5-phosphatase in NIH3T3 cells using antisense RNA (33). They showed that 5-phosphatase depletion causes I(1,4,5)P3, I(1,3,4,5)P4, IP5, and IP6 levels to increase. Studies of IP3K-deficient mice indicate a phenotypic role for I(1,3,4,5)P4 production, in neuronal and T lymphocyte function (34–36). Consist with our data in flies, loss of IP3K B in mice did not decrease IP5 levels (IP6 was not shown) in labeled cells (35), although one cannot exclude the possible compensation by other IP3K isoforms. Is the Ipk2/Ipk1 pathway a common route of IP5 and IP6 synthesis in all eukaryotes? Based on our data and the evolutionary conservation of Ipk2/Ipk1 across species, the parsimonious answer is "yes". However, some studies in the literature challenge the exclusivity of this pathway. Substrate specificity studies of human Ipk2 indicate that a catalytic preference for I(1,3,4,6)P4 5-kinase versus I(1,4,5)P3 6-/3-kinase activities (22). Accordingly, human Ipk2 would act downstream of the I(1,3,4)P3 5-/6-kinase. Other studies of human Ipk2 show in vitro that human Ipk2 is a dual-specificity kinase that converts I(1,4,5)P3 to IP5 (23). Heterologous expression of human Ipk2 does not appear to restore IP5 synthesis in ipk2 deficient yeast (22). Thus it may be that humans, but not other metazoans, have evolved a distinct more complex pathway reminiscent of the metazoan model shown in Fig. 1A. It is also possible that different cell-types or tissues utilize more than one pathway to generate these important messengers.
It is also interesting that while Ipk1 and Ipk2 are conserved among all eukaryotes, the I(1,3,4)P3 5-/6-kinase is not found in yeast or flies. In mammalian cells, I(1,3,4)P3 5-/6-kinase alteration through overexpression or RNAi effects tumor necrosis factor induced apoptosis possibly through alteration of higher IPs (37). In plants, Shi et al. (38) provide evidence for a role for higher IP synthesis by disruption of the maize 5-/6-kinase open reading frame, which results in a 30% reduction in IP6 levels in seeds (38). Plant Ipk2 may therefore contribute to this pathway through phosphorylation of the D-5 position of I(1,3,4,6)P4 to generate I(1,3,4,5,6)P5. Since dmIpk2 also has 5-kinase activity toward I(1,3,4,6)P4 with a relative affinity that is comparable to the human and plant Ipk2 it is possible it serves a similar role in flies. Although, our sequence analysis of the Drosophila genome did not reveal a I(1,3,4)P3 5-/6-kinase gene product argues against this hypothesis.
Our data is consistent with a phospholipase C-dependent sequential phosphorylation of I(1,4,5)P3 to IP6. Drosophila possesses three different phospholipase C isoforms including NorpA, Plc21C, and Small wing (Sl) (39). Studies that examine these homologs may elucidate whether IP5 and IP6 are synthesized from I(1,4,5)P3 and provide an understanding of how their synthesis is regulated. Phenotypic analysis of our mutant flies and cell lines are ongoing and may expose functional roles for higher IP synthesis in eukaryotes. It is also important to note that there are phospholipase C-independent routes for IP6 synthesis reported in the literature. In the slime mold Dictyostelium, deletion of the sole phospholipase C gene does not significantly change higher IP synthesis (40). Biochemical evidence for phospholipase C-independent IP synthesis has been reported from plants and slime mold through the sequential phosphorylation of I(3)P or inositol (41–43).
It is tempting to speculate that Drosophila Ipk2 and Ipk1 comprise a nuclear inositol signaling pathway that regulates nuclear processes similar to those in budding yeast. The evolutionarily conservation of the nuclear localization of Ipk2 from a variety of species is consistent with a conserved functional role. IP4 and IP5 in yeast are implicated as messengers that mediate transcriptional events (8), possibly through the regulation of chromatin remodeling (11, 12). IP6 production is implicated in regulation at the nuclear pore and is required for the efficient export of mRNA from the nucleus (7, 44). The localization of dmIpk2 to the nucleosol and not bulk chromatin provides the first evidence indicating that the majority of Ipk2 is not physically associated with chromatin. It is possible that activation of signaling promotes the movement of a minor pool of Ipk2 to chromatin, or that local production of IP4 or IP5 in the nucleosol activates transcriptional programs or chromatin remodeling. The generation of GFP-dmIpk2 transgenic flies will facilitate future studies aimed at examining potential dynamic changes in Ipk2 localization on polytene chromosomes during transcription.
If Ipk2 has an important role in regulating nuclear, or cytoplasmic function in Drosophila, we may observe phenotypic changes in transgenic flies. Currently, we have not observed a gross effect on development in either RNAi depleted (60% knockdown of cellular IP production) or overexpressing flies (5-fold increase in IP production). This may not be such a surprise since: 1) Ipk2 is conditionally essential in yeast; 2) several mutant Ipk2 lines with impaired but not complete loss of function in many cases appear normal; and 3) overexpression of Ipk2 in yeast has no observable phenotype. These data indicate that only a small amount of Ipk2 is required for regulation of nuclear function and that overproduction is well tolerated. Thus, our future phenotypic analysis will focus on specific tissues and/or the complete loss of function of Ipk2 through genetic mutation or deletion.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Pharmacology and Cancer Biology, Howard Hughes Medical Institute, Duke University Medical Center, DUMC 3813, Durham, NC 27710. Tel.: 919-681-6414; Fax: 919-668-0991; E-mail: yorkj{at}duke.edu.
1 The 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.
2 B. Elliot and V. Bankaitis, personal communication.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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